Dissertations / Theses: 'PCSK1' – Grafiati (2024)

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Author: Grafiati

Published: 7 July 2024

Last updated: 7 July 2024

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1

Choquet, Hélène. "Contribution du gène PCSK1 aux formes monogéniques et polygéniques d'obésité." Phd thesis, Université du Droit et de la Santé - Lille II, 2010. http://tel.archives-ouvertes.fr/tel-00576415.

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Quatre études de liaison génome entier ont mis en évidence une région commune de5,6 Mb dans la région du chromosome 5q15 liée à des traits associés à l'obésité, cette région incluant le gène de la prohormone convertase 1 (PCSK1). Une mutation Pc1 chez la souris a été associée à l'obésité, l'hyperphagie et à une augmentation de l'efficacité du métabolisme. La déficience complète en PCSK1 a été associée à une forme récessive rare d'obésité chezl'homme, et depuis 1997 seuls trois patients présentant cette déficience ont été décrits dans la littérature. Les porteurs de mutations délétères PCSK1 présentent des phénotypes sévères,incluant l'obésité, des hypoglycémies post-prandiales et des problèmes intestinaux ethormonaux. Contrairement aux observations faites chez la souris, les membres des famillesporteurs hétérozygotes ont été considérés comme cliniquement sains. Toutes ces études ontdésigné PCSK1 comme un gène candidat important pour l'obésité.Dans un premier temps, la contribution du gène PCSK1 au risque d'obésitépolygénique a été évaluée chez 13,659 individus d'origine européenne issus de huit cohortescas contrôles ou familiales indépendantes. Neuf variants fréquents couvrant 92% de lavariabilité génétique du locus ont été génotypés. Les méta-analyses des huit études pour levariant commun rs6232 et pour le cluster rs6234-rs6235 ont montré une associationreproductible avec l'obésité chez l'adulte et chez l'enfant (P=7.27x10-8 et P=2.31x10-12respectivement). Le rs6232 était associé à une augmentation du risque d'obésité de 34%, alorsque le cluster rs6234-rs6235 augmentait le risque d'obésité de 22%. Les analysesfonctionnelles ont montré une diminution significative de 10,4% de l'activité catalytique de laprotéine PC1/3 pour le N221D, et une diminution non significative de l'activité catalytique dela protéine PC1/3 pour le cluster Q665E/S690T.L'implication du gène PCSK1 dans l'obésité monogénique a ensuite été entreprise parle séquençage des exons de PCSK1 chez 845 sujets obèses non-consanguins d'origineeuropéenne,. Huit nouvelles mutations non-synonymes ont été identifiées. L'étude des conséquences fonctionnelles des mutations détectées sur la protéine PC1/3 a montré que62.5% de ces mutations détectées étaient prédites délétères par les analyses in silico et 87.5%de ces mutations avaient un effet sur l'auto-activation ou sur l'activité enzymatique de PC1/3in vitro. Dans le but d'estimer le degré de pénétrance pour ces sept mutations pathogéniques,6,060 obèses et 6,274 sujets minces ont été génotypés, démontrant un enrichissem*nt par sixde ces mutations PCSK1 chez les sujets obèses (P=0.007). Cette étude a mis en évidence pourla première fois une augmentation du risque d'obésité chez les porteurs hétérozygotes de mutations perte de fonction du gène PCSK1, confirmant un mode de transmission codominantde l'obésité avec une pénétrance incomplète. La pénétrance de l'obésité a été105estimée à 54.5% pour les porteurs hétérozygotes de mutations délétères PCSK1. Unedéficience partielle en PCSK1 pourrait expliquer environ 0.83% des formes extrêmesd'obésité et représenter la seconde forme la plus fréquente d'obésité monogénique après ladficience en MC4R.Pour conclure, en plus des formes syndromiques très rares d'obésité dues à unedéficience complète en PCSK1, ce travail a permis de démontrer le rôle des variants codantsfréquents non-synonymes dans le risque d'obésité, ainsi que l'importance longtempsinsoupçonné d'une déficience partielle en PCSK1 dans les formes monogéniques d'obésité.

2

Choquet, Hélène. "Contribution du gène PCSK1 aux formes monogéniques et polygéniques d’obésité." Thesis, Lille 2, 2010. http://www.theses.fr/2010LIL2S012/document.

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Quatre études de liaison génome entier ont mis en évidence une région commune de5,6 Mb dans la région du chromosome 5q15 liée à des traits associés à l’obésité, cette région incluant le gène de la prohormone convertase 1 (PCSK1). Une mutation Pc1 chez la souris a été associée à l’obésité, l’hyperphagie et à une augmentation de l’efficacité du métabolisme. La déficience complète en PCSK1 a été associée à une forme récessive rare d’obésité chezl’homme, et depuis 1997 seuls trois patients présentant cette déficience ont été décrits dans la littérature. Les porteurs de mutations délétères PCSK1 présentent des phénotypes sévères,incluant l’obésité, des hypoglycémies post-prandiales et des problèmes intestinaux ethormonaux. Contrairement aux observations faites chez la souris, les membres des famillesporteurs hétérozygotes ont été considérés comme cliniquement sains. Toutes ces études ontdésigné PCSK1 comme un gène candidat important pour l’obésité.Dans un premier temps, la contribution du gène PCSK1 au risque d’obésitépolygénique a été évaluée chez 13,659 individus d’origine européenne issus de huit cohortescas contrôles ou familiales indépendantes. Neuf variants fréquents couvrant 92% de lavariabilité génétique du locus ont été génotypés. Les méta-analyses des huit études pour levariant commun rs6232 et pour le cluster rs6234-rs6235 ont montré une associationreproductible avec l’obésité chez l’adulte et chez l’enfant (P=7.27x10-8 et P=2.31x10-12respectivement). Le rs6232 était associé à une augmentation du risque d’obésité de 34%, alorsque le cluster rs6234-rs6235 augmentait le risque d’obésité de 22%. Les analysesfonctionnelles ont montré une diminution significative de 10,4% de l’activité catalytique de laprotéine PC1/3 pour le N221D, et une diminution non significative de l’activité catalytique dela protéine PC1/3 pour le cluster Q665E/S690T.L’implication du gène PCSK1 dans l’obésité monogénique a ensuite été entreprise parle séquençage des exons de PCSK1 chez 845 sujets obèses non-consanguins d’origineeuropéenne,. Huit nouvelles mutations non-synonymes ont été identifiées. L’étude des conséquences fonctionnelles des mutations détectées sur la protéine PC1/3 a montré que62.5% de ces mutations détectées étaient prédites délétères par les analyses in silico et 87.5%de ces mutations avaient un effet sur l’auto-activation ou sur l’activité enzymatique de PC1/3in vitro. Dans le but d’estimer le degré de pénétrance pour ces sept mutations pathogéniques,6,060 obèses et 6,274 sujets minces ont été génotypés, démontrant un enrichissem*nt par sixde ces mutations PCSK1 chez les sujets obèses (P=0.007). Cette étude a mis en évidence pourla première fois une augmentation du risque d’obésité chez les porteurs hétérozygotes de mutations perte de fonction du gène PCSK1, confirmant un mode de transmission codominantde l’obésité avec une pénétrance incomplète. La pénétrance de l’obésité a été105estimée à 54.5% pour les porteurs hétérozygotes de mutations délétères PCSK1. Unedéficience partielle en PCSK1 pourrait expliquer environ 0.83% des formes extrêmesd’obésité et représenter la seconde forme la plus fréquente d’obésité monogénique après ladficience en MC4R.Pour conclure, en plus des formes syndromiques très rares d’obésité dues à unedéficience complète en PCSK1, ce travail a permis de démontrer le rôle des variants codantsfréquents non-synonymes dans le risque d’obésité, ainsi que l’importance longtempsinsoupçonné d’une déficience partielle en PCSK1 dans les formes monogéniques d’obésité
Four whole genome studies basing on positional cloning approach revealed a region ofchromosome 5q linked to traits related to obesity, this region contained the gene coding forthe prohormone convertase 1 named PCSK1. Pc1 mutation in mice has been associated withobesity, hyperphagia and increased metabolic efficiency. In human, PCSK1 deficiency is amonogenic form of obesity. The first case of complete PCSK1 deficiency has been identifiedin 1997 and since two other cases were discovered. Deleterious PCSK1 mutations carrierswere either hom*ozygous or compound heterozygous and presented severe phenotypes, such asobesity, intestinal troubles and endocrine disorders. Surprisingly, the family members whowere heterozygous for these mutations appeared clinically unaffected. Overall of these studieshighlighted PCSK1 as a candidate gene for obesity.We have therefore decided to assess the contribution of PCSK1 gene to polygenicobesity risk. To assess the contribution of PCSK1 to polygenic obesity risk, we genotyped tagsingle nucleotide polymorphisms in a total of 13,659 European individuals from eightindependent case-control or family-based cohorts. The non-synonymous variants rs6232,encoding N221D, and cluster rs6234-rs6235, encoding the Q665E-S690T pair, wereconsistently associated with obesity in adults and children (P=7.27 x 10-8 and P=2.31 x 10-12,respectively). Functional analysis revealed a significant impairment of the N221D mutant onPC1/3 protein catalytic activity.In continuity of this study we decided to assess the involvement of PCSK1 gene inmonogenic obesity, knowing that only three cases of complete PCSK1 deficiency have beenreported up to now. The objectives of this study were to evaluate the prevalence of rarePCSK1 mutations contributing to human obesity and to investigate the mode of inheritance ofobesity in the context of PCSK1 deficiency. We sequenced exons of the PCSK1 gene in 845non-consanguineous extremely obese subjects of European origin and we identified eightnovel PCSK1 non-synonymous mutations in eight carriers, all heterozygous. Wecharacterized the functional consequences of the detected mutations on PC1/3 protein and wefound that 62.5% of mutations detected were predicted to be deleterious in silico and werevealed that 87.5% of mutations had an effect on the autoactivation or on the enzymaticactivity of PC1/3 in vitro. In order to estimate the degree of penetrance for the sevenpathogenic mutations, we genotyped 6,060 obese and 6,274 lean subjects. We assessed a 6-fold enrichment of these PCSK1 mutations in obese subjects (P = 0.007). We provided thefirst evidence of an increased obesity risk in heterozygous carriers of loss of functionmutations in PCSK1 gene, confirming a co-dominant mode of transmission of obesity withincomplete penetrance for this gene. The penetrance of obesity was estimated to 54.5% for108heterozygous carriers of deleterious PCSK1 mutations. Partial PCSK1 deficiency mightexplain ~ 0.83% of extreme obesity.To conclude, in addition of the syndromic forms of obesity due to a complete PCSK1deficiency, we provided the strong evidence of the contribution of common non-synonymousvariants in obesity risk and we highlighted that a partial PCSK1 deficiency is associated withan increased risk of obesity

3

Guerardel, Audrey. "Analyse de deux gènes candidats physiologiques et positionnels de l'obésité humaine CART et PCSK1." Lille 2, 2005. http://www.theses.fr/2005LIL2S012.

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L'obésité commune est une maladie multifactorielle, dont l'émergence récente comme maladie de masse, est liée à l'occidentalisation du mode de vie. Elle est la conséquence d'une diminution de l'activité physique et d'un accès illimité à une alimentation calorique. Néanmoins, les facteurs génétiques influencent la prédisposition individuelle, comme l'attestent des études familiales et l'identification de formes monogéniques d'obésité. Les déterminants des formes polygéniques fréquentes (95%) sont encore peu caractérisés, même si des études récentes ont montré notamment le rôle de certains gènes dans la signalisation de l'insuline (ENPP1) et les voies métaboliques de neurotransmetteurs (GABA, sérotonine), qui prédisposeraient à l'obésité dans un environnement peu favorable. L'implication des facteurs génétiques dans les maladies polygéniques tel que l'obésité est abordée par des approches directes d'analyses de gènes candidats physiologiques et indirectes par l'étude de gènes candidats positionnels localisés dans des régions chromosomiques de liaison à des traits phénotypiques. Deux études " génome entier " sur des familles caucasiennes françaises ont permis de souligner l'importance du locus 5cen-q comme région de susceptibilité à la variation du taux de leptine et à l'obésité. Parmi les nombreux gènes localisés dans cette région ; les gènes CART (5q12-q13) et PCSK1 (5q15-q21) sont exprimés au niveau du système nerveux central dans des régions impliquées dans le contrôle de la prise alimentaire et le maintien de l'homéostasie énergétique et sont des acteurs de la voie de la leptine. L'analyse de 5,4 kb du gène CART (Cocaine and Amphetamine Regulated Transcript), a permis l'identification d'un SNP de la région promotrice (SNP-3608T>C) qui pourrait être un composant des déterminants génétiques de l'obésité polygénique. Ce polymorphisme se révèle également être associé, dans une population générale, avec les sous-fractions de cholestérol et les apolipoprotéines plasmatiques suggérant une implication du gène CART dans l'athérogénèse. Ce SNP aurait également un effet dans le remodelage de la masse osseuse corticale dans une population de femmes danoises. Le gène PCSK1 (Proprotein Convertase Subtilisin/kexin type 1) code une endoprotéase à sérine de type subtilisine, impliquée dans la maturation de pro-hormones et de neuropeptides précurseurs tels que la proinsuline et POMC. Des mutations du gène PCSK1 responsables d'une forme monogénique rare d'obésité sévère de l'enfant ont été décrites. L'analyse de ce gène dans un contexte polygénique a permis l'identification de SNP fréquents (dont une mutation exonique non-synonyme) associés à l'obésité adulte et/ou infantile. L'approche génétique a validé des hypothèses physiologiques et a amélioré notre compréhension des voies métaboliques impliquées en suggérant l'effet de ces deux gènes dans l'obésité polygénique et le rôle pléiotropique du gène CART
Common obesity is a multifactorial disease, whose recent increase, is related to the modernization of life. This epidemic is the consequence of a physical inactivity and an unlimited access to over-nutrition and consumption of caloric food. Nevertheless, many familial studies and the identification of monogenic forms of obesity indicate that genetic factors are also involved. All determinants of the polygenic forms are still unknown, recent studies show the role of genes in the signalling of insulin (ENPP1) and metabolic pathways of neurotransmitters (GABA, serotonin) which would predispose to obesity in a sedentary, high calorie lifestyle. The identification of genetic factors in the polygenic diseases such as obesity is assessed by direct studies of physiological genes and by indirect analyses with positional candidate genes located in chromosomal regions of linkage to phenotype traits. Two genome wide-scans on French Caucasian families show the importance of the locus 5cen-q. Among many genes located in this region ; CART (5q12-q13) and PCSK1 (5q15-q21) genes are expressed in the central nervous system (principally in the hypothalamus) and are involved in the control of food intake and the regulation of energy homeostasis. The analysis of a 5,4 Kb region of the CART gene (Cocaine and Amphetamine Regulated Transcript), including the promoter, 3 exons, introns and the 3'UTR, resulted in the identification of a promoter SNP (SNP-3608T>C) which is associated with the polygenic obesity. In a general population, this polymorphism is also associated, with subfractions of plasma cholesterol and apolipoproteins which suggests that the CART gene maybe implicated in lipid metabolism and atherogenesis. Within a Danish study of menopausal women, the SNP-3608T>C was shown to effect remodelling of the bone mass (on arm BMD). PCSK1 (Proprotein Convertase Subtilisin/kexin type 1) Gene code for a neuroendocrine member of the family of subtilisin-like proprotein convertases and is important for the maturation of pro-hormones and neuropeptides precursors such as the proinsulin and POMC. PCSK1 gene mutations are responsible for a number of rare monogenic forms of severe obesity. The analysis of this gene in a polygenic context enabled the identification of frequent mutations including a non-synonymous exonic variant which is associated with adult and/or childhood polygenic obesity. The genetic approach validates physiological hypotheses and improves current understanding of metabolic pathways, and suggests a pleiotropic effect of the CART gene and that the CART and PCSK1 genes are implicated in polygenic obesity

4

Folon, Lise. "Étude de l'impact des variants génétiques rares sur l'obésité monogénique." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. https://pepite-depot.univ-lille.fr/ToutIDP/EDBSL/2023/2023ULILS059.pdf.

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L'obésité est une maladie multifactorielle complexe avec une forte composante génétique. Contrairement à l'obésité commune qui est une maladie polygénique, les formes d'obésité monogénique sont causées par un seul variant génétique rare avec un effet fort et délétère. Ces formes sont rares, précoces et généralement sévères, affectant environ 5% des individus atteints d'obésité. La plupart des mutations rares liées aux formes monogéniques de l'obésité ont été identifiées dans les gènes de la voie leptine-mélanocortine, qui est essentielle pour la régulation de la prise alimentaire. L'identification de ces gènes est cruciale dans la compréhension de la physiopathologie de l'obésité et l'élaboration de nouveaux traitements.J'ai tout d'abord étudié les variants rares hétérozygotes du gène PCSK1 qui code l'enzyme prohormone convertase 1 (PC1/3). PC1/3 est impliqué dans la voie leptine-mélanocortine. Les mutations bialléliques de PCSK1 causent une obésité précoce avec une endocrinopathie sévère. Les patients déficients en PCSK1 (hétérozygotes ou hom*ozygotes) peuvent dorénavant être traités avec des injections de setmélanotide pour perdre du poids. Or l'impact des variants rares hétérozygotes de PCSK1 sur l'obésité, et leur pertinence en médecine de précision sont encore mal définis. Dans l'étude RaDiO incluant 9320 participants, 65 variants rares hétérozygotes de PCSK1 ont été identifiés et analysés in vitro. Ces variants ont été classés en cinq groupes suivant la sévérité de leur impact sur l'activité enzymatique de PC1/3. Les résultats d'analyses d'association ont révélé que les variants rares induisant une perte totale de fonction augmentaient fortement le risque d'obésité et l'indice de masse corporelle (IMC) alors que les variants des autres groupes avec un effet partiel ou neutre sur l'activité de PC1/3 n'avaient aucun effet sur l'adiposité. Nous avons observé que les outils de prédiction in silico étaient peu fiables pour détecter les mutations entraînant une perte totale de fonction.Dans un second temps, je me suis intéressée aux variants rares du gène DYRK1B. Bien que ce gène ne soit pas impliqué dans la voie leptine-mélanocortine, des variants pathogènes de DYRK1B ont été décrits chez plusieurs patients avec une obésité centrale, un diabète de type 2 (DT2) et une maladie des artères coronaires. Cependant, l'impact des variants rares de DYRK1B n'a pas été évalué à grande échelle. A partir de l'étude RaDiO incluant 9353 participants, 65 variants rares dans DYRK1B ont été détectés. Après une analyse in vitro de chaque variant, nous avons identifié 20 variants pathogènes ou probablement pathogènes (P/LP) d'après les critères de l'American College of Medical Genetics and Genomics. Parmi ces variants P/LP, six montraient un effet entrainant une perte totale de fonction de DYRK1B (P/LP-full). Les analyses d'association ont montré que les variants P/LP-full de DYRK1B étaient fortement associés à une augmentation de l'IMC et de la glycémie à jeun, et à un risque accru d'obésité et de DT2, alors que les variants P/LP n'avaient qu'un effet modeste sur l'adiposité et aucun impact sur l'homéostasie glucidique.En conclusion, l'utilisation de la génétique fonctionnelle a permis de mettre en évidence que seuls les variants hétérozygotes de PCSK1 et DYRK1B avec une perte de fonction totale causaient une obésité monogénique. Pour DYRK1B, l'obésité est en plus associée à un DT2. Ces résultats soulignent l'importance cruciale de déterminer in vitro l'impact fonctionnel des mutations en vue du diagnostic génétique et de l'éventuel choix d'un traitement approprié. Nous avons démontré que les tests de prédiction in silico ne sont pour l'heure pas assez précis
Obesity is a complex multifactorial disease with a strong genetic component. Unlike common obesity, which is a polygenic disease, monogenic forms of obesity are caused by a single rare genetic variant with a strong and deleterious effect. These monogenic forms are rare, early-onset and generally very severe, affecting around 5% of individuals with obesity. Most rare mutations associated with monogenic obesity are found in genes within the leptin-melanocortin pathway, which is crucial for the regulation of food intake. Identifying these genes is crucial for understanding the pathophysiology of obesity and developing new treatments.I initially studied rare heterozygous variants of the PCSK1 gene, which encodes the prohormone convertase 1 (PC1/3) enzyme. PC1/3 is involved in the leptin-melanocortin pathway. Biallelic mutations in PCSK1 cause early-onset obesity with severe endocrinopathy. Patients with PCSK1 deficiency (heterozygous or hom*ozygous) can now be treated with setmelanotide injections to promote weight loss. However, the impact of rare heterozygous variants of PCSK1 on obesity and their relevance in precision medicine are still not well-defined. In the RaDiO study, which included 9,320 participants, 65 rare heterozygous variants of PCSK1 were identified and assessed in vitro. These variants were classified into five groups based on the severity of their impact on the enzymatic activity of PC1/3. Association analysis results revealed that rare variants inducing a complete loss of function significantly increased the risk of obesity and body mass index (BMI), whereas variants in other groups with partial or neutral effects on PC1/3 activity had no impact on adiposity. We observed that in silico prediction tools were unreliable in detecting mutations leading to a complete loss of function.Subsequently, I focused on rare variants of the DYRK1B gene. Although this gene is not directly involved in the leptin-melanocortin pathway, pathogenic variants of DYRK1B have been described in several patients with central obesity, type 2 diabetes (T2D), and coronary artery disease. However, the impact of rare DYRK1B variants has not been assessed on a large scale. In the RaDiO study, which included 9,353 participants, 65 rare variants in DYRK1B were detected. Following in vitro analysis of each variant, we identified 20 pathogenic or likely pathogenic variants (P/LP) according to the criteria of the American College of Medical Genetics and Genomics. Among these P/LP variants, six showed an effect leading to a complete loss of function of DYRK1B (P/LP-full). Association analyses showed that P/LP-full variants of DYRK1B were strongly associated with increased BMI and fasting glucose levels, as well as a heightened risk of obesity and T2D, whereas P/LP variants had only a modest effect on adiposity and no impact on glucose homeostasis.In conclusion, the use of functional genetics has demonstrated that only heterozygous variants of PCSK1 and DYRK1B with a complete loss of function cause monogenic obesity. For DYRK1B, obesity is additionally associated with T2D. These results underscore the critical significance of assessing the functional impact of mutations in vitro for genetic diagnosis and the potential selection of appropriate treatments. We have demonstrated that in silico prediction tests are currently not precise enough

5

Bhat, Mamatha. "Expression of PCSK9 in Hepatocellular Carcinoma." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106271.

