PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement.

BACKGROUND AND AIMS: Proprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury. METHODS: Carotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knock-out (PCSK9-/-) and wild type littermate (PCSK9+/+) mice. RESULTS: In PCSK9-/- mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9+/+ mice. When compared with PCSK9-/-, lesions of PCSK9+/+ mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9-/-, when compared to PCSK9+/+ cells, showed higher levels of α-smooth muscle actin (α-SMA; 2.24 ± 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 ± 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA(-54 ± 14%; p < 0.01). PCSK9-/- cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9-/- SMCs, led to a downregulation of α-SMA (-56 ± 2%; p < 0.01), MHC-II (-45% ± 25.5 fold: p = 0.06) and calponin (-25% ± 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 ± 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9-/- was significantly lower compared to PCSK9 reconstituted cells. CONCLUSIONS: Taken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall.

PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling.

In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis.

IDOL N342S Variant, Atherosclerosis Progression and Cardiovascular Disorders in the Italian General Population.

Inducible degrader of the low density lipoprotein receptor (IDOL), is an E3 ubiquitin ligase that negatively modulates low density lipoprotein receptor (LDL-R) expression. Genome-wide association studies (GWAS) indicated that genetic variants in IDOL gene contributes to variation in LDL-C plasma levels and the detailed analysis of a specific locus resulted in the identification of the functional common single nucleotide polymorphism (SNP) rs9370867 (c.G1025A, p.N342S) associates with increased LDL-R degradation and increased LDL-C levels. These findings, however, were not confirmed in two other independent cohorts and no data about the impact of this variant on atherosclerosis progression and cardiovascular risk are available. Aim of this study was to investigate the association between a functional variant in IDOL and atherosclerosis progression in an Italian general population. 1384 subjects enrolled in the PLIC study (Progression of Lesions in the Intima of Carotid) were genotyped by Q-PCR allelic discrimination and the association with anthropometric parameters, plasma lipids and the carotid intima media thickness (cIMT) and the impact on cardiovascular disease (CVD) incidence were investigated. The N342S variant was not associated with changes of the plasma lipid profile among GG, AG or AA carriers, including total cholesterol (249±21, 249±19 and 248±21 mg/dl respectively), LDL-C (158±25, 161±22 and 160±23 mg/dL), cIMT (0.74±0.14, 0.75±0.17 and 0.77±0.15 mm) and CVD incidence. In agreement, the expression of LDLR and the uptake of LDL was similar in macrophages derived from GG and AA carriers. Taken together our findings indicate that the N342S variant does not impact plasma lipid profile and is not associated with atherosclerosis progression and CVD in the general population, suggesting that other variants in the IDOL gene might be functionally linked with cholesterol metabolism.

PCSK9 inhibition for the treatment of hypercholesterolemia: promises and emerging challenges.

Hypercholesterolemia, is a prominent risk factor for cardiovascular disease (CVD). Undestanding of the biochemical mechanisms that regulate the expression of the low density lipoproteins receptor (LDLR) and the hepatic clearance of LDL cholesterol (LDL-C) paved the way to the statin therapy as the gold standard for CVD prevention. The discovery of proteins that regulate - at a post-translational level - the activity of the LDLR has been a major breakthrough in developing new cholesterol-lowering drugs. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key modulator of the LDLR degradation in the liver. Genetic studies confirmed that in humans PCSK9 mutations associate with hypercholesterolemia and hypocholesterolemia (gain-of-function or loss-of-function variants respectively). Moreover, PCSK9 is up-regulated by statin treatment and limits the efficacy of these agents. These findings led to the development of PCSK9 inhibitors. Anti-PCSK9 monoclonal antibodies showed encouraging results and are currently being evaluated in phase III clinical trials. The aim of this short review is to describe the new frontier of PCSK9 inhibition in the treatment of hypercholesterolemia. Emphasis here is given to critical emerging issues linked to PCSK9 physiology and pharmacology, which will require future investigation to definitely address the potential of anti-PCSK9 drugs in clinical practice.

HDL: to treat or not to treat?

Several studies have shown an inverse relationship between HDL cholesterol (HDL-C) levels and the risk of cardiovascular disease. Low HDL-C levels are commonly present in subjects with diabetes, metabolic syndrome, or obesity. These observations have suggested that increasing HDL concentrations might help in decreasing the cardiovascular disease risk. However, despite initial positive results, some recent data from clinical trials with HDL-raising therapies failed to confirm this hypothesis; in addition, data from Mendelian randomization analyses showed that nucleotide polymorphisms associated with increased HDL-C levels did not decrease the risk of myocardial infarction, further challenging the concept that higher HDL-C levels will automatically translate into lower cardiovascular disease risk. Differences in the quality and distribution of HDL particles might partly explain these findings, and in agreement with this hypothesis, some observations have suggested that HDL subpopulation levels may be better predictors of cardiovascular disease than simple HDL-C levels. Thus, it is expected that increased HDL-C levels may be beneficial when associated with an improvement in HDL function, suggesting that pharmacological approaches able to correct or increase HDL functions might produce more reliable clinical benefits.

