Proprotein convertase subtilisin/kexin type 9 (PCSK9) has gained attention as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels. This novel protease causes the degradation of hepatic low density lipoprotein receptors. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C levels and cardiovascular risk. Previous studies have demonstrated that statins upregulate PCSK9 mRNA expression in cultured cells and animal models. In light of these observations, we studied the effect of atorvastatin on circulating PCSK9 protein levels in humans using a sandwich ELISA to quantitate serum PCSK9 levels in patients treated with atorvastatin or placebo for 16 weeks. We observed that atorvastatin (40 mg/day) significantly increased circulating PCSK9 levels by 34% compared with baseline and placebo and decreased LDL-C levels by 42%. These results suggest that the addition of a PCSK9 inhibitor to statin therapy may result in even further LDL-C decreases. The serum protease proprotein convertase subtilisin/ kexin type 9 (PCSK9) has gained tremendous attention as a potential key regulator of serum low density lipoprotein cholesterol (LDL-C) levels (1-3). PCSK9 is a protease made by the liver that acts to degrade hepatic low density lipoprotein receptors (LDLRs) (4-10). The mechanism by which PCSK9 degrades LDLRs is extremely complex and is only beginning to be understood. It was recently suggested that the protease itself does not have to be proteolytically active to cause degradation of the LDLR but rather binds to the LDLR and subsequently targets it for intracellular destruction within the hepatocyte (11-13). Regardless of the exact mechanism, the result of LDLR levels being decreased is that the liver has a decreased ability to bind LDL from the circulation and serum LDL-C levels increase. Therefore, mutations in PCSK9 can have dramatic effects on serum LDL-C levels in humans.Patients with gain-of-function mutations of PCSK9 manifest severe familial hypercholesterolemia and accompanying increased cardiovascular risk (14-17). These mutations in PCSK9 account for ?10-25% of familial dominant hypercholesterolemia cases that could not be explained by mutations in either the LDLR or apolipoprotein B (apoB) (14-17). In contrast, heterozygous subjects with loss-of-function mutations in PCSK9, including mutations that prevent the self-cleavage and secretion of the protein itself, have significantly decreased levels of serum LDL-C and dramatically decreased cardiovascular risk (18)(19)(20). Approximately 2% of African-Americans carry such mutations, with an accompanying 80-90% decreased risk of serious cardiovascular events (18). Recently, the first compound heterozygote for PCSK9 loss-of-function mutations was described. This subject, a healthy 32 year old female, had an extremely low serum LDL-C level of 14 mg/dl (20).Interestingly, statins have been shown to increase the activity/nuclear t...

This article is available online at http://www.jlr.org Proprotein convertase subtilisin kexin type 9 (PCSK9) has been recognized as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels ( 1-7 ). PCSK9 is a protease made and secreted by the liver into the plasma, which then binds to and degrades hepatic LDL receptors (LDLR) (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The mechanism by which PCSK9 degrades LDLR is complex. Recent studies suggest that after self-cleavage and secretion, PCSK9 does not have to be enzymatically active to cause degradation of the LDLR ( 19-21 ). Rather, PCSK9 binds to the LDLR and subsequently targets it for lysosomal destruction within the hepatocyte ( 8,(19)(20)(21). This concept of how PCSK9 acts to decrease hepatic LDLR levels is supported by recent fi ndings that disruption of the binding of PCSK9 to the LDLR using anti-PCSK9 antibody results in preserved LDLR and decreased [22][23][24].Several different PCSK9 mutations have been reported in humans. Patients with gain-of-function mutations of PCSK9 present with severe familial hypercholesterolemia and accompanying increased cardiovascular risk (25)(26)(27)(28)(29). In contrast, individuals with loss-of-function mutations in PCSK9, including mutations which prevent the selfcleavage and secretion of the protein, have signifi cantly decreased levels of serum LDL-C and lower cardiovascular risk (30)(31)(32). Approximately 3% of African-Americans are heterozygous for such mutations ( 30 ). Of note, a compound heterozygote for PCSK9 loss-of-function mutations was recently described. The subject, a healthy 32-year-old female, had a serum LDL-C level of 14 mg/dl ( 32 ). A second Abstract Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of serum LDL-cholesterol (LDL-C) levels. PCSK9 is secreted by the liver into the plasma and binds the hepatic LDL receptor (LDLR), causing its subsequent degradation. We fi rst demonstrated that a moderate dose of atorvastatin (40 mg) increases PCSK9 serum levels, suggesting why increasing statin doses may have diminished effi cacy with regard to further LDL-C lowering. Since that initial observation, at least two other groups have reported statin-induced PCSK9 increases. To date, no analysis of the effect of high-dose atorvastatin (80 mg) on PCSK9 over time has been conducted. Therefore, we studied the time course of atorvastatin (80 mg) in human subjects. We measured PCSK9 and lipid levels during a 2-week lead-in baseline period and every 4 weeks thereafter for 16 weeks. We observed that atorvastatin (80 mg) caused a rapid 47% increase in serum PCSK9 at 4 weeks that was sustained throughout 16 weeks of dosing. Importantly, while PCSK9 levels were highly correlated with total cholesterol (TC), LDL-C, and triglyceride (TG) levels at baseline, atorvastatin (80 mg) completely abolished all of these correlations. Together, these results further suggest an explanation for why increasing doses of statins fail to achieve proportional LDL-C lowering. Abbreviations: HDL-...

