The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding.

Caveolin-1 (CAV1) and Cavin-1 are components of caveolae, both of which interact with and influence the composition and stabilization of caveolae. CAV1 is associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein (BMP) type 2 receptor (BMPR2) is localized in caveolae associated with CAV1 and is commonly mutated in PAH. Here, we show that BMP/Smad signaling is suppressed in pulmonary microvascular endothelial cells of CAV1 knockout mice. Moreover, hypoxia enhances the CAV1/Cavin-1 interaction but attenuates the CAV1/BMPR2 interaction and BMPR2 membrane localization in pulmonary artery endothelial cells (PAECs). Both Cavin-1 and BMPR2 are associated with the CAV1 scaffolding domain. Cavin-1 decreases BMPR2 membrane localization by inhibiting the interaction of BMPR2 with CAV1 and reduces Smad signal transduction in PAECs. Furthermore, Cavin-1 knockdown is resistant to CAV1-induced pulmonary hypertension in vivo. We demonstrate that the Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling and is a promising target for the treatment of PAH.

Cavin-2 promotes fibroblast-to-myofibroblast trans-differentiation and aggravates cardiac fibrosis.

AIMS: Transforming growth factor ß (TGF-ß) signalling is one of the critical pathways in fibroblast activation, and several drugs targeting the TGF-ß/Smad signalling pathway in heart failure with cardiac fibrosis are being tested in clinical trials. Some caveolins and cavins, which are components of caveolae on the plasma membrane, are known for their association with the regulation of TGF-ß signalling. Cavin-2 is particularly abundant in fibroblasts; however, the detailed association between Cavin-2 and cardiac fibrosis is still unclear. We tried to clarify the involvement and role of Cavin-2 in fibroblasts and cardiac fibrosis. METHODS AND RESULTS: To clarify the role of Cavin-2 in cardiac fibrosis, we performed transverse aortic constriction (TAC) operations on four types of mice: wild-type (WT), Cavin-2 null (Cavin-2 KO), Cavin-2flox/flox , and activated fibroblast-specific Cavin-2 conditional knockout (Postn-Cre/Cavin-2flox/flox , Cavin-2 cKO) mice. We collected mouse embryonic fibroblasts (MEFs) from WT and Cavin-2 KO mice and investigated the effect of Cavin-2 in fibroblast trans-differentiation into myofibroblasts and associated TGF-ß signalling. Four weeks after TAC, cardiac fibrotic areas in both the Cavin-2 KO and the Cavin-2 cKO mice were significantly decreased compared with each control group (WT 8.04 ± 1.58% vs. Cavin-2 KO 0.40 ± 0.03%, P < 0.01; Cavin-2flox/flox , 7.19 ± 0.50% vs. Cavin-2 cKO 0.88 ± 0.44%, P < 0.01). Fibrosis-associated mRNA expression (Col1a1, Ctgf, and Col3) was significantly attenuated in the Cavin-2 KO mice after TAC. α1 type I collagen deposition and non-vascular αSMA-positive cells (WT 43.5 ± 2.4% vs. Cavin-2 KO 25.4 ± 3.2%, P < 0.01) were reduced in the heart of the Cavin-2 cKO mice after TAC operation. The levels of αSMA protein (0.36-fold, P < 0.05) and fibrosis-associated mRNA expression (Col1a1, 0.69-fold, P < 0.01; Ctgf, 0.27-fold, P < 0.01; Col3, 0.60-fold, P < 0.01) were decreased in the Cavin-2 KO MEFs compared with the WT MEFs. On the other hand, αSMA protein levels were higher in the Cavin-2 overexpressed MEFs compared with the control MEFs (2.40-fold, P < 0.01). TGF-ß1-induced Smad2 phosphorylation was attenuated in the Cavin-2 KO MEFs compared with WT MEFs (0.60-fold, P < 0.01). Heat shock protein 90 protein levels were significantly reduced in the Cavin-2 KO MEFs compared with the WT MEFs (0.69-fold, P < 0.01). CONCLUSIONS: Cavin-2 loss suppressed fibroblast trans-differentiation into myofibroblasts through the TGF-ß/Smad signalling. The loss of Cavin-2 in cardiac fibroblasts suppresses cardiac fibrosis and may maintain cardiac function.