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Hepatocellular carcinoma (HCC) is an often fatal condition due to late diagnosis, resistance to existing anticancer agents, as well as underlying liver disease that can limit the use of hepatotoxic chemotherapy. Proprotein convertases (PCs) are serine proteases that convert a variety of growth factors, cell surface glycoproteins, receptors and metalloproteinases into their active forms, thus regulating the biological activity of these proteins. PCs have been found to be upregulated in various malignancies. Growth factors implicated in HCC, such as IGF-1, HGF, VEGF and PDGF, have all been shown to be converted into their active forms by PCs. In this study, I explored the hypothesis that expression of proprotein convertases, specifically PCSK9, furin and PC5, is elevated in HCC. This was evaluated through construction of a Tissue Microarray and staining for these proteins. We found that PCSK9 expression was significantly downregulated in HCC tumours associated with poorer survival. PCSK9 is upregulated in the context of liver regeneration and has been involved in cholesterol metabolism, with development of monoclonal antibodies against PCSK9 to treat hypercholesterolemia. Its altered expression in aggressive HCC tumours potentially indicates that HCC is able to modulate its local microenvironment to enable a constant energy supply. There has recently been a move in oncology research to study suppression of suppress tumour growth by modifying energy supply and metabolism (for eg, metformin in prostate and breast cancer). Further confirmation at the mRNA level is required to confirm the altered expression of PCSK9, however this appears to be a promising finding and potential chemotherapeutic target.
Contexte et hypothèses: Le carcinome hépatocellulaire (CHC) est le 5e cancer le plus courant dans le monde entier et la 3ème cause de décès par cancer dans le monde entier, avec une survie médiane à 5 ans de 8,9%. La reconnaissance tardive en raison du manque de biomarqueurs pour détecter la maladie résécable, une résistance aux agents anticancéreux, ainsi qu'une maladie du foie sous-jacente limitant l'utilisation de chimiothérapie hépatotoxique sont des facteurs qui diminuent le taux de survie. Les proprotéines convertases (PCs) sont des sérine-protéases qui convertissent une variété de facteurs de croissance, glycoprotéines de surface cellulaire, les récepteurs, et les métalloprotéinases à leurs formes actives, contrôlant ainsi l'activité biologique de ces protéines. On a démontré l'expression augmentée de PCs dans de diverses tumeurs malignes. On a prouvé que les facteurs de croissance impliqués dans le CHC, tels que l'IGF-1, HGF, VEGF et PDGF, sont convertis à leurs forme actives par les PC. Notre hypothèse est que l'expression de proprotéines convertases est élevée dans le CHC, permettant l'activation de différentes protéines essentielles dans le développement et la progression du CHC. L'objectif de recherche était d'évaluer l'expression des PCs PCSK9, furine et PC5 dans le CHC par rapport aux stroma environnant, zones péri-cirrhotiques, et foie normal afin de déterminer si un gradient d'expression existe. PCSK9 en particulier est connu comme étant plus exprimé chez le foie régénérateur post-hepatectomie. Les diapositives de pathologie de CHC stockés dans le département de pathologie du CUSM ont été examinés par une pathologiste, et les zones appropriées (tumeur de CHC, interface de tumeur et du foie, le foie cirrhotique, et d'autres échantillons d'hépatite et de foie normal) dans les blocs de tissu correspondants ont été creusés et ont été incorporées dans un microarray de tissu (TMA). Des lignes cellulaires de CHC etablies, dont le HepG2 et le Huh7, avec des profils d'expression de PC connus, ont été incorporées sous forme de pastilles de cellules dans la TMA, afin de servir de témoins positifs et négatifs. La TMA a été sectionnée en diapositives, qui ont été colorées avec des anticorps de la PCSK9, furine et PC5. On a découvert que le niveau d'expression de PCSK9 était diminuée dans les CHC avec un pire prognostique. L'expression augmentée de PCSK9 dans les CHC plus aggressifs pourrait indiquer un rôle du PCSK9 dans la tumorigenèse, directement ou indirectement. Il se peut que les CHCs plus aggressifs sont capables de modifier l'environnement local pour apprivoiser l'énergie métabolique, et que le PCSK9 permet que le cholestérol soit utilisé comme source d'énergie. La confirmation de son importance fonctionnelle avec mRNA pourrait potentiellement mener au développement de chimiothérapie ciblée avec des anticorps contre le PCSK9 (stratégie en étude pour l'hypercholestérolémie). Compte tenu des options chimiothérapeutiques actuellement limitées pour le CHC, une telle constatation pourrait améliorer la prise en charge clinique du CHC.

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DA, DALT LORENZO. "IMPACT OF PCSK9 ON EXTRAHEPATIC TISSUES." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/813080.

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Introduzione e scopo: la proproteina convertasi subtilisina kexin tipo 9 (PCSK9) è una glicoproteina di 692 amminoacidi che appartiene alla famiglia delle proproteine convertasi. È prodotta principalmente dal fegato dal quale viene secreta nel torrente circolatorio. PCSK9 interagisce con diversi recettori della famiglia dell’LDLr, inclusi VLDLr, LRP1 ma anche con CD36, e guida la loro degradazione lisosomiale. La mancanza di PCSK9 determina quindi una maggiore espressione dei recettori della famiglia LDLr e favorisce l'accumulo di lipidi nei tessuti extraepatici. L’eccesso di lipidi cellulari è associato a disfunzione mitocondriale e danni tissutali in diversi organi, tra cui il pancreas e il cuore. Per questo motivo ci siamo chiesti se la mancanza di PCSK9 circolante e localmente prodotto possa influenzare l'accumulo di lipidi nei tessuti extraepatici come il pancreas e il cuore influenzandone la funzionalità.Metodi: Animali WT, Pcsk9 KO, albumin CRE PCSK9LoxP / LoxP KO condizionali (privi di PCSK9 selettivamente nel fegato e quindi non presentando proteina PCSK9 rilevabile in circolazione) e topi maschi Doppi KO LDLr-Pcsk9 di 2 mesi sono stati nutriti per 20 settimane con SFD o HFD. I livelli plasmatici di GTT, ITT, insulina e peptide C, morfologia del pancreas e accumulo di colesterolo nelle isole pancreatiche sono stati studiati nei diversi modelli animali. Inoltre su questi topi sono state eseguite analisi ecocardiografiche del cuore e test funzionali. La respirazione mitocondriale è stata studiata in condizioni di riposo e in seguito a condizioni massime di accoppiamento e disaccoppiamento in tutti i modelli di topo, seguite da caratterizzazione delle proteine mitocondriali mediante western blot e analisi di metabolomica approfondita.Risultati: Il metabolismo glucidico è significativamente ridotto nei topi Pcsk9 KO alimentati sia con una dieta standard che con una dieta ricca di grassi per 20 settimane rispetto agli animali WT; la sensibilità all'insulina, tuttavia, non viene alterata. Un'analisi dettagliata della morfologia del pancreas dei topi Pcsk9 KO rispetto ai controlli ha rivelato isole più grandi con un maggiore accumulo di esteri del colesterolo, che si associa ad aumentati livelli intracellulari di insulina e alla diminuzione dei livelli plasmatici di insulina e C-peptide. Questo fenotipo è stato completamente ripristinato nei topi Pcsk9 / Ldlr DKO, il che implica che l’aumentata espressione di LDLr potrebbe spiegare il fenotipo osservato. Da notare che i topi privi di PCSK9 circolante non presentavano un fenotipo alterato, indicando così che PCSK9 circolante e di origine epatica non influisce sulla funzione delle cellule beta e sulla secrezione di insulina. In parallelo, una caratterizzazione dettagliata della funzionalità cardiaca, ha rivelato che i topi Pcsk9 KO mostrano un fenotipo caratteristico dello scompenso cardiaco con frazione di eiezione conservata. Inoltre, i topi Pcsk9 KO presentano una ridotta resistenza alla corsa senza difetti muscolari accoppiata a importanti adattamenti nel metabolismo cardiaco e nella funzionalità mitocondriale dovuti all'accumulo di colesterolo cardiaco. Un fenotipo simile è stato osservato negli LDLr Doppi KO confermando un effetto indipendente dall'espressione dell’LDLr. Il fenotipo cardiaco risulta completamente ristabilito nel modello KO selettivo del fegato escludendo così il coinvolgimento del PCSK9 circolante nello sviluppo dell'insufficienza cardiaca con frazione di eiezione conservata. Studi traslazionali hanno mostrato che i soggetti umani portatori del polimorfismo di perdita di funzione R46L mostravano un aumento della massa ventricolare sinistra senza alterazioni nella frazione di eiezione rispetto ai controlli con BMI R46R abbinati.Conclusione / Discussione: PCSK9 prodotto localmente nel pancreas e nel cuore limita l'accumulo di lipidi in modo dipendente da LDLr nel pancreas e in modo LDLr indipendente nel cuore contribuendo così a mantenere l'omeostasi dei tessuti. La carenza genetica di PCSK9 porta allo sviluppo di intolleranza al glucosio e insufficienza cardiaca con frazione di eiezione conservata nei modelli murini e nell'uomo.
Background and Aim: Proprotein convertase subtilisin Kexin type 9 (PCSK9) is a 692-amino acid glycoprotein that belongs to the family of proprotein convertases. It is produced mainly by the liver and secreted into the circulation. PCSK9 interacts with several receptors of the LDLr family, including VLDLr, LRP1 but also with CD36, and drives their degradation in the lysosome. As a consequence, PCSK9 deficiency results in increased expression of LDLr family receptors and favors lipid accumulation in extrahepatic tissues. Lipids overload is associated with mitochondrial dysfunction and tissue damage in different organs including the pancreas and the heart. We wondered whether the lack of both circulating and locally produced PCSK9 may affect lipid accumulation on extrahepatic tissues such as the pancreas and the heart those affecting their functionality.Methods: 2-months old WT, Pcsk9 KO, Albumin CRE PCSK9LoxP/LoxP conditional KO (lacking PCSK9 production selectively in the liver and thus presenting undetectable PCSK9 protein in the circulation) and Double KO LDLr-Pcsk9 male mice were fed for 20 weeks with SFD or HFD. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Moreover, echocardiographic analysis of the heart and functional tests were performed on these mice. Mitochondrial respiration was investigated under resting conditions and following maximal coupling and uncoupling conditions in all mice models followed by mitochondrial protein profiling by western blotting and extensive metabolomic analysis.Results: Glucose clearance was significantly reduced in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying that increased LDLR could explain the phenotype observed. Of note mice lacking circulating PCSK9 did not present an impaired phenotype, thus indicating that circulating, liver-derived PCSK9 does not impact beta-cell function and insulin secretion. In parallel, a detailed characterization of heart function revealed that Pcsk9 KO displays a phenotype characteristic of heart failure with preserved ejection fraction. Moreover, PCSK9 KO mice present a reduced running resistance without muscular defects coupled to major adaptations in cardiac metabolism and mitochondrial functionality due to heart cholesterol accumulation. A similar phenotype was observed in LDLr Double KO confirming an effect independent of LDLr expression. The cardiac phenotype is completed reverted in the liver selective KO model thus excluding the involvement of circulating PCSK9 in the development of Heart Failure with preserved Ejection Fraction. Translational studies showed that human subjects carrying the R46L loss of function polymorphism displayed increased left ventricular mass without alterations in ejection fraction compared to R46R BMI-matched controls.Conclusion/Discussion: PCSK9 locally produced in the pancreas and the heart affects limits lipid accumulation in an LDLr dependent manner in the pancreas and an LDLr independent manner in the heart thus contributing to maintaining tissue homeostasis. Genetic PCSK9 deficiency leads to the development of glucose intolerance and heart failure with preserved ejection fraction in mice models and humans.

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Stefan, Elias. "Familjär hyperkolesterolemi (FH) – analys av prevalens i Stockholm och hälsoekonomiska konsekvenser av tidigdiagnostik och behandling." Thesis, Uppsala universitet, Institutionen för farmaci, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-434844.

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Background: Familial hypercholesterolemia (FH) is a genetic disorder estimated to affect 0,4 % of the world's population (1 in 250). Patients with FH have abnormally high LDL-cholesterol. Aim: The aim of this study was to estimate the prevalence of FH in Stockholm County and to evaluate the health economic impact of diagnosing people with FH early in life. Methods: Two algorithms were used to estimate the number of people with high LDLcholesterol. The first method applied data on cholesterol measurements from patients in Stockholm County between 2006-2008 and a modified version of Dutch Lipid Clinic Network. The second method was based on dispensed prescriptions of ezetimibe, lomitapide, evolucumab and alirocumab during 2019. A health economic model was created to estimate the economical outcome of diagnosing and treating patients early before undergoing a cardiovascular event. Results: The prevalence of FH in Stockholm County was estimated to 0.63 %, corresponding to a total of 12 000 individuals. The accumulated costs over 20 years for FH is estimated to be more than 1,1 billion SEK for diagnosed and treated patients, and 1,7 billion SEK for undiagnosed and untreated patients. Conclusions: The prevalence of FH in Stockholm County is probably higher than previously suggested. Early diagnosis and treatment is an investment for society and necessary for the patients to prevent cardiovascular events and improve quality of life.

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Giunzioni,I. "MACROPHAGE EXPRESSION OF PCSK9 INFLUENCES ATHEROSCLEROSIS DEVELOPMENT." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229332.

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Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) regulates low‐density lipoprotein (LDL) cholesterollevels by binding and degrading hepatic LDL receptor (LDLR), thus promoting atherosclerosis. Little is knownof PCSK9 effect in macrophages and whether this contributes to the development of the atheroma.To test the effect of human (h) PCSK9 expression on atherosclerosis we developed transgenic miceexpressing hPCSK9 on wild type (WT), LDLR‐/‐ or apoE‐/‐ background. We first demonstrated that bothmPCSK9 and hPCSk9 are expressed at the mRNA level and secreted in the culture medium by MPM. As inhepatocytes, hPCSK9 reduced LDLR levels in the murine macrophage cell line J774A.1 and in inflammatoryMPM. On the contrary, hPCSK9 did not reduce LRP1 expression, another member of the LDL‐R gene familyinvolved in the development of atherosclerosis through its effects on macrophage inflammatory responsesand promotion of cell survival. To test the effects of PCSK9 in the atheroma, we fed our transgenic mice ahigh fat diet for 8 weeks. As expected, serum cholesterol levels were increased by 2 fold in hPCSK9tgcompared to WT mice (325±64 vs. 158±44 mg/dl, respectively, p<0.05) and hPCSK9 was detected inatherosclerotic plaques of hPCSK9 tg. In contrast, there was no effect of PCSK9 expression in apoE‐/‐ miceon serum cholesterol levels compared with apoE‐/‐ controls (1066±161 vs. 964±188 mg/dl, respectively, NS).Surprisingly, hPCSK9 expressing apoE‐/‐ mice showed increased proximal aorta lesion area. Lesioncomposition analysis revealed that lesions of PCSK9/apoE‐/‐ mice have a higher content of Ly6Chigh positivecells (6.7±0.2%) compared to apoE‐/‐ controls (5.7±0.4%). Moreover, analysis of spleen lysates revealed anincrease in the percentage of Ly6Chigh positive cells in hPCSK9tg compared to control apoE‐/‐, suggesting adirect effect of PCSK9 in macrophage inflammation and plaque development. On the contrary, despite anincrease in both cholesterol levels and lesion size in hPCSK9 tg/LDLR‐/‐ compared to LDLR‐/‐, no differencesin Ly6Chigh positive cells were found between the two groups. To study the effect of macrophage PCSK9 inthe atheroma, bone marrow cells from PCSK9/apoE‐/‐ or apoE‐/‐ mice were transplanted into apoE‐/‐recipients. hPCSK9 was detected in serum and lesions from PCSK9/apoE‐/‐ mice but there was no effect ofPCSK9 expression on serum cholesterol levels compared with apoE‐/‐ controls. Interestingly, lesioncomposition analysis showed significantly higher levels of Ly6Chigh positive cells in recipients ofhPCSK9/apoE‐/‐ bone marrow cells compared to controls (7.4±1.5% vs. 5.6±1.1%, respectively, p<0.05). Totest whether the effects of hPCSK9 on inflammation were dependent on binding and degradation of LDLR inmacrophages, we transplanted bone marrow cells from PCSK9/LDLR‐/‐ or LDLR‐/‐ mice into LDLR‐/‐ recipients.We observed that, despite a large amount of PCSK9 accumulated in the serum of transgenic mice, nearlyundetectable levels were found in the plaque. No differences were found between the two groups in termsof cholesterol levels, lesion size and Ly6Chigh positive cells between the two groups.Our results show for first time that human PCSK9 expression in macrophages directly influencesatherosclerotic plaque composition in the absence of changes in serum cholesterol levels, suggesting adirect effect of PCSK9 in macrophage inflammation and plaque development. The effect on inflammation isdependent on LDLR since no effects in lesion composition were found in its absence.

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Kourimate, Sanae. "Pcsk9 : régulation et implication dans le syndrome métabolique." Nantes, 2008. https://archive.bu.univ-nantes.fr/pollux/show/show?id=4ac185ba-f999-45ff-9241-4278a9699b5c.

