OBJECTIVE—Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor thereby elevating plasma low-density lipoprotein cholesterol levels ...and the risk of coronary heart disease. Thus, the use of PCSK9 inhibitors holds great promise to prevent heart disease. Previous work found that PCSK9 is involved in triglyceride metabolism, independently of its action on low-density lipoprotein receptor, and that other yet unidentified receptors could mediate this effect. Therefore, we assessed whether PCSK9 enhances the degradation of CD36, a major receptor involved in transport of long-chain fatty acids and triglyceride storage.
APPROACH AND RESULTS—Overexpressed or recombinant PCSK9 induced CD36 degradation in cell lines and primary adipocytes and reduced the uptake of the palmitate analog Bodipy FL C16 and oxidized low-density lipoprotein in 3T3-L1 adipocytes and hepatic HepG2 cells, respectively. Surface plasmon resonance, coimmunoprecipitation, confocal immunofluorescence microscopy, and protein degradation pathway inhibitors revealed that PCSK9 directly interacts with CD36 and targets the receptor to lysosomes through a mechanism involving the proteasome. Importantly, the level of CD36 protein was increased by >3-fold upon small interfering RNA knockdown of endogenous PCSK9 in hepatic cells and similarly increased in the liver and visceral adipose tissue of Pcsk9 mice. In Pcsk9 mice, increased hepatic CD36 was correlated with an amplified uptake of fatty acid and accumulation of triglycerides and lipid droplets.
CONCLUSIONS—Our results demonstrate an important role of PCSK9 in modulating the function of CD36 and triglyceride metabolism. PCSK9-mediated CD36 degradation may serve to limit fatty acid uptake and triglyceride accumulation in tissues, such as the liver.
BACKGROUND:Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and ...atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined.
METHODS:Adcy9-inactivated (Adcy9) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtgAdcy9 and CETPtgAdcy9), were submitted to an atherogenic protocol (injection of an AAV8 adeno-associated virus serotype 8 expressing a PCSK9 proprotein convertase subtilisin/kexin type 9 gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated.
RESULTS:Adcy9 mice had a 65% reduction in aortic atherosclerosis compared to WT (P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9 mice compared to WT animals (P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9 mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9 (P<0.01 for all). Aortic endothelium from Adcy9 mice allowed significantly less adhesion of splenocytes compared to WT (P<0.05). Adcy9 mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume (P<0.01 for both). Feed efficiency was increased in Adcy9 compared to WT mice (P<0.01), which was accompanied by prolonged cardiac RR interval (P<0.05) and improved nocturnal heart rate variability (P=0.0572). Adcy9 inactivation–induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtgAdcy9 mice (P>0.05 versus CETPtgAdcy9).
CONCLUSIONS:Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.
DNA methylation and histone acetylation inhibitors are widely used to study the role of epigenetic marks in the regulation of gene expression. In addition, several of these molecules are being tested ...in clinical trials or already in use in the clinic. Antimetabolites, such as the DNA-hypomethylating agent 5-azacytidine (5-AzaC), have been shown to lower malignant progression to acute myeloid leukemia and to prolong survival in patients with myelodysplastic syndromes. Here we examined the effects of DNA methylation inhibitors on the expression of lipid biosynthetic and uptake genes. Our data demonstrate that, independently of DNA methylation, 5-AzaC selectively and very potently reduces expression of key genes involved in cholesterol and lipid metabolism (e.g. PCSK9, HMGCR, and FASN) in all tested cell lines and in vivo in mouse liver. Treatment with 5-AzaC disturbed subcellular cholesterol homeostasis, thereby impeding activation of sterol regulatory element-binding proteins (key regulators of lipid metabolism). Through inhibition of UMP synthase, 5-AzaC also strongly induced expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and promoted triacylglycerol synthesis and cytosolic lipid droplet formation. Remarkably, complete reversal was obtained by the co-addition of either UMP or cytidine. Therefore, this study provides the first evidence that inhibition of the de novo pyrimidine synthesis by 5-AzaC disturbs cholesterol and lipid homeostasis, probably through the glycerolipid biosynthesis pathway, which may contribute mechanistically to its beneficial cytostatic properties.
Background: The anticancer drug 5-azacytidine acts through an incompletely understood mechanism.
Results: 5-Azacytidine reprograms the glycerolipid biosynthesis pathway and prevents activation of master transcription factors that regulate lipid homeostasis.
Conclusion: 5-Azacytidine deeply modifies how cells manage cholesterol and lipid synthesis.
Significance: The findings unravel important insights into the mechanism of 5-azacytidine and highlight new potential cancer therapeutics.
