The effect of PCSK9 immunization on the hepatic level of microRNAs associated with the PCSK9/LDLR pathway

IntroductionMicroRNAs (miRNAs) are a class of gene expression epigenetic regulators that play roles in regulating genes involved in cholesterol homeostasis, including low-density lipoprotein receptor (LDLR) and PCSK9; therefore, miRNAs have been suggested as potential therapeutic targets for treating cardiometabolic disorders. Thus, the present study aimed to assess the effect of immunotherapy with the PCSK9 peptide vaccine on the hepatic expression levels of microRNAs associated with the LDLR pathway, including miRNA-27a, miRNA-30c, and miRNA-191, in normal vaccinated mice.Material and methodsPCSK9 immunogenic peptide and 0.4% alum adjuvant were mixed at a 1 : 1 ratio and used as a vaccine formulation. Male albino mice were randomly assigned to the vaccine or control group. Mice in the vaccine group were injected four times at two-week intervals with a PCSK9 peptide vaccine, and mice in the control group were injected with phosphate-buffered saline (PBS). Animal livers were sampled 2 weeks after the last injection to assess miRNA expression levels. The hepatic expression levels of miRNA-27a, miRNA-30c, and miRNA-191 were evaluated by SYBR Green real-time PCR, quantified by a comparative (2^–ΔΔCT) method (fold change (FC)) and normalized to U6 small nuclear RNA (U6snRNA) expression as an internal control.ResultsThe hepatic expression level of miRNA-27a was significantly lower in mice following immunotherapy with the PCSK9 peptide vaccine compared to the control group (FC: 0.731 ±0.1, p = 0.027). Also, there was a borderline significantly lower hepatic expression level of miRNA-30c in the vaccinated group compared to the control (FC: 0.569 ±0.1, p = 0.078). However, no significant differences were found in the hepatic expression level of miRNA-191 between the two studied groups (FC: 0.852 ±0.1, p = 0.343).ConclusionsAccording to the findings, the PCSK9 peptide vaccine could effectively reduce the hepatic expression level of miRNA-27a and may be helpful in the management of LDL-C level and atherosclerosis, which may be mediated through the LDLR pathway.     

PCSK9 inhibitors for prevention of atherosclerotic cardiovascular disease

The discovery of proprotein convertase subtilisin kexin-type 9 (PCSK9) and the development of inhibitors of PCSK9 function appear to mark an epochal advance in clinical lipidology. PCSK9 is a circulating protein that binds to low-density lipoprotein (LDL) receptors and facilitates their lysosomal degradation following internalization in cells. Blocking PCSK9 thus increases the recycling of LDL receptors and results in more receptors on the cell surface, particularly in the liver, thereby lowering LDL levels. In this Roundtable, we discuss the recent large cardiovascular outcomes trials in which evolocumab and alirocumab, monoclonal antibodies directed against PCSK9, successfully reduced major cardiovascular events. We discuss the safety of these drugs as well as the safety of maintaining very low LDL cholesterol levels. Finally, we address pragmatic considerations affecting the use of PCSK9 inhibitors in clinical practice.     

The molecular basis of familial hypercholesterolemia in Lebanon: Spectrum of LDLR mutations and role of PCSK9 as a modifier gene

Autosomal dominant hypercholesterolemia (ADH), a major risk for coronary heart disease, is associated with mutations in the genes encoding the low‐density lipoproteins receptor (LDLR), its ligand apolipoprotein B (APOB) or PCSK9 (Proprotein Convertase Subtilin Kexin 9). Familial hypercholesterolemia (FH) caused by mutation in the LDLR gene is the most frequent form of ADH. The incidence of FH is particularly high in the Lebanese population presumably as a result of a founder effect. In this study we characterize the spectrum of the mutations causing FH in Lebanon: we confirm the very high frequency of the LDLR p.Cys681X mutation that accounts for 81.5 % of the FH Lebanese probands recruited and identify other less frequent mutations in the LDLR. Finally, we show that the p.Leu21dup, an in frame insertion of one leucine to the stretch of 9 leucines in exon 1 of PCSK9, known to be associated with lower LDL‐cholesterol levels in general populations, is also associated with a reduction of LDL‐cholesterol levels in FH patients sharing the p.C681X mutation in the LDLR. Thus, by studying for the first time the impact of PCSK9 polymorphism on LDL‐cholesterol levels of FH patients carrying a same LDLR mutation, we show that PCSK9 might constitute a modifier gene in familial hypercholesterolemia. © 2009 Wiley‐Liss, Inc.     