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Proprotein convertase subtilisin/kexin type 9 (PCSK9) est une serine protéase, jouant un rôle important dans la régulation des niveaux de cholestérol circulant. Les mutations gains de fonctions de PCSK9 sont associées à une hypercholestérolémie autosomale dominante due à une élévation des concentrations en Low Density Lipoprotein associées au cholestérol (LDLc). A l'inverse, des mutations perte de fonction de PCSK9, sont associées à une diminution de ces concentrations et à une réduction de 88% du risque d'apparition de maladies coronariennes. Dans le réticulum endoplasmique, Pro-PCSK9 subit un autoclivage indispensable à sa maturation et à sa sécrétion. PCSK9 circulante se lie au domaine EGF-A du récepteur aux LDL (LDLR) et entraîne sa dégradation lysosomale. Tout comme le LDLR, PCSK9 est positivement régulé par les statines via le Sterol Responsive element Binding Protein -2 (SREBP2). Les statines sont très utilisées en clinique pour leurs effets hypocholestérolémiants, ajouter des inhibiteurs de PCSK9 pourrait améliorer leur efficacité. Le premier objectif a été de caractériser la régulation négative de PCSK9 par le récepteur nucléaire Peroxisome Proliferator Activated Receptor alpha (PPARα) et ses agonistes synthétiques : les fibrates. Les résultats obtenus in vitro sur des lignées d’hépatocytes humains révèlent que l’activation de PPARα réprime la transcription de PCSK9 -via une répression de son promoteur- suppriment son activation par les statines, et potentialise l’effet des statines sur l’activité du LDLR. De plus, ces résultats révélèrent que la furine et PC5/6A -deux protéines convertases qui dégradent PCSK9- sont également régulées positivement par PPARα. Au-delà de PCSK9, cette étude a permis d’identifier une nouvelle famille d’enzyme régulée par les fibrates : les pro-protéines convertases. La deuxième partie de mes travaux de thèse a porté sur la mise au point d’un test flurorométrique de dosage de l’activité autocatalytique de PCSK9. La spécificité de notre approche a été de considérer non pas la protéine recombinante purifiée, qui semble dépourvue d’activité pour une raison inconnue, mais la forme endogène des hépatocytes. Après avoir validé ce test sur des hépatocytes primaires de souris PCSK9-/-, je l’ai appliqué à l’étude de divers mutants de PCSK9. Enfin, suite aux travaux du laboratoire sur la régulation de PCSK9 par l'insuline, j’ai également caractérisé l’expression de PCSK9 dans divers modèles animaux de diabète et d’insulinorésistance. PCSK9 est une cible thérapeutique sérieuse pour diminuer le LDL-c. D'après ces résultats, l’inhibition transcriptionnelle de PCSK9 par les fibrates semble être très prometteuse. Cependant, en clinique les fibrates sont préférentiellement utilisés pour leurs propriétés hypotriglycéridémiantes, puisque leurs effets sur le cholestérol restent limités. Existerait-il in vivo un mécanisme inhibiteur de cette voie? L'identifier serait une des perspectives qu'ouvre cette thèse
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the serine protease family. Gain of function mutations within PCSK9 are associated with dominant forms of familial hypercholesterolemia. Inversely, humans harbouring loss of function mutations have a significant plasma LDLc reduction and a 88% decrease of the risk of coronary heart disease. In the endoplasmic reticulum Pro-PCSK9 undergoes an autocalytique clivage that is crucial for its secretion. Then, this secreted protein binds to the EGF-A domain of the LDLR and targets it to the lysosomes rather than to the cell surface. Both PCSK9 and the LDLR are up-regulated by statins via SREBP2. Using PCSK9 inhibitors may optimize the effects of this hypocholesterolemic drug. The first aim of my thesis was to investigate in vitro the mechanisms of PCSK9 repression by the fibrates which are PPARα synthetic agonists. Activation of PPARα down-regulates PCSK9 transcription at the promoter level and increase the expression of two others Proprotein convertases: furin and PC5/6A which are known to degrade PCSK9. Fibrates counteracts PCSK9 induction by statins and amplifies the effects of this hypocholesterolemic drugs on the LDLR acitivity. The second part of my studies was based on measuring the endogenous cleavage activity of PCSK9, using a fluorogenic peptide corresponding to the cleavage site of Pro-PCSK9. After validation of the specificity of this assay on mice primary hepatocytes from PCSK9-/-, I applied it to the test of several PCSK9 variants. The final part of my studies dealt about the characterisation of PCSK9 expression in diabetic and insulin resistant animal models. PCSK9 is an attractive therapeutic target for lowering plasma LDLc levels. This study clearly showed that PCSK9 transcriptional inhibition by fibrates might be envisaged in combination with statins. However, in vivo, in humans, the fibrates are rather known for their hypotriglyceridemic properties. The limited effect of fibrates on lowering LDLc might be explained by a counteracting pathway. Identifying this pathway is one of the promising perspectives of this thesis

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CANCLINI, LAURA. "PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 PREFERENTIALLY ASSOCIATES WITH A SPECIFIC LDL SUBFRACTION: A DETAILED CHARACTERIZATION AND STUDY OF THE EFFECTS OF ANTI-PCSK9 MABS TREATMENT." Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/947250.

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BACKGROUND AND AIM: Proprotein Convertase Subtilysin/Kexin Type 9 is a key regulator of LDL-C levels. Nevertheless, its physiological modulation is not fully understood. It is unclear whether PCSK9 has biological effects other than degrading the LDLR; consensus is lacking also on how PCSK9 is transported in the bloodstream, and whether this influences PCSK9 function. There are several conflicting data about PCSK9’s possible association with different lipoprotein subtypes. The biological function of the lipoprotein-bound PCSK9, also, is still a matter of debate. Due to its role in modulating plasma cholesterol levels, several approaches have been developed to modulate PCSK9 activity. Anti-PCSK9 mAbs represent the most advanced clinical available strategy for PCSK9 pharmacological inhibition, followed by the siRNA approach which seems promising. In this context, our aims were: 1) to establish a valid experimental procedure for the study of the possible association between PCSK9 and plasma lipoproteins. This would help us reach a clear idea about PCSK9’s lipoprotein compartmentation. 2) To characterize the lipoprotein fraction that binds PCSK9; 3) to address whether anti-PCSK9 mAbs therapy interferes with the PCSK9-LPs association, with resulting biological consequences. The capacity of PCSK9 to bind different lipoproteins raises the possibility that changes in the lipoprotein compartmentalization could regulate PCSK9 activity, and mAbs possible effects on such interaction may have biological consequences thus affecting their own pharmacological action. The study of such regulatory mechanisms could represent a potential avenue for developing new cholesterol-lowering drugs. METHODS: To reach our first aim, we compared several well-established methods in the lipoprotein isolation technique to clarify whether PCSK9 associates differently with circulating lipoproteins. The lipoprotein fractions collected were quantified for their cholesterol, triglycerides, PCSK9, apoB, apo AI and Lp(a) content using clinical grade reactives or ELISA. They were further characterized through immunoblotting, agarose gel electrophoresis, transmission electron microscopy (TEM), lipidomic and mass spectrometry. Following this, we analyzed PCSK9’s lipoprotein distribution among OptiPrep UC-isolated lipoproteins, obtained from anti-PCSK9 mAbs treated subjects before therapy (T0), and 1 (T1), 3 (T3) and 6 (T6) months after the beginning of treatment. The collected lipoprotein fractions were further characterized as described above. RESULTS AND CONCLUSION: Based on our results, the PCSK9-LDL association exists and is sensitive to high salt concentrations. OptiPrep UC and FPLC are both suitable methods for the study of this association. We believe that PCSK9 circulates mostly as a free type of protein, and partially (approx. 10-20% of total recovered PCSK9) associated through its active form with a light LDL subfraction resembling an IDL. We believe that this association originates in the circulation, as we did not find PCSK9 in the VLDL-containing fractions. PCSK9 did not associate with Lp(a) or HDL. Anti-PCSK9 mAbs administration induced a large decrease in LDL-C levels, - from 140,3±64,8 mg/dL to 51,5±35,6 mg/dL before and after treatment, respectively - parallel with a drastic increase in PCSK9 plasma levels - from 409,4±123,3 ng/mL to 3802,3±1001,0 ng/mL (n=22); despite this, they did not affect the PCSK9-LDL association but instead induce a more than 10-fold increase in the absolute quantity of LDL-bound PCSK9. Interestingly, the amount of PCSK9 recovered in the LDL fractions after therapy was about 242,3±164,8 ng/mL, like that of plasma of untreated subjects; this suggested that the LDL-bound PCSK9 may be inactive. Preliminary data obtained from Inclisiran-treated subjects suggest that the PCSK9- LDL association is maintained also when reducing both LDL and PCSK9 levels, therefore reinforcing the idea that the PCSK9-LDL association might be biologically meaningful.

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Dewpura, Thilina. "Importance of phosphorylation in PCSK9 processing, stability and function." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28604.

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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein regulating the degradation of low density lipoprotein receptor. Single nucleotide polymorphisms in PCSK9 associate with both hyper- and hypo-cholesterolemia; studies show significant reduction in risk of coronary heart disease for 'loss of function' PCSK9 carriers. We used a combination of mass spectrometry and radiolabeling to report that PCSK9 is phosphorylated at two sites, Ser47 in its propeptide, and Ser688 in its C-terminus. Site directed mutagenesis (SDM) demonstrated that a Golgi casein kinase-like kinase was responsible for PCSK9 phosphorylation based on the consensus site, SXE/S(p). PCSK9 phosphorylation is cell-type specific; phosphorylation status did not affect PCSK9 processing or secretion. Phosphorylated PCSK9 propeptide is protected against proteolysis. Immunoblotting demonstrated that PCSK9 mutants engineered by SDM to prevent phosphorylation at either site (substitution to Ala) or in combination resulted in significantly increased LDLR levels in HuH7 cells by up to -25%. PCSK9 mutants engineered by SDM to mimick phosphorylation (substitution to Asp/Glu) at the N-terminus, but not at the C-terminus or in combination, promoted LDLR degradation significantly more than wild-type. Far western analysis demonstrated that preventing PCSK9 phosphorylation promoted its interaction with the endogenous inhibitor Annexin A2.

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Roubtsova, Anna. "Partial hepatectomy and liver regeneration in PCSK9 knockout mice." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112356.

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The proprotein convertase subtilisin/kexin type 9, PCSK9, belongs to the proprotein convertase (PC) family. Human mutations in the gene encoding PCSK9 lead to either familial hyper- or hypocholesterolemia, resulting from a gain or loss of function, respectively. Mice lacking PCSK9 are viable and show a 42% decrease in plasma cholesterol levels. The enzyme triggers the degradation of the low density lipoprotein receptor (LDLR) through a partially unknown mechanism.
PCSK9 is very abundant in the liver and intestine during development and adulthood. Hepatocytes have a capacity to reproduce themselves and, upon injury, can repopulate the liver. For a better understanding of the role of PCSK9 in the liver, partial hepatectomy was performed on Pcsk9 +/+, Pcsk9+/- and Pcsk9-/- mice. The absence of PCSK9 resulted in defective liver regeneration, while wild type (WT) and heterozygous mice had no phenotype. Regeneration defects could be prevented by a high cholesterol diet. PCSK9 deficiency, by contributing to maintaining low circulating cholesterol levels may thus hamper liver regeneration. This knowledge is critical for the analysis of future PCSK9 inhibitors expected to be developed in the near future.
Key words. Proprotein convertase subtilisin/kexin 9 (PCSK9), a familial hyper- or hypocholesterolemia, low density lipoprotein receptor, knockout mouse model, partial hepatectomy.

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Torrinha,JoséMariadeQueiroze.LencastreFleming. "Inibidores PCSK9: nova estratégia para o tratamento da hipercolesterolemia." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5293.

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
As doenças cardiovasculares são uma das principais causas de morbilidade e mortalidade a nível mundial. Neste enquadramento, um dos principais fatores de risco associado às doenças cardiovasculares é a hipercolesterolemia. As opções farmacológicas existentes para o tratamento e prevenção desta dislipidemia centram-se, sobretudo, no uso de fármacos como as estatinas, a ezetimiba, os fibratos, o ácido nicotínico e as resinas sequestradoras de ácidos biliares. Porém, esta abordagem farmacológica no combate da hipercolesterolemia caracteriza-se pelo prolongado período terapêutico decorrendo daí possíveis efeitos laterais a longo prazo, pela intolerância a grupos terapêuticos observada em alguns doentes (como acontece com as estatinas) ou, ainda, pela eficácia limitada de alguns grupos terapêuticos (como é o caso, dos fibratos), o que suscita preocupação. Os avanços científicos no conhecimento dos processos que envolvem a hipocolesterolemia e a incessante procura de fármacos mais seguros e eficazes impulsionou o desenvolvimento dos inibidores da pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), afirmando-se como uma nova e promissora estratégia terapêutica. Os níveis plasmáticos elevados de colesterol proveniente das lipoproteínas de baixa densidade (C-LDL) são um fator de risco, no desenvolvimento de aterosclerose e de doença vascular isquémica. O recetor LDL (R-LDL) é essencial no metabolismo do colesterol, uma vez que ao se ligar ao C-LDL é capaz de eliminá-lo da circulação. É aqui, que reside o principal foco de interesse desta nova estratégia terapêutica, uma vez que a PCSK9 promove a degradação do recetor R-LDL, conduzindo a uma redução da depuração de LDL, aumentando os níveis de colesterol LDL. Desta forma, a inibição da atividade da PCSK9, veio revelar-se como uma nova abordagem potencialmente interessante para o desenvolvimento de novos fármacos destinados à redução do C-LDL. Os anticorpos monoclonais humanos contra PCSK9 estão em desenvolvimento clínico e são, neste momento, a aposta mais promissora de inibição da PCSK9. Até ao momento, os resultados dos ensaios clínicos demonstraram a eficácia destas moléculas com redução do C-LDL na ordem dos 60%. Adicionalmente, os seus efeitos na redução do C-LDL são aditivos aos das estatinas e até à data, não mostraram qualquer efeito tóxico a nível muscular, como acontece com estas últimas, sendo fármacos bem tolerados e aparentemente seguros.
Cardiovascular diseases are a major cause of morbidity and mortality worldwide. In this context, one of the main risk factor associated with cardiovascular disease is hypercholesterolemia. The treatment and prevention of this dyslipidemia is mainly focused on the use of drugs such as statins, ezetimibe, fibrates, nicotinic acid and bile acid sequestrants. However, the pharmacological approach in hypercholesterolemia treatment is characterized by prolonged therapeutic period elapsing possible long-term side effects, by the intolerance to treatment in some patients (as is the case of statins), or by the limited efficacy of various drugs/pharmaceuticals (as for example of fibrates), which raise concerns. Scientific advances in the understanding of hypercholesterolemia and the constant need for safer and more effective drugs prompted the development of the convertase pro-protein subtilisin inhibitor/kexin type 9 (PCSK9), as a promising new therapeutic strategy. Elevated plasma LDL cholesterol (LDL-C) levels are a risk factor for atherosclerosis and ischemic vascular disease. The LDL receptor (LDL-R) has an essential role in the cholesterol metabolism, since it binds to LDL-C removing it from circulation. Here, lies the main focus of interest of this novel therapeutic strategy, since PCSK9 promotes LDL-R the degradation, leading to a reduction of LDL clearance, increasing levels of LDL cholesterol. Therefore, inhibition of PCSK9 activity is a potentially interesting new approach for the development of new drugs to reduce LDL-C. Human monoclonal antibodies against PCSK9 are in clinical development and are presently the most promising strategy for the inhibition of PCSK9. At the moment, results of clinical trials show the efficacy of these molecules with reductions efficiencies of LDL-C in the order of 60%. Furthermore, this LDL-C reduction are additive to those of statins and until now have not shown any toxic effect in muscle, as observed with statins, and have a good record for safety and tolerability.

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Vila, Belmonte Àlex. "Estimación de la prevalencia de la Hipercolesterolemia Familiar y de las características clínicas asociadas en Cataluña." Doctoral thesis, Universitat de Girona, 2019. http://hdl.handle.net/10803/670206.

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Familial hypercholesterolemia (FH) is the most frequent hereditary cause of premature coronary heart disease.The aim of this doctoral thesis is to determine the prevalence of subjects with FH phenotype (FH-P); to describe their clinical characteristics and the lipid-lowering therapy that they follow; as well as to estimate the number and type of patients eligible for treatment with proprotein convertase subtilisin/kexin type 9 inhibitors (iPCSK9) according to the different indication criteria of the scientific societies and the National Health System (NHS). This research was done based on data from real-world clinical practice.Our study estimates that at least one in 192 and one in 425,774 people of the general population have heterozygous and hom*ozygous FH-P, respectively. Weobserve that it is underdiagnosed and treated suboptimally. The number of possible candidates to receive iPCSK9 ranges, depending on which of the different scientific societies’ recommendations are used.
La hipercolesterolemia familiar (HF) es la causa hereditaria más frecuente de enfermedad coronaria prematura.Esta tesis doctoral pretende determinar la prevalencia de sujetos con fenotipo de HF (F-HF); describir sus características clínicas y el tratamiento hipolipemiante que realizan; así como estimar el número y tipo de pacientes candidatos a recibir inhibidores de la proproteína convertasa subtilisina/kexina tipo 9 (iPCSK9) según los diferentes criterios de indicación de las sociedades científicas y el Sistema Nacional de Salud (SNS). La investigación se ha basado en datos de la práctica clínica real. Nuestro estudio estima que al menos una de cada 192 y una de cada 425.774 personas en la población general presentan F-HF heterocigota y hom*ocigota, respectivamente. Se observa que está infradiagnosticada y tratada de forma subóptima. El número de posibles candidatos a recibir iPCSK9 varía, en función de las recomendaciones de las diferentes sociedades científicas que se utilizan.

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Shore, Robert. "A functional characterisation of the PCSK6 locus associated with handedness." Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/15719.

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Humans display a 90% population level bias towards right-handedness, implying the vast majority of people have a left-hemisphere dominant for motor control. Although handedness presents a weak, but very consistent heritability across the literature (estimated to be approximately 25%), to date few genetic loci associated with this complex trait have been identified and replicated in subsequent studies. One such gene which has been found to be associated with handedness and subsequently replicated is PCSK6, most recently through a quantitative GWAS (P < 0.5*10−8, Brandler et al. (2013)). Interestingly, PCSK6 is known to activate Nodal, a morphogen involved in a highly conserved bilaterian pathway known to regulate left-right body axis determination. Here I present the first molecular characterisation of a handedness-associated region by conducting a detailed functional analysis of the PCSK6 locus, combining genetic analysis, in silico prediction and molecular assays to investigate how common genetic variants influence handedness-related phenotypes. Specifically, I defined the associated locus to be 12.7 kb in size, spanning a predicted 1.8 kb bidirectional promoter which controls the expression of both an antisense long non-coding RNA (lncRNA), and a novel short PCSK6 isoform. A series of luciferase-expressing constructs were generated to characterise the promoter, identifying a minimal sequence capable of driving transcription in a sense strand direction. I have demonstrated experimentally that one of the top associated markers in previous GWA studies, rs11855145, directly creates/disrupts a suspected transcription factor bind site in the vicinity of this bidirectional promoter. Further functional studies of the genetic variation within PCSK6 may help explain the molecular regulatory mechanisms affecting gene expression. This project provides a model for assays to study other GWAS-nominated candidate genes, and in particular for establishing the role of noncoding variants. The findings from this study support the role of common variants in influencing complex phenotypes, such as handedness.

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Sarkar, Samantha Khadija. "The Regulation of PCSK9 Structure and Function Through Lipoprotein Interactions." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39099.

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Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a negative regulator of the low-density lipoprotein receptor, and PCSK9 inhibition has become an important cholesterol-lowering therapeutic strategy. PCSK9 also associates with LDL particles, and evidence suggests that the activity of PCSK9 may be regulated by LDL binding. We have investigated the biochemistry of the interaction between PCSK9 and lipoproteins. Through mutagenesis and in-vitro binding assays, we found conserved motifs in the PCSK9 N-terminus that play a role in LDL binding. Through secondary structure studies using circular dichroism and computational modelling, we determined that the N-terminal region of the PCSK9 prodomain undergoes an environment-dependent structural shift that affects the ability of PCSK9 to bind LDL. We also found that the commonly found loss-of-function polymorphism R46L is capable of modulating this structural shift. Importantly, we found a surface-exposed region of the PCSK9 prodomain that maps a cluster of gain-of-function mutations (L108R, S127R, and D129G) that severely disrupt LDL binding. Through gel shift assays and density gradient centrifugation, we observed that PCSK9 shows remodeling-dependent ability to bind different classes of lipoprotein particles in vitro, binding strongly to LDL and IDL but showing barely detectable association to VLDL. Further, in human plasma, we found that lipoprotein-bound populations of PCSK9 shifted in response to differences in lipoprotein profiles between normolipidemic and hypercholesterolemic or hypertriglyceridemic subjects. Overall, elucidation of how lipoproteins regulate PCSK9 activity will reveal new targets for designing cholesterol-lowering therapeutics.

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Haas, Mary Elizabeth. "Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in Secondary Hyperlipidemias." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493577.