Statins and skeletal muscles toxicity: from clinical trials to everyday practice.

The mechanism(s) underlying the occurrence of statin-induced myopathy are ill defined, but the results of observational studies and clinical trials provide compelling evidence that skeletal muscle toxicity is a frequent, dose-dependent, adverse event associated with all statins. It has been suggested that reduced availability of metabolites produced by the mevalonate pathway rather than intracellular cholesterol lowering per se might be the primary trigger of toxicity, however other alternative explanations have gained credibility in recent years. Aim of this review is: (i) to describe the molecular mechanisms associated to statin induced myopathy including defects in isoprenoids synthesis followed by altered prenylation of small GTPase, such as Ras and Rab proteins; (ii) to present the emerging aspects on pharmacogenetics, including CYP3A4, OATP1B1 and glycine amidinotransferase (GATM) polymorphisms impacting either statin bioavailability or creatine synthesis; (iii) to summarize the available epidemiological evidences; and (iii) to discuss the concepts that would be of interest to the clinicians for the daily management of patients with statin induced myopathy. The interplay between drug-environment and drug-drug interaction in the context of different genetic settings contribute to statins and skeletal muscles toxicity. Until specific assays/algorithms able to combine genetic scores with drug-drug-environment interaction to identify patients at risk of myopathies will become available, clinicians should continue to monitor carefully patients on polytherapy which include statins and be ready to reconsider dose, statin or switching to alternative treatments. The beneficial effects of adding agents to provide the muscle with the metabolites, such as CoQ10, affected by statin treatment will also be addressed.

Targeting PCSK9 for hypercholesterolemia.

Dyslipidemias are a predominant risk factor for cardiovascular disease. Biological and genetic research has led to the identification of several genes and proteins that may be pharmacologically targeted to improve lipoprotein profiles and possibly cardiovascular outcomes in patients with dyslipidemia. The observation that proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates the levels of circulating low-density lipoprotein C (LDL-C) by enhancing the degradation of the hepatic low-density lipoprotein receptor (LDLR) prompted the search for drugs that inhibit PCSK9 activity. Several approaches to inhibiting PCSK9 activity have been proposed; these involve inhibitory antibodies, small molecules, and gene silencing. To date, the most promising and advanced approach relates to monoclonal antibodies, which can decrease LDL cholesterol by 65-70%, even as an add-on therapy to a maximal dose of a statin. Phase III studies and large, event-driven clinical trials are ongoing and will fully address the viability and role of these drugs in clinical practice.

Gene silencing approaches for the management of dyslipidaemia.

The key role of dyslipidaemias in determining cardiovascular risk has been well established, and statins often provide effective therapeutic management. However, many patients do not achieve recommended lipid levels despite maximal therapy, and some cannot tolerate high-dose statin therapy. Recently, genetic insights into mechanisms underlying regulation of lipoprotein metabolism have expanded the potential targets of drug therapy and led to the development of novel agents, including development of gene silencing approaches. These therapeutic options include the modulation of synthesis in the liver, maturation in the circulation, and catabolism of lipoproteins. In this review, we discuss the pharmacological consequences of silencing apolipoprotein B, apolipoprotein (a), microRNA 33, proprotein convertase subtilisin/kexin type 9, and apolipoprotein C-III. New potential targets such as other microRNAs, diacylglycerol acyl transferase-1, and angiopoietin-like protein 3 are also presented. The pharmacological consequences of gene silencing and the advancement of these therapeutic approaches in clinical development will be examined.

Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis.

The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2ß and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2ß and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

Proprotein convertase subtilisin kexin type 9 (PCSK9) secreted by cultured smooth muscle cells reduces macrophages LDLR levels.

