Inhibition of PCSK9 does not improve lipopolysaccharide-induced mortality in mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that targets LDL receptors (LDLRs) for degradation in liver. Blocking the interaction of PCSK9 with the LDLR potently reduces plasma LDL cholesterol levels and cardiovascular events. Recently, it has been suggested that inhibition of PCSK9 might also improve outcomes in mice and humans with sepsis, possibly by increasing LDLR-mediated clearance of endotoxins. Sepsis is a complication of a severe microbial infection that has shared pathways with lipid metabolism. Here, we tested whether anti-PCSK9 antibodies prevent death from lipopolysaccharide (LPS)-induced endotoxemia. Mice were administered PCSK9 antibodies prior to, or shortly after, injecting LPS. In both scenarios, the administration of PCSK9 antibodies did not alter endotoxemia-induced mortality. Afterward, we determined whether the complete absence of PCSK9 improved endotoxemia-induced mortality in mice with the germ-line deletion of Pcsk9 Similarly, PCSK9 knockout mice were not protected from LPS-induced death. To determine whether low LDLR expression increased LPS-induced mortality, Ldlr-/- mice and PCSK9 transgenic mice were studied after injection of LPS. Endotoxemia-induced mortality was not altered in either mouse model. In a human cohort, we observed no correlation between plasma inflammation markers with total cholesterol levels, LDL cholesterol, and PCSK9. Combined, our data demonstrate that PCSK9 inhibition provides no protection from LPS-induced mortality in mice.

Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1.

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.

Increased Hepatic Lipogenesis Elevates Liver Cholesterol Content.

Cardiovascular diseases (CVDs) are the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD) and dyslipidemia is considered at least partially responsible for the increased CVD risk in NAFLD patients. The aim of the present study is to understand how hepatic de novo lipogenesis influences hepatic cholesterol content as well as its effects on the plasma lipid levels. Hepatic lipogenesis was induced in mice by feeding a fat-free/high-sucrose (FF/HS) diet and the metabolic pathways associated with cholesterol were then analyzed. Both liver triglyceride and cholesterol contents were significantly increased in mice fed an FF/HS diet. Activation of fatty acid synthesis driven by the activation of sterol regulatory element binding protein (SREBP)-1c resulted in the increased liver triglycerides. The augmented cholesterol content in the liver could not be explained by an increased cholesterol synthesis, which was decreased by the FF/HS diet. HMGCoA reductase protein level was decreased in mice fed an FF/HS diet. We found that the liver retained more cholesterol through a reduced excretion of bile acids, a reduced fecal cholesterol excretion, and an increased cholesterol uptake from plasma lipoproteins. Very low-density lipoproteintriglyceride and -cholesterol secretion were increased in mice fed an FF/HS diet, which led to hypertriglyceridemia and hypercholesterolemia in Ldlr-/- mice, a model that exhibits a more human like lipoprotein profile. These findings suggest that dietary cholesterol intake and cholesterol synthesis rates cannot only explain the hypercholesterolemia associated with NAFLD, and that the control of fatty acid synthesis should be considered for the management of dyslipidemia.

PCSK9-deficiency does not alter blood pressure and sodium balance in mouse models of hypertension.

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.