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Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) has emerged over the past decade as an important regulator of plasma cholesterol and cardiovascular disease risk. PCSK9 promotes degradation of low density lipoprotein (LDL) receptors, thereby decreasing LDL clearance. Accordingly, patients with gain-of-function mutations in PCSK9 have increased LDL cholesterol and increased risk of cardiovascular disease. Conversely, PCSK9 inhibitors recently approved by the FDA are effective in reducing LDL cholesterol.While the contribution of PCSK9 to familial hypercholesterolemia is well-established, less is understood about the role of PCSK9 in diseases in which hyperlipidemia results secondary to an initial disease insult. Hormonal regulation of PCSK9 is also incompletely understood. Understanding the regulators of PCSK9 and specific diseases in which it contributes to hypercholesterolemia is important for identifying additional mechanisms via which PCSK9 can be manipulated, and for choosing patient populations in which PCSK9 inhibitors will be effective.Here, we investigate PCSK9 in two diseases of secondary hyperlipidemia. In nephrotic syndrome, damage to kidney podocytes causes extreme proteinuria and hypercholesterolemia of unclear etiology. We show that plasma cholesterol and PCSK9 are dramatically elevated in mice made nephrotic by nephrotoxic serum treatment or podocyte apoptosis. Moreover, knockout of Pcsk9 protects mice from the effects of nephrotic syndrome on plasma lipids, particularly increased LDL cholesterol. Similarly, nephrotic patients show decreased plasma PCSK9 and cholesterol upon disease remission. Second, loss of adipose tissue in lipodystrophy results in low levels of the hormone leptin. Treatment of lipodystrophic patients with leptin reduces LDL cholesterol through unknown mechanisms. We used this background of hypoleptinemia to investigate the effects of leptin on PCSK9. We found that in female lipodystrophic patients, leptin treatment reduced plasma PCSK9, correlating with decreased LDL cholesterol. Similarly, in male leptin-deficient ob/ob mice, leptin also decreased plasma PCSK9 but did not affect plasma lipids. Our data show that PCSK9 is a novel regulator of hypercholesterolemia in nephrotic syndrome, suggesting that PCSK9 inhibitors may be an important therapy for this patient population with ambiguous treatment options. They also show that leptin can suppress PCSK9 expression, which may explain the observed decreases in LDL cholesterol upon leptin treatment of lipodystrophic patients.
Medical Sciences

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Ricci,C. "PCSK9 AND INFLAMMATION: IN VITRO STUDY ON HEPATOCYTES AND MACROPHAGES." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/541464.

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TNF-alpha induces proprotein convertase subtilisin kexin type 9 (PCSK9) expression in hepatic HepG2 cell line, through the activation of suppressor of cytokine signaling 3 (SOCS3):Background. The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by pro-inflammatory cytokines, including the tumor necrosis factor- (TNF-). SOCS3 is also implicated in hypertriglyceridemia associated to insulin-resistance (IR). Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) levels are frequently found to be positively correlated to IR and plasma very low-density lipoprotein-triglycerides (VLDL-TG) concentrations. Aim. The present study aimed to investigate the possible role of TNF- and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. Methods and results. TNF- induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein levels, with no effect on its promoter activity. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels while an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fatty-acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD-1), and apo-B. These responses were associated with significant increase of SCD-1 protein, activation of SREBP-1, accumulation of cellular TG and secretion of apoB. HepG2SOCS3 show lower phosphorylation levels of IRS-1 Tyr896 and Akt Ser473 in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, FAS and apoB mRNA. Conclusions. Our data candidate PCSK9 as a gene involved in lipid metabolism regulated by pro-inflammatory cytokine TNF-, in a SOCS3 dependent manner.Proprotein subtilisin/kexin type 9 (PCSK9) induces pro-inflammatory response in macrophages:Background. Intraplaque release of inflammatory cytokines and chemokines from macrophages is directly implicated in atherogenesis, by inducing the proliferation and migration of media smooth muscle cells (SMCs) to the neointima. PCSK9 is present and released by SMCs within the atherosclerotic plaque but its role within the vascular wall is still unknown. Aim. In the present study, we tested the hypothesis of a pro-inflammatory effect of PCSK9 on macrophages. Methods and results. The pro-inflammatory effect of PCSK9 was assessed on THP-1-derived macrophages, exposed to different concentrations (0.250 ÷ 2.5 µg/ml) of human recombinant PCSK9 (hPCSK9). After exposure for 24h to 2.5 µg/ml PCSK9, a significant induction of IL-1β (8.17±2.88 fold), IL-6 (36.4±19.3 fold), TNF-α (67.4±25.9 fold), CXCL2 (42.6±0.0 fold), and MCP1 (17.0±6.8 fold), were observed. Importantly, physiological concentration of PCSK9 (0.250 µg/ml) also elicited a significant pro-inflammatory effect. Similar results were observed in human primary macrophages, where 2.5 µg/ml of hPCSK9 increased IL-1β (14.61±2.47 fold), IL-6 (4.17±0.86 fold), TNF-α (4.51±2.08 fold), CXCL2 (2.58±0.26 fold), and MCP-1 (1.76±0.07 fold) gene expression and the released of TNF-α (+82.3%) and IL-6 (+41.8%) in cultured media, as determined by ELISA assay. Co-culture experiments of HepG2 overexpressing hPCSK9 and THP1 macrophages also showed the induction of mRNA TNF-α (1.89±0.35 fold), IL-1β (2.03±0.29 fold), MCP-1 (4.82±1.26 fold) and CXCL2 (5.40±0.61 fold). Finally, the effect of hPCSK9 on TNF-α mRNA in murine LDLR-/- bone marrow macrophages (BMM) was significantly impaired as compared to wild-type BMM (5.44±0.28 fold vs 35.4±2.7 fold for LDLR-/- and wild-type, respectively). Conclusions. The present study provided evidences of a pro-inflammatory action of PCSK9 on macrophages, mainly dependent by the LDLR. The pathophysiological relevance of this effect still needs to be determined.

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BOTTA, MARGHERITA. "DECIPHERING THE ROLE OF ADIPOKINES ON PCSK9 REGULATION: EXPERIMENTAL EVIDENCE." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/695060.

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Background: Adipose tissue is an endocrine organ secreting active molecules, namely, adipokines. In a condition of dysfunctional visceral fat depots, adipokines may be detrimental for the cardiovascular system. The present study was aimed at evaluating some of the molecular mechanisms linking adipokines and the expression of proprotein convertase subtilisin/kexin 9 (PCSK9), the key regulator of low-density lipoprotein receptor and also involved in triglycerides (TG) metabolism. This latter evidence come from genetic studies reporting high levels of TG in patients with gain of function (GOF) mutations in PCSK9 gene while, in those carrying loss of function (LOF) mutations, LDL-C reduction associates with reduced fasting and post-prandial TG levels. Leptin is a cytokine-like hormone produced mainly by white adipose tissue and playing a role not only in satiety but also in regulating blood pressure, endothelial function, glucose homeostasis, insulin sensitivity. Resistin is an adipokine produced by adipose tissue in mice and by monocytes in human; it is involved in inflammation and insulin resistance. Both leptin and resistin are dysregulated in obesity. In order to study the complex biosystem of TG metabolism we have developed and characterized an in vitro tool allowing to study the impact of genetic variants of lipoprotein lipase (LPL), one of the major regulators of TG levels. We believe that this in vitro tool could be of interest to study the effect of PCSK9 on TG regulation. Materials: Human epatocarcinoma cell line (HepG2); silencing RNA (siRNA) anti-PCSK9 and anti-STAT3; qPCR; Western blot; enzyme-linked immunosorbent assay (ELISA); PCSK9 promoter luciferase reporter assay with plasmids pGL3-PCSK9-D4 (wild type), pGL3-PCSK9-SREmut (mutated on SRE element), pGL3-PCSK9-HNFmut (mutated on HNF element), pGL3-PCSK9-D1 (wild type) and pGL3-PCSK9-D1-STAT3mut (mutated on STAT3). C57BL6J mice, ob/ob mice (mice lacking leptin). Results: In a clinical setting, a positive association between circulating levels of leptin and PCSK9 (p=0.044 and β=0.295) was found in patients with BMI<25 Kg/m2. In the same cohort and according to BMI strata, no associations were found between resistin and PCSK9. When the link between leptin and PCSK9 was investigate in ob/ob mice lacking leptin, the hepatic expression of PCSK9 was significantly lower compared to the one of wild type mice. In C57BL6 mice a high-fat-diet resulted in raised levels of serum leptin and increased hepatic expression of PCSK9. In HepG2 cells, leptin and resistin induced (i) PCSK9 gene expression by +95% and +150%, respectively, and (ii) PCSK9 promoter activity via the involvement of Sterol Regulatory Element motif. Indeed, a mutation in HNF-1 motif did not alter leptin- and resistin-driven luciferase activity. The mandatory role of signal transducer and activator of transcription 3 (STAT3) in the relationship between adipokines and PCSK9 has been confirmed by silencing STAT3. After silencing, leptin- and resistin were not able to regulate the mRNA expression of PCSK9. Although not fully characterized, the involvement of STAT3 in the PCSK9 promoter activity has been tested by inserting a mutation on the STAT3 responsive element. Apolipoprotein (APOB) and microsomal triglycerides transfer protein (MTP) mRNA were increased by leptin (+57% and +60%, respectively) and resistin (+ 50% for both), an effect dependent on PCSK9, as demonstrated by using siRNA anti-PCSK9. As a further confirmation, a significant increment in APOB was found in response to PCSK9 overexpression. In order to study the complex biosystem of TG metabolism, which is also a target of PCSK9, we have initially developed and characterized an in vitro tool allowing to evaluate the impact of genetic variants of LPL, an enzyme involved in TG metabolism. This technique allowed to distinguished between LPL mutations leading to a reduced secretion or a reduced activity despite a normal synthesis. Conclusions This work describes the relationship between PCSK9 expression and the adipokines leptin and resistin. The molecular basis beyond these findings require the involvement of STAT3 pathway and the activation of SRE motif at PCSK9 promoter level. Overexpression of PCSK9 led to an increment of gene expression of APOB, whereas silencing PCSK9 corresponded to an opposite effect i.e. a decrement in gene expression. The tool developed to study LPL mutations could be of help in evaluating PCSK9 variants associated with raised TG levels.

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Ramin-Mangata, Stéphane. "Le rôle du récepteur aux LDL et de PCSK9 dans le diabète de type 2." Thesis, La Réunion, 2020. http://www.theses.fr/2020LARE0005.

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Les statines sont des médicaments hypolipémiants largement prescrits dans le cadre des maladies cardiovasculaires. Elles inhibent la synthèse endogène de cholestérol et induisent l’activation de l’expression du LDLR par le facteur de transcription SREBP-2. Ce sont des médicaments dont le bénéfice est indiscutable d’un point de vue cardiovasculaire. Néanmoins, l’action des statines est limitée par la proprotéine convertase subtilisin kexin type 9 (PCSK9), l’inhibiteur naturel du récepteur aux LDL (LDLR), qui est également sous la dépendance de SREBP-2. Ainsi, de nouvelles stratégies hypolipémiantes ciblant la forme circulante de PCSK9 ont été développées et autorisées. Ce sont les inhibiteurs de PCSK9. Bien que bénéfique, l’utilisation de statines à forte dose et à long terme induit dans certains cas l’apparition d’un diabète de type 2 chez les individus prédisposés. De plus, des variants génétiques « perte de fonction » de PCSK9 sont associés à l’apparition du diabète de type 2. Les effets des inhibiteurs de PCSK9 sur la survenue du diabète de type 2 à long terme ne sont pas connus à ce jour. Ainsi, nous avons émis l’hypothèse que l’engorgement en cholestérol des cellules β pancréatiques associé à des niveaux très élevés d’abondance du LDLR à la membrane suite à l’utilisation des statines et d’inhibiteurs de PCSK9 induit un dysfonctionnement cellulaire, une altération de la sécrétion d’insuline et à terme le diabète de type 2. Les objectifs de mes travaux de thèse ont été (i) de déterminer la modulation des taux de PCSK9 par les statines chez des individus diabétiques de type 2, (ii) d’étudier si les niveaux circulants réduits en PCSK9 seraient prédictifs de la survenue du diabète de type 2 et enfin (iii) d’évaluer l’effet des statines, de PCSK9 et des inhibiteurs de PCSK9 sur la sécrétion d’insuline par les cellules β. Au moyen de trois cohortes de patients, nous avons montré que la concentration plasmatique de PCSK9 est augmentée chez les diabétiques de types 2 et que les niveaux de PCSK9 circulants réduits sont négativement associés à une résistance à l’insuline et à une altération de la glycémie à jeun. En utilisant des sections de pancréas humains et des lignées de cellules β pancréatiques humaines, nous avons démontré pour la première fois que PCSK9 est exprimé, synthétisé et sécrété uniquement par les cellules β pancréatiques au niveau des îlots de Langerhans. Nous n’avons pas observé d’effet de PCSK9 et de ses inhibiteurs sur la sécrétion d’insuline en réponse au glucose. L’ensemble des résultats de mes travaux de thèse indiquent que l’inhibition de PCSK9 n’aura vraisemblablement pas d’effet pro-diabétogène, ce qui est rassurant pour le développement de ces nouvelles thérapies hypocholestérolémiants
Statins are lipid-lowering drugs widely prescribed to prevent cardiovascular diseases. They inhibit the endogenous synthesis of cholesterol and thereby increase LDLR gene expression by activating the SREPB-2 transcription factor. The positive effects of statins regarding cardiovascular diseases are undisputable. However, their action is limited by the proprotein convertases subtilisin kexin type 9 (PCSK9), the natural inhibitor of the LDL receptor (LDLR), which is also activated by the SREBP-2 transcription factor. As a result, novel lipid-lowering strategies targeting circulating PCSK9 have emerged and have been approved recently. These are the PCSK9 inhibitors. Despite their well-established beneficial effects, the use of high doses of statins for long-term treatments induces in rare instances the onset of type 2 diabetes in predisposed individuals. In addition, “loss of function” genetic variants of PCSK9 are associated with an increased risk of type 2 diabetes. The effects of long term use of PCSK9 inhibitors on the risk of type 2 diabetes remain to be established. Thus, we hypothesized that cholesterol overload of insulin secreting pancreatic beta cells induced by the overexpression of the LDLR at their plasma membranes following treatment with statins and PCSK9 inhibitors may cause cell dysfunction, lower insulin secretion, and ultimately type 2 diabetes. The aims of my thesis were (i) to determine the circulating levels of PCSK9 and their modulation by statins in patients with type 2 diabetes, (ii) to determine if reduced circulating PCSK9 levels are predictive of new onset type 2 diabetes and finally (iii) to investigate the effect of statins, PCSK9, and PCSK9 inhibitors on beta cell function. Using three cohorts of patients, we showed that circulating PCSK9 plasma levels are increased in patients with type 2 diabetes and that reduced circulating PCSK9 levels are negatively associated with insulin resistance and elevated fasting blood glucose. In human pancreatic sections and human pancreatic beta cell lines, we showed for the first time that PCSK9 is expressed, synthesized and secreted only by beta cells in pancreatic islets. We did not find any significant effect of PCSK9 or PCSK9 inhibitors on glucose stimulated insulin secretion. Altogether, my thesis works underpin that the use of PCSK9 inhibitors in the clinic will probably not be diabetogenic. This is reassuring regarding the development of these new lipid-lowering therapies

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Nguyen, My-Anh. "Characterization of PCSK9-mediated LDLR Degradation in Hepatic and Fibroblast Cells." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26114.

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The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

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Sarkar, Samantha Khadija. "Asociation of PCSK9 with Low Density Lipoproteins (LDL) in the Regulation of LDL-Cholesterol Levels." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32825.

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Proprotein Convertase Subtilisin / Kexin Type-9 (PCSK9) has emerged as a major regulator of plasma cholesterol levels. PCSK9 is secreted mainly from the liver and circulates as a plasma protein. PCSK9 binds cell surface low-density lipoprotein (LDL) receptors and mediates their degradation upon endocytosis in the liver. This decreases the liver’s ability to clear LDL-cholesterol from the blood. PCSK9 is also capable of binding LDL particles themselves; this interaction inhibits the ability of PCSK9 to bind and mediate LDLR degradation in cultured hepatic cells, but its effect on PCSK9 function in vivo remains unknown. A disordered N-terminal region of the PCSK9 prodomain is necessary for binding to isolated LDL particles in vitro. This N-terminal region is also autoinhibitory to PCSK9-LDL receptor binding. We hypothesized that the N-terminal of the PCSK9 prodomain plays a role in an allosteric mechanism that regulates PCSK9 function. Through mutagenesis studies, we found that both a conserved stretch of acidic residues and an adjacent conserved stretch of hydrophobic residues are crucial for the PCSK9-LDL interaction; the hydrophobicity of the residue at position 38 (Tyr) within the conserved acidic stretch was also found to be important for this. Helical wheel modeling of the prodomain N-terminal sequence revealed the potential for a lipid-ordered amphipathic helix to form, which may aid PCSK9 docking onto LDL. Replacing residues A44 and L41 with helix-disrupting proline residues abolished LDL binding. Co-pelleting ultracentrifugation assays also show that wild-type PCSK9 is capable of associating with liposomes, while the A44P mutation disrupts this lipid association. The A44P-PCSK9 mutation, showing an 80-90% decrease in LDL association but with LDL receptor binding and degrading functions intact, may serve as an important tool in future studies investigating the PCSK9-LDL interaction in vivo. Elucidation of the mechanism by which LDL-binding naturally inhibits PCSK9 activity may also help to develop new anti-PCSK9 therapeutics in the future.

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Molin, Tor. "Evolokumabs effekt och kostnadseffektivitet hos patienter utan familjär kolesterolemi." Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-85420.

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Atherosclerosis is the underlying cause for many serious cardiovascular diseases which causes over 50 % of all deaths in Sweden. Atherosclerotic plaque builds up in the vessel walls over decades that will eventually lead to a complete block of an artery or cause thrombosis when the plaque ruptures, this leads to myocardial infarction and stroke. A major contributing factor to the buildup of plaque is cholesterol, especially low density lipoprotein, LDL. LDL can oxidize and start an inflammation process and together with cells from the immune system form the basis for a plaque. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein which main function is to regulate the amount of LDL-receptors available by promoting their degradation. PCSK9 causes degradation of the receptors with the effect that more LDL is left in the blood stream. Evolocumab is a monoclonal antibody with PCSK9 as the target, with PCSK9 neutralised less LDL will be left in the blood stream which will help prevent atherosclerosis. This is a complex and expensive way of treating atherosclerosis, the primary treatment is statins and secondarily cholesterol absorbtion inhibitors, bile acid sequestrants and fibrates. The purpose of this study was to examine evolocumabs effect on cardiovascular events in high risk patients and for which patient groups it is cost-efficient. The method used was searching the medical database PubMed with the keywords “evolocumab” and “evolocumab cost-effectiveness”. 5 articles was analysed and they showed that evolocumab lowered LDL-cholesterol with 55-60 % and significantly improved the lipid profile of patients. The hazard risk was lowered by 20-25 % for serious cardiovascular events and a 10 % lower rate of death was assessed after 5 years of treatment. Since no large studies have followed up on patients for over 5 years and measured cardiovascular events and deaths it is hard to know for sure how efficient evolocumab is at preventing death. The price of evolocumab is 50000 SEK for one year of treatment and to assess which patients should be treated proved difficult due to the facts that the Swedish national regions have a side deal with the drug manufacturer Amgen in which they get compensation if evolocumab would prove to not be cost-efficient enough and there is no fixed acceptable price per quality adjusted life year (QALY) in Sweden.
Ateroskleros är grund till många allvarliga hjärt- och kärlsjukdomar och ligger bakom mer än hälften av alla dödsfall i Sverige. Sjukdomen innebär att plack byggs upp i kärlväggarna under flera decennier och till slu*t täpper igen ett kärl eller brister och en blodpropp bildas vilket leder till hjärtinfarkt och stroke. En bidragande faktor till bildningen av aterosklerotiska plack är kolesterol främst i form av lågdensitetlipoprotein (LDL), oxiderat LDL bidrar till att en inflammationsprocess startar i kärlet. Proprotein konvertas subtilisin/kexin typ 9 (PCSK9) är ett protein vars uppgift är att reglera antalet LDL-receptorer genom att binda in till receptorerna och ”märka” ut dem för nedbrytning. Evolokumab är en antikropp mot PCSK9 vars effekt ökar antalet LDL-receptorer vilket i sin tur minskar mängden LDL i blodet. Evolokumab är ett nytt och dyrt läkemedel, ateroskleros behandlas istället främst med statiner men också med kolesterolabsoptionshämmare, resiner och fibrater. Syftet med arbetet var att undersöka evolokumabs effekt att minska risk för hjärt- och kärlsjukdomar samt för vilka patientgrupper det är kostnadseffektivt. Metoden som användes var att söka artiklar på PubMed med sökorden ”Evolocumab” och ”Evolocumab cost effectiveness”. Fem artiklar undersöktes och de visade att evolokumab sänker LDL-kolesterolet i blodet med 55-60 % och även andra lipidvärden förbättras betydligt. Riskminskningen att drabbas av hjärt- och kärlsjukdomar bedömdes vara 20-25 % och riskminskningen att dö till följd av dessa sjukdomar uppskattades till 10 % efter 5 års behandling för patienter med hög risk att drabbas av dessa sjukdomar. Standardbehandling med evolokumab ger en kostnad på 50000 kr per år för läkemedlet och angående vilka patienter som bör få Evolokumab är det svårt att göra en gränsdragning då det i Sverige finns en sidoöverenskommelse som innebär att tillverkaren kompenserar landstingen om behandlingen inte är kostnadseffektiv samt att i Sverige finns inget fast värde för ett kvalitetsjusterat levnadsår (QALY).