Abstract only Polymorphisms in the ADCY9 gene, coding for adenylate cyclase type 9, determine atherosclerotic cardiovascular responses to the CETP inhibitor dalcetrapib in patients. ADCY9 is broadly ...expressed and involved in various immune cell functions and inflammatory responses. Our objective is to determine the role of ADCY9 in the development of atherosclerosis in the absence of CETP. We used 8-12 week-old wild-type (WT, n=25) or Adcy9 -inactivated ( Adcy9 Gt , n=21) male mice. To induce hypercholesterolemia and atherosclerosis, mice were infected with an adeno-associated virus coding for a gain-of-function mutant of Pcsk9 ( Pcsk9 D377Y ) and were fed an atherogenic high-cholesterol diet for 16 weeks in absence or presence of dalcetrapib treatment (200 mg/kg/day). Percent atherosclerotic lesion area (PALA) in the whole aorta was quantified using en face Oil Red O plaque staining. We used VCAM-1 immunofluorescence staining (n=6 per group) in atherosclerotic lesions of the aortic root to quantify inflammation. We also measured aortic root accumulation of macrophages and vascular smooth muscle cells by immunofluorescence imaging of CD68 and smooth muscle actin (SMA), respectively, in plaque lesions. We also quantified blood leukocytes in normocholesterolemic WT and Adcy9 Gt mice by flow cytometry analysis of CD45 + cells. In hypercholesterolemic animals, Adcy9 Gt mice showed a 60% decrease in atherosclerotic lesions (PALA: 2.7±0.5%) compared to WT mice (6.5±0.7%, P<0.01). Dalcetrapib treatment, in these mice without CETP, did not modify significantly the reduction (63%) of PALA in Adcy9 Gt mice (2.9±0.4%) compared to WT (7.6±1.6%, P<0.01). Macrophage content was reduced by 55% from 17.8±2.8% in WT to 7.2%±1.9% in Adcy9 Gt mice (P<0.05), while SMA staining was similar. VCAM-1 expression also tended to be decreased, from 5.7±3.3% in WT to 2.0±0.4% in Adcy9 Gt mice (P=0.0598). CD45 + blood leukocytes were reduced by 35.4% in Adcy9 Gt compared to WT normocholesterolemic mice (P<0.01). Adcy9 inactivation in mice (without CETP) results in large reductions of atherosclerosis and plaque macrophage and in decreased VCAM-1 expression. These results support the ADCY9 genotype-dependent clinical effects of dalcetrapib.
Abstract only Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the cholesteryl ester transfer protein (CETP) inhibitor dalcetrapib on cardiovascular events, ...atherosclerosis imaging and body weight variation. The underlying mechanisms responsible for the interactions between ADCY9 and CETP have not yet been determined. Adcy9 -inactivated ( Adcy9 Gt/Gt ) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETP Gt and CETP WT ), were submitted to an atherogenic protocol (injection of an AAV8 expressing a PCSK9 gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, cell adhesion, vasorelaxation, telemetry and adipose tissue MRI were evaluated. Adcy9 Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P <0.01). CD68-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9 Gt/Gt mice compared to WT animals ( P <0.05 for both). Adcy9 inactivation did not change counts of blood monocytes and their subsets. Splenocytes showed reduced adhesion to native aortic endothelium from Adcy9 Gt/Gt mice ( P <0.05 vs WT). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9 Gt/Gt mice (versus WT, P <0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase and endothelial-dependent hyperpolarization pathways all contributed to the improvement of vasodilatation in Adcy9 Gt/Gt versus WT ( P <0.01 for all). Adcy9 Gt/Gt mice gained more weight than WT with the atherogenic diet, and this was associated with an increase in whole body adipose tissue volume ( P <0.05 for both). Feed efficiency was increased in Adcy9 Gt/Gt compared to WT mice ( P <0.05), which was accompanied by improved nocturnal heart rate variability ( P =0.0572) and prolonged cardiac RR interval ( P <0.05). Adcy9 inactivation-induced effects on atherosclerosis, endothelium-dependent vasodilation, weight gain and feed efficiency were lost in CETP Gt mice ( P >0.05 vs CETP WT ). Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, improved endothelial function and autonomic tone.
