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Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19 ± 0.77-fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function., (© 2016. Published by The Company of Biologists Ltd.)

Aims/hypothesis: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a critical regulator of cholesterol homeostasis. PCSK9 inhibitors are being actively developed to lower LDL-cholesterol levels. However, there are conflicting data regarding the consequences of Pcsk9 deficiency on glucose homeostasis in mouse models. Here, we analysed in humans the association between the PCSK9 p.R46L loss-of-function (LOF) variant and (1) glucose homeostasis variables; (2) type 2 diabetes status; and (3) the risk of 9 year incident type 2 diabetes in a prospective study., Methods: PCSK9 p.R46L was genotyped in 4630 French participants from the Data from an Epidemiological Study on the Insulin Resistance Syndrome (DESIR) prospective study and in 1342 French participants with type 2 diabetes. The association between p.R46L and metabolic traits or type 2 diabetes risk was assessed through linear or logistic regression models adjusted for age, sex and BMI. The association between p.R46L and incident type 2 diabetes was assessed using a Cox regression model adjusted for sex, age and BMI at baseline., Results: Significant associations (p < 10(-6)) were found between p.R46L and lower total cholesterol (-0.394 mmol/l), LDL-cholesterol (-0.393 mmol/l) and apolipoprotein B concentrations (-0.099 g/l). However, no significant association was observed between p.R46L and markers of glucose homeostasis (including fasting glucose, fasting insulin, HbA1c, HOMA-B, HOMA-IR) or type 2 diabetes risk. Furthermore, no significant association between p.R46L variant and risk of incident type 2 diabetes was observed in DESIR., Conclusions/interpretation: The PCSK9 p.R46L LOF variant was not associated with impaired glucose homeostasis in humans. These data are reassuring regarding the safety of PCSK9 inhibitors.

Purpose of Review: Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9., Recent Findings: The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons., Summary: Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors.

Objective: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is highly expressed in the kidney, where its function remains unclear. In vitro data suggested that PCSK9 could impair the trafficking of the epithelial Na channel (ENaC). Here, we aimed at determining the consequences of PCSK9-deficiency on blood pressure, sodium balance and ENaC function in vivo in mice., Methods: Blood pressure was measured using non-invasive tail-cuff system or radiotelemetry under basal conditions in male and female PCSK9(+/+) and PCSK9(-/-) mice, as well as in models of hypertension: l-NAME (2 mg/kg/day), angiotensin II (1 mg/kg/day) and deoxycorticosterone acetate (DOCA)-salt in male mice only. Plasma and urine electrolytes (Na(+), K(+), Cl(-)) were collected under basal conditions, after DOCA-salt and amiloride treatment. Renal expression of ENaC subunits was assessed by western blotting., Results: PCSK9-deficiency did not alter both basal blood pressure and its increase in salt-insensitive (l-NAME) and salt-sensitive (Ang-II and DOCA-salt) hypertension models. Plasma PCSK9 concentrations were increased by 2.8 fold in DOCA-salt-induced hypertension. The relative expression of the cleaved, active, 30-kDa αENaC subunit was significantly increased by 32% in kidneys of PCSK9(-/-) mice under basal, but not under high-Na(+) diet or DOCA-salt conditions. Amiloride increased urinary Na(+) excretion to similar level in both genotypes, indicating that ENaC activity was not affected by PCSK9-deficiency., Conclusions: Despite an increase of cleaved αENaC under basal condition, PCSK9(-/-) mice display normal sodium balance and blood pressure regulation. Altogether, these data are reassuring regarding the development of PCSK9 inhibitors in hypercholesterolemia., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Objective: Transintestinal cholesterol excretion (TICE) is an alternate pathway to hepatobiliary secretion. Our study aimed at identifying molecular mechanisms of TICE., Approach and Results: We studied TICE ex vivo in mouse and human intestinal explants, and in vivo after bile diversion and intestinal cannulation in mice. We provide the first evidence that both low-density lipoprotein (LDL) and high-density lipoprotein deliver cholesterol for TICE in human and mouse jejunal explants at the basolateral side. Proprotein convertase subtilisin kexin type 9 (PCSK9)(-/-) mice and intestinal explants show increased LDL-TICE, and acute injection of PCSK9 decreases TICE in vivo, suggesting that PCSK9 is a repressor of TICE. The acute repression was dependent on the LDL receptor (LDLR). Further, TICE was increased when mice were treated with lovastatin. These data point to an important role for LDLR in TICE. However, LDLR(-/-) mice showed increased intestinal LDL uptake, contrary to what is observed in the liver, and tended to have higher TICE. We interpret these data to suggest that there might be at least 2 mechanisms contributing to TICE; 1 involving LDL receptors and other unidentified mechanisms. Acute modulation of LDLR affects TICE, but chronic deficiency is compensated for most likely by the upregulation of the unknown mechanisms. Using mice deficient for apical multidrug active transporter ATP-binding cassette transporter B1 a and b, and its inhibitor, we show that these apical transporters contribute significantly to TICE., Conclusions: TICE is operative in human jejunal explants. It is a metabolically active process that can be acutely regulated, inversely related to cholesterolemia, and pharmacologically activated by statins.