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Bigolin, Paola. "Ipercolesterolemia familiare: dal genotipo al fenotipo ed implicazione terapeutiche dei nuovi farmaci biologici ipocolesterolemizzanti." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3426245.

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AIMS: Familial Hypercholesterolemia (FH) is a frequent genetic cause of early coronary artery disease, and is still under-diagnosed and under-treated. With the advent of PCSK9 inhibitors as adjunctive therapy to maximal lipid-lowering therapy, a significant reduction in cholesterol levels of low-density lipoprotein (LDL-C) and cardiovascular events was observed, while maintaining a good safety and tolerance profile. Ultrasonography (US) detects Achilles tendon (AT) xanthomas in patients (pts) with FH. Given the recent introduction of new therapies, there are no studies in the literature that evaluate the efficacy and safety of this therapy over a period of more than 3 years. We analysed the potential associations between FH genotype, clinical phenotype and ultrasonographic AT findings, evaluating the contribution of AT US to identify individuals with an FH-causing mutation. We also analysed the long-term efficacy and safety of additional therapy with PCSK9 inhibitors, comparing treatment with evolocumab and that with alirocumab. SUBJECTS AND METHODS: Genetic screening, clinical and biochemical parameters in 194 pts with possible, probable or definite clinical diagnosis of FH according to the Dutch Lipid Clinic Network Score (DLCNS); 71 pts underwent bilateral AT US.RESULTS: 43 pts carriers of null allele (NA) and 62 of defective (DEF) allele for LDL receptor while 33 pts with no known mutations (NM) for FH. Presence of xanthomas and gerontoxon, total and LDL-cholesterol (NA vs DEF vs NM: 326.5+97.7 mg/dl, 316.9+93.9 mg/dl, 211.1+76.3mg/dl, p<0.000) at diagnosis were significantly higher in NA pts than other subgroups. AT thickness was significantly different among the three groups (p< 0,005) and 78.2%, 72.4% and 31.6% had USX in NA, DEF and NM carriers respectively (p=0.002). Among the 52 pts positive for FH-causing mutations, 16 pts had a clinical diagnosis either possible or probable and in nine pts the presence of USX was clinically undetected and thereby not considered for DLCNS calculation. Tendon ultrasound was able to show a prevalence of 51% of tendon xanthomas in comparison to the prevalence of alterations detected only by physical examination, which was 10,2%.Following the addition of treatment with PCSK9 inhibitors, a mean reduction in LDL-C levels from 169 ± 30 mg / dl to 46 ± 16 was obtained, compared to the traditional maximal lipid-lowering treatment, ie in percentage terms a reduction of 72.4%. Compared to baseline LDL-C levels, this corresponds to an average reduction of 86.9%. There were no statistically significant differences between treatment with evolocumab and that with alirocumab in terms of reduction of LDL-C levels. In the course of traditional maximal lipid-lowering therapy, the goal of LDL cholesterol was obtained based on the personal level of cardiovascular risk in 0% of cases, while with the addition of PCSK9 inhibitors, 100% of subjects achieved therapeutic goal. No statistically significant differences were found following the introduction of PCSK9 inhibitor treatment with regard to CPK and transaminase levels. Over the years we have observed that LDL-C levels remained substantially stable. CONCLUSIONS: Genotypic functional characterization is associated with different phenotypic clinical features, AT thickness and presence of US xanthomas. AT ultrasonography may help reclassifying as definite FH, patients with DLCN score of possible/probable FH. Achilles tendon ultrasound has a greater sensibility than standard physical exam. It discloses a noticeable higher prevalence of tendon xanthomas (51%) in comparison to clinical evaluation (10,2%). This exam allows to look in a more integrated way at the cardiovascular and tendon complications in everyeach patient. It also suggests, when xanthomas are found, the necessity to adopt a stronger lipid-lowering therapy.In our study, in subjects with FH, it emerged that PCSK9 inhibitor therapy (evolocumab or alirocumab), in addition to maximal lipid-lowering therapy, results in a significant reduction of LDL-C levels, allowing the totality of patients to achieve the therapeutic goal of LDL-C related to its cardiovascular risk. Further studies are needed to confirm mainly the persistence of long-term efficacy and safety of PCSK9 inhibitor therapy and to evaluate on a larger scale whether there are differences between evolocumab and alirocumab in terms of efficacy in reducing LDL-C and cardiovascular risk.
INTRODUZIONE e SCOPI dello STUDIO: L’Ipercolesterolemia Familiare (FH) è un disordine del metabolismo lipidico su base genetica, rara in omozigosi (1/250000) ma coinvolgente 1 soggetto ogni 250 abitanti nella forma eterozigote. Il fenotipo lipidico è caratterizzato da livelli molto elevati di colesterolo delle lipoproteine a bassa densità (LDL) dalla nascita e da un rischio elevato di aterosclerosi che predispone ad eventi clinici cardiovascolari (CHD) precoci. La FH è causata da mutazioni nei geni che codificano per proteine chiave coinvolte nelle vie metaboliche che riguardano il recettore delle LDL (LRL-R) e il suo ciclo metabolico, con conseguente diminuzione dell’uptake cellulare delle LDL e conseguente aumento delle concentrazioni plasmatiche del colesterolo LDL (LDL-C). Tra i geni coinvolti sono note mutazioni con perdita di funzione nel gene LDLR, mutazioni nel gene dell’apolipoproteina B (ApoB) che alterano il dominio di legame dell’ApoB con LDL-R, mutazioni con guadagno di funzione nel gene per la proteina convertasisubtilisina/kexina tipo 9 (PCSK9).Tra le mutazioni del LDL-R si riconoscono cinque classi funzionali, una delle quali è chiamata allele nullo e normalmente determina un difetto nella sintesi del recettore con conseguente funzione recettoriale quasi completamente abolita (<5% rispetto alla norma). Le restanti sono legate un’alterata sintesi della proteina dovuta ad alterazioni della sequenza amminoacidica che comporta difetti nel trasporto del recettore, nel legame tra ligando e recettore, nella localizzazione dello stesso a livello della superficie cellulare e infine nel riciclaggio.Allo scopo di stabilire una diagnosi clinica, sono raccomandati i criteri del Dutch Lipid Clinic Network (DLCN) che permettono di fare diagnosi di FH considerando cinque aspetti anamnestici, clinici e bioumorali. La formazione precoce di gerontoxon, xantelasmi e xantomi sono markers clinici suggestivi per indirizzare verso la diagnosi di FH. L’utilizzo dell’ecografia consente di valutare con maggiore accuratezza lo spessore tendineo, aumentato nel caso in cui siano presenti accumuli lipidici. Dal momento che il tendine di Achille si è rivelato essere la più comune localizzazione per lo sviluppo di xantomi, la valutazione ecografica di questo distretto consente di aumentare notevolmente la sensibilità (fino al 75%) nella diagnosi di FH, a discapito di una relativa perdita di specificità nei confronti di altre forme di ipercolesterolemia.Con l’avvento degli inibitori di PCSK9 come terapia aggiuntiva a una terapia ipolipemizzante massimale, si è osservata una riduzione significativa dei livelli di colesterolo delle lipoproteine a bassa densità (LDL-C) e degli eventi cardiovascolari, mantenendo un buon profilo di sicurezza e tolleranza. In tale contesto si inserisce il nostro studio con la valutazione della mappatura genetica dell’Ipercolesterolemia Familiare in relazione al fenotipo clinico, l’approfondimento dell’utilità dell’impiego dell’ecografia dei tendini achillei come strumento di approfondimento diagnostico, l’analisi di dati di efficacia e sicurezza della terapia addizionale con inibitori di PCSK9.SOGGETTI e METODI:194 soggetti con diagnosi possibile, probabile o certa di FH, in accordo con i criteri del DLCN, sono stati sottoposti a screening genetico e valutazione delle caratteristiche cliniche e bioumorali; di 168 pazienti (pz) ad ora è disponibile il risultato dello screening genetico; 101 pz sono stati sottoposti ad ecografia bilaterale dei tendini achillei; gli xantomi ecografici sono stati definiti come presenza di uno spessore tendineo >6,15 mm in almeno un tendine e/o presenza di formazioni ipoecogene; 20 pazienti con FH eterozigote in trattamento con nuovi farmaci biologici ipocolesterolemizzanti (inibitori PCSK9).RISULTATI: Dei 168 pz con risultato dello screening genetico in particolare 105 pz presentavano mutazione del gene per il recettore delle LDL in forma eterozigote, di cui 43 portatori di allele nullo (NA) e 62 di allele difettivo (DEF); in 33 pz non sono state individuate mutazioni per FH (NM). La prevalenza di xantomi obiettivi e gerontoxon, insieme ai livelli di colesterolo totale e LDL basali sono risultati significativamente maggiori nei soggetti NA rispetto agli altri sottogruppi (xantomi obiettivi: NA vs DEF vs NM 71,4 vs 48,5 vs 30,7 %: p<0,001 Anova; LDL: NA vs DEF vs NM 326,5+97,7 vs 316,9+93,9 vs 211,1+76,3 mg/dl: p<0,001 Anova).Dei 101 pazienti di cui si disponeva del risultato degli esami bioumorali e dell’ecografia tendinea la prevalenza di xantomi obiettivi e gerontoxon, insieme ai livelli di colesterolo totale e LDL basali sono risultati significativamente maggiori nei soggetti NA rispetto agli altri sottogruppi (xantomi obiettivi: NA vs DEF vs NM 26,1 vs 13,8 vs 0,0 %: p=0,054 Anova; LDL: NA vs DEF vs NM 316+117 vs 321+109 vs 199+44 mg/dl: p<0,001 Anova). Lo spessore dei tendini achillei è risultato significativamente diverso tra i tre gruppi (NA vs DEF vs NM 7,64±2,06 vs 7,65±4,02 vs 5,67±0,75 mm: p<0,005 Anova) e la prevalenza di xantomi ecografici era del 78,2%, 72,4% e 31,6% nei soggetti portatori di NA, DEF e NM rispettivamente (p=0,002). Il solo esame obiettivo rilevava la presenza di xantomi tendinei achillei nel 10,2% dei soggetti, mentre l’ecografia tendinea rivelava una prevalenza di lesioni tendinee pari al 51%.Nell’ambito dei 74 pz sottoposti ad ecografia dei tendini achillei di cui si dispone attualmente del risultato dello screening genetico, sono stati considerati i 52 pz con mutazioni responsabili di FH; tra questi 36 pz avevano una diagnosi clinica certa di FH secondo i criteri DLCN (punteggio >8), mentre vi erano 16 pz con diagnosi possibile (punteggio tra 3 e 5) o probabile (punteggio tra 6 e 8). Di questi 16 pz 1 mostrava xantomi evidenziabili clinicamente mentre 10 presentavano lo xantoma ecografico. Nel sottogruppo dei 20 pz trattati con PCSK9, in seguito all’aggiunta di trattamento con inibitori di PCSK9 si è ottenuta, rispetto al trattamento ipolipemizzante tradizionale massimale, una riduzione media dei livelli di LDL-C da 169 ± 30 mg/dl a 46 ± 16, ossia in termini percentuali una riduzione del 72,4% (valore minimo 41,5% e massimo 87,5%). Rispetto ai livelli basali di LDL-C, ciò corrisponde a una riduzione media pari all’86,9%. Non sono emerse differenze statisticamente significative tra il trattamento con evolocumab e quello con alirocumab in termini di riduzione dei livelli di LDL-C. In corso di terapia ipolipemizzante tradizionale massimale si otteneva l’obiettivo di colesterolo delle LDL previsto in base al personale livello di rischio cardiovascolare nello 0% dei casi, mentre con l’aggiunta della terapia con inibitori di PCSK9 il 100% dei soggetti raggiungeva l’obiettivo terapeutico. Non si sono dimostrate differenze statisticamente significative in seguito all’introduzione del trattamento con inibitori di PCSK9 per quanto riguarda i livelli di CPK e di transaminasi. Nel corso degli anni abbiamo osservato che i livelli di LDL-C si mantenevano sostanzialmente stabili.CONCLUSIONI:La caratterizzazione genotipica funzionale si conferma essere associata a fenotipi clinici diversi, anche in termini di spessori tendinei e prevalenza di xantomi ecografici, confermando come il paziente con allele nullo presenti una maggiore aggressività clinica della patologia.L’ecografia del tendine di Achille risulta più sensibile rispetto all’esame obiettivo classico, rilevando una prevalenza di xantomi tendinei notevolmente maggiore rispetto a quella rilevata mediante il solo esame obiettivo. Tale esame consente di guardare in modo integrato alle complicanze tendinee e vascolari nel singolo paziente, suggerendo, ove siano presenti xantomi, un trattamento ipolipemizzante più intensivo.I risultati preliminari di questo studio suggeriscono inoltre come l’ecografia dei tendini di Achille possa essere uno strumento da considerare nell’aiutare a riclassificare quei pazienti per in cui il DLCN score è compatibile con diagnosi possibile o probabile di FH. Tale strumento potrebbe inoltre rivelarsi un valido alleato per il clinico, aiutandolo nel raggiungimento di una diagnosi sempre più precoce, ed una garanzia per il paziente di ricevere quanto prima il trattamento farmacologico più adeguato alla sua fascia di rischio.L’utilizzo degli anticorpi monoclonali anti-PCSK9, evolocumab ed alirocumab, rappresenta un approccio terapeutico innovativo, caratterizzato da elevato profilo di sicurezza ed altamente efficacie in associazione alla terapia massimale attualmente disponibile nei pazienti eterozigoti per FH. Ulteriori studi sono necessari per confermare principalmente la persistenza di efficacia e sicurezza a lungo termine della terapia con inibitori di PCSK9 e per valutare su larga scala se vi siano differenze tra evolocumab e alirocumab in termini di efficacia nella riduzione dei livelli di LDL-C e del rischio cardiovascolare.

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Mishra, Priyambada. "Study of Inhibitory Effect of Epididymal Cres on Pc4/Pcsk4 Activity." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19954.

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PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

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Denis, Nicholas. "Quantitative Subcellular Analysis of the Effects of the Enigmatic Protein PCSK9." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20133.

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PCSK9 is the third gene implicated in autosomal dominant hypercholesterolemia, due to its role in promoting the degradation of the low density lipoprotein receptor (LDLR). Little is known regarding the mechanism by which it promotes the degradation of LDLR, nor the effects PCSK9 has on other cellular proteins. I report here the first quantitative subcellular proteomic study of proteins affected by the expression of a variant of PCSK9. I show that the expression levels of 293 proteins were affected by the expression of the PCSK9-ACE2-V5 construct. Of particular interest, is a protein involved in receptor recycling, EHBP1, which shows reduced protein levels by both PCSK9-ACE2-V5 and the PCSK9-D374Y mutant. I show that an EHBP1 binding protein, EHD4, binds with PCSK9 and LDLR. These results establish novel effects of PCSK9 on liver cell protein levels, of which some relating to endosomal sorting are shown to bind to PCSK9 and LDLR in complex, providing insight into the mechanism of PCSK9 mediated LDLR degradation.

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Vilaplana, Saiz Marta. "Design and synthesis of new potentially inhibitors of PCSK9 and KRAS proteins." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673609.

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This thesis shows the research carried out on two specific diseases: familial hypercholesterolemia, which is included within the cardiovascular diseases group, and cancer. These diseases are the major cause of deaths in Spain and worldwide. The study of familial hypercholesterolemia is of great interest, as it is mainly caused by a protein called PCSK9 whose atypical functioning leads to an increase of LDL-C in blood. Thus, the study of new diseases results in the study of new therapeutic targets. Therefore, a thorough investigation on the PCSK9 protein has been conducted by means of molecular modelling analysis. Also, the synthesis of potentially PCSK9 inhibitors new compounds has been performed. The modelling studies have been based on the research of new binding sites and in de novo synthesis of new inhibitors. On the other side, the experimental synthesis of new compounds has been conducted by the preparation of pyrrolo[2,3-d]pyrimidines and finally, by the preparation of a series of compounds analogues of Combretastatin A4 through modification in the two aromatic rings and the fixation of its configuration with the introduction of a cycle. In the case of cancer, the study is based mainly on the research of a key protein in cell proliferation, growth and cell signalling. KRAS protein is either active or mutated in most common cancers, being the main cause of pancreas and colorectal cancer. Research on this target is of high chemical and biological relevance since no drugs have been found to act directly on said target. Molecular modelling studies are performed in order to find the binding site of a reference product whose inhibiting capacity over the protein is known. Furthermore, the synthesis of a series of symmetric pyrazoles with different structural modifications has also been carried out. Additionally, biological WST-8 / CCK8 assay have been performed on some of the synthetized compounds, specifically on two Combretastatin A4 analogues, in an endothelial cell line that allows studying both endothelial dysfunction and cardiovascular diseases. Research on the MAPK signalling pathway has been performed with pyrazoles structure derivatives.

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Ngqaneka, Thobile. "The impact of Niacin on PCSK9 levels in vervet monkeys (Chlorocebus aethiops)." University of Western Cape, 2020. http://hdl.handle.net/11394/7931.

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Magister Pharmaceuticae - MPharm
Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and strokeremain a major cause of death globally. Various deep-rooted factors influence CVDdevelopment; these include but are not limited to elevated blood lipids, high blood pressure,obesity and diabetes. A considerable number of proteins are involved directly and indirectly inthe transport, maintenance and elimination of plasma lipids, including high and low-densitylipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in theremoval of LDL particles from systemic circulation. One such mechanism is associated withthe gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which hasbecome an exciting therapeutic target for the reduction of residual risk of CVDs. Currently,statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop moreeffective LDL-C-lowering drugs that might supplement statins. This study was aimed atcontributing to the generation of knowledge regarding the effect of niacin in reducing LDLlevels through PCSK9 interaction.

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Balzarotti,G. "PCSK9 (PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9)AND GLUCOSE METABOLISM: WHICH CONNECTION?" Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/543205.