A review of PCSK9 inhibition and its effects beyond LDL receptors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an integral role in the degradation of low-density lipoprotein receptors (LDL-R), making it an intriguing target for emerging pharmacotherapy. Two PCSK9 inhibitors, alirocumab and evolocumab, have been approved and are available in the United States and European Union. However, much of the PCSK9 story remains to be told. The pipeline for additional pharmacotherapy options is rich with several compounds under development, using alternative strategies for inhibiting PCSK9. Perhaps, more intriguing is the interaction between PCSK9 and non-LDL-R targets, including mediators of inflammation and immunological processes, which remain under intense investigation. This review will discuss the currently available PCSK9 inhibitors, the development of novel approaches to PCSK9 modulation, and the potential non-LDL-R-mediated effects of PCSK9 inhibition.     

PCSK9 regulates apoptosis in human neuroglioma u251 cells via mitochondrial signaling pathways

Proprotein convertase subtilisin/kexin type 9 (PCSK9), belongs to a family of proprotein convertases (PCs), encodes a neural apoptosis-regulated convertase 1. However, the precise role of PCSK9 during glioma cells apoptosis has not been reported. Therefore, we examined the effects of knockdown and overexpression of PCSK9 on apoptosis of human neuroglioma U251 cells, and investigated the underlying mechanisms of apoptosis. We found that PCSK9 regulated cells proliferation as determined by CCK-8 and Hoechst staining analysis. In addition, western blot results showed that PCSK9 siRNA promote apoptosis via activation of caspase-3 and down-regulation of the anti-apoptotic proteins, XIAP and p-Akt, while PCSK9 overexpression inhibited apoptosis. Moreover, PCSK9 siRNA improved the ratio of Bax/Bcl-2 which leads to the release of cytochrome c, while PCSK9 overexpression decreased it. Taken together, these data demonstrate that PCSK9 may regulate apoptosis through mitochondrial pathway and is expected to be a promising therapeutic strategy for the malignant glioma.     

Proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor use in the management of resistant hypercholesterolemia induced by mitotane treatment for adrenocortical cancer

We report the case of a patient with probable heterozygous familial hypercholesterolemia and mitotane-induced resistant hypercholesterolemia, despite combination therapy with rosuvastatin and ezetimibe. The patient was managed with the addition of evolocumab. Use of a proprotein convertase subtilisin-kexin type 9 inhibitor, should be considered in patients who develop mitotane-related hypercholesterolemia that cannot be managed with conventional lipid-lowering treatment.     

PCSK9抑制剂在冠状动脉粥样硬化性心脏病 治疗中的应用

低密度脂蛋白胆固醇（LDL-C）血浆水平升高是动脉粥样硬化性心血管疾病（ASCVD）的主要危险因素。他汀类药物虽然能够有效降低 LDL-C水平,但仍有部分患者无法达到降脂目标或者无法耐受。目前大量研究表明前蛋白转化酶枯草溶菌素9（PCSK9）与血浆LDL-C紧密相关,抑制PCSK9可以有效降低LDL-C水平及ASCVD发生率。本文主要对PCSK9机制以及PCSK9抑制剂治疗冠状动脉粥样硬化性心脏病的新研究进展作一综述。     

Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is at a locus for autosomal dominant hypercholesterolemia, and recent data indicate that the PCSK9 gene is involved in cholesterol biosynthesis. Mutations within this gene have previously been found to segregate with hypercholesterolemia. In this study, DNA sequencing of the 12 exons of the PCSK9 gene has been performed in 51 Norwegian subjects with a clinical diagnosis of familial hypercholesterolemia where mutations in the low-density lipoprotein receptor gene and mutation R3500Q in the apolipoprotein B-100 gene had been excluded. Two novel missense mutations were detected in the catalytic subdomain of the PCSK9 gene. Two patients were heterozygotes for D374Y, and one patient was a double heterozygote for D374Y and N157K. D374Y segregated with hypercholesterolemia in the two former families where family members were available for study. Our findings support the notion that mutations in the PCSK9 gene cause autosomal dominant hypercholesterolemia.     

