Plasma PCSK9 levels are significantly modified by statins and fibrates in humans

Abstract

Background: Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a secreted glycoprotein that is transcriptionally regulated by cholesterol status. It modulates levels of circulating low density lipoprotein cholesterol (LDLC) by negatively regulating low density lipoprotein receptor (LDLR) levels. PCSK9 variants that result in 'gain of function' have been linked to autosomal dominant hypercholesterolemia, while significant protection from coronary artery disease has been documented in individuals who carry 'loss of function' PCSK9 variants. PCSK9 circulates in human plasma, and we previously reported that plasma PCSK9 is positively correlated with total cholesterol and LDLC in men.

Results: Herein, we report the effects of two lipid-modulating therapies, namely statins and fibrates, on PCSK9 plasma levels in human subjects. We also document their effects on endogenous PCSK9 and LDLR expression in a human hepatocyte cell line, HepG2, using immunoprecipitation and immunoblot analyses. Changes in plasma PCSK9 following fenofibrate or gemfibrozil treatments (fibric acid derivatives) were inversely correlated with changes in LDLC levels (r = -0.558, p = 0.013). Atorvastatin administration (HMGCoA reductase inhibitor) significantly increased plasma PCSK9 (7.40%, p = 0.033) and these changes were inversely correlated with changes in LDLC levels (r = -0.393, p = 0.012). Immunoblot analyses of endogenous PCSK9 and LDLR expression by HepG2 cells in response to statins and fibrates showed that LDLR is more upregulated than PCSK9 by simvastatin (2.6x vs 1.5x, respectively at 10 muM), while fenofibrate did not induce changes in either.

Conclusion: These results suggest that in vivo (1) statins directly increase PCSK9 expression while (2) fibrates affect PCSK9 expression indirectly through its modulation of cholesterol levels and (3) that these therapies could be improved by combination with a PCSK9 inhibitor, constituting a novel hypercholesterolemic therapy, since PCSK9 was significantly upregulated by both treatments.

Figure 1

Relationship between percent change in plasma PCSK9 and LDL-cholesterol levels following fibrate treatment. PCSK9 and LDL-C were measured pre- and post-fibrate therapy as described in Methods. Pre-treatment levels were set as 100%.

Figure 2

Effect of increasing concentrations of fenofibrate on PCSK9 and LDLR protein expression in HepG2 cells. Cells were grown in DMEM + 10% FBS and supplemented with increasing concentrations of fenofibrate for 24 hours at 37°C. 50 μg of total cell lysates (A and C) and 100 μl cell media (B) were analyzed by immunoblotting (IB), or immunoprecipitation (IP) followed by IB, respectively, as described in Methods. A and B were IB with anti-hPCSK9 Ab and C with anti-hLDLR Ab. The protein signals were quantified by densitometry using Syngene Chemigenius 2XE imager and Gene Tools software. All values were made relative to values from untreated cells set as 1 and presented as mean ± SEM (n = 3).

Figure 3

Relationship between percent change in plasma PCSK9 and LDL-cholesterol levels following statin treatment. PCSK9 and LDL-C were measured pre- and post-statin therapy as described in Methods. Pre-treatment levels were set as 100%.

Figure 4

Effect of increasing concentrations of simvastatin on PCSK9 and LDLR expression in HepG2 cells. HepG2 cells were grown in DMEM + 10% FBS and supplemented with increasing concentrations of simvastatin for 24 hours at 37°C. 50 μg of total cell lysates (A and C) and 100 μl cell media (B) were analyzed by immunoblotting (IB), or immunoprecipitation (IP) followed by IB, respectively, as described in Methods. A and B were IB with anti-hPCSK9 Ab and C with anti-hLDLR Ab. The protein signals were quantified by densitometry using Syngene Chemigenius 2XE imager and Gene Tools software. All values were made relative to values from untreated cells set as 1 and presented as mean ± SEM (n = 3). * Indicates significant differences from untreated control (p < 0.05) using student's T-test.