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PCSK9 (proprotein convertase subtilisin/kexin type 9) and glucose metabolism: which connection?Background: PCSK9 (proprotein convertase subtilisin/kexin type 9), is a protein, mainly synthesized and secreted by the liver, which binds to specific target proteins and escorts them towards lysosomes for degradation. The best defined activity of PCSK9 is its ability to modulate the hepatic uptake of LDL cholesterol (LDL-C), by enhancing the intracellular degradation of the LDL receptor (LDLR). In humans, several mutations in PCSK9 gene were described, both “gain-of-function” mutations associated to hypercholesterolemia and “loss of function” mutations linked to low LDL-C levels [1]. These findings suggest PCSK9 inhibitors as a promising class of drugs for the treatment of patients with severe hypercholesterolemia or at very high cardiovascular risk. However, some gaps regarding the potential role of PCSK9 in targeting the LDLR in organs other than the liver are still open. Indeed, the LDLR is abundantly expressed in pancreatic β-cells, where it plays a key role in the uptake of plasma LDL particles [2]. Therefore, further investigations are needed to better clarify the physiological role of PCSK9, also in light of its pharmacological targeting.Methods: WT, PCSK9 KO, LDLR KO, PCSK9/LDLR DKO, albumin (Alb)Cre+/PCSK9LoxP/LoxP (liver selective PCSK9 KO mice) and AlbCre-/PCSK9LoxP/LoxP mice were fed a HFD (High Fat Diet – 45% Kcal fat) or SFD (Standard Fat Diet – 10% Kcal fat) for 12 or 20 weeks. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology and cholesterol accumulation in pancreatic islets were studied in the different animal models. Results: Glucose clearance was significantly impaired in PCSK9 KO mice fed a SFD or a HFD for 20 weeks compared to WT animals, with both diet. On the contrary, insulin sensitivity was not affected as both animals showed a similar decrease in plasma glucose levels following insulin injection (ITT). Plasma insulin and C-peptide levels were reduced in PCSK9 KO mice compared to WT and accordingly fasting and refeeding experiments showed increased plasma glucose but reduced insulin levels in PCSK9 KO compared to controls. A detailed analysis of pancreas morphology of PCSK9 KO mice vs WT littermates revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels. This phenotype was completely reverted in PCSK9/LDLR DKO mice implying the LDLR as the PCSK9 target responsible for the phenotype observed. Further studies in AlbCre+/PCSK9LoxP/LoxP, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9 does not impact β-cells function and insulin secretion. Conclusion:The PCSK9/LDLR axis affects β-cells function and control insulin secretion. Our data indicate that this effect is independent of circulating PCSK9, and is probably related to local effects of PCSK9 suggesting the possibility that anti-PCSK9 antibodies or liver specific therapies, such as siRNAs, might have a limited impact on LDLR expression in pancreas and β-cells dysfunction.

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Moreau, François. "Rôle de PCSK9 et conséquences des chirurgies bariatriques sur le métabolisme intestinal du cholestérol." Thesis, Nantes, 2017. http://www.theses.fr/2017NANT4069/document.

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L’intestin est un acteur majeur du métabolisme du cholestérol de part son rôle dans dans l’absorption, la sécrétion des lipoprotéines et l’efflux transintestinal de cholestérol (TICE). De plus, c’est le second organe majeur, après le foie, à exprimer la Proproteine Convertase Subtilisine Kexine de type 9 (PCSK9), un inhibiteur naturel du récepteur aux LDL. Notre analyse des mécanismes moléculaires impliqués dans l’ hypocholestérolémie induite par les chirurgies bariatriques montre que la sleeve gastrectomie induit une hypocholestérolémie transitoire et modérée lié aux modifications de prise alimentaire. En revanche, le by-pass Roux en Y (RYGB) réduit fortement la cholestérolémie, stimule significativement son élimination fécale en induisant le TICE et en réduisant l’absorption intestinale de cholestérol. La seconde partie de ma thèse visait à répondre à une controverse autour de la faculté de l’intestin à sécréter PCSK9. In vivo (souris) et ex vivo (souris et homme), il ne semble pas que les cellules intestinales matures sécrètent PCSK9. En revanche, nous confirmons que la lignée humaine Caco2 est capable de sécréter PCSK9 mais que cette sécrétion est abolie lorsque les cellules deviennent matures. Les mécanismes responsables de cette perte de sécrétion restent mal définies mais sont dues au moins à deux paramètres: 1) une réduction du contenu intracellulaire induite par un catabolisme lysosomal accru; 2) une modification posttraductionelle de PCSK9 (glycosilation) altérant les voies de sécrétion post-Golgiennes. Les cellules Caco2 constituent un outil précieux pour disséquer les mécanismes et partenaires protéiques nécessaires à la sécrétion de PCSK9. Leurs identifications pourraient permettre de développer de nouveaux inhibiteurs pour réduire la sécrétion de PCSK9, réduire l’hypercholestérolémie et lutter plus efficacement contre les maladies cardiovasculaires
The intestine is a major actor of cholesterol metabolism from its role in absorption, secretion of lipoproteins and transintestinal cholesterol efflux (TICE). In addition, it is the second major organ, after the liver, to express the Proproteine Convertase Subtilisin Kexin type 9 (PCSK9), the natural inhibitor of the LDL receptor. Our analysis of molecular mechanism involved in hypocholesterolemia induced by bariatric surgeries shows that the gastrectomy sleeve induces a transient and moderate hypocholesterolemia linked to the modification of the food intake. In contrast, the Roux-Y by-pass (RYGB) strongly reduces cholesterol, significantly stimulates its fecal elimination by inducing TICE and decreasing intestinal absorption of cholesterol. The second part of my thesis consisted to elucidate the controversy around the faculty of the intestine to secrete PCSK9. In vivo (mice) and ex vivo (mice and human), it seems that mature enterocytes can’t secrete PCSK9. On the other hand, we confirm that the Caco2, an human intestinal cell line, is capable of secreting PCSK9, but this secretion is abolished when the cells become mature. Mechanisms responsible for this loss of secretion remain poorly defined and are, at least, due to: 1) a reduction in the intracellular content induced by increased lysosomal catabolism; 2) a post-translational modification of PCSK9 (glycosilation) altering post-Golgi secretion pathways. Caco2 cells are a powerfull tool for identify the mechanisms and partners required for the secretion of PCSK9. Their identifiers allow the development of new inhibitors to reduce the secretion of PCSK9, reduce hypercholesterolemia and fight more effectively against cardiovascular diseases

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Lasica, Rick, and Ashley Loy. "Cost-Effectiveness Analysis of PCSK9 Inhibitors for the Treatment of Heterozygous Familial Hypercholesterolemia." The University of Arizona, 2017. http://hdl.handle.net/10150/624203.

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Class of 2017 Abstract
Objectives: To determine the cost-effectiveness of proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors with high-intensity statins compared to high-intensity statins alone for the treatment of heterozygous familial hypercholesterolemia (HeFH).Methods: A Markov model was built through TreeAge Pro to model two groups: patients taking PCSK9 inhibitors with high-intensity statins or high-intensity statins alone. For each group, there were five health states that patients could be in: well, unstable angina, myocardial infarction, ischemic stroke, or death. The data used in the model were extracted from published clinical trials evaluating PCSK9 inhibitors and statins.Results: For the primary analysis, the overall cost and effectiveness was $31,390.93 and 23.01 for the statin alone group and $362,798.50 and 24.32 for the PCSK9 with statin group, respectively. The incremental cost, incremental QALY, and incremental cost-effectiveness ratio (ICER) was $331,407.60, 1.31 QALYs, and $252,833.60/QALY, respectively.Conclusions: Since the calculated ICER was higher than the pre-established threshold of $150,000, the results from our primary analysis suggest that treatment of patients with HeFH with a PCSK9 inhibitor and a high-intensity statin is not cost effective, compared to treatment with a high-intensity statin alone. However, when certain parameters (cost of PSCK9 and mortality rate) were adjusted in the secondary analyses, these agents appear to be cost-effective.

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GRENI, FEDERICO. "Variabilità fenotipica nell'emocromatosi: studio di due potenziali modificatori genetici in PCSK7 e GNPAT." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/140994.

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Introduction and aim: Hereditary hemochromatosis (HH) is a genetic disease characterized by a progressive iron overload in different tissues. hom*ozygosity for the p.C282Y mutation is the most frequent genotype associated with the disease and it is directly responsible for an inappropriate production of hepcidin, the main regulator of iron homeostasis. Several evidences indicated that p.C282Y hom*ozygous genotype has an incomplete penetrance due to the combined action of genetic and acquired modifier factors. Recently, the attention was focused on GNPAT rs11558492 and PCSK7 rs236918 single nucleotide polymorphisms (SNPs). The aim of my thesis was to analyse the role of these potential genetic modifiers in an Italian cohort of p.C282Y hom*ozygotes.Materials and methods: Patients: 298 patients (205 males and 93 females) and 169 healthy controls. Exclusion criteria were: alcohol intake >50 g/day in men and >30 g/day in women, chronic hepatitis, inflammatory status. SNPs genotyping was performed by ARMS-PCR or PCR-RFLP. Random samples were confirmed by direct sequencing. Patients and controls allelic and genotypic frequencies were compared to EVS database and analysed according to serum ferritin levels (SF), liver iron concentration (LIC) measured by liver biopsy or magnetic resonance, iron removed (IR) and liver fibrosis histologically assessed by Ishak score (IS). Fisher’s exact test, chi-squared test and t-test were used to perform statistical comparisons between groups and averages of considered variables.Results: GNPAT rs11558492 analysis. Our results demonstrated that: a. allelic and genotypic frequencies were comparable among patients, controls and EVS data. No significant differences were found even considering two subgroups of males only with extreme phenotypes (SF <1000 mcg/L, IR <5 g and/or LIC <100 mcmol/g vs SF >2000 mcg/L, IR >10 g and/or LIC> 50 mcmol/g); b. according to iron indices, allelic and genotypic frequencies did not significantly differ neither among patients nor compared to controls, limited to SF; c. similarly, minor allele (G) frequency did not differ between patients with absent/mild fibrosis and patients with severe fibrosis/cirrhosis (20.5% vs 25%). PCSK7 rs236918 analysis. Our study demonstrated that: a. minor allele (C) frequency was higher in patients with severe fibrosis/cirrhosis than in patients with absent/mild fibrosis (21.9% vs 7.1%; p=0.003); b. C-allele carriers were more likely to have worse liver staging scores than wild-type patients (OR=2.77, p=0.0018; ORmale-only=2.56, p=0.0233); c. PCSK7 genotype has a direct effect on severe fibrosis/cirrhosis (OR=3.11, p=0.0157) and a mild nonsignificant indirect effect mediated through SF and IR (mediation analysis: 22% and 28%, respectively).Conclusions: Our results demonstrated that: a. GNPAT rs11558492 is not a major modifier of iron status in HH patients and controls, and is not associated with severe fibrosis/cirrhosis in HH patients. b. PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian HH patients.

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Alghamdi, Rasha Hassen. "Development of Inhibitors of Human PCSK9 as Potential Regulators of LDL-Receptor and Cholesterol." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30492.

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Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) is the ninth member of the Ca+2-dependent mammalian proprotein convertase super family of serine endoproteases that is structurally related to the bacterial subtilisin and yeast kexin enzymes. It plays a critical role in the regulation of lipid metabolism and cholesterol homeostasis by binding to and degrading low-density lipoprotein-receptor (LDL-R) which is responsible for the clearance of circulatory LDL-cholesterol from the blood. Owing to this functional property, there is plenty of research interest in the development of functional inhibitors of PCSK9 which may find important biochemical applications as therapeutic agents for lowering plasma LDL-cholesterol. The catalytic domain of PCSK9 binds to the EGF-A domain of LDL-R on the cell surface to form a stable complex and re-routes the receptor from its normal endosomal recycling pathway to the lysosomal compartments leading to its degradation. Owing to these findings, we propose that selected peptides from PCSK9 catalytic domain, particularly its disulphide (S-S) bridged loop1 323-358 and loop2 365-385, are likely to exhibit strong affinity towards the EGF-A domain of LDL-R. Several regular peptides along with corresponding all- dextro and retro-inverse peptides as well as the gain-of-function mutant variants were designed and tested for their regulatory effects towards LDL-R expression and PCSK9-binding in human hepatic HepG2 and mouse hepatic Hepa1c1c7 cells. Our data indicated that disulfide bridged loop1-hPCSK9323-358 and its H357 mutant as well as two short loop2-hPCSK9372-380 and its Y374 mutant peptides modestly promote the LDL-R protein levels. Our study concludes that specific peptides from the PCSK9 catalytic domain can regulate LDL-R and may be useful for development of novel class of therapeutic agents for cholesterol regulation.

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Thain, Katherine Roberta. "Identifying functional single nucleotide polymorphisms in two candidate genes (PROC and PCSK9) in sepsis." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44755.

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Genetic variation contributes to outcome from sepsis. A large number of associations have been observed between genetic variants and sepsis outcome, however, identification of causal single nucleotide polymorphisms (SNPs), or their mechanisms of action, have not been successfully elucidated. The aims of this project are to identify causal variants in two candidate genes and determine whether these variants are involved in the mechanisms leading to altered outcomes in sepsis. The known pathophysiology of sepsis is complex and involves dysregulation of several systemic processes, including the coagulation and inflammatory systems. Based on this knowledge, and known literature on genetic variation in coagulation genes, PROC was chosen as a candidate gene in which to search for causal SNPs. In addition, based on the known role of lipids in sepsis, as well as the already identified causal SNPs in the PCSK9 gene, PCSK9 was selected as a second candidate gene to test the hypothesis that genetic variation in lipid mediators alters outcome in sepsis. Two intronic SNPs were found in the PROC gene (rs2069915 and rs2069916) that are in high linkage disequilibrium and appear to modify untranslated mRNA, leading to lower concentrations of circulating protein C in individuals hom*ozygous for the major alleles of these SNPs. Furthermore, in the PCSK9 gene, an intronic SNP (rs644000) was found that appears to mark known Loss-of-Function and Gain-of-Function coding SNPs, and was associated with outcome in two cohorts of patients with septic shock, and with a reduction of cytokine levels in a subset of these patients. Additionally, using murine genetic Pcsk9 knock-out and pharmacologic inhibition strategies in a murine model of systemic bacteremia, a markedly attenuated global, cardiovascular and inflammatory cytokine response to lipopolysaccharide administration was observed. Furthermore, increased endotoxin clearance was measured after PCSK9 knock-out. Together these results indicate that reduction of PCSK9 activity in both mice and humans reduces the inflammatory response and improves outcome in septic shock.The work presented here furthers the understanding of the role played by non-coding SNPs in protein expression and has implications for a new, potentially personal, drug strategy for sepsis patients in intensive care units.

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Honorato, Aldrina Laura da Silva Costa. "Investigação de mutações no gene PCSK9 em famílias com diagnóstico clínico de Hipercolesterolemia Familiar." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17138/tde-07122018-091850/.

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A hipercolesterolemia familiar (HF) é uma alteração de origem genética comum que pode se manifestar clinicamente desde o nascimento e provoca um aumento nos níveis plasmáticos de LDL-colesterol (LDL-c), xantomas e doença coronária prematura. Sua detecção e tratamento precoce reduzem a morbidade e mortalidade coronária. A identificação e rastreamento em cascata familiar usando níveis de LDL-c e detecção genética é a estratégia mais aconselhável e rentável para descoberta de novos casos. O tratamento crônico com estatinas reduz o risco cardiovascular da população em geral, contudo, estudos clínicos com estatinas revelam risco cardiovascular residual mesmo após correção das concentrações de LDL-c. Com o surgimento de novas drogas e mais recentemente um inibidor da enzima pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), este estudo enfatizou na investigação específica para aqueles acometidos com defeitos genéticos nessa enzima, por ser de frequência ainda mais rara e pouco estudada, necessitando de melhor investigação na população em estudo a fim de rastrear a ocorrência de mutações patológicas na PCSK9. O objetivo desse estudo foi identificar e caracterizar mutações e/ou deleções patológicas no gene PCSK9 em pacientes com Hipercolesterolemia Familiar provenientes do Hospital das Clínicas de Ribeirão Preto da FMRP/USP selecionados para o teste genético. Foi feito o rastreamento de mutações pelo método Hight Resolution Melting (HRM), de forma prática, rápida e eficiente, onde mutações detectadas foram seqüenciadas. Foram identificadas 7 mutações não patogênicas, caracterizando que a população estudada não apresenta Hipercolesterolemia Familiar associada a mutações no gene PCSK9, fato que não exclui o diagnóstico por outros defeitos genéticas associados a doença.
Familial hypercholesterolemia (FH) is an alteration of common genetic origin that can manifest clinically from birth and which causes an increase in the LDL-cholesterol plasma levels (LDL-c), xanthomas and premature coronary disease. Its early detection and treatment reduce morbidity and coronary mortality. The identification and tracking in familial cascade using levels of LDL-c and genetic detection is the most advisable and profitable strategy to find new cases. The chronic treatment with statins reduces the cardiovascular risk in the population in general. However, clinic studies on statins show a residual cardiovascular risk even after the correction of LDL-c concentrations. With the appearance of new drugs and, more recently, of a proprotein convertase subtilisin/kexin type 9 enzyme inhibitor (PCSK9), this study highlighted the specific investigation for those stricken by genetic defects in this enzyme, once it is even rarer and understudied and needs further investigation in the study\'s population aiming at tracking the occurrence of a pathological mutation in the PCSK9. This study aimed at identifying and characterizing mutations and/or pathological deletions in the PCSK9 gene in patients with Familial Hypercholesterolemia from the RPMS/USP Ribeirão Preto Clinical Hospital which were selected for the genetic test. We performed the mutation tracking by using the High Resolution Melting (HRM) method in a practical, fast and efficient way, where the mutations detected were sequenced. We identified 7 non-pathogenic mutations, showing that the population studied does not present Familial Hypercholesterolemia associated to mutations in the PCSK9 gene, which doesn\'t exclude the diagnosis by other genetic defects associated to the disease.

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Duff, Christopher. "Post-translational regulation of the low-density lipoprotein receptor : PCSK9 as a therapeutic target." Thesis, University of Leeds, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.749285.

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Matyas, Angela. "The Functional Characterization of PCSK9's Binding Interactions with LDL and the LDL Receptor." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40592.

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Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.

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Cho, Elizabeth. "Using Pharmacogenetics to Find Treatment for Familial Hypercholesterolemia Patients with Both apoB and PCSK9 Mutations." Scholarship @ Claremont, 2019. https://scholarship.claremont.edu/scripps_theses/1229.

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Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

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COGGI, DANIELA. "RELATIONSHIP BETWEEN PLASMA LEVELS OF PCSK9, VASCULAR EVENTS AND MARKERS OF SUBCLINICAL ATHEROSCLEROSIS AND INFLAMMATION." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/811217.