L'hypercholestérolémie familiale autosomique dominante (ADH) est un trouble génétique caractérisé par des taux élevés de lipoprotéine de basse densité (LDL) plasmatique. Un niveau élevé de LDL ...plasmatique est connu pour contribuer au développement de l’athérosclérose, une cause majeure des crises cardiaques et des accidents vasculaires cérébraux. Le récepteur des LDL (LDLR) est la principale voie d’élimination des particules de LDL. En revanche, la proprotéine convertase subtilisine/kexine de type 9 (PCSK9), une glycoprotéine sécrétée par le foie, se lie au LDLR et augmente sa dégradation dans les lysosomes, ce qui entraîne une augmentation de LDL plasmatique et un risque plus élevé de maladie cardiovasculaire. En outre, des mutations de-perte-de fonction de PCSK9 peuvent considérablement réduire les niveaux de LDL plasmatiques et réduire le risque de maladie coronarienne jusqu'à ~ 88%. Toutes ces découvertes ont fait de PCSK9 une cible importante pour le traitement de l'hypercholestérolémie. Des anomalies génétiques du LDLR, de PCSK9 ou de l’apolipoprotéine B (apoB), le ligand du LDLR, peuvent provoquer l'ADH, mais dans certaines familles ADH il n'a pas été possible d'identifier de mutation de ces gènes, suggérant que d'autres anomalies génétiques pourraient également être impliquées dans la maladie. ;
Dans la présente thèse, qui repose sur deux études (articles), nous avons étudié les protéines d’interaction de PCSK9 (premier article, chapitre 2) et l'effet de PCSK9 sur l'athérosclérose (deuxième article, chapitre 3). Dans notre première étude, l'analyse par spectrométrie de masse des protéines interagissant avec PCSK9 a révélé que la Golgi glycoprotéine 1 (GLG1) est une nouvelle protéine d’interaction de PCSK9. Leur co-immunoprécipitation révélée par immunobuvardage et leur co-localisation par microscopie confocale par immunofluorescence ont confirmé que GLG1 est un partenaire de PCSK9. De plus, nos résultats ont montré que GLG1 interagit aussi avec le LDLR et l'apoB. En utilisant un modèle murin, nous avons montré des taux sanguins plus faibles de PCSK9, de cholestérol et de triglycérides chez les souris knockdown GLG1. De plus, le déficit en GLG1 a réduit l'activité de la protéine de transfert des triglycérides microsomales (MTP) et induit l'agrégation périnucléaire de l'apoB, réduisant ainsi la sécrétion d'apoB. Dans notre deuxième étude, nous avons développé un modèle d'athérosclérose chez la souris pour étudier l'effet de l'absence de PCSK9 sur les plaques d’athérosclérose. Nous avons montré que la surexpression d'un mutant gain-de-fonction de PCSK9 dans le foie de souris a accéléré le développement de plaques d'athérosclérose dans la racine aortique et que celles-ci ont ensuite été réduites en induisant la régulation négative de PCSK9 en utilisant le système Tet-on.;
En conclusion, nous avons contribué à l'identification d'une nouvelle protéine interagissant avec PCSK9, GLG1, qui régule le taux plasmatique de cholestérol et représente une cible potentielle pour le traitement de l'hypercholestérolémie. Nous avons également démontré que la modulation du gène PCSK9 régule directement le niveau de plaques d'athérosclérose dans la racine de l'aorte. Ces études aideront à développer des thérapies efficaces pour réduire l'hypercholestérolémie et le risque de maladie cardiovasculaire
Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by high plasma low-density lipoprotein (LDL) cholesterol levels. Elevated plasma LDL level is known to contribute to the development of atherosclerosis, a leading cause of heart attack and stroke. Liver LDL receptor (LDLR) acts as a primary pathway for endocytosis and clearance of LDL particles. In contrast, PCSK9, a liver-secreted glycoprotein, binds to LDLR and enhances its lysosomal degradation, resulting in increased plasma LDL concentrations and a higher risk of cardiovascular disease. Genetic defects in LDLR, PCSK9, and apolipoprotein B (apoB), the ligand of LDLR, can cause ADH, but in some ADH-families no mutations can be found in these genes, suggesting that other gene defects may also be involved in ADH. Furthermore, loss-of-function mutations in PCSK9 can greatly reduce plasma LDL levels and lower risk of coronary heart disease by up to ~88%. All these findings have made PCSK9 an attractive target for the treatment of hypercholesterolemia.;
In the present thesis, which is based on two studies (articles), we investigated protein interactors of PCSK9 (first article, chapter 2) and the effect of PCSK9 on atherosclerosis (Second article, chapter 3). In our first study, mass spectrometry analysis of PCSK9 interacting proteins revealed that Golgi glycoprotein 1 (GLG1) is a novel PCSK9 interactor. Co-immunoprecipitation, Western blotting, and colocalization by confocal immunofluorescence microscopy confirmed that GLG1 is an endogenous PCSK9 binding partner. We also demonstrated that LDLR and apoB interact with GLG1. Using a mouse model, we found lower levels of circulating PCSK9, cholesterol, and triglycerides in Glg1 knockdown mice. Moreover, GLG1 deficiency reduced microsomal triglyceride transfer protein (MTP) activity and induced perinuclear aggregation of apoB, thereby, reducing apoB secretion. In our second study, we developed a mouse model of atherosclerosis to investigate the effect of PCSK9 modulation on the regression of atherosclerotic plaques. We showed that overexpression of a PCSK9 gain-of-function in mouse liver accelerated the development of atherosclerotic lesions in the aortic root, which were then reduced by inducing PCSK9 downregulation using a Tet-on system. ;
In conclusion, we have contributed to the identification of a novel PCSK9 interacting protein, GLG1, which regulates plasma level of cholesterol and represents a potential target for hypercholesterolemia treatment. We also demonstrated that PCSK9 gene modulation directly regulates the level of atherosclerotic plaques in the aortic root. We showed in our study that the wild-type mice, overexpressing PCSK9-D377Y in an inducible manner, is a useful mouse model for understanding the molecular role of PCSK9 on atherosclerotic plaques development. These studies will help to develop effective therapies to reduce hypercholesterolemia and the risk of cardiovascular disease.
PDF
