Plasma Proprotein Convertase Subtilisin/kexin Type 9 Concentrations Research Articles

Maternal Obesity Modulates Cord Blood Concentrations of Proprotein Convertase Subtilisin/Kexin-type 9 Levels.

In utero exposure to maternal obesity or diabetes is considered a pro-inflammatory state. To evaluate whether cord blood proprotein convertase subtilisin/kexin-type 9 (PCSK9), which is regulated by inflammation and metabolic derangements, is elevated in neonates born to overweight, obese, or diabetic mothers. A retrospective study in full-term neonates born between 2010 and 2023, at Brigham and Women's Hospital. There were 116 neonates included in our study, of which 74 (64%) were born to overweight/obese mothers and 42 (36%) were born to nonoverweight/nonobese mothers. Neonates born to overweight/obese mothers had significantly higher cord blood concentrations of PCSK9 compared with neonates born to nonoverweight/nonobese group (323 [253-442] ng/mL compared with 270 [244-382] ng/mL, P = .041). We found no significant difference in cord blood concentrations of PCSK9 between neonates of diabetic mothers compared with neonates of nondiabetic mothers. In multivariate linear regression analysis, higher cord plasma PCSK9 concentration was significantly associated with maternal overweight/obesity status (b = 50.12; 95% CI, 4.02-96.22; P = .033), after adjusting for gestational age, birth weight, male sex, and intrauterine growth restriction. Neonates born to mothers with overweight/obesity have higher cord blood PCSK9 concentrations compared with the nonoverweight/nonobese group, and higher cord blood PCSK9 concentrations were significantly associated with maternal overweight/obesity status, after adjusting for perinatal factors. Larger longitudinal studies are needed to examine the role of PCSK9 in the development of metabolic syndrome in high-risk neonates born to overweight, obese, or diabetic mothers.

Association of circulating proprotein convertase subtilisin/kexin type 9 concentration with coagulation abnormalities in patients with primary membranous nephropathy

Objectives The aims of the study were to explore the potential associations between plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) and coagulation indexes in patients with primary membranous nephropathy (PMN). Methods A total of 87 patients diagnosed with PMN were enrolled in our study. 30 healthy participants were recruited to match PMN participants. Plasma PCSK9 concentrations were tested by enzyme-linked immunosorbent assay (ELISA). Correlations between PCSK9 and coagulation abnormalities in patients with PMN were analyzed using univariate and multiple linear regression analysis. Results Plasma PCSK9 levels in patients with PMN were significantly higher than that in healthy controls [232.0 (143.5, 359.5) ng/mL vs. 166.8 (129.7, 199.7) ng/mL; p = 0.001]. Plasma levels of PCSK9 were positively correlated with factor VIII, factor IX, factor XI, log-transformed tissue factor, protein C and protein S (r = 0.267, p = 0.013; r = 0.496, p < 0.001; r = 0.217, p = 0.045; r = 0.584, p < 0.001; r = 0.372, p = 0.001; r = 0.282, p = 0.011). In multiple linear regression analysis, PCSK9 concentration was independently and positively correlated with factor VIII, factor IX, and tissue factor (β = 0.186, p = 0.047; β = 0.325, p = 0.001; β = 0.531, p < 0.001; respectively). PCSK9 concentration was independently and negatively correlated with PT (β= −0.343, p = 0.011). Conclusion Plasma PCSK9 levels had good positive correlations with procoagulant clotting factors and negative correlations with PT in PMN, which might provide novel information with regard to PCSK9 and hypercoagulability in PMN.

