Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis

Abstract

The functions of caveolae and͞or caveolins in intact animals are beginning to be explored. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in several postnatal vascular paradigms. First, increasing levels of Cav-1 do not increase caveolae number in the endothelium in vivo. Second, despite a lack of quantitative changes in organelle number, endothelial-specific expression of Cav-1 impairs endothelial nitric oxide synthase activation, endothelial barrier function, and angiogenic responses to exogenous VEGF and tissue ischemia. In addition, VEGF-mediated phosphorylation of Akt and its substrate, endothelial nitric oxide synthase, were significantly reduced in VEGF-treated Cav-1 transgenic mice, compared with WT littermates. The inhibitory effect of Cav-1 expression on the Aktendothelial nitric oxide synthase pathway was specific because VEGFstimulated phosphorylation of mitogen-activated protein kinase (ERK1͞2) was elevated in the Cav-1 transgenics, compared with littermates. These data strongly support the idea that, in vivo, Cav-1 may modulate signaling pathways independent of its essential role in caveolae biogenesis.nitric oxide ͉ caveolae ͉ VEGF ͉ signal transduction C aveolae are 50-to 100-nm flask-shaped invaginations of the plasma membrane (1) and are present in most mammalian cells. The putative functions of caveolae include cholesterol transport (2, 3), endocytosis (4), potocytosis (5), and signal transduction (6-9). Recent insights into the physiological roles of caveolae and their primary coat proteins, caveolins, have been dissected in genetically modified mice (10, 11). Caveolin-1 (Cav-1) and Cav-3 are dispensable during vascular and organ development but are essential for caveolae formation in specialized cells including most endothelia, adipocytes, and skeletal͞cardiac myocytes. In vitro data have shown that signaling molecules can potentially interact with Cav-1, and that interactions with Cav-1 can increase or decrease the fidelity or magnitude of signaling (12)(13)(14). The ability of proteins to localize in caveolae, in addition to direct interactions of proteins with caveolins, has led to the hypothesis that caveolae may compartmentalize signaling in the plasma membrane and that the interactions (direct and indirect) between resident proteins and Cav-1 may fine-tune the signaling cascades.Physiologically, the loss of caveolae results in impairment of cholesterol homeostasis (15), insulin sensitivity (16), nitric oxide (NO) (10, 11), calcium signaling (10), and cardiac function (17). These studies validate the in vivo importance of caveolae and caveolins beyond cell-based studies that are largely hampered by operational definitions of biochemical fractions containing caveolins and the lack of specificity inherent in reagents that remove cellular cholesterol. Although caveolin knockout mice are useful to delineate the importance of caveolae in a given response, there are additional questions that are difficult to dis...

Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

“…12 In mouse models where angiogenesis was stimulated by ischemia or an adenovirus encoding vascular endothelium growth factor (VEGF), endothelial-specific transgenesis of the caveolin-1 gene decreased neovascularization. 13 In agreement with these results, siRNA-mediated down-regulation of caveolin-1 increased EC transmigration in vitro subjected to angiogenic conditions. 15,16 Similarly, the number of caveolae in the EC of capillaries regressing on anti-VEGF treatment increases dramatically.…”

Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis

“…On days 14 to 28 after surgery, TNFR2-KO mice showed various degrees of clinical phenotype compared with C57BL/6 and TNFR1-KO mice (Figure 1c), based on the clinical scoring system that we described recently. 30,31 Four of 10 R2-KO mice had severe necrosis of the feet (Figure 1d). To precisely determine functional defects in TNFR2-KO mice, blood flow was measured and ischemic and nonischemic limb perfusion were measured before and after surgery and 3 days, 2 weeks, and 4 weeks after surgery.…”

Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

“…After the incubation in oxygenated Krebs buff er and stimulations with Ach or sodium nitroprusside, vascular rings were immediately frozen in liquid nitrogen, homogenized in 6% trichloroacetic acid solution, and centrifuged at 2,000 g for 15 min at 4 ° C, as previously described ( 15 ). The supernatants were applied to a cGMP enzyme -immunoassay system (GE Healthcare), and pellets were used to determine protein content.…”

“…In all of the strains, the sensitivity of the soluble guanylyl cyclase was normal because the increases in cGMP production in response to the NO donor sodium nitroprusside (1 M for 3 min) were similar. channels; these eff ects are rescued by breeding global Cav-1 KO to EC-specifi c Cav-1 transgenic (TG) mice ( 15 ) to reconstitute Cav-1 (called Cav-1 RC) back into the endothelium ( 11,16 ). We show that EC-specifi c Cav-1 reconstitution corrects the abnormal vasomotion in vessels and the cardiac and pulmonary abnormalities observed in Cav-1 KO mice, demonstrating that the diverse cardiovascular phenotypes in these mice are attributable to caveolae in the endothelium and not to other cell types that express Cav-1.…”