Caveolin-1 is upregulated in hepatic stellate cells but not sinusoidal endothelial cells after liver injury

Abstract

Sinusoidal endothelial cells (SEC) and hepatic stellate cells (HSC) are closely associated specialized vascular cells residing in the hepatic sinusoid. These cells have been shown to play important roles in many different pathophysiologic processes, in particular in liver fibrosis/cirrhosis and portal hypertension. Caveolin-1 functions as a scaffolding protein, and has a variety of functions including in many disease states, such as liver cirrhosis. Although previous studies have shown that in the injured rat liver, caveolin-1 is upregulated, the precise cells in which remains unclear. Therefore, the purpose of this study was to clarify the cell type (or types) in which caveolin-1 is expressed in normal and injured rat liver. We have utilized both detailed immunohistochemical labeling with cell specific markers as well as cell isolation techniques (isolating sinusoidal endothelial cells, HSCs, and hepatocytes) in normal and injured (bile duct ligation) rat liver. We show here that in the normal liver caveolin-1 is expressed predominantly in HSCs and SECs but after liver injury there is upregulation of caveolin-1 in HSCs, but not in SECs. These data have functional implications for the cells in which caveolin-1 is regulated.

Keywords: Immunoblotting; Immunohistochemistry; Nitric oxide; Signaling; eNOS.

Figure 1.

Caveolin-1 is upregulated after liver injury. In (A), total RNA was isolated from normal and injured (BDL) rat liver. RNA was reverse transcribed to cDNA followed by RT-PCR to detect caveolin-1 mRNA expression, which is presented graphically (n=3, (*p <0.05 vs normal). In (B), Liver tissue was homogenized and prepared from normal and injured (BDL) rat liver as in Methods. Proteins were separated by SDS PAGE and analyzed by immunoblotting. In the upper panel, a representative immunoblot is shown. In the lower panel, specific signals were scanned and quantitated and normalized to β-actin (n=3, *p < 0.01 vs normal).

Figure 2.

Caveolin-1 remains unchanged in SECs after liver injury. SECs were isolated from normal (NSEC) and injured (BDL, BSEC) rat liver and cultured in collagen coated dishes for 24 hours as in Methods. In (A), total RNA was extracted and cDNA was prepared after reverse transcription and used for real time PCR analysis to determine caveolin-1 mRNA expression, which is presented graphically (n=3, *p < 0.01 vs NSEC). In (B), total protein lysates were prepared from the same cells and caveolin-1 expression was detected by immunoblot analysis, (representative immunoblots are shown in upper panel). In the middle panel, specific signals were quantitated and normalized to β-actin and eNOS. The lowest panel depicts GRK2 expression, known to be upregulated in only injured SECs (Liu et al., 2005), was used as a control (n=3, *p<0.05 vs NSEC).

Figure 3.

Caveolin-1 is upregulated in injured HSCs. HSCs were isolated from normal and injured (BDL, BHSC) rat liver and cultured. In (A), total RNA was extracted and cDNA was prepared, followed by RT-PCR to detect caveolin-1 mRNA expression, which is presented graphically (n=3, *p < 0.01 vs HSC). In (B), total protein lysates were prepared from the same cells and caveolin-1 expression was detected by immunoblot analysis (representative immunoblots are shown in upper panel). In the middle panel, specific signals were quantitated, normalized to β-actin and the data presented graphically (n=3, *p<0.05 vs HSC). In (C), HSCs were grown for 1, 3 and 5 days in culture and processed as in (B). The upper panel shows a representative immunoblot while the middle panel depicts quantitative analysis of blots. In both (B) and (C) the lowest panels depict expression of smooth muscle α actin, known to be upregulated only in injured HSCs (Rockey et al., 1992), was used as a control (n=3, *p<0.05 vs day1).

Figure 4.

Hepatocytes have a minimal expression of caveolin-1. Hepatocytes were isolated from normal and injured (BDL) rat liver and cultured for 24 hours. In (A), total RNA was extracted and cDNA was prepared, followed by RT-PCR to detect caveolin-1 mRNA expression, which is presented graphically (n=3, p > 0.05 vs normal). In (B), total protein lysates were prepared from normal and injured (BDL) hepatocytes and caveolin-1 expression was detected by immunoblot analysis (a representative immunoblot is shown in the upper panel). In the lower panel, specific signals were quantitated, normalized to β-actin, and the data presented graphically (n=3, p > 0.05 vs normal).

Figure 5.

Immunohistochemical detection of caveolin-1 in normal and injured rat liver tissue. Immunoflorescence staining to detect caveolin-1 and desmin (A), VE cadherin (B), or albumin (C) was performed in normal and BDL rat liver sections. Desmin expression is restricted to HSCs and smooth muscle cells, VE cadherin to endothelial cells, whereas albumin expression is restricted to hepatocytes. In each series, the upper panels depict representative normal liver sections, while the lower panels depict representative injured (BDL) liver sections. The scale bar represents 100 μm.