New Insights Into the Regulation of Lipoprotein Metabolism by PCSK9: Lessons From Stable Isotope Tracer Studies in Human Subjects.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a convertase enzyme mostly produced by the liver. It is a key regulator of LDL metabolism because of its ability to enhance degradation of the LDL receptor. PCSK9 also regulates the metabolism of lipoprotein(a) [Lp(a)] and triglyceride-rich lipoproteins (TRLs). Its key role in modulating atherosclerotic cardiovascular disease (ASCVD) is supported by genetic studies and clinical outcome trials. Kinetic studies provide mechanistic insight into the role of PCSK9 in regulating the physiology and pathophysiology of plasma lipids and lipoproteins. Kinetic data have demonstrated that plasma PCSK9 concentration is inversely associated with the clearance of LDL in men. Gain-of-function mutations of PCSK9 markedly increase plasma LDL-cholesterol concentrations due to impaired LDL-apoB catabolism. Conversely, PCSK9 deficiency results in low LDL-cholesterol associated with enhanced LDL-apoB clearance. Inhibition of PCSK9 with monoclonal antibodies (such as evolocumab or alirocumab) lowers plasma LDL-cholesterol and apoB levels chiefly by upregulating the catabolism of LDL particles in healthy individuals. As monotherapy, PCSK9 inhibitor reduced Lp(a) concentrations by decreasing the production rate. However, as combination therapy, it reduced the plasma concentration of Lp(a) by increasing the fractional catabolism of Lp(a) particles. In statin-treated patients with high Lp(a), PCSK9 inhibition lowers plasma Lp(a) concentrations by accelerating the catabolism of Lp(a) particles. The effect of PCSK9 inhibition on TRL metabolism has been studied in healthy individuals and in patients with type 2 diabetes. These findings suggest that PCSK9 appears to play a less important role in TRL than LDL metabolism. Kinetic studies of PCSK9 inhibition therapy on lipoprotein metabolism in diverse high risk patient populations (such as familial hypercholesterolemia) and new therapeutic combination also merit further investigation.

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts.

BackgroundThe proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves’ orbitopathy (GO) and whether it may be a legitimate target for treatment.MethodsThe PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1β (IL-1β)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1β-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA.ResultsThe PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1β-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS).ConclusionsPCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Association of circulating PCSK9 concentration with cardiovascular metabolic markers and outcomes in stable coronary artery disease patients with or without diabetes: a prospective, observational cohort study

BackgroundWhether plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels is a predictor for cardiovascular outcomes has currently been controversial. No data is currently available regarding the relation of PCSK9 to cardiovascular metabolic markers (CVMMs) and major adverse cardiovascular events (MACEs) in stable coronary artery disease (CAD) patients with diabetes or without diabetes.MethodsA total 1225 untreated patients with stable CAD were consecutively enrolled and their baseline plasma PCSK9 levels were determined by ELISA. Patients were divided into high and low PCSK9 groups according to PCSK9 median. All patients followed up for the occurrence of MACEs and received standard therapy after admission. The associations of PCSK9 with CVMMs and MACEs were evaluated.ResultsPCSK9 levels were positively correlated with multiple CVMMs including total cholesterol, low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol and hemoglobin A1c at baseline (all p < 0.05). During a median follow-up of 3.3 years, 103 (8.4%) events occurred. PCSK9 levels were higher in patients with events compared to those without (p < 0.05). The Kaplan–Meier analysis displayed that patients in high PCSK9 group had lower event-free survival than that in low group (p < 0.05). Multivariable Cox regression analysis revealed that PCSK9 levels were independently associated with MACEs in diabetic patients (adjusted hazard ratio [HR]: 1.361, 95% confidence interval [CI]: 1.037–1.785, p < 0.05). When added the combination of PCSK9 levels and diabetic status to stratifying factors, patients in high PCSK9 group appeared to have extremely high risk of subsequent MACEs with diabetes (adjusted HR: 5.233, 95% CI: 2.546–10.757, p < 0.01).ConclusionsThe present study firstly showed that elevated PCSK9 levels were related to multiple CVMMs and MACEs in stable CAD with diabetes, suggesting that plasma PCSK9 measurement could help to identify diabetic patients with CAD at higher cardiovascular risk. More studies may be needed to confirm our findings.

Does 24-h Activity Cycle Influence Plasma PCSK9 Concentration? A Systematic Review and Meta-Analysis.

