Early steatohepatitis in hyperlipidemic mice with endothelial-specific gain of TRPC3 function precedes changes in aortic atherosclerosis

Abstract

Nonalcoholic fatty liver disease (NAFLD) and its more advanced form nonalcoholic steatohepatitis (NASH) are the most common chronic liver diseases in developed countries. Moreover, NAFLD and NASH are considerable risk factors for atherosclerosis, the most frequent vascular pathology in these and other metabolic diseases. Despite this strong connection, current knowledge of the relationship between NAFLD/NASH and atherosclerosis is scarce. Recently, we studied hyperlipidemic Apoe knockout mice with endothelial-specific gain of transient receptor potential canonical 3 channel function (TgESTRPC3/ApoeKO) and found that these animals had increased burden of advanced aortic atherosclerosis (16 wk on high-fat diet) compared with nontransgenic ApoeKO littermate controls (non-Tg/ApoeKO), whereas early lesions (10 wk on high-fat diet) were not different. Here, we report that at the early stage when differences in aortic atherosclerosis are not yet manifest, the livers of TgESTRPC3/ApoeKO mice show steatosis, fibrosis, and altered hepatic enzymes compared with non-Tg/ApoeKO animals. Because differences in liver pathology were noticeable long before differences in atherosclerosis were evident, our studies suggest that TRPC3-related endothelial mechanisms that promote steatohepatitis may also contribute to atherosclerosis progression. In vitro, downregulation of TRPC3 in liver sinusoid endothelial cells reduces their susceptibility to endoplasmic reticulum stress-induced apoptosis, suggesting that a proapoptotic effect of TRPC3 may add to other fibrogenic factors in vivo. These novel findings show a positive association between augmented expression of an endothelial TRPC channel, development of early steatohepatitis, and atherosclerotic burden in a hyperlipidemic mouse model of NAFLD fed conventional Western-type diet.

Keywords: NAFLD; NASH; TRPC channels; atherosclerosis; endothelium.

Fig. 1.

Apoe knockout mice with endothelial-specific gain of transient receptor potential canonical 3 function (TgESTRPC3/ApoeKO) or their nontransgenic littermates (non-Tg/ApoeKO) were maintained on a high-fat diet (HFD) for 10 (top panels) or 16 (bottom panels) wk. At the end of the diet periods mice were killed and liver sections (10 μm) prepared and stained with oil-red O (hematoxylin and eosin counterstaining) to evaluate lipid content. The bar graphs show values for hepatic triglycerides and cholesterol content (means ± SE, n = 8) for these groups of mice after the indicated diet periods. *P < 0.001 vs. controls.

Fig. 2.

Aortic root sections from TgESTRPC3/ApoeKO mice (n = 10) or their transgenic littermates (non-Tg/ApoeKO, n = 10) that were maintained on a high-fat diet for 10 or 16 wk, were subjected to histomorphometric analysis to determine total plaque area as in Ref. . Shown are mean values and corresponding SE.

Fig. 3.

TgESTRPC3/ApoeKO or their nontransgenic littermates (non-Tg/ApoeKO) were maintained on a high-fat diet for 10 (top panels) or 16 (bottom panels) wk. At the end of the diet periods mice were killed and liver sections (10 μm) prepared and stained with Mason's trichrome to evaluate collagen content. Fibrotic areas (blue staining) are indicated by arrows. The bar graph shows relative fibrosis area (% of total liver area) as means ± SE, n = 8. *P < 0.001 vs. controls.

Fig. 4.

In A, liver sections (10 μm) from TgESTRPC3/ApoeKO mice or nontransgenic littermates (non-Tg/ApoeKO) that were maintained on a high-fat diet for 10 or 16 wk were stained for in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the number of apoptotic (TUNEL⁺) cells were counted and normalized per mm² of section area. Shown are means ± SE (n = 5), *P < 0.05 vs. corresponding control. In B, liver sinusoid endothelial cells isolated from ApoeKO mice (mLSECs) were incubated in EBM-2 (Control) or EBM-2 containing oxidized LDL (oxLDL, 50 μg/ml) or tunicamycin (Tun, 10 μg/ml) for 24 h and processed for TUNEL assay (black bars). Alternatively, mLSECs were transfected with scrambled oligonucleotides (gray bars) or with siRNA specific for TRPC3, 6, or 7 (siT3, siT6, or siT7; empty bars) and 48 h posttransfection subjected to the above described treatments and processed for TUNEL assay. Shown are means ± SE (n = 3). *P < 0.001 vs. corresponding control. Differences for “siT3+oxLDL” and “siT3+Tun” vs. corresponding scrambled-transfected controls have P < 0.0001. In the presence of siT6 or siT7, the effect of oxLDL and Tun was not different from corresponding scrambled-transfected controls.