Jason S. Troutt

Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9

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.

High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol

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 first demonstrated that a moderate dose of atorvastatin (40 mg) increases PCSK9 serum levels, suggesting why increasing statin doses may have diminished efficacy 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.

Circulating Proprotein Convertase Subtilisin Kexin Type 9 Has a Diurnal Rhythm Synchronous With Cholesterol Synthesis and Is Reduced by Fasting in Humans

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 (>18 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.

Serum PCSK9 is associated with multiple metabolic factors in a large Han Chinese population

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that regulates cholesterol metabolism through low-density lipoprotein receptor (LDLR) degradation. Gain-of-function and loss-of-function mutations within PCSK9 gene lead to hypercholesterolemia or hypocholesterolemia respectively. Studies in the U.S. and Canada reported a correlation between multiple metabolic factors and circulating PCSK9 concentrations. However, there is no data available on circulating PCSK9 levels in Chinese. A sandwich ELISA assay was applied to measure serum PCSK9 levels in a Chinese population of 2719 adults from Nanjing district, China, which represents a large and uniform ethnic population of Han Chinese. Serum PCSK9 levels ranged from 12.85 to 222.50 ng/ml with a mean concentration of 69.35 ng/ml in this population. Serum PCSK9 levels were slightly higher in women than in men. Compared to premenopausal women, postmenopausal women had significantly higher PCSK9 levels. Serum PCSK9 levels were correlated with multiple metabolic variables including age, BMI, total cholesterol, LDL cholesterol, triglycerides, fasting blood glucose, systolic blood pressure (SP) and diastolic blood pressure (DP) in this population. After stepwise regression analysis, there was a significant positive association between serum PCSK9 levels and total cholesterol, triglycerides and SP in men. In women, there was a positive correlation between PCSK9 levels and total cholesterol, age and DP. Our study indicates that the serum PCSK9 level may be a biomarker of metabolic status and cardiovascular disease.

Effects of currently prescribed LDL-C-lowering drugs on PCSK9 and implications for the next generation of LDL-C-lowering agents

BackgroundDuring the past decade, proprotein convertase subtilisin kexin type 9 (PCSK9) has been identified as 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, causing its subsequent degradation. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia that manifests with dramatically increased serum levels of LDL-C, while loss-of-function mutations in PCSK9 are associated with significantly decreased LDL-C and cardiovascular risk.ResultsInitial studies in animals and cultured cells demonstrated that statins increased PCSK9 mRNA expression, resulting in many research groups exploring the effect of statins on PCSK9 levels in humans. We first reported that statins increased human PCSK9 circulating protein levels. Additional researchers subsequently confirmed these observations, further prompting many laboratories including our own to examine the effect of other lipid lowering medications on PCSK9 levels. Our observation that fenofibrate (200 mg/day) significantly increased PCSK9 levels was confirmed by another laboratory, and an additional group demonstrated that ezetimibe also increased PCSK9 levels.ConclusionsIt has become clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels. These observations almost certainly explain why these agents are not more effective in lowering LDL-C and suggest that efforts should be made toward the development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through decreasing or inhibiting PCSK9.

Fenofibrate treatment increases human serum proprotein convertase subtilisin kexin type 9 levels

Over the past several years, proprotein convertase subtilisin kexin type 9 (PCSK9) has gained significant attention as a key regulator of serum LDL-cholesterol (LDL-C) levels. 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 and cardiovascular risk. Our laboratory was the first to demonstrate that atorvastatin increases PCSK9 serum levels, an observation that has since been confirmed by at least two other groups. In light of these observations, we studied the effect of another common lipid-lowering medication, fenofibrate, on circulating PCSK9 protein levels in patients treated with fenofibrate or placebo for 12 weeks. We observed that fenofibrate (200 mg per day) significantly increased circulating PCSK9 levels by 25% compared with baseline. Placebo treatment, in comparison, had no effect on PCSK9 levels. Interestingly, fenofibrate-induced increases in serum PCSK9 levels were highly correlated with fenofibrate-induced changes in HDL-C and triglyceride levels, as well as with fenofibrate-induced changes in LDL-C levels. These results suggest an explanation for why fibrates do not achieve as much LDL-C lowering as might otherwise be expected and indicate that the addition of a PCSK9 inhibitor to fibrate therapy may result in additional beneficial LDL-C lowering.