PCSK9 is present in the circulation in 2 forms: a mature form composed of the PCSK9 prodomain attached to its catalytic domain and a form that is the result of furin cleavage at the Arg218-Gln219 peptide bond. 10,11 Commercially available and proprietary ELISA methods have been capable of measuring Background-The secreted protein proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising new target for lowering plasma low-density lipoprotein cholesterol and preventing cardiovascular disease (CVD). The relationship between circulating PCSK9 and incident CVD in the general population is unknown. We investigated whether serum PCSK9 concentration is associated with incident CVD in a prospective cohort study of 4232 men and women 60 years of age at the time of recruitment. Methods and Results-Incident CVD was recorded by matching to national registries. After 15 years of follow-up, a total of 491 incident events (fatal and nonfatal myocardial infarctions, unstable angina, deaths from coronary heart disease, fatal and nonfatal ischemic strokes) were recorded. Cox proportional hazards model was used to calculate hazard ratios with 95% confidence intervals. Baseline serum PCSK9 concentration predicted incident CVD; concentration in quartile 4 compared with quartile 1 was associated with a hazard ratio of 1.69 (95% confidence interval, 1.30-2.19) after adjustment for sex. Further adjustment for low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lipoprotein(a), triglycerides, hypertension, diabetes mellitus, smoking, overweight, obesity, physical inactivity, and statin use resulted in a decrease in the hazard ratio to 1.48 (95% confidence interval, 1.12-1.95). Continuing medical education (CME) credit is available for this article. Go to http://cme.ahajournals.org to take the quiz. Received July 15, 2015; accepted February 12, 2016. Conclusions-Serum Leander et al PCSK9 and Incident Cardiovascular Events 1231total PCSK9 concentrations. 12 Total PCSK9 concentration in the circulation is reportedly influenced by common and rare PCSK9 gene variants, sex, use of statins, and diurnal variation. [13][14][15] Several demographic and metabolic parameters appear to correlate with serum PCSK9, including plasma LDL cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein B (apoB), insulin, glucose, fibrinogen, C-reactive protein, smoking, and body mass index. 13,14 Reported correlations have typically been weak and inconsistent, with the exception of LDL cholesterol, apoB, and triglycerides, for which correlations were weak but consistent. Considering that PCSK9 has convincingly been shown to regulate LDL cholesterol via the LDL receptor, we hypothesized that the serum PCSK9 concentration would be associated with future CVD events. There are to date few published studies examining whether the circulating PCSK9 concentrations can predict CVD risk. Therefore, we undertook the present investigation. Methods Study DesignParticipants in this cohort study were randomly selected f...

Objective-To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. Methods and Results-We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid-binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (Ͼ18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. Conclusion-Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9. Key Words: circulating PCSK9 Ⅲ cholesterol synthesis Ⅲ LDL cholesterol Ⅲ diurnal rhythm Ⅲ growth hormone Ⅲ cholesterol-lowering drugs Ⅲ lipoproteins G enetic variants of proprotein convertase subtilisin kexin type 9 (PCSK9) influence plasma low-density lipoprotein (LDL) cholesterol in humans, accounting for both hypercholesterolemia and hypocholesterolemia and altered coronary risk. 1,2 PCSK9 modulates the number of LDL receptors (LDLRs) by triggering the degradation of LDLRs. 3 Gain-offunction mutations in the PCSK9 gene produce a phenotype of familial hypercholesterolemia, 1 whereas loss-of-function mutations reduce plasma LDL cholesterol levels. 2 Circulating PCSK9 is largely derived from the liver, 4,5 and plasma levels relate to the hepatic expression of PCSK9. 6 -9 It is still unclear to what extent PCSK9 is physiologically regulated and if such regulation may influence plasma LDL cholesterol levels in humans. Novel therapies aiming at lowering serum LDL cholesterol by interfering with PCSK9 activity are under development. 10 Fasting plasma levels of PCSK9 correlate positively with LDL cholesterol levels in healthy and diabetic patients 7,[11][12][13][14] ; however, in the most extensive study, 7 with Ͼ3000 subjects, PCSK9 levels only predicted 7% of the variation in LDL cholesterol. In animals, the hepatic gene expression of PCSK9 is partly under hormonal control: treatment with glucagon or high-dose estrogen reduces PCSK9 mRNA and increases the number of LDLRs, 15 whereas insulin 15,16 and growth hormone (GH) 17 both increase PCSK9 mRNA levels in rat liver.The gene expressions of PCSK9, the LDLR and 3-hydroxy-3-...