Elevated plasma triglyceride (TG) levels are an independent risk factor for cardiovascular disease (CVD). Proprotein convertase subtilisin-kexin 9 (PCSK9) – a protein therapeutically targeted to lower plasma cholesterol levels – might regulate plasma TG-rich lipoprotein (TRL) levels. We provide a timely and critical review of the current evidence for a role of PCSK9 in TRL metabolism by assessing the impact of PCSK9 gene variants, by reviewing recent clinical data with PCSK9 inhibitors, and by describing the potential mechanisms by which PCSK9 might regulate TRL metabolism. We conclude that the impact of PCSK9 on TRL metabolism is relatively modest, especially compared to its impact on cholesterol metabolism. [ABSTRACT FROM AUTHOR]

The role of PCSK9 in Alzheimer's disease (AD) is controversial. We compared cerebrospinal fluid (CSF) PCSK9 concentrations in 36 AD and 31 non-AD patients. CSF PCSK9 levels did not differ between AD and non-AD groups (2.80 versus 2.62 ng/mL). However, PCSK9 CSF levels were increased in AD and non-AD patients with other neurodegenerative process (non-AD ND, n = 20) compared to patients without neurodegenerative disorders (non-ND, n = 11): 2.80 versus 2.30 (p < 0.005) and 2.83 versus 2.30 ng/mL (p = NS), respectively. CSF PCSK9 were positively correlated with AD biomarkers (Aβ1-42, T-tau, and P-tau). PCSK9 concentrations in CSF are increased in neurodegenerative disorders rather than specifically in AD. [ABSTRACT FROM AUTHOR]

Abstract Background and aims Some studies suggested that proprotein convertase subtilisin kexin type 9 (PCSK9) is linked to liver steatosis severity and non-alcoholic steatohepatitis (NASH). We aimed to assess whether circulating PCSK9 levels are associated with either liver fat content (LFC) or histological markers of NASH in high-risk patients. Methods We present results from three cross-sectional studies from two French Hospitals: Dijon and Numevox (departments of Endocrinology) and Angers (department of Hepatology). Only patients without lipid-lowering therapy were included. All 132 patients had type 2 diabetes in Dijon, compared to 55/224 in Numevox (25%) and 39/122 in Angers (32%). LFC was assessed on MRI (Dijon and Numevox), and NASH lesion on liver biopsy (Angers). Additionally, we included mRNA results from 138 overweight patients of a Belgian Hospital (Antwerp). Results While circulating levels of PCSK9 were positively correlated with total cholesterol, LDL-C, triglycerides and non-HDL-C in all 3 cohorts, no significant association was found between PCSK9 and transaminases. Furthermore, no association was found between plasma PCSK9 levels and LFC in both Numevox (β adjusted = 0.71 ± 1.33, p = 0.60) and Dijon (β adjusted = −1.03 ± 0.90, p = 0.25). There was no correlation between circulating PCSK9 and histological liver lesions: steatosis severity (β adjusted = −3.95 ± 2.75, p = 0.15), NASH activity score (β adjusted = −0.31 ± 0.17, p = 0.082), lobular (β = −0.067 ± 0.055, p = 0.22) or portal inflammation (β = −0.088 ± 0.079, p = 0.27), ballooning (β = −0.025 ± 0.065, p = 0.70) and fibrosis (β = −0.17 ± 0.11, p = 0.12). Finally, hepatic PCSK9 mRNA levels were not correlated with NASH histological severity. Conclusions Circulating PCSK9 concentrations are not associated with the severity of liver steatosis or histological markers of NASH. These data are reassuring regarding the clinical use of PCSK9 inhibitors in cardiovascular diseases. Highlights • PCSK9 is a master regulator of cholesterol metabolism. • The link between PCSK9 and liver steatosis is unclear. • Plasma PCSK9 levels are not associated with steatosis severity assessed by MRI. • Hepatic PCSK9 expression is not correlated with severity of NASH. • These results are reassuring regarding the clinical use of PCSK9 inhibitors. [ABSTRACT FROM AUTHOR]