OBJECTIVE: Proprotein convertase subtilisin kexin type 9 (PCSK9) is an important regulator of hepatic low-density lipoprotein (LDL)-cholesterol levels. Although PCSK9 is mainly of hepatic origin, extra-hepatic tissues significantly contribute to PCSK9 production and, potentially, local regulation of LDL receptor expression. METHODS AND RESULTS: In the present study we show that, among vascular cells, PCSK9 is expressed in smooth muscle cells (SMCs) but not in endothelial cells, macrophages and monocytes. PCSK9 was also detectable in human atherosclerotic plaques. Conditioned media from SMCs significantly reduced LDLR expression in human macrophage and in the macrophage cell line J774. Co-culture experiments also demonstrated the influence of SMCs on LDLR expression in J774. PCSK9 released from SMCs directly regulated LDLR expression in macrophages as demonstrated by retroviral overexpression or knockdown of PCSK9 with small interfering RNA and by using recombinant PCSK9. Moreover, the proteolytic activity of PCSK9 was not required for LDLR downregulation since cultured media containing either the catalytic inactive PCSK9 or PCSK9 WT had a similar effect on LDLR in J774. Finally, conditioned media from SMCs affected ß-VLDL cholesterol uptake and PCSK9 expression reduced both LDLR and LDL uptake in J774. CONCLUSIONS: Taken together our data indicate that PCSK9 secreted by human SMCs is functionally active and capable of reducing LDLR expression in macrophages. A possible direct role for this protein in foam cell formation and atherogenesis is suggested.

Novel biotinylated bile acid amphiphiles: micellar aggregates formation and interaction with hepatocytes.

Amphiphilic bile acids linked through an oligoethylene glycol to a biotin moiety were synthesized and shown to create micellar structures in aqueous environment, interact with avidin and be efficiently incorporated into hepatocyte cells, suggesting their potential as a drug delivery system against liver diseases.

Circulating soluble receptor for advanced glycation end products is inversely associated with body mass index and waist/hip ratio in the general population.

Advanced glycation end products, AGEs, and its specific receptor, RAGE, are involved in vascular complications. A role for the soluble form of RAGE (sRAGE), which acts as a decoy for AGE, has been documented in patients with diabetes but no information is available in non-diabetic subjects. The aim of this study was to investigate the association of plasma levels of sRAGE with cardiometabolic risk factors in the general population. In addition we evaluated the relation of the common -374A/T polymorphism of RAGE with plasma levels of sRAGE. One hundred and seventy-six healthy subjects free of diabetes or coronary artery disease untreated for hypertension, dyslipidemia or cardiometabolic related diseases were randomly selected for this study from the general population. Plasma sRAGE were negatively and significantly correlated with BMI, waist/hip circumference ratio and fasting glycemia, while a positive correlation was observed with apolipoprotein A-I. These correlations were observed mainly in women who showed significantly higher sRAGE levels (1744+/-660 pg/mL vs 1414+/-649 pg/mL; P<0.05). In a stepwise regression analysis waist circumference was independently associated with sRAGE and, when waist circumference was excluded, BMI was independently associated with sRAGE. Finally in overweight subjects (BMI>25 kg/m(2)) plasma sRAGE was significantly lower compared to lean subjects (1460+/-640 pg/mL vs 1710+/-693 pg/mL; P<0.05). In healthy subjects plasma levels of sRAGE were negatively correlated with BMI and waist/hip ratio supporting a possible protective role for these proteins before any evidence of diabetic or vascular complications.

Dihydrotestosterone decreases tumor necrosis factor-alpha and lipopolysaccharide-induced inflammatory response in human endothelial cells.

CONTEXT: An increasing body of evidence suggests that testosterone may exert beneficial effects on the development of atherosclerosis. It was suggested that testosterone may act after conversion into estradiol and activation of the estrogen receptors; however, a direct role of androgens on the vascular wall has been proposed. OBJECTIVE: We investigated the effects of dihydrotestosterone on the proinflammatory response observed in human endothelial cells. DESIGN: Human endothelial cells isolated from umbilical cords were incubated with lipopolysaccharide or TNFalpha in the presence or absence of dihydrotestosterone (DHT). mRNA and cellular proteins were processed for gene expression studies, and transient transfection experiments were performed to investigate molecular mechanisms involved in the effects observed. SETTING: These studies took place at the Department of Pharmacological Sciences, University of Milan, Milan, Italy. RESULTS: Lipopolysaccharide and TNFalpha induced VCAM-1 and ICAM-1 mRNA and protein expression, as detected by real-time quantitative PCR, fluorescence-activated cell sorting, and confocal microscopy, but this effect was inhibited when cells were incubated with DHT. In addition, DHT inhibited mRNA expression of IL-6, MCP-1, CD40, TLR4, PAI-1, and Cox-2 and the release of cytokines and chemokines such as GRO, granulocyte-macrophage colony-stimulating factor, and TNF. The DHT effect was counteracted by bicalutamide, an antagonist of the androgen receptor. Furthermore, when cells were cotransfected with a Cox-2 promoter or a 3X-NF-kappaB luciferase reporter vector and a plasmid expressing the human androgen receptor, DHT treatment inhibited the increase of the luciferase activity observed with TNFalpha. CONCLUSION: DHT could positively regulate endothelial function through the control of the inflammatory response mediated by nuclear factor-kappaB in endothelial cells.