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Contesto e scopo: La proproteina convertasi subtilisina/kexina di tipo 9 (PCSK9), uno dei principali regolatori del metabolismo del recettore delle LDL, è stata associata allo sviluppo di aterosclerosi. Diversi studi hanno confermato tale associazione attraverso vie lipidiche e non lipidiche. Tuttavia, le relazioni dirette tra PCSK9 circolante e marcatori di aterosclerosi subclinica e clinica sono ancora da chiarire. Pertanto, abbiamo valutato le relazioni tra i livelli plasmatici di PCSK9 ed alcuni indici di aterosclerosi subclinica (marcatori di imaging) e clinica (eventi vascolari; EV). Un altro obiettivo è stato l'identificazione dei determinanti indipendenti di PCSK9, con particolare attenzione ai lipidi e ai biomarcatori infiammatori. Infine, abbiamo anche valutato la relazione tra alcuni marcatori di imaging e quattro SNPs del gene PCSK9, noti per essere associati alla presenza di bassi livelli di colesterolo LDL. Per validare i risultati ottenuti in quest’ultima parte, le analisi genetiche sono state replicate in una coorte indipendente reclutata nel Regno Unito (UK). Metodi: Lo studio è stato realizzato sfruttando le banche dati, biobanche e la banca di immagini dello studio IMPROVE. 3,703 soggetti europei (54-79 anni; 48% uomini), privi di EV al basale e definiti ad alto rischio per la presenza di almeno tre fattori di rischio vascolare, sono stati reclutati e seguiti per 36 mesi. PCSK9 è stata misurata tramite ELISA e trasformata in logaritmo prima delle analisi. I marcatori di imaging convenzionali [spessore medio-intimale carotideo (cIMT, dall’inglese intima-media thickness) e dimensione della placca carotidea] ed emergenti [cambiamento di cIMT nel tempo, ecolucenza dello spessore del complesso medio intimale della carotide comune misurato in zone libere da placca (PF CC-IMTmean), ecolucenza della placca più grande rilevata in tutto l'albero carotideo e punteggio di calcio carotideo (cCS, dall’inglese carotid calcium score)] sono stati misurati su scansioni ultrasonografiche conservate nella banca di immagini. In particolare, l'ecolucenza è stata misurata in termini di mediana della scala dei grigi (GSM, dall’inglese grey scale median) della distribuzione dei pixel di una specifica regione d’interesse, mentre il cCS è stato calcolato come somma delle lunghezze dei coni d’ombra acustici generati dal calcio all'interno delle placche carotidee. I lipidi sono stati misurati con metodi enzimatici (ad eccezione del colesterolo LDL che è stato calcolato con la formula di Friedewald). Tra i marcatori infiammatori, la proteina C reattiva ad alta sensibilità (hs-PCR) è stata misurata con la turbidimetria, mentre il conteggio dei globuli bianchi (WBC, dall’inglese white blood cells) e la formula leucocitaria sono stati misurati localmente. Tutti i soggetti dello studio IMPROVE e della coorte UK (n=22,179; 48 % uomini) sono stati genotipizzati.Risultati: Nell'analisi univariata, PCSK9 correlava positivamente con colesterolo totale, LDL e HDL e con trigliceridi e basofili (tutte le p <0.0001), mentre correlava negativamente con neutrofili ed eosinofili (entrambe le p=0.04). Le correlazioni positive osservate con hs-PCR e con il conteggio dei WBC erano solo vicine alla significatività statistica (p=0.060 e 0.064, rispettivamente). Le terapie con fibrati o statine (positivamente; entrambe le p <0.0001), così come sesso maschile e storia familiare di diabete (negativamente; entrambe le p <0.05) erano i predittori indipendenti più forti dei livelli plasmatici di PCSK9. Nell'analisi non aggiustata, si osservava una correlazione negativa tra PCSK9 e variabili basali di cIMT (IMTmean, IMTmax, IMTmean-max, e PF CC-IMTmean), una correlazione negativa tra PCSK9 e la variazione di cIMT nel tempo (Fastest-IMTmax-progr) e cCS (tutte le p ≤0.01), mentre si osservava un trend positivo tra PCSK9 e GSM sia del PF CC-IMTmean che della placca carotidea (entrambe le p ≤0.0001). Il cCS (positivamente) e il GSM del PF CC-IMTmean (positivamente) erano associati significativamente (o vicini alla significatività) a PCSK9 in diversi modelli multivariati (tutte le p ≤0.064). Tutte le correlazioni osservate all’analisi univariata tra PCSK9 e le variabili basali di cIMT, Fastest-IMTmax-progr e GSM della placca carotidea perdevano la significatività statistica dopo aggiustamento delle stesse per età, sesso, latitudine ed altri potenziali confondenti. Durante il follow-up [mediana (intervallo interquartile): 3.01 (2.98; 3.12) anni], sono stati registrati 215 EV: 125 coronarici, 73 cerebrali e 17 EV periferici. Tra questi, 37 erano eventi hard (infarto miocardico, morte improvvisa ed ictus). Nell'analisi non aggiustata, PCSK9 era associata positivamente ad eventi combinati e coronarici (entrambe le p <0.01), ma non ad eventi cerebrovascolari. Anche in questo caso, tuttavia, tutte le associazioni osservate perdevano la significatività statistica dopo aggiustamento delle analisi per età, sesso e stratificazione per latitudine. La mancanza di associazione con EV era confermata anche nel modello aggiustato per tutti i fattori confondenti considerati e nelle analisi focalizzate sugli eventi hard. Per quanto riguarda il ruolo delle varianti genetiche, nessuno dei quattro SNPs considerati correlava con cIMT (IMTmean, IMTmax, IMTmean-max) quando l'analisi era effettuata nei soggetti reclutati nello studio IMPROVE. La variante rs11591147, invece, correlava negativamente con l’IMTmax misurato nella popolazione UK (p=0.002). Combinando le quattro varianti genetiche in uno score, la relazione con cIMT era non significativa nello studio IMPROVE, mentre era negativa e significativa nella popolazione UK (tutte le p <0.01).Conclusioni: I livelli plasmatici di PCSK9 non sono associati a EV. Per quanto riguarda i marcatori dell'aterosclerosi subclinica, i livelli plasmatici di PCSK9 non sono associati né alla dimensione della lesione, né all'ecolucenza della placca carotidea, ma sono associati all'ecolucenza dello spessore della parete carotidea e al carotid calcium score. Ulteriori studi sono pertanto necessari per comprendere meglio il ruolo di tale proproteina nell'ecolucenza dello spessore della parete carotidea e nel carotid calcium score. La terapia con fibrati o statine, così come il sesso maschile e la storia familiare di diabete sono i predittori indipendenti più forti di PCSK9 circolante. È stata inoltre confermata l'associazione, precedentemente osservata, tra PCSK9 circolante e alcuni marcatori lipidici ed infiammatori. La relazione tra i livelli plasmatici di PCSK9 ed altri marcatori infiammatori (neutrofili, basofili ed eosinofili) merita ulteriori indagini, così come merita ulteriori indagini l’associazione tra le quattro varianti genetiche di PCSK9 selezionate e il cIMT nella coorte britannica, in quanto lascia intravvedere un possibile ruolo di SNPs o polimorfismi genici di PCSK9 nell’aterosclerosi e nelle strategie della sua prevenzione.
Background and purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the main regulators of LDL receptor metabolism, has been associated with atherosclerosis development. Several studies have confirmed such association through both lipid and non-lipid pathways. However, the direct relationships between circulating PCSK9 and markers of subclinical and clinical atherosclerosis are still matter of debate. Therefore, we investigated the relationships between plasma PCSK9 levels and some indexes of subclinical (imaging markers) and clinical (vascular events; VEs) atherosclerosis. Another objective was the identification of the independent determinants of PCSK9, with particular attention to lipids and inflammatory biomarkers. Finally, we also assessed the relationship between some imaging markers and four SNPs of the PCSK9 gene, known to be associated with the presence of low levels of LDL-cholesterol. In order to validate the results obtained in this last part, the genetic analyses were replicated in an independent cohort recruited in the United Kingdom (UK). Methods: The study was carried out taking advantage of databases, biobanks and imaging-bank of the IMPROVE study. 3,703 European subjects (54-79 years; 48% men), free of VEs at baseline and defined at high risk for the presence of at least three vascular risk factors, were recruited and followed-up for 36 months. PCSK9 was measured by ELISA and log-transformed prior to analyses. Conventional imaging markers [carotid intima-media thickness (cIMT) and carotid plaque-size], and emerging imaging markers [cIMT change over time, echolucency of the intima-media thickess of common carotid measured in plaque free areas (PF CC-IMTmean), echolucency of the biggest plaque detected in the whole carotid tree, and carotid calcium score (cCS)] were measured on ultrasonographic scans stored in the imaging-bank. In particular, echolucency was measured in terms of grey scale median (GSM) of pixels distribution of a specific region of interest, whereas cCS was calculated as sum of lengths of acoustic shadow cones generated by calcium within carotid plaques. Lipids were measured with enzymatic methods (except for LDL-cholesterol, which was calculated by Friedewald's formula). Among inflammatory markers, high-sensitivity C-reactive protein (hs-CRP) was measured by turbidimetry, whereas white blood cells (WBC) count and the leukocyte formula had already been measured locally. All the IMPROVE study and UK (n=22,179; 48% men) subjects have been genotyped.Results: In the univariate analysis, PCSK9 was positively correlated with total, LDL-, and HDL-cholesterol, and with triglycerides and basophils (all p <0.0001), whereas was negatively correlated with neutrophils and eosinophils (both p=0.04). The positive correlations observed with hs-CRP and WBC count were just close to the statistical significance (p=0.060 and 0.064, respectively). Fibrates or statins therapies (positively; both p <0.0001), as well as male sex and family history of diabetes (negatively; both p <0.05) were the strongest independent predictors of plasma PCSK9 levels. In the unadjusted analysis, a negative correlation was observed between PCSK9 levels and basal cIMT variables (i.e. carotid IMTmean, IMTmax, IMTmean-max, and PF CC-IMTmean), a negative correlation between PCSK9 and cIMT change over time (Fastest-IMTmax-progr) and cCS (all p ≤0.01), whereas a positive trend was observed between PCSK9 and GSM of both PF CC-IMTmean and carotid plaque (both p ≤0.0001). The cCS (positively) and the GSM of PF CC-IMTmean (positively) were significantly (or almost significantly) associated with PCSK9 in several multivariate models (all p ≤0.064). All correlations observed in the univariate analysis between PCSK9 and basal cIMT variables, Fastest-IMTmax-progr and GSM of carotid plaque lost the statistical significance after adjustment for age, sex, latitude, and other potential confounders. During the follow-up [median (interquartile range): 3.01 (2.98; 3.12) years], 215 VEs were recorded: 125 coronary, 73 cerebral and 17 peripheral VEs. Among these, 37 were hard events (i.e. myocardial infarction, sudden death and stroke). In the unadjusted analysis, PCSK9 was positively associated with combined and coronary events (both p <0.01), but not with cerebrovascular events. Also in this case, however, all the associations observed lost the statistical significance after adjustment of the analyses for age, sex, and stratification for latitude. The lack of association with VEs was confirmed also in the model adjusted for all confounding factors considered, and in the analyses focused on hard events. With regard to the role of genetic variants, none of the four SNPs considered was correlated with cIMT (i.e. IMTmean, IMTmax, IMTmean-max) when the analysis was performed in the subjects recruited in the IMPROVE study. The rs11591147 variant, by contrast, was negatively correlated with IMTmax measured in the UK population (p=0.002). By combining the four genetic variants in a score, the relationship with cIMT was not significant in the IMPROVE study, whereas was negative and significant in the UK population (all p <0.01).Conclusions: Plasma PCSK9 levels are not associated with VEs. Regarding markers of subclinical atherosclerosis, PCSK9 levels are associated neither with lesion size, nor with carotid plaque echolucency, but are associated with echolucency of carotid wall thickness and with carotid calcium score. Therefore, further studies are needed to better understand the role of such circulating proprotein in carotid wall thickness echolucency and in carotid calcium score. Fibrates or statins therapies, as well as male sex and family history of diabetes are the strongest independent predictors of PCSK9 levels. The associations, previously observed, between circulating PCSK9 and some lipid and inflammatory markers have been confirmed. The relationship between plasma levels of PCSK9 and other inflammatory markers (neutrophils, basophils and eosinophils) deserves further investigation, as does the association between the four selected PCSK9 variants and cIMT in the UK cohort, as it suggests a possible role of PCSK9 SNPs or gene polymorphisms in atherosclerosis and in its preventive strategies.

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Ly, Kévin. "Élucidation et identification des différents interacteurs impliqués dans le mécanisme de régulation du LDLR par la protéine PCSK9." Thèse, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/9786.

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Résumé : Les maladies cardiovasculaires représentent la principale cause de mortalité mondiale, soit le tiers des décès annuels selon l’Organisation mondiale de la Santé. L’hypercholestérolémie, caractérisée par une élévation des niveaux plasmatiques de lipoprotéines de faible densité (LDL), est l’un des facteurs de risque majeur pour les maladies cardiovasculaires. La proprotéine convertase subtilisine/kexine type 9 (PCSK9) joue un rôle essentiel dans l’homéostasie du cholestérol sanguin par la régulation des niveaux protéiques du récepteur LDL (LDLR). PCSK9 est capable de se lier au LDLR et favorise l’internalisation et la dégradation du récepteur dans les lysosomes. L’inhibition de PCSK9 s’avère une cible thérapeutique validée pour le traitement de l’hypercholestérolémie et la prévention des maladies cardiovasculaires. Par contre, plusieurs mécanismes responsables de la régulation et la dégradation du complexe PCSK9-LDLR n’ont pas encore été complètement caractérisés comme la régulation par la protéine annexin A2 (AnxA2), un inhibiteur endogène de PCSK9. De plus, plusieurs évidences suggèrent la présence d’une ou plusieurs protéines, encore inconnues, impliquées dans le mécanisme d’action de PCSK9. Celles-ci pourraient réguler l’internalisation et le transport du complexe PCSK9-LDLR vers les lysosomes. Les objectifs de cette thèse sont de mieux définir le rôle et l’impact de l’AnxA2 sur la protéine PCSK9 en plus d’identifier de nouveaux partenaires d’interactions de PCSK9 pour mieux caractériser son mécanisme d’action sur la régulation des niveaux de LDLR. Nous avons démontré que l’inhibition de PCSK9 par l’AnxA2 extracellulaire s’effectue via sa liaison aux domaines M1+M2 de la région C-terminale de PCSK9 et nous avons mis en évidence les premières preuves d’un contrôle intracellulaire de l’AnxA2 sur la traduction de l’ARNm de PCSK9. Nos résultats révèlent une liaison de l’AnxA2 à l’ARN messager de PCSK9 qui cause une répression traductionnelle. Nous avons également identifié la protéine glypican-3 (GPC3) comme un nouveau partenaire d’interaction extracellulaire avec le PCSK9 et intracellulaire avec le complexe PCSK9-LDLR dans le réticulum endoplasmique des cellules HepG2 et Huh7. Nos études démontrent que GPC3 réduit l’activité extracellulaire de PCSK9 en agissant comme un compétiteur du LDLR pour la liaison avec PCSK9. Une meilleure compréhension des mécanismes de régulation et de dégradation du complexe PCKS9-LDLR permettra de mieux évaluer l’impact et l’efficacité des inhibiteurs de la protéine PCSK9.
Abstract : Cardiovascular disease is the leading cause of global mortality, responsible for one third of global deaths, according to the latest statistics from World Health Organization. Hypercholesterolemia, characterized by increased plasma low-density lipoprotein (LDL) cholesterol, is a major determinant of cardiovascular disease risk. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol homeostasis by regulating LDL receptor (LDLR) protein levels. PCSK9 binds to the LDLR and promotes its internalization and degradation in late endosomal/lysosomal compartments. Inhibition of PCSK9 action on LDLR has emerged as a novel therapeutic target for hypercholesterolemia and the prevention of cardiovascular disease. Annexin A2 (AnxA2) was reported as an endogenous extracellular inhibitor of PCSK9 activity upon cell-surface LDLR degradation and mechanisms of PCSK9’s regulation by AnxA2. However, its role on PCSK9 regulation still need better characterization in hepatocellular carcinoma cell lines. Moreover, many evidences suggest the presence of additional unknown interaction partners involve in the LDLR regulation and degradation mediated by PCSK9. These unknown partners could regulate the internalization and trafficking of the PCSK9-LDLR complex to lysosomes. The objectives of this thesis are to better define the role and impact of AnxA2 on PCSK9 and to identify novel PCSK9 interacting partners that participate and regulate the PCSK9-LDLR complex formation and degradation. We demonstrated that PCSK9 inhibition by extracellular AnxA2 occurs via its interaction with the M1+M2 modules of PCSK9’s C-terminal region. Most importantly, we revealed a new role of intracellular AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Our results suggest a translational repression from the binding of AnxA2 to PCSK9’s mRNA. Also, we successfully identified a novel and functional interaction between glypican-3 (GPC3) and PCSK9. We demonstrated the extracellular GPC3 interaction with PCSK9 and the intracellular GPC3 with both PCSK9 and LDLR in human hepatocellular carcinoma cell lines HepG2 and Huh7. Our studies revealed that extracellular GPC3 can act as an endogenous competitive binding partner of PCSK9 to the LDLR, and hence reducing its activity towards LDLR degradation. The continued understanding of PCSK9 interactions is critical, from a mechanistic point of view as well as from the optimization of therapeutic interventions.

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Roalkvam, Ness Tonje. "PCSK9-hemmere som kolesterolsenkere- koster det mer enn det smaker? : Hvilken evidens finnes det for kostnadseffektivitet?" Thesis, Umeå universitet, Farmakologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-121836.

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Concepción, Carrillo Luis Edgar, and Esquivel Brandon Iturbe. "Descripción de la expresión de PCSK9 en pacientes del CMLALM con alta sospecha de hipercolesterolemia familiar." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2021. http://hdl.handle.net/20.500.11799/111123.

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La Hipercolesterolemia Familiar (HF) se define como una enfermedad hereditaria autosómica dominante del metabolismo de las lipoproteínas, caracterizada por concentraciones plasmáticas de colesterol LDL (cLDL) entre 350 y 550 mg/dL, en la forma heterocigota, y mayores de 550 mg/dL en la hom*ocigota,Este trastorno se debe a un grupo de errores genéticos que resultan en niveles anormalmente elevados de colesterol LDL (c-LDL), que causan deposición de placas ateroscleróticas en las arterias incrementando el riesgo de infarto agudo de miocardio en población joven. Se sabe que existen tres mutaciones genéticas asociadas: del receptor de LDL (rLDL), de la apolipoproteína B (ApoB) y de la pro-proteína convertasa subtilisina/kexina 9 (PCSK9).El proyecto consistió en un estudio prospectivo, descriptivo, clínico, transversal en el que se realizó medición de la expresión del gen PCSK9, ya que se ha demostrado que la pérdida de la función de este se asocia a niveles disminuidos de cLDL. Se reportó y describieron los casos sospechosos de hipercolesterolemia familiar en la consulta externa de dislipidemias y diabetes del Centro Médico “Lic. Adolfo López Mateos”, contribuyendo con un estudio que representó a una parte de la población mexiquense, con el fin de difundir el uso de los criterios de sospecha clínica.Aplicando la correlación de Spearman se halló una correlación negativa entre la expresión del gen PCSK9 y los niveles de colesterol LDL sin demostrar significancia tras el análisis estadístico, probablemente debido a la población, a variables como la edad, localización sociodemográfica, factores medio ambientales u otros que no fueron considerados dentro del análisis estadístico. Además tras realizar una regresión lineal para predecir los niveles de colesterol LDL, se encontró que los niveles de triglicéridos tienen valor predictor para colesterol LDL, más que la expresión del gen PCSKS9.
Ciprés Grupo Médico CGM SC

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Boyer, Marjorie. "Impact d'interventions non pharmacologiques sur les biomarqueurs émergents du risque cardiovasculaire : fonctionnalité des HDL et PCSK9." Doctoral thesis, Université Laval, 2018. http://hdl.handle.net/20.500.11794/32490.

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Les maladies cardiovasculaires (MCV) représentent la première cause de mortalité au monde, malgré des progrès significatifs dans la prise en charge des facteurs de risque traditionnels. De nouveaux biomarqueurs émergent, par exemple la capacité d’efflux du cholestérol (CEC) des HDL qui serait associée aux MCV indépendamment des concentrations de cholestérol HDL. Un autre biomarqueur est la proprotéine convertase subtilisine/kexine de type 9 (PCSK9), qui augmente les niveaux de cholestérol LDL en dégradant le récepteur aux LDL. Les facteurs qui influencent la CEC des HDL et la quantité de PCSK9 sont peu connus. L’objectif général de cette thèse est de déterminer si des interventions non pharmacologiques telles que la modification des habitudes de vie et la chirurgie bariatrique peuvent influencer ces biomarqueurs. Afin de répondre à cet objectif, nous avons étudié trois populations à risque : (1) 86 patients avec maladie coronarienne, (2) 117 hommes avec obésité abdominale et dyslipidémie, et (3) 69 hommes et femmes atteints d’obésité sévère. Les deux premières cohortes ont suivi une intervention de modification des habitudes de vie pendant 1 an visant à instaurer un minimum de 150 minutes d’activité physique par semaine et à améliorer la qualité nutritionnelle de leur diète. La cohorte de patients avec obésité sévère a subi une chirurgie bariatrique de type dérivation biliopancréatique avec commutation duodénale. Les La CEC des HDL a été mesurée à l’aide d’essais cellulaires dans les deux premières cohortes, alors que le niveau de PCSK9 a été mesuré par ELISA dans les trois cohortes. Dans tous les cas, les mesures ont été effectuées au début et à la fin des interventions. Nous avons montré que chez les patients coronariens et les hommes avec obésité abdominale, l’intervention de modification des habitudes de vie a augmenté la CEC des HDL, mais a eu un effet mineur sur les niveaux de PCSK9. En revanche, chez les hommes avec obésité abdominale les niveaux de PCSK9 sont diminués par un repas riche en gras. Les améliorations de la CEC des HDL à la suite de l’intervention de modification des habitudes de vie étaient principalement expliquées par l’augmentation des concentrations de l’apolipoprotéine A1 et du cholestérol HDL. La chirurgie bariatrique a augmenté la concentration de PCSK9 en phase aiguë, mais l’a diminué à long terme. Ces résultats suggèrent que le niveau de PCSK9 seraient modifiés par des changements drastiques comme une importante perte de poids à la suite d’une chirurgie bariatrique. En conclusion, les interventions non pharmacologiques semblent moduler positivement la CEC des HDL et modestement la PCSK9 dans des populations à haut risque cardiovasculaire, ce qui pourrait en partie expliquer l’impact bénéfique de ces interventions sur le risque cardiovasculaire.
Cardiovascular disease (CVD) is the leading cause of death in the world, despite significant progress in the management of traditional CVD risk factors. New biomarkers are emerging, such as the HDL cholesterol efflux capacities (HDL-CEC) that are associated with CVD, independently of HDL cholesterol levels. Another biomarker is proprotein convertase subtilisin/kexin type 9 (PCSK9), which increases low-density lipoprotein (LDL) cholesterol levels by degrading the LDL receptor. Little is known about the factors that influence HDLCEC and PCSK9 physiological variation. The overall objective of this thesis is to determine whether improvements in lifestyle and bariatric surgery can influence these biomarkers. To achieve this objective, we have studied three populations at risk: 86 patients with coronary artery disease, 117 men with abdominal obesity and dyslipidemic and 69 men and women with severe obesity. The first two cohorts followed a 1-year lifestyle modification program aimed at achieving a minimum of 150 minutes of aerobic physical activity weekly and improving diet quality. We measured HDL-CEC using cell assays in both populations before and after the interventions. The cohort of patients with severe obesity underwent bariatric surgery of biliopancreatic diversion with duodenal switch. PCSK9 levels were measured by ELISA in all cohorts, at the beginning and at the end of the interventions. Our results showed that in coronary patients and men with abdominal obesity, the interventions led to minor changes in PCSK9 levels. In contrast, in men with abdominal obesity PCSK9 levels were acutely decreased by a high-fat meal. Bariatric surgery increased PCSK9 concentration in the acute phase, but decreased in the long term. These results suggest that PCSK9 levels could be modified following drastic interventions such as significant weight loss following bariatric surgery. Lifestyle modification program induced significant increases of HDL-CEC in coronary patients and men with abdominal obesity. In addition, improvements in HDLCEC following lifestyle modification interventions were mainly explained by increases in apolipoprotein A1 and HDL cholesterol levels. In conclusion, non-pharmacological interventions appear to positively modulate HDL-CEC and modestly influence PCSK9 in populations at high cardiovascular risk, which could partly explain the beneficial impact of these interventions on cardiovascular risk.

