Caveolin-1 provides palliation for adverse hepatic reactions in hypercholesterolemic rabbits

Abstract

Caveolins are an essential component of cholesterol-rich invaginations of the plasma membrane known as caveolae. These flask-shaped, invaginated structures participate in a number of important cellular processes, including vesicular transport, cholesterol homeostasis, and signal transduction. We investigated the effects of CAV-1 on mitochondrial biogenesis and antioxidant enzymes in hypercholesterolemia-affected target organs. A total of eighteen male New Zealand white rabbits were divided into three groups: a normal-diet group, an untreated hypercholesterolemia-induced group, and a hypercholesterolemia-induced group that received intravenous administration of antennapedia-CAV-1 (AP-CAV-1) peptide every 2 days for 2 weeks. Serum biochemistry, CAV-1 distribution, neutral lipid distribution, mitochondrial morphology, biogenesis-mediated protein content, oxidative stress balance, antioxidant enzyme levels, and apoptotic cell death of liver tissue were analysed. Hepatic and circulating cholesterol and low-density lipoprotein cholesterol (LDL-C) levels differed significantly between the three groups (P<0.05). Immunohistochemical staining intensity of CAV-1 was greater in AP-CAV-1-treated rabbits than in untreated rabbits, especially in the vicinity of the liver vasculature. The high levels of neutral lipids, malondialdehyde, peroxisome proliferator-activated receptor-γ coactive 1α (PGC-1α), and nuclear respiratory factor-1 (NRF-1) seen in untreated hypercholesteremic animals were attenuated by administration of AP-CAV-1 (P<0.05). In addition, mitochondria in animals that received treatment exhibited darker electron-dense matrix and integrated cristae. Furthermore, the levels of ROS modulator 1 (Romo1) and superoxide dismutase (SOD)-2, as well as catalase activity were significantly lower in CAV-1-treated hypercholesterolemic rabbits (P<0.05). AP-CAV-1 treatment also restored mitochondrial respiratory chain subunit protein content (OXPHOS complexes I-V), thereby preserving mitochondrial function (P<0.05). Furthermore, AP-CAV-1 treatment significantly suppressed apoptotic cell death, as evidenced by a reduction in the number of TUNEL-positive cells. Our results indirectly indicate that CAV-1 mediates the negative effects of PGC-1α on hepatic mitochondrial respiratory chain function, promotes the antioxidant enzyme defence system, and maintains mitochondrial biogenesis.

Figure 1. CAV-1 distribution and neutral lipid accumulation in liver tissue after 8 weeks on the normal diet and high-cholesterol diet with or without CAV-1 treatment.

(A) Note that CAV-1 is mainly expressed in the vicinity of the liver vasculature. Staining intensity was greater in CAV-1-treated rabbits (CAV-1 group) than in untreated hypercholesterolemic rabbits (control group). (B) Quantification of CAV-1 IHC staining in rabbit liver tissue per ×400 field/5 fields per animal, n = 6 rabbits for each group. Neutral lipid accumulation in the liver cells, indicated by exterior images (C) and oil-red O staining (D), was significantly higher in the control group than in the CAV-1 group. (E) High-cholesterol diet induced a significant increase in malondialdehyde levels in liver tissue. The data represent those from at least 3 independent experiments (magnification 400×, n = 6 for each group, Bar = 1 µm). * and † are significantly different from the normal and control groups, respectively, P<.05.

Figure 2. Changes in the mtDNA copy numbers in the control and CAV-1 groups.

A significant decrease in mtDNA copy number/cell was observed in the CAV-1 group compared with the normal group by SigmaPlot t-tests (* P<0.05). Values are mean ± SD from at least 3 independent experiments, n = 6 for each group.

Figure 3. CAV-1 reduces mitochondrial biogenesis and rescues morphological abnormalities in hypercholesterolaemic rabbits.

Expression levels of PGC-1α (A) and NRF-1 (B) proteins were higher in the control group but not in the CAV-1 group (magnification 400×, Bar = 1 µm). (C) Quantification of mitochondrial biogenesis marker PGC-1α and NRF-1 IHC stain per ×400 field/5 fields per animal, n = 6 rabbits for each group. * P<.05 compared with normal group. (D) Electron microscopy reveals a more condensed cytoplasm, with a dark electron-dense matrix and integrated cristae after CAV-1 treatment (magnification 3000×, n = 6 for each group, Bar = 0.5 µm). The data represent those from at least 3 independent experiments.

Figure 4. CAV-1 expression prevents Romo1-derived-ROS generation and increases the activity of antioxidant proteins.

(A) Levels of the ROS modulator 1 (Romo1), mitochondrial antioxidant enzymes SOD2 (B) and catalase (C) were detected by immunohistochemistry. AP-CAV-1 treatment resulted in reduced expression of Romo1 protein and activity of SOD2 and catalase. (D) Quantification of Romo1, SOD2 and catalase staining. * and † are significantly different from the normal and control groups, respectively, P<.05. The data represent those from at least 3 independent experiments (magnification 400×, n = 6 for each group, Bar = 1 µm).

Figure 5. CAV-1 restored mitochondrial function, anti-oxidative capacity and mitochondrial respiratory chain subunits.

(A) and (B), Immunoblots and densitometry of the mitochondrial complex subunits (I, II, III, IV and V) from 8-wk normal or high-cholesterol fed rabbits with or without CAV-1 treatment. (C) Representative immunoblot showing levels of mitochondrial biogenesis marker and antioxidant enzymes in normal rabbits, untreated rabbits, and treated hypercholesterolaemic rabbits. (D) Columns represent average values over three independent experiments. Density from the normal group was set as 1. * and † are significantly different from the normal and control groups, respectively, P<.05. β-actin was used as a loading control. Values are means ± SD.

Figure 6. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay and Hoechst staining analysed by immunofluorescence.

(A) Signals of TUNEL-positive cells (arrows), which were upregulated when the rabbits were fed a high-cholesterol diet for 8 weeks, were higher in the control group than in the normal and CAV-1 groups. TUNEL staining has green fluorescence (Alexa Fluror 488nm), and the nuclei are stained with Hoechst dye (blue). (B) Quantification of TUNEL IHF stain in rabbit liver tissue per ×400 field/7 fields per animal, n = 6 rabbits for each group. A significantly suppressed number of TUNEL-positive cells were observed in the CAV-1 group. * and † are significantly different from the normal and control groups, respectively, P<.01. Values are mean ± SD from at least 3 independent experiments, n = 6 for each group.