Higher plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) concentration has been associated with a higher risk of atherosclerotic cardiovascular disease (ASCVD). Animal and human studies have examined the relationship between 24-h activity cycles (24-HAC) and PCSK9, but conflicting results exist. Therefore, this review aimed to examine the relationship between 24-HAC and plasma PCSK9 concentration in animals and humans.Three databases (PubMed, CINAHL, and Web of Science) were searched for eligible articles. Descriptive data were summarized using network meta-analysis. The effect size was estimated using pairwise meta-analysis. The interventions designed to increase moderate to vigorous physical activities (MVPA) did not significantly change plasma PCSK9 concentration (Hedges' g = 0.137; p = 0.337). However, the effect was influenced by statin therapy and intervention delivery mode. Specifically, physical activity interventions in conjunction with statin therapy significantly increased plasma PCSK9 concentration (Hedges' g = 0.275; p = 0.007). Supervised exercise training significantly increased plasma PCSK9 concentration (Hedges' g = 0.630; p = 0.001), but physical activity counseling did not (p = 0.845). The effects of MVPA on plasma PCSK9 may be moderated by statin therapy, intervention delivery mode, or other potential unknown mechanistic factors. Thus, caution should be taken when using plasma PCSK9 as an outcome indicator for physical activity interventions aimed at decreasing the risk of ASCVD. Graphical abstract.

Changes in PCSK9 and LDL cholesterol concentrations by everolimus treatment and their effects on polymorphisms in PCSK9 and mTORC1.

The purpose of this study was to evaluate the effects of concentrations of proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein (LDL) cholesterol by the mammalian target of rapamycin (mTOR) inhibitor everolimus and their effects on genetic polymorphisms in the PCSK9 and mTORC1 genes in 53 renal transplant recipients. Prior to and on day 15 after everolimus administration, the concentrations of everolimus in blood and PCSK9 and LDL cholesterol in plasma were evaluated. Additionally, mTORC1 (rs2536T>C and rs2295080T>G) and PCSK9 (rs505151G>A, rs562556G>A, and rs11593680C>T) polymorphisms were analyzed. Mean PCSK9 plasma concentrations on day 15 after everolimus treatment were significantly higher than those before treatment (295 versus 214ng/mL, respectively; p = 0.004). Significant correlations between the area under the blood concentration-time curves (AUC)0-12 on day 15 of everolimus treatment and the change rate in PCSK9 concentrations were found (r = 0.316, p = 0.021). However, there were no significant correlations between the change rate in PCSK9 and LDL cholesterol concentrations. The change rate in PCSK9 concentrations by everolimus treatment was significantly greater in patients with the mTORC1 rs2295080G allele than the T/T genotype (p = 0.006); however, there were no significant differences between PCSK9 rs505151G>A and rs11583680C>T genotypes. In multivariate analyses, patients with mTORC1 rs2295080G (p = 0.010), higher everolimus AUC0-12 (p = 0.006), and female sex (p = 0.029) showed higher change rates of PCSK9 following everolimus therapy. Administration of everolimus significantly elevated plasma PCSK9 concentrations, potentially causing everolimus-induced hyperlipidemia.

PCSK9 and atherosclerosis burden in the coronary arteries of patients undergoing coronary angiography

AimsTo investigate the association between plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) concentrations, current acute coronary syndrome (ACS), coronary artery disease (CAD) presence, severity and extension and the burden of coronary calcifications in patients with suspected CAD. Methods and resultsOne hundred and one patients, with or without current ACS, were recruited for this cross-sectional study. CAD presence was defined based on either the presence or absence of at least one significant (≥50%) CAD lesion (SCAD). CAD severity was classified according to the absence of coronary lesions, the presence of non-significant (<50%) CAD (MCAD) or SCAD in at least one major coronary artery. Patients with one, two or three significantly diseased major coronary arteries were defined as 1-SCAD, 2-SCAD and 3-SCAD, respectively. The cumulative length of SCAD lesions and the amount of calcifications in coronary arteries were estimated. Plasma PCSK9 concentrations were higher in patients with SCAD as compared to those without (p = .012). A significant increase in plasma PCSK9 concentrations was observed with greater CAD severity (p = .042). Higher plasma PCSK9 concentrations were found in 3-SCAD patients as compared to either 2-SCAD or 1-SCAD (p < .001). PCSK9 increased with the cumulative length of SCAD lesions and the burden of calcifications (p < .05 for both comparisons). Multivariable adjustment abolished the association between PCSK9 and either CAD presence or severity, but not the association between PCSK9 and the number of significantly diseased vessels, SCAD lesion length and the burden of coronary calcifications. ACS was associated with a borderline significant increase of plasma PCSK9 concentrations among patients not taking statins (p = .05). ConclusionCirculating PCSK9 concentrations discriminate patients with greater coronary atherosclerotic lesion extension and calcification, and are increased in patients with current ACS.