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Chu, Ge. "PCSK9 and Its Variants: An Unbiased Global Proteomic Study to Identify Interactors and Effects on Protein Trafficking." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32988.

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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that promotes degradation of low-density lipoprotein receptors. Gain- and loss-of-function variants of PCSK9 cause hypercholesterolemia and hypocholesterolemia, respectively. Although it has been a decade since the discovery of PCSK9, its effect in terms of global protein changes and interactions still require further understanding. This study provided a global outlook at the protein changes caused by PCSK9 and its variants in human hepatic HUH7 cell line. First, a proteomics-based method for protein subcellular distribution analysis has been developed. Second, through secretome analyses, six apolipoproteins and six proteins involved in the coagulation pathway were found with >2-fold changes between wild type PCSK9 and its variants. Third, through secreted interactome analyses, a list of 159 PCSK9 interactor candidates was identified. Two interacting proteins, FASN and PSMD2, were validated and demonstrated with dynamic interacting patterns between PCSK9 and its variants.

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Lyne, Michael Harvey. "Cloning and characterisation of a meiosis-specific gene, pck1, from the fission yeast Schizosaccharomyces pombe." Thesis, University of Exeter, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260618.

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Garçon, Damien. "Effet intestinal de PCSK9 au delà du métabolisme du cholestérol : focus sur la lipémie postprandiale et l'allergie alimentaire." Thesis, Nantes, 2020. http://www.theses.fr/2020NANT1011.

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PCSK9 (ProProtein Convertase Subtilisin Kexin Type 9) est le 3e gène responsable de l’hypercholestérolémie familiale. En effet, PCSK9 est un inhibiteur naturel du récepteur au LDL. Les patients présentant des mutations gain de fonction pour PCSK9 sont à très haut risque concernant les maladies cardiovasculaires. En plus de son impact sur le métabolisme du cholestérol, PCSK9 joue un rôle dans un autre facteur de risque cardiovasculaire : la lipémie postprandiale. Ce phénomène caractérisé par une élévation des triglycérides plasmatiques après un repas est facteur de risque des maladies cardiovasculaires dans certaines pathologies notamment chez les patients diabétiques de type 2. Il a été montré que les modèles murins déficients pour PCSK9 ont une réduction de leur lipémie postprandiale. Lors de ma thèse, nous avons montré par l’utilisation de modèle de souris déficiente, l’inhibition de PCSK9 par anticorps anti-PCSK9 et le développement d’un modèle original de déficience intestinale de PCSK9 que la forme circulante de PCSK9 est cruciale dans le phénomène de lipémie postprandiale. Au delà du métabolisme des lipides, il a été montré que PCSK9 joue un rôle dans les réponses inflammatoires, notamment au cours d’un choc septique. Lors de ma thèse, nous avons observé l’impact de la déficience et de l’inhibition de PCSK9 sur le développement de l’allergie alimentaire. Nous avons mis en évidence que l’absence de PCSK9 protège de l’apparition des symptômes de l’allergie. Ma thèse a donc permis de mettre en lumière un rôle de PCSK9 au delà du métabolisme du cholestérol et au niveau intestinal
PCSK9 (ProProtein Convertase Subtilisin Kexin Type 9) is the 3rd gene responsible for familial hypercholesterolemia. Indeed, PCSK9 is a natural inhibitor of the LDL receptor. Patients with PCSK9 gain function mutations are at very high risk for cardiovascular disease. In addition to its impact on cholesterol metabolism, PCSK9 plays a role in another cardiovascular risk factor: postprandial lipemia. This phenomenon, characterized by a rise in plasma triglycerides after a meal, is a risk factor for cardiovascular disease in certain pathologies, particularly in patients with type 2 diabetes. It has been shown that mouse models deficient in PCSK9 have a reduction in their postprandial lipemia. During my thesis, we showed by using deficient mouse models, inhibition of PCSK9 by anti-PCSK9 antibodies and the development of an original model of intestinal PCSK9 deficiency that the circulating form of PCSK9 is crucial in the phenomenon of postprandial lipemia. Beyond lipid metabolism, PCSK9 has been shown to play a role in inflammatory responses, particularly during septic shock. In my thesis, we observed the impact of PCSK9 deficiency and inhibition on the food allergy development. We showed that the absence of PCSK9 protects against the onset of allergy symptoms. My thesis has therefore highlighted the role of PCSK9 beyond cholesterol metabolism and at the intestinal level

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Langhi, Cédric. "Implication de PCSK9 dans les maladies métaboliques : régulation par les acides biliaires et rôle fonctionnel dans le pancréas." Nantes, 2009. https://archive.bu.univ-nantes.fr/pollux/show/show?id=8ce2a36e-ee33-47ea-b8e8-fc89d736a8f4.

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Abstract:

PCSK9 (proprotein convertase subtilisin kexin type 9) est le 3ème gène impliqué dans l'hypercholestérolémie familiale dominante, après les mutations des gènes codant pour le récepteur aux LDL (LDLR) et pour son ligand l’apo-B. PCSK9 agit comme un inhibiteur de l'expression hépatique du LDLR par un mécanisme post-traductionnel. En se liant au domaine extracellulaire du LDLR à la surface des membranes plasmiques, PCSK9 induit l'internalisation du LDLR et sa dégradation dans les lysosomes. Ainsi les mutations de PCSK9 associées à l'hypercholestérolémie sont des mutations gain de fonction. A l'inverse, les mutations perte de fonction de PCSK9 induisent une hypocholestérolémie et une protection contre les maladies cardiovasculaires. Le développement d'inhibiteurs de PCSK9 est donc un enjeu thérapeutique majeur dans la prise en charge des hypercholestérolémies. Dans ce contexte, la 1ère partie de ma thèse a consisté à étudier la régulation transcriptionnelle de PCSK9 par les acides biliaires et le récepteur nucléaire FXR (Farnesoid X Receptor). De nombreux gènes du métabolisme lipidique sont régulés par FXR et l’activation de FXR a des effets bénéfiques dans les troubles métaboliques. Les résultats obtenus dans des lignées d'hépatocytes humains montrent que l'activation de FXR réprime l'expression de PCSK9, ce qui s'accompagne d'une augmentation de l'activité du LDLR mesurée in vitro. Ceci suggère que l'utilisation d’agoniste de FXR pour réprimer PCSK9 et ainsi potentialiser l'action des statines pourrait être utile dans le traitement de l'hypercholestérolémie. On sait à présent que le métabolisme du cholestérol intervient dans la régulation de la sécrétion d’insuline par les cellules β du pancréas et pourrait ainsi jouer un rôle dans la physiopathologie du diabète de type 2. La 2nde partie de ma thèse s'est intéressée au rôle de PCSK9 dans la fonction insulino-sécrétrice des cellules β. A partir d’ilots isolés du pancréas humains et de souris, je démontre que PCSK9 est exprimée dans les cellulesdelta. Par ailleurs, PCSK9 diminue l'expression du LDLR au sein des ilots de Langerhans, probablement via une action endocrine En revanche, l'invalidation de PCSK9 chez la souris ne semble pas perturber l'homéostasie du glucose ex vivo et in vivo, ni la survie des cellules β en réponse à un traitement par la streptozotocine
PCSK9 (proprotein convertase subtilisin kexin type 9) is the 3rd gene implicated in autosomic familial hypercholesterolemia with the LDL Receptor (LDLR) gene and its ligand apo-B. PCSK9 acts as a post-transcriptional inhibitor of hepatic LDLR expression. Gain of function mutations of PCSK9 are associated with hypercholesterolemia. By contrast, loss of function mutations of PCSK9 induce hypocholesterolemia and a protection against cardiovascular diseases. Therefore, development of PCSK9 inhibitors is a promising therapeutical approach to treat hypercholesterolemia. In this context, the 1st part of my thesis consisted of studying transcriptional regulation of PCSK9 by bile acids and the nuclear receptor FXR (Farnesoid X Receptor). FXR regulates many genes involved in lipid metabolism, and FXR activation may have beneficial effects in metabolic diseases. My results in human hepatocytes cell lines show that FXR activation represses PCSK9 expression. Such PCSK9 repression is correlated with the induction of LDLR activity in vitro. These findings suggest that FXR agonists may be used in combination with statins to amplify their hypocholestrolemic action in hyperlipidemic patients. It is now admitted that cholesterol metabolism modulates insulin secretion by pancreatic β cells and might interfere with the development of type 2 diabetes. The 2nd part of my thesis focussed on the role of PCSK9 in the β cells function. Using isolated pancreatic islets from humans and mice, I show that PCSK9 is expressed in delta cells of islets of Langehrans. PCSK9 is able to downregulate LDLR expression in the whole islet, probably acting in an endocrine manner. However, PCSK9-deficiency does not alter glucose homeostasis ex vivo and in vivo in mice, as well as β cell survival upon streptozotocin treatment

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Bélanger, Jasmin Stéphanie. "A putative role for PCSK9 in synaptic remodelling and plasticity in response to brain injury: implications for Alzheimer's disease." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96705.

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Abstract:

Since the association of the ε4 allele of the apolipoprotein E (apoE) withAlzheimer's disease (AD) risk, growing evidences support a role for cholesterolmetabolism in the pathophysiology of this disease. Many genes involved in lipidmetabolism have now been studied and associate with the risk of AD. PCSK9 is aproprotein convertase recently identified as the third gene linked to familialhypercholesterolemia. It is a key regulator of plasma cholesterol concentrations byenhancing the degradation of cell surface low-density-lipoprotein receptor (LDLR). Thepresent project derives from the global hypothesis that in the brain, PCSK9 may play arole in cholesterol homeostasis by regulating the expression of the LDLR proteins undernormal and especially, neurodegenerative conditions.A first study was conducted to evaluate potential variations in PCSK9 expressionin the brain of autopsy-confirmed AD compared to age-matched control subjects. Agenetic association study was also performed to determine the effect of five commonPCSK9 polymorphisms on AD risk and modulation of gene expression. Using theentorhinal cortex lesion (ECL) model in a second study, a role for PCSK9 in reactivesynaptogenesis was evaluated in response to brain damage in this in vivo paradigm inmice. A third study investigated in an in vitro model of reactive neuronal plasticity, theeffect of PCSK9 on synaptogenesis and remodelling processes in response to neuronalinjury.The results show a cortical and hippocampal upregulation of PCSK9 expression inthe brain of end-stages AD patients which do not result from the five studied geneticvariants. No correlations were observed for PCSK9 with markers of AD pathology;suggesting an involvement of PCSK9 in response to neurodegeneration. Consistent withthis idea, PCSK9 levels were increased during the active phase of neuronal membraneremodelling following ECL. In vitro, overexpression of PCSK9 in normal or neuronallikecells undergoing post-injury reactive plasticity caused increased synaptic densitywhich supports a role for PCSK9 in synaptogenesis and plasticity. While PCSK9 wasfound to negatively affect the LDLR levels in AD brains and both the LDLR and apoER2during reactive plasticity in vitro, levels of the LDLR in ECL mice was not affected byPCSK9 but instead, together with apoE, levels were upregulated in the early phase ofsynaptic remodelling.Together, these findings indicate that PCSK9 plays an important role incompensatory neuronal repair associated with age, brain injury or chronic degeneration asfound in AD. Its expression in the brain possibly regulates cholesterol homeostasis and/orsignalling pathways mediated by the apoE/LDLR pathway or other members of theLDLR family during axonal and synaptic remodelling. These findings are consistent withthe relationship that exists between lipid homeostatic processes and AD pathology andindicate that PCSK9 may be a new player in the regulation of these processes that worthsfurther investigation in a context of neurodegenerative disorders.
Depuis la découverte que l'allèle ε4 de l'apolipoprotein E (apoE) est associé avec un risque plus élevé de développer la maladie d'Alzheimer (MA), un nombre grandissant d'études démontre que le cholestérol joue un rôle important dans les mécanismes pathophysiologiques reliés à cette maladie. Plusieurs gènes impliqués dans le métabolisme du cholestérol ont fait l'objet d'études et démontré une associationgénétique avec la MA. PCSK9 est une proprotein convertase qui récemment, fut associée à l'hypercholestérolémie familiale. Cette convertase est un régulateur principal des niveaux de cholestérol plasmatique par sa capacité à promouvoir la dégradation d'un récepteur de surface, le récepteur des lipoprotéines à faible densité (LDLR). Les travaux de ce projet sont basés sur l'hypothèse initiale que dans le cerveau, PCSK9 pourrait également jouer un rôle dans l'homéostasie du cholestérol en contrôlant l'expression protéique des récepteurs de LDL sous des conditions normales et neurodégénératives. Le premier volet expérimental avait pour objectif de comparer les niveaux d'expression de PCSK9 dans des cerveaux de patients identifiés à la MA ou considérés comme contrôles sains. De plus, une étude d'association génétique a été effectué afin de déterminer l'effet de polymorphismes de PCSK9 sur le risque de la MA ainsi que sur le contrôle de l'expression de PCSK9. Dans la deuxième étude, l'utilisation d'un modèle murin de lésion du cortex entorhinal a permis d'évaluer le rôle de PCSK9 dans des processus de synaptogénèse suite à un dommage neuronal. La troisième étude avait pour objectif de déterminer les effets de PCSK9, lorsque surexprimé, sur le remodelage et plasticité neuronale en réponse à une lésion dans un modèle cellulaire. Les résultats de ce projet ont démontré entre autre, une augmentation des niveaux d'expression de PCSK9 dans les régions du cortex frontal ainsi que l'hippocampe de cerveaux obtenus de patients Alzheimer en fin de maladie. Cette augmentation n'est pas causée par l'un des cinq variants génétiques de PCSK9 étudiés et aucune corrélation a été observé entre cette convertase et les marqueurs pathologiques caractérisant la MA; ce qui suggère l'implication de PCSK9 en réponse à la neurodégénération. Conformément à cette hypothèse, les niveaux de PCSK9 sont également augmentés lors de la phase active de remodelage membranaire suite à la lésion du cortex entorhinal. La surexpression de PCSK9 dans des cellules de type neuronal a causé une augmentation de la densité synaptique tant sous des conditions normales que lorsque les cellules sont activement en mode de réparation suivant la lésion, supportant ainsi un rôle pour PCSK9 dans les mécanismes de synaptogénèse et de plasticité synaptique. Alors que les niveaux de récepteurs aux LDL (LDLR) dans les cerveaux Alzheimer en plus des récepteurs de type apoER2 lors de processus de plasticité in vitro sont affectés de façon négative par PCSK9, les niveaux de LDLR suite à la lésion du cortex entorhinal ne semblent pas affectés par PCSK9 mais plutôt, parallèlement à apoE et PCSK9, sont augmentés dans la phase active de remodelage synaptique. Globalement, ces résultats indiquent que PCSK9 joue un rôle important dans les mécanismes compensatoires de réparation neuronal associés au vieillissem*nt, à un dommage cérébrale ou à des conditions chroniques de dégénération tel qu'observées lors de la MA. Son expression dans le cerveau réflète possiblement une régulation de l'homéostasie du cholestérol ou des voies de signalisation par le contrôle de récepteurs de surface aux LDL lors de remodelage membranaires et synaptiques en réponse à un dommage neuronal. Ces résultats sont en accord avec le lien existant entre les processus d'homeostasies lipidiques et les mécanismes pathologiques associés à la MA et indiquent que PCSK9 pourrait être un nouveau joueur participant à ce phénomène. Ainsi, PCSK9 mérite de plus amples investigations dans un contexte de maladies neurodégénératives.

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Bourbiaux, Kévin. "Développement de peptides structurés pour l’inhibition de l’interaction PCSK9/LDLR et le rétablissem*nt de l’absorption cellulaire du LDL-c." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTS009.

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Abstract:

La proprotéine convertase subtilisine/kexine de type 9 (PCSK9) régule la concentration des récepteurs des lipoprotéines de basse densité (LDLR) au niveau de la membrane cellulaire et par conséquent le taux de LDL-cholestérol dans le système vasculaire. PCSK9 est donc une cible essentielle dans le traitement des maladies cardiovasculaires (MCV). A ce jour, les anticorps monoclonaux anti-PCSK9 associés aux statines est la seule thérapie disponible ciblant PCSK9 en dépit des risques d’immunogénicité, une administration sous-cutanée contraignante et un coût élevé. Néanmoins, de petit* peptides possédant des structures tridimensionnelles très stables se sont avérés être des inhibiteurs prometteurs de l’interaction entre PCSK9 et le LDLR induisant une augmentation significative de l’absorption du LDL dans les cellules. A partir de ces séquences, nous avons synthétisé des peptides stabilisés par des agrafes, avec un patch poly-lysine (technologie SIP) à l’extrémité C-terminale mais également des séquences chimériques afin de développer des analogues très actifs et résistants à la protéolyse. Nous avons obtenu des composés mille fois plus affins pour PCSK9 que le peptide de référence Pep2-8, capables de rétablir l’absorption du LDL dans les cellules pour de très faibles concentrations (IC50 = 175 nM). Les structures tridimensionnelles des composés clés ont été étudiées par dichroïsme circulaire (CD) et résonance magnétique nucléaire (RMN) afin d’étudier leurs relations structure-activité
Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as a regulator of low density lipoprotein receptor (LDLR) on the cell membrane and therefore plays a major role in cardiovascular diseases (CVD). To date, only monoclonal antibodies (mAbs) to PCSK9 are used associated with statins in therapies, despite a potential immunogenicity, a restrictive mode of administration and a high cost. Beside, small peptides with discrete three-dimensional structures were found to inhibit the interaction between PCSK9 and the LDLR, increasing the LDL-uptake. Starting from these sequences, we used various strategies incorporating staples and/or C-terminal lysine patches (SIP technology), synthesizing chimeric sequences to develop highly potent compounds resistant to enzymatic degradation. We obtained derivatives that have a 1000-fold stronger affinity than the parent peptide with high biological activities (IC50 = 175 nM). The three-dimensional structures of key compounds were extensively studied by circular dichroim (CD) and nuclear magnetic resonance (NMR) to investigate their structure-activity relationship

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SCRIMALI, Chiara. "A novel therapeutic strategy to cure the hom*ozygous Familial Hypercholesterolemia with residual LDL receptor activity." Doctoral thesis, Università degli Studi di Palermo, 2020. http://hdl.handle.net/10447/395446.

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