Novel mutations of the PCSK9 gene cause variable phenotype of autosomal dominant hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH) is a frequent (1/500) monogenic inherited disorder characterized by isolated elevation of LDL leading to premature cardiovascular disease. ADH is known to result from mutations at two main loci: LDLR (encoding the low density lipoprotein receptor), and APOB (encoding apolipoprotein B100), its natural ligand. We previously demonstrated that ADH is also caused by mutations of the PCSK9 (proprotein convertase subtilisin/kexin type 9) gene that encodes Narc-1 (neural apoptosis-regulated convertase 1). However, the role of this novel disease locus as a cause of hypercholesterolemia remains unclear. In the present study, we analysed the PCSK9 coding region and intronic junctions in 130 adult or pediatric patients with ADH, previously found as being non LDLR/non APOB mutation carriers. Four novel heterozygous missense variations were found: c.654A>T (p.R218S), c.1070G>A (p.R357H), c.1405C>T (p.R469W), and c.1327G>A (p.A443T). All mutations were absent in 340 normolipidemic controls. Except for the A443T, all mutations are nonconservative and modify a highly conserved residue. Segregation with hypercholesterolemia is incomplete in one pedigree. Type and severity of hyperlipidemia and of cardiovascular disease could vary among subjects from the same family. Finally, the proband carrying the R357H mutation exhibited very high plasma cholesterol during pregnancy, whereas the proband carrying the p.R469W mutation exhibited a severe phenotype of hypercholesterolemia in combination with a LDLR mutation resulting from a frameshift at residue F382 (1209delC). These observations suggest that variations in PCSK9 are a rare cause of non LDLR/non APOB ADH (approximately 2.3%) and that additional environmental or genetic factors may contribute to the phenotype caused by PCSK9 missense mutations in humans.     

绞股蓝总苷对PCSK9基因表达及辛伐他汀降血脂作用的影响

目的:探讨绞股蓝总苷(gypenosides,GPs)对大鼠肝脏前蛋白转化酶枯草溶菌素9(PCSK9)基因表达及辛伐他汀的降血脂作用的影响。方法:采用高脂饲料喂饲建立大鼠高脂血症模型。60只健康雄性SD大鼠随机分为正常对照组(control组)、高脂模型组(model组)、辛伐他汀组(Simvastatin组)、GPs组和GPs与辛伐他汀联合用药组(combined组)。除正常对照组喂食普通饲料外,其余3组大鼠均喂食高脂饲料。将GPs溶于0.3%羧甲基纤维素钠(CMC-Na)溶液中,用灌胃方式给药。Control组和model组灌0.3%CMC-Na(1mL/100 g),GPs组灌GPs 160 mg·kg~(-1)·d~(-1),simvastatin组灌辛伐他汀5mg·kg~(-1)·d~(-1),combined组灌两者联合剂量。实验8周后,处死大鼠。取腹腔动脉血,测定血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C);称大鼠体重及肝脏组织湿重,测定肝指数;取肝脏用4%多聚甲醛固定,石蜡包埋,HE染色作常规形态学检测;另取肝脏提取总RNA,real-time PCR测定PCSK9和低密度脂蛋白受体(LDLR)的mRNA表达;提取肝脏总蛋白,Western blot测定PCSK9和LDLR蛋白的表达。结果:成功建立高脂血症大鼠模型。与model组大鼠比较,simvastatin组、GPs组以及combined组TC、TG和LDL-C水平均明显下降(P0.05),各组HDLC水平有不同程度的上升(P0.05)。与model组大鼠比较,simvastatin组、GPs组以及combined组肝指数均明显下降(P0.05)。肝组织病理结果显示,高脂血症性大鼠出现脂肪肝病变;Simvastatin组、GPs组以及combined组大鼠肝细胞脂肪变性程度有不同程度减轻,尤以combined组效果显著。与model组比较,simvastatin组PCSK9和LDLR的mRNA表达均明显升高,GPs组以及combined组PCSK9的mRNA表达明显降低(P0.05),GPs组LDLR的mRNA表达变化不明显,combined组LDLR的mRNA表达明显升高(P0.05)。与model组比较,Simvastatin组PCSK9和LDLR的蛋白表达均明显升高;GPs组和combined组的PCSK9蛋白表达明显降低,LDLR蛋白表达明显升高(P0.05)。结论:GPs能抑制肝脏PCSK9表达,增加LDLR表达量;与辛伐他汀联用可增强其降血脂和减轻肝脏脂肪病变的效果。     