Cavin-2 loss exacerbates hypoxia-induced pulmonary hypertension with excessive eNOS phosphorylation and protein nitration.

Pulmonary hypertension (PH) is associated with a poor prognosis even in recent years. Caveolin-1 (CAV1), a caveolae-associated protein, is a causal gene in PH. Cavin-2, one of the other caveolae-associated proteins, forms protein complexes with CAV1 and influences each other's functions. However, the role of Cavin-2 in PH has not been thoroughly investigated. To clarify the role of Cavin-2 in PH, we exposed Cavin-2-deficient (Cavin-2 KO) mice to hypoxia. A part of the analyses was confirmed in human pulmonary endothelial cells (HPAECs). After 4-week 10% O2 hypoxic exposure, we performed physiological, histological, and immunoblotting analyses. Right ventricular (RV) systolic pressure elevation and RV hypertrophy were exacerbated in Cavin-2 KO mice with hypoxia-induced PH (Cavin-2 KO PH mice). The vascular wall thickness of pulmonary arterioles was aggravated in Cavin-2 KO PH mice. Cavin-2 loss reduced CAV1 and induced sustained endothelial nitric oxide synthase (eNOS) hyperphosphorylation in the Cavin-2 KO PH lungs and HPAECs. NOx production associated with eNOS phosphorylation was also increased in the Cavin-2 KO PH lung and HPAECs. Furthermore, the nitration of proteins, including protein kinase G (PKG), was raised in the Cavin-2 KO PH lungs. In conclusion, we revealed that Cavin-2 loss exacerbated hypoxia-induced PH. Our results suggest that Cavin-2 loss leads to sustained eNOS hyperphosphorylation in pulmonary artery endothelial cells via CAV1 reduction, resulting in Nox overproduction-mediated nitration of proteins, including PKG, in smooth muscle cells.

Energy-sparing by 2-methyl-2-thiazoline protects heart from ischaemia/reperfusion injury.

AIMS: Cardiac ischaemia/reperfusion (I/R) injury remains a critical issue in the therapeutic management of ischaemic heart failure. Although mild hypothermia has a protective effect on cardiac I/R injury, more rapid and safe methods that can obtain similar results to hypothermia therapy are required. 2-Methyl-2-thiazoline (2MT), an innate fear inducer, causes mild hypothermia resulting in resistance to critical hypoxia in cutaneous or cerebral I/R injury. The aim of this study is to demonstrate the protective effect of systemically administered 2MT on cardiac I/R injury and to elucidate the mechanism underlying this effect. METHODS AND RESULTS: A single subcutaneous injection of 2MT (50 mg/kg) was given prior to reperfusion of the I/R injured 10 week-old male mouse heart and its efficacy was evaluated 24 h after the ligation of the left anterior descending coronary artery. 2MT preserved left ventricular systolic function following I/R injury (ejection fraction, %: control 37.9 ± 6.7, 2MT 54.1 ± 6.4, P < 0.01). 2MT also decreased infarct size (infarct size/ischaemic area at risk, %: control 48.3 ± 12.1, 2MT 25.6 ± 4.2, P < 0.05) and serum cardiac troponin levels (ng/mL: control 8.9 ± 1.1, 2MT 1.9 ± 0.1, P < 0.01) after I/R. Moreover, 2MT reduced the oxidative stress-exposed area within the heart (%: control 25.3 ± 4.7, 2MT 10.8 ± 1.4, P < 0.01). These results were supported by microarray analysis of the mouse hearts. 2MT induced a transient, mild decrease in core body temperature (°C: -2.4 ± 1.4), which gradually recovered over several hours. Metabolome analysis of the mouse hearts suggested that 2MT minimized energy metabolism towards suppressing oxidative stress. Furthermore, 18F-fluorodeoxyglucose-positron emission tomography/computed tomography imaging revealed that 2MT reduced the activity of brown adipose tissue (standardized uptake value: control 24.3 ± 6.4, 2MT 18.4 ± 5.8, P < 0.05). 2MT also inhibited mitochondrial respiration and glycolysis in rat cardiomyoblasts. CONCLUSIONS: We identified the cardioprