Endothelial-specific overexpression of caveolin-1 accelerates atherosclerosis in apolipoprotein E-deficient mice

Abstract

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Genetic ablation of Cav-1 on an apolipoprotein E knockout background inhibits the progression of atherosclerosis, whereas re-expression of Cav-1 in the endothelium promotes lesion expansion. Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect because loss of Cav-1 abolishes both the caveolae organelle as well as the Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mouse that overexpresses Cav-1 in the endothelial cells in an apolipoprotein E-deficient background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduced endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1 in vivo.

Figure 1

Characterization of Cav-1 endothelial-specific transgenic mice. A: Construct used for generating the Cav-1 transgenic mice. B: Expression of the transgene, endogenous Cav-1, and ApoE alleles. C: Cav-1 protein levels from mouse aorta. D: Western blot analyses (left panel) and densitometry (right panel) of Cav-1, Cav-2, actin, HSP90, and eNOS in lung endothelial cell extract prepared from wild-type (WT), ApoE^−/−, and ApoE^−/−Cav-1^TG mice. Results for two representative mice are shown for each genotype.

Figure 2

Endothelial-specific overexpression of Cav-1 enhances the progression of atherosclerosis. Oil Red O staining of aortas from mice with the indicated phenotypes. Atheroma formation was significantly increased in ApoE^−/−Cav-1^TG mice (n = 12) compared with ApoE^−/− mice (n = 12). Similar results were observed in females [ApoE^−/−Cav-1^TG mice (n = 9); ApoE^−/− mice (n = 6)].

Figure 3

Endothelial-specific overexpression of Cav-1 does not influence body weight, cholesterol, triglycerides, and lipoprotein profiles. A: Body weight of ApoE^−/− (n = 12) and ApoE^−/− Cav-1^TG (n = 12) mice before and after 12 weeks of being fed a high-cholesterol diet. B: Fasting cholesterol levels of ApoE^−/− (n = 12) and ApoE^−/−Cav-1^TG (n = 12) mice before and after 12 weeks of being fed a high-cholesterol diet. C: Fasting triglyceride levels from both groups of mice before and after 12 weeks of being fed a high-cholesterol diet. D: Lipoprotein profiles from ApoE^−/− and ApoE^−/−Cav-1^TG. VLDL, very low-density cholesterol; IDL, intermediate-density cholesterol; LDL, low-density cholesterol; HDL, high-density cholesterol.

Figure 4

Endothelial-specific overexpression of Cav-1 increases VCAM-1 expression in the artery wall. Western blot analyses (left panel) and densitometry (right panel) of eNOS, ICAM-1, VCAM-1, Akt, and HSP90 in aortic extracts prepared from ApoE^−/− and ApoE^−/−Cav-1^TG mice fed with a high-cholesterol diet for 12 weeks. Results from three representative mice are shown for each genotype. The data represent the mean ± SEM of triplicate samples. *P < 0.05 compared with ApoE^−/−.

Figure 5

Impaired cell proliferation, migration, and NO release in Cav-1^TG ECs. A: EC proliferation measured as viable cell number at different time points. Proliferation of Cav-1^TG ECs was reduced. The data represent the mean ± SEM of three independent experiments. *P < 0.05 compared with wild-type (WT) ECs. B: Migration of MLECs was examined in Transwells using FBS as a chemoattractant. Migration of Cav-1^TG ECs was reduced compared with that of wild-type ECs. The data represent the mean ± SEM of three separate experiments. *P < 0.05 compared with ECs. C: Basal production of NO in ECs measured as NO₂⁻ in the medium over a 24-hour period was determined by chemiluminescence. Cav-1 overexpression reduces NO₂⁻ accumulation in the medium. The data represent the mean ± SEM of three separate experiments. *P < 0.05 compared with ECs. Representative Western blot analyses (right panel) of Cav-1, actin, eNOS, and phospho (p)-eNOS from cell lysates of compared with and Cav-1^TG MLECs used in the NO release experiments.