Circulating Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Predicts Future Risk of Cardiovascular Events Independently of Established Risk Factors

Background —The secreted protein proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising new target for lowering of plasma low-density lipoprotein (LDL) cholesterol and prevention of 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 4,232 men and women aged 60 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 number of 491 incident events (fatal and non-fatal myocardial infarctions, unstable angina, deaths from coronary heart disease, fatal and non-fatal ischemic strokes) were recorded. Cox Proportional Hazards model was used to calculate Hazard ratios (HR) with 95% confidence intervals (CI). Baseline serum PCSK9 concentration predicted incident CVD; concentration in quartile 4, as compared to quartile 1, was associated with a HR of 1.69 (95% CI 1.30-2.19) after adjustment for sex. Further adjustment for LDL cholesterol, high-density lipoprotein cholesterol, lipoprotein(a), triglycerides, hypertension, diabetes, smoking, overweight, obesity, physical inactivity and statin use resulted in a decrease of the HR: 1.48 (95% CI 1.12-1.95). Conclusions —Serum PCSK9 concentration is associated with future risk of CVD even after adjustments for established CVD risk factors. Further studies are needed to confirm this observation.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). Conclusions— Serum PCSK9 concentration is associated with future risk of CVD even after adjustments for established CVD risk factors. Further studies are needed to confirm this observation.

Circulating PCSK9 Predicts Future Risk of Cardiovascular Events Independently of Established Risk Factors

Background —The secreted protein proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising new target for lowering of plasma low-density lipoprotein (LDL) cholesterol and prevention of 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 4,232 men and women aged 60 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 number of 491 incident events (fatal and non-fatal myocardial infarctions, unstable angina, deaths from coronary heart disease, fatal and non-fatal ischemic strokes) were recorded. Cox Proportional Hazards model was used to calculate Hazard ratios (HR) with 95% confidence intervals (CI). Baseline serum PCSK9 concentration predicted incident CVD; concentration in quartile 4, as compared to quartile 1, was associated with a HR of 1.69 (95% CI 1.30-2.19) after adjustment for sex. Further adjustment for LDL cholesterol, high-density lipoprotein cholesterol, lipoprotein(a), triglycerides, hypertension, diabetes, smoking, overweight, obesity, physical inactivity and statin use resulted in a decrease of the HR: 1.48 (95% CI 1.12-1.95). Conclusions —Serum PCSK9 concentration is associated with future risk of CVD even after adjustments for established CVD risk factors. Further studies are needed to confirm this observation.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). Conclusions— Serum PCSK9 concentration is associated with future risk of CVD even after adjustments for established CVD risk factors. Further studies are needed to confirm this observation.

Isolation and characterization of the circulating truncated form of PCSK9

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.

Common and Low-Frequency Genetic Variants in the PCSK9 Locus Influence Circulating PCSK9 Levels

Objective —Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that influences plasma low-density lipoprotein concentration and susceptibility to coronary heart disease. Circulating PCSK9 levels show considerable interindividual differences, but the factors responsible for this variation are largely unknown. Methods and Results—We analyzed circulating PCSK9 levels in 4 cohorts of healthy, middle-aged Swedes (n=5722) and found that PCSK9 levels varied over ≈50-fold range, showed a positive relationship with plasma low-density lipoprotein–cholesterol concentration, and were associated with plasma triglyceride, fibrinogen, insulin, and glucose concentrations. A genome-wide association study conducted in 2 cohorts (n=1215) failed to uncover common genetic variants robustly associated with variation in circulating PCSK9 level. As expected, the minor allele of the PCSK9 R46L variant was in all cohorts associated with reduced PCSK9 levels and decreased plasma low-density lipoprotein–cholesterol concentrations, but no relationship was observed with the plasma triglyceride concentration. Further mapping of the PCSK9 locus revealed a common polymorphism (rs2479415, minor allele frequency 43.9%), located ≈6 kb upstream from PCSK9, which is independently associated with increased circulating PCSK9 levels. Conclusion—Common and low-frequency genetic variants in the PCSK9 locus influence the pronounced interindividual variation in circulating PCSK9 levels in healthy, middle-aged white (predominantly Swedish) subjects.