Plasma PCSK9 correlates with apoB-48-containing triglyceride-rich lipoprotein production in men with insulin resistance

Intestinal triglyceride (TG)-rich lipoproteins (TRLs) are important in the pathogenesis of atherosclerosis in insulin resistance (IR). We investigated the association of plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) concentrations with apoB-48-containing TRL metabolism in 148 men displaying various degrees of IR by measuring in vivo kinetics of TRL apoB-48 during a constant-fed state after a primed-constant infusion of L-[5,5,5-D3]leucine. Plasma PCSK9 concentrations positively correlated with TRL apoB-48 pool size (r = 0.31, P = 0.0002) and production rate (r = 0.24, P = 0.008) but not the fractional catabolic rate (r = -0.04, P = 0.6). Backward stepwise multiple linear regression analysis identified PCSK9 concentrations as a positive predictor of TRL apoB-48 production rate (standard β = +0.20, P = 0.007) independent of BMI, age, T2D/metformin use, dietary fat intake during the kinetic study, and fasting concentrations of TGs, insulin, glucose, LDL cholesterol, or C-reactive protein. We also assessed intestinal expression of key genes involved in chylomicron processing from duodenal samples of 71 men. Expression of PCSK9 and HMG-CoAR genes was positively associated (r = 0.43, P = 0.002). These results support PCSK9 association with intestinal secretion and plasma overaccumulation of TRL apoB-48 in men with IR.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Plasma proprotein convertase subtilisin/Kexin type 9 (PCSK9) is associated with Lp(a) in non-CAD type 2 diabetic patients

Type 2 diabetes mellitus (T2DM) is associated with elevated plasma apolipoprotein B, Lp(a), triglycerides and reduced HDL-C. The present study was conducted to investigate whether plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of LDL-receptor (LDLr) expression in hepatocytes, was associated with diabetic dyslipidemia. We developed a sandwich ELISA using recombinant human PCSK9 protein and a polyclonal antibody pre-coated onto a microplate specific directed against human PCSK9. We measured circulating PCSK9 concentrations in a cohort of patients with T2DM ( n = 50) compared to an age- and sex-matched control group ( n = 50). Plasma apolipoprotein B, Lp(a) triglycerides, LDL-C, HDL-C and LDL-r were measured in both groups. Plasma PCSK9 levels were significantly elevated in T2DM compared to controls (44.61 ± 14.44 and 33.22 ± 11.79 ng/mL respectively, P < 0.0001). However, LDL-r levels did not differ between the two groups. No significant correlation of PCSK9 with lipid parameters was found. Remarkably PCSK9 was positively correlated with Lp(a) in whole population ( r = +0.254, P < 0.0138) and T2DM group ( r = +0287, P = 0.0474) but not in controls. Lp(a) levels has been proposed as a contributing factor to the accelerated development of macrovascular complications in T2DM and the synergic effect with PCSK9 may explain the enhanced atherogenicity in type 2 diabetic patients.

Effect of cilostazol on plasma levels of proprotein convertase subtilisin/kexin type 9.