Proteinase inhibitor 9 is reduced in human atherosclerotic lesion development

Background

Granzyme B, a proapoptotic serine protease, is abundant in advanced, unstable atherosclerotic plaques, and it is suggested to contribute to plaque instability by inducing vascular smooth muscle cells apoptosis and by degrading plaque extracellular matrix. Proteinase inhibitor 9, the only known endogenous inhibitor of granzyme B in humans, confers protection against granzyme-B-induced apoptosis. However, the role of proteinase inhibitor 9 in atherosclerotic lesion development has yet to be determined. We hypothesized that atherosclerotic lesions have lower proteinase inhibitor 9 expression levels that will increase their susceptibility to granzyme-B-induced apoptosis.

Methods

Serial sections of human coronary arteries exhibiting different stages of lesion development were assessed by immunohistochemistry for proteinase inhibitor 9, α-smooth muscle cells actin, granzyme B, CD8, and active caspase-3. Frozen samples were analyzed by Western blot to evaluate total proteinase inhibitor 9 levels.

Results

Vascular smooth muscle cells express less proteinase inhibitor 9 as disease severity increases, and a significant difference in proteinase inhibitor 9 expression is observed between medial and intimal smooth muscle cells. High granzyme B levels colocalize with CD8+ cells and foam cells in the shoulder region and necrotic core area of advanced lesions. In advanced lesions, increased expression of activated caspase-3 in intimal SMC was associated with reduced proteinase inhibitor 9 expression in the presence of granzyme B.

Conclusion

Reduced proteinase inhibitor 9 expression in human vascular smooth muscle cells is associated with atherosclerotic disease progression and is inversely related to the extent of apoptosis within the intima. Reduced proteinase inhibitor 9 expression may contribute to increased smooth muscle cell susceptibility to granzyme-B-induced apoptosis within the plaque.     

Pharmacodynamic relationship between PCSK9, alirocumab,and LDL-C lowering in the ODYSSEY CHOICE I trial

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Case reports of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition nonresponse

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, a novel class of monoclonal antibodies, reduces low-density lipoprotein cholesterol levels and improves cardiovascular outcomes. Given the short time frame, these agents have been available for use; reports of nonresponse to the PCSK9 inhibitor therapy are scarce in literature. We describe 2 cases with substantially lesser than expected low-density lipoprotein cholesterol lowering on PCSK9 therapy. Nonresponse to PCSK9 inhibition was attributed to autosomal recessive hypercholesterolemia (secondary to low-density lipoprotein receptor adaptor protein 1 mutation) and plasmapheresis after PCSK9 inhibitor drug injections. Additional PCSK9 inhibitor nonresponders are likely to emerge as the use of these agents increases overtime.     

