Hepatic caveolin-1 is enhanced in Cyp27a1/ApoE double knockout mice

Abstract

Sterol 27-hydroxylase (CYP27A1) is involved in bile acid synthesis and cholesterol homoeostasis. Cyp27a1^(-/-)/Apolipoprotein E^(-/-) double knockout mice (DKO) fed a western diet failed to develop atherosclerosis. Caveolin-1 (CAV-1), the main component of caveolae, is associated with lipid homoeostasis and has regulatory roles in vascular diseases. We hypothesized that liver CAV-1 would contribute to the athero-protective mechanism in DKO mice. Cyp27a1^(+/+)/ApoE^(-/-) (ApoE KO), Cyp27a1^(+/-)/ApoE^(-/-) (het), and DKO mice were fed a western diet for 2 months. Atherosclerotic plaque and CAV-1 protein were quantified in aortas. Hepatic Cav-1 mRNA was assessed using qPCR, CAV-1 protein by immunohistochemistry and western blotting. Total hepatic and plasma cholesterol was measured using chemiluminescence. Cholesterol efflux was performed in RAW264.7 cells, using mice plasma as acceptor. CAV-1 protein expression in aortas was increased in endothelial cells of DKO mice and negatively correlated with plaque surface (P < 0.05). In the liver, both CAV-1 protein and mRNA expression doubled in DKO, compared to ApoE KO and het mice (P < 0.001 for both) and was negatively correlated with total hepatic cholesterol (P < 0.05). Plasma from DKO, ApoE KO and het mice had the same efflux capacity. In the absence of CYP27A1, CAV-1 overexpression might have an additional athero-protective role by partly overcoming the defect in CYP27A1-mediated cholesterol efflux.

Keywords: atherosclerosis; cholesterol efflux; cholesterol metabolism; liver.

Figure 1

CAV‐1 protein expression in mice aorta and heart with and without atherosclerotic plaques. Aorta (A, D, G), atrium (B, E, H) and ventricles (C, F, I) sections were stained for CAV‐1 by IHC. (A) Almost no positive staining was observed in vascular cells making up plaques. In ApoE KO mice, CAV‐1 expression, which is localized around vessels in both the (B) atrium and (C) ventricles (black arrows), is increased. In DKO mice, higher CAV‐1 expression was seen in (D) endothelial cells (black arrows), with low expression in both (E) atrium and (F) ventricles. (G–I) are negative controls; 400× magnification.

Figure 2

Genotype‐induced effect on expression of CAV‐1 in the liver. (A) Cav‐1 mRNA expression was quantified by real‐time PCR, using β Actin as internal standard. CAV‐1 protein was quantified by WB (B) and IHC (C). Data are shown as mean ± SD. For statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Genotype‐induced effect on expression of CAV‐1 in the liver. Representative WB (A) and IHC (B) of CAV‐1 in liver. CAV‐1 was detected as a 22‐KDa protein. The IHC staining localized to sinusoids (black arrows) and around micro/macrovesicular steatosis (black arrowheads); 400× magnification; scale bar = 20 μm.

Figure 4

Beneficial effects of hepatic CAV‐1 on atherosclerosis protection in DKO mice. Atherosclerosis is mainly driven by high circulating cholesterol concentration, low HDL‐C and high LDL‐C. In plasma of DKO mice fed WD, the low levels of LDL‐C can be attributed to increased SREBP2 in the liver, which increases LDLR expression and LDL‐C uptake. CAV‐1 is also associated with the LDLR and the process of influx of LDL from the circulation to the liver. In DKO mice, SHP is downregulated, suppressing the inhibitory effect on SREBP1c and CYP7A1 as well as increasing TG and BA biosynthesis. The increased hepatic CAV‐1 expression appears to provide additional beneficial effects in promoting hepatic TG accumulation via increased DGAT expression and increasing hepatic gene expression of lipogenic markers (ACC and FAS). Pparγ was also increased in DKO mice and correlated positively with Cav‐1 mRNA expression, supporting its role in upregulating CAV‐1. The lower TNFα, in combination with higher CAV‐1 expression in the liver further supports the potential anti‐inflammatory role for CAV‐1. In the absence of 27‐OHC in DKO mice, the rate‐limiting enzyme of cholesterol biosynthesis, HMGR is suppressed and cholesterol production is increased. Cholesterol is still metabolized to BA via the classical pathway of BA synthesis initiated by CYP7A1. The reduced transhepatic BA flux in DKO mice results in lower circulating LDL‐C, higher HDL‐C and TG concentrations; this leads to reduced atherosclerotic plaque formation. Green and red boxes represent up‐ and downregulation respectively.