The protein complex proprotein convertase subtilisin/kexin type 9 (PCSK9) serves as an important target for the prevention and treatment of atherosclerosis and lipid homeostasis. This study investigated the effect of cilostazol on plasma PCSK9 concentrations. We performed a post hoc analysis of two prospective, double-blind, randomized controlled trials including 115 patients of whom 61 received cilostazol 200 mg/day and 54 received placebo for 12 weeks. Linear regression analysis was performed to determine the associations between several parameters and changes in PCSK9 levels. Use of cilostazol, but not placebo, significantly increased plasma PCSK9 concentrations, high-density lipoprotein cholesterol levels, and number of circulating endothelial progenitor cells (EPCs), and decreased triglyceride levels with a trend toward an increase in total cholesterol (TC) levels. A reduction in hemoglobin A1C and an increase in plasma vascular endothelial growth factor and adiponectin levels with cilostazol treatment were also found. Changes in the number of circulating EPCs were positively correlated and the TC concentrations were inversely correlated with changes in the PCSK9 levels. After adjusting for changes in levels of TC and numbers of circulating EPCs and history of metabolic syndrome, use of cilostazol remained independently associated with changes in plasma PCSK9 levels. In conclusion, cilostazol treatment was significantly and independently associated with an increase in plasma PCSK9 levels in patients with peripheral artery disease or at a high risk of cardiovascular disease regardless of background statin use and caused an improvement in some metabolic disorders and levels of vasculo-angiogenic biomarkers.

Association Between Plasma Proprotein Convertase Subtilisin/Kexin Type 9 and the Presence of Metabolic Syndrome in a Predominantly Rural-Based Sub-Saharan African Population.

The prevalence of metabolic syndrome (MetS) has increased dramatically in low- and middle-income countries. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in low-density lipoprotein receptor degradation, but its relationship with metabolic parameters is still poorly understood. We aimed to investigate the association between plasma PCSK9 and metabolic parameters in a Kenyan cohort. Total plasma PCSK9 levels were measured in 2016 by an in-house enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody. The International Diabetes Federation (IDF) 2009 consensus statement criteria were used to assess the presence of MetS. In 1338 Kenyans, 11% of the cohort had ≥3 MetS criteria. Total plasma PCSK9 concentration was significantly higher in subjects with MetS than in the non-MetS group (166.8 ± 4.4 vs. 148.0 ± 1.3, P < 0.0001). A progressive increase in circulating PCSK9 was observed when subjects were stratified according to the number of MetS criteria (<3, 3, 4, or 5) [P of the analysis of variance (ANOVA) <0.0001]. In a model corrected for age, sex, lifestyle factors, and body mass index, PCSK9 concentration was a significant predictor of all MetS criteria taken individually, except for waist circumference. Plasma PCSK9 levels were significantly associated with low-density lipoprotein cholesterol, but the strongest association was seen with triglycerides even after multiple adjustments. The presence of MetS was significantly associated with the PCSK9 concentration. Further studies are needed to provide a molecular connection between PCSK9 and insulin, as well as triglyceride metabolism.

Association between plasma proprotein convertase subtisilin/kexin type 9 concentration and coronary artery calcification.

Background Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to be related to several risk factors and diseases such as inflammatory markers and coronary artery disease. The aim of present study was to investigate whether plasma PCSK9 concentration was associated with coronary artery calcification. Methods A total of 403 consecutive untreated patients with angina-like chest pain, who received electron beam computed tomography, were enrolled and a coronary artery calcification score (CACS) was also measured. The baseline clinical characteristics were collected and blood sample was taken after 12-h fasting. The plasma PCSK9 concentrations were determined by ELISA in all patients, and the relationship between plasma PCSK9 concentrations and CACS was investigated. Results Patients with coronary artery calcification (CACS > 0) had significant higher plasma PCSK9 concentrations compared with those (CACS = 0) without coronary artery calcification (258.58 ± 69.53 ng/mL vs. 202.53 ± 52.17 ng/mL, P < 0.001). Patients with highest PCSK9 concentrations had the highest CACS. Multivariable linear regression analysis suggested that PCSK9 was independently associated with coronary artery calcification ( P = 0.002) after adjusting for traditional cardiovascular risk factors. Furthermore, the area under the curve for the plasma PCSK9 concentration in predicting coronary artery calcification was 0.736 (95% CI: 0.687-0.785, P < 0.001), with a sensitivity of 66% and specificity of 70%. Conclusion A positive association between plasma PCSK9 concentration and coronary artery calcification in untreated patients with angina-like chest pain was observed in our study, suggesting that further investigation may be needed in order to confirm our primary findings and explore the clinical implications.

Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA.

The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.