Lipid metabolism in cells of the atherosclerotic human aorta. Experiments in primary culture

Bulletin of Experimental Biology and Medicine -     

广东汉族人PCSK9基因E670G位点多态性与冠心病相关性研究

目的通过对中国广东地区冠心病（CAD）患者和健康个体前蛋白转化酶枯草杆菌蛋白酶/西布曲明9a型（PCSK9）基因E670G位点单核苷酸多态性（SNP）的研究,分析其在CAD患者和健康人中的分布,以及与冠心病和血脂代谢的相关性。结论采集经冠脉造影确诊的CAD患者血液标本165例,健康人血液标本180例,分别提取基因组DNA模板,采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法分析PCSK9基因E670G位点SNP,并用基因测序法验证结果。血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）浓度水平采用酶法测定。结果中国广东地区CAD患者和健康人群在该位点的等位基因频率、基因型频率分布差异无统计学意义（P〉0.05）。CAD患者组和对照组之间TC、LDL-C、HDL-C水平差异具有统计学意义（P〈0.05）,TG水平差异无统计学意义（P〉0.05）。CAD患者组中AA和AG基因型之间LDL-C、HDL-C的差异具有统计学意义（P〈0.05）,TC、TG的差异没有统计学意义（P〉0.05）;与非670G携带者比较,670G携带者血LDL-C水平增高,血HDL-C水平降低。AG基因型的冠脉病变支数明显高于AA基因型,其差异具有统计学意义（P〈0.05）。结论 PCSK9基因E670G多态性与血LDL-C、HDL-C水平,以及冠心病病变严重程度相关联。     

Impact of bariatric surgery on circulating PCSK9 levels as a marker of cardiovascular disease risk: a meta-analysis

IntroductionThis systematic review and meta-analysis focuses on PCSK9 changes in obese patients following bariatric surgery.MethodsA systematic literature search in four databases was performed. Comprehensive Meta-Analysis (CMA) V2 software used to conduct the meta-analysis. Studies were evaluated regarding heterogeneity in design, populations under investigation, and treatment duration using a random-effects model and the generic inverse variance weighting approach. A random-effect meta-regression approach was used to investigate the association with the estimated effect size.ResultsThe results of the meta-analysis on 4 trials including 260 individuals demonstrated a remarkable decline of PCSK9 after bariatric surgery (WMD = –57.34 ng/ml, 95% CI: –87.97, –26.71, p < 0.001; I² = 96.25%). Consistently, a significant decrease of LDL-C after bariatric surgery (WMD = –22.57 mg/dl, 95% CI: –27.5, –17.574, p < 0.001; I² = 86.35%) was observed.ConclusionsPCSK9 is reduced significantly after bariatric surgery. The decrease of PCSK9 might be utilized as an independent surrogate marker of improvement of atherosclerotic cardiovascular disease risk after bariatric surgery.     

Prognostic impact of cascade screening for familial hypercholesterolemia on cardiovascular events

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disorder mainly caused by mutations in the low-density lipoprotein (LDL) receptor or associated genes, resulting in elevated serum cholesterol levels and an increased risk of premature atherosclerotic cardiovascular disease (ASCVD).ObjectiveWe aimed to evaluate the prognostic impact of cascade screening for FH.MethodsWe retrospectively investigated the health records of 1050 patients with clinically diagnosed FH, including probands and their relatives who were cascade-screened, who were referred to our institute. We used Cox models that were adjusted for established ASCVD risk factors to assess the association between cascade screening and major adverse cardiac events (MACE). The median period of follow-up evaluating MACE was 12.3 years (interquartile ranges [IQR] = 9.1–17.5 years), and MACE included death associated with ASCVD, or acute coronary syndrome.ResultsDuring the observation period, 113 participants experienced MACE. The mean age of patients identified through cascade screening was 18-years younger than that of the probands (38.7 yr vs. 57.0 yr, P < 0.0001), with a lower proportion of ASCVD risk factors. Interestingly, patients identified through cascade screening under milder lipid-lowering therapies were at reduced risk for MACE (hazard ratio [HR] = 0.67; 95%CI = 0.44 to 0.90; P = 0.0044) when compared with the probands, even after adjusting for those known risk factors, including age, and prior ASCVD.ConclusionsThe identification of patients with FH via cascade screening appeared to result in better prognosis.