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

Abstract

Caveolin-1 (Cav-1) is the principal structural component of caveolae organelles in smooth muscle cells, adipocytes, fibroblasts, epithelial cells, and endothelial cells (ECs). Cav-1-deficient (Cav-1 knockout [KO]) mice are viable and show increases of nitric oxide (NO) production in vasculature, cardiomyopathy, and pulmonary dysfunction. In this study, we generated EC-specific Cav-1-reconstituted (Cav-1 RC) mice and reexamined vascular, cardiac, and pulmonary phenotypes. Cav-1 KO pulmonary arteries had decreased smooth muscle contractility and increased endothelial NO synthase activation and hypotension; the latter two effects were rescued completely in Cav-1 RC mice. Cav-1 KO mice exhibited myocardial hypertrophy, pulmonary hypertension, and alveolar cell hyperproliferation caused by constitutive activation of p42/44 mitogen-activated protein kinase and Akt. Interestingly, in Cav-1 RC mice, cardiac hypertrophy and pulmonary hypertension were completely rescued, whereas alveolar hyperplasia was partially recovered because of the lack of rescue of Cav-1 in bronchiolar epithelial cells. These results provide clear physiological evidence supporting the important role of cell type-specific Cav-1 expression governing multiple phenotypes in the vasculature, heart, and lung.

Figure 1.

Characterization of pulmonary artery function in Cav-1 KO and Cav-1 RC mice. (A) Cav-1 (green) and smooth muscle α-actin (red) localization in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries in cross sections by immunostaining (B and C). Bar, 20 μm. Insets show higher magnification images of section. Nuclei (blue) are detected via DAPI labeling. PE- (B) and high K⁺–induced (C) contractions in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. Ach-induced relaxations in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. The arteries were precontracted with PE (D) or high K⁺ (E), and Ach-induced relaxations were examined. (F) cGMP production in Cav WT, KO, and RC pulmonary arteries. *, P < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Data are expressed as the mean ± the SEM.

Figure 2.

eNOS localization and activation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. (A and B) eNOS (A and B, green), Cav-1 (A, red), and GM130 (B, red) expression in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries in situ by whole-mount immunostaining. Bar, 100 nm. Nuclei (blue) are detected via DAPI labeling (C) caveolins, eNOS, HSP90; Akt protein expression and eNOS (Ser¹¹⁷⁹) and Akt (Ser⁴⁷³) phosphorylation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. These blots are representative of four individual experiments. (D) Summary of eNOS and Akt phosphorylation-isolated vessels. Results are shown as phosphoprotein/total protein for eNOS and Akt, respectively. *, P, < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Data are expressed as the mean ± the SEM.

Figure 3.

Heart and lung morphology and hemodynamics in WT, Cav-1 KO, and Cav-1 RC mice. (A–C) Hearts and lungs were stained with hematoxylin and eosin. (A) Low-magnification pictures of heart slices. (B) Cardiac muscle (top) and wall thickness of main coronary arteries (bottom). (C) Alveolar area (top) and large bronchioles (B, bottom) and arteries (A) in lung. (D and E). Cav-1 expression in WT, Cav-1 KO, and Cav-1 RC hearts (D) and lungs (E). Arrows delineate Cav-1 expression in Cav-1 RC mice. Nuclei (blue) are detected via DAPI labeling. These figures are representative of 4–6 experiments. Basal vascular permeability (F), right ventricle (RV) systolic pressure under nonstimulated and dobutamine-stimulated conditions (G), and mean systemic blood pressure (G) in Cav-1 WT, KO, and RC pulmonary arteries. Data are the means ± the SEM. n = 4–6 mice per group. *, P < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Bars: (A) 1 mm; (B–E) 200 μm.

Figure 4.

Enhanced Akt and p42/44 ERK phosphorylation and fibrogenic gene expression in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. (A and B) Total caveolins, eNOS, HSP90, Akt, p42/44, p38, and JNK protein expression and phosphor-Akt (Ser⁴⁷³), p42/44 (Thr²⁰²/Tyr²⁰⁴), p38 (Thr¹⁸⁰/Tyr¹⁸²) in tissue extracts. (A) Typical Western blots from six individual experiments; (B) the summary of these experiments by quantitative imaging. Akt (Ser⁴⁷³) phosphorylation (C) and p42/44 (Thr²⁰²/Tyr²⁰⁴) phosphorylation (D) in main coronary arteries of the three strains of mice, respectively. Akt (Ser⁴⁷³) phosphorylation (E) and p42/44 (Thr²⁰²/Tyr²⁰⁴) phosphorylation (F) in pulmonary arteries (top) and lung parenchyma (middle showing large airway and bottom showing alveoli). Photos are representative from four separate experiments. Quantitative analysis of PECAM-1 and E-cad (G) and total nuclei (H) in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Expression of TGF-β1, and interstitial collagen genes (I) by RT-PCR (compared with GAPDH) in total RNA isolated from hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Data are the means ± the SEM. n = 4 mice per group.