Plasma PCSK9 concentrations during the course of nondiabetic chronic kidney disease: Relationship with glomerular filtration rate and lipid metabolism

The association between proprotein convertase subtilisin/kexin type 9 (PCSK9), a critical regulator of low-density lipoprotein (LDL) metabolism, and kidney function is a matter of debate. We aimed to assess the association of circulating PCSK9 concentrations with both glomerular filtration rate (eGFR) and serum lipid parameters in nondiabetic patients with chronic kidney disease (CKD). Fasting plasma PCSK9 concentrations were measured by ELISA in 94 nondiabetic nondialysis CKD (ND-CKD) patients not receiving statins, at different stages of CKD. Plasma PCSK9 levels were associated neither to eGFR (P=.770) nor to proteinuria (P=.888) at several stages of CKD. In addition, plasma PCSK9 levels did not vary significantly between the different CKD stages. Plasma PCSK9 concentrations were positively correlated with apolipoprotein B (r=0.221; P=.03) and triglycerides (r=0.211; P=.04) but not with total cholesterol, calculated LDL-cholesterol, HDL cholesterol, lipoprotein(a), or CRP. In a homogeneous population of nondiabetic subjects without lipid-lowering therapy, plasma PCSK9 concentrations are not associated to eGFR at several stages of CKD. These data suggest that kidney function per se does not impact significantly PCSK9 metabolism.

Predicting proprotein convertase subtilisin kexin type-9 loss of function mutations using plasma PCSK9 concentration

Low plasma proprotein convertase subtilisin kexin type-9 (PCSK9) concentration has been associated with loss of function (LOF) PCSK9 mutations in several studies. However, the current standard for detection of these LOF mutations is through gene sequencing. Gene sequencing is labor intensive and expensive. Identifying a simple test to help predict PCSK9 LOF mutations would help to better target subjects requiring gene sequencing. Determine the diagnostic accuracy of plasma PCSK9 concentration in detecting PCSK9 LOF mutations using genotyping as the gold standard. We carried out a retrospective analysis of 1412 French-Canadian participants of the Quebec Child and Adolescent Health and Social Survey who had been screened for the PCSK9 R46L and Leucine insertion (InsLEU) LOF mutations by genotyping. Their plasma PCSK9 concentrations were measured using a well-described enzyme-linked immunosorbent assay. We used the Youden index to determine the optimal cutoff for PCSK9 concentration to predict mutation status. We further investigated the use of low-density lipoprotein cholesterol (LDL-C) concentration in combination with plasma PCSK9 concentration to refine the prediction of mutation status. Plasma PCSK9 had a moderate accuracy (area under the curve, 0.71) in detecting the PCSK9 R46L mutation with a sensitivity of 71% and specificity of 70% at cutoff of 70ng/mL. Combining PCSK9 and LDL-C increased the diagnostic accuracy for the detection of the R46L mutation (area under the curve, 0.75). However, plasma PCSK9 concentration was no better than chance at detecting PCSK9 InsLEU mutation. This analysis suggests that plasma PCSK9 combined with LDL-C concentrations might be useful to predict certain PCSK9 LOF mutations such as the R46L mutation but may fail to predict others such as the InsLEU mutation.

Impact of a 1-year lifestyle modification program on plasma lipoprotein and PCSK9 concentrations in patients with coronary artery disease

Patients with coronary artery disease (CAD) are characterized by an impaired cardiometabolic risk profile including high levels of atherogenic apolipoprotein (apo) B-containing lipoprotein levels. Genetic studies have highlighted a critical role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in lipoprotein metabolism and CAD risk. To determine whether improving dietary quality and increasing physical activity levels improve parameters of the cardiometabolic risk profile such as plasma apoB and PCSK9 levels in patients with CAD. We recruited 86 men aged between 39 and 80years (82 of them on statins) undergoing coronary artery bypass graft (CABG) surgery. These patients participated in a 1-year lifestyle modification program aiming at achieving a minimum of 150minutes/week of physical activity and improving diet quality by following dietary guidelines. We used magnetic resonance imaging to measure visceral adipose tissue and a modified Bruce protocol to measure fitness levels before and after the intervention. Plasma apoB and low-density lipoprotein cholesterol levels were not modified by the intervention (-3.0%, P=.08 and 1.3%, P=.56, respectively), whereas non-HDL cholesterol decreased by 4.5% (P=.04) and triglycerides by 13% (P=.002). In contrast, PCSK9 levels increased by 5.2% after the intervention (P=.05). HDL cholesterol and apolipoprotein A-I levels also increased (+12%, P<.0001 and+6%, P<.0001, respectively). PCSK9 levels increased with improvements in fitness (r=0.23, P=.04) and visceral fat mobilization (r=-0.23, P=.04). In post-CABG patients, a lifestyle modification program lead to significant improvements in some parameters of the lipoprotein profile but unexpectedly increased plasma PCSK9 levels.

Dietary high oleic canola oil supplemented with docosahexaenoic acid attenuates plasma proprotein convertase subtilisin kexin type 9 (PCSK9) levels in participants with cardiovascular disease risk: A randomized control trial

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a novel circulating protein which plays an important role in regulation of cholesterol metabolism by promoting hepatic LDL receptor degradation. However, the action of dietary fat composition on PCSK9 levels remains to be fully elucidated. The objective was to investigate the action of different dietary oils on circulating PCSK9 levels in the Canola Oil Multicenter Intervention Trial (COMIT). COMIT employed a double-blinded crossover randomized control design, consisting of five 30-d treatment periods. Diets were provided based on a 3000Kcal/d intake, including a 60g/d treatment of conventional canola oil (Canola), a high oleic canola/DHA oil blend (CanolaDHA), a corn/safflower oil blend (CornSaff), a flax/safflower oil blend (FlaxSaff) or a high oleic canola oil (CanolaOleic). Plasma PCSK9 levels were assessed using ELISA at the end of each phase. Lipid profiles (n=84) showed that CanolaDHA feeding resulted in the highest (P<0.05) serum total cholesterol (TC, 5.06±0.09mmol/L) and LDL-cholesterol levels (3.15±0.08mmol/L) across all five treatments. CanolaDHA feeding also produced the lowest (P<0.05) plasma PCSK9 concentrations (216.42±8.77ng/mL) compared to other dietary oil treatments. Plasma PCSK9 levels positively correlated (P<0.05) with serum TC, LDL-cholesterol, apolipoprotein A, and apolipoprotein B levels but did not correlate to HDL-cholesterol levels. Results indicate that post-treatment response in PCSK9 may be altered with the CanolaDHA diet. In conclusion, the elevated LDL-C levels from a DHA oil treatment may not be relevant for the observed decline in PCSK9 levels.

Docosahexaenoic Acid Attenuates Cardiovascular Risk Factors via a Decline in Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Plasma Levels.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that regulates cholesterol metabolism by promoting LDL receptor degradation in the liver and has recently been proposed as a therapeutic target in the management of hyperlipidaemia. We investigated the impact of dietary fat on the metabolism of sterols and on plasma PCSK9 concentrations to explore likely clinical usefulness. In a post hoc analysis of a double-blind randomised crossover controlled feeding trial, the Canola Oil Multicenter Intervention Trial (COMIT), volunteers (n = 54) with at least one condition related to metabolic syndrome consumed diets with one of the following treatment oils in beverages: (1) conventional canola oil (Canola); (2) canola oil rich in docosahexanoic acid (DHA) (CanolaDHA); and (3) high-oleic acid canola oil (CanolaOleic). The enrichment in oleic acid resulted in lower plasma cholesterol concentration compared with diets enriched in DHA. Contrarily, DHA-enriched oil significantly decreased plasma PCSK9 and triacylglycerols levels, but increased circulating levels of sterols. The variations in lathosterol, sitosterol, and campesterol indicate that plasma PCSK9 levels are sensitive to changes in cholesterol synthesis and/or absorption. There was a significant correlation between plasma PCSK9 levels and plasma triacylglicerol and apolipoprotein B levels, which was not affected by dietary fat. Therefore, our results suggest that the impact of dietary fats should not be discarded as complementary treatment in the management of patients with hyperlipidaemia. These findings should be considered in the analysis of ongoing studies and may represent a cautionary note in the treatment of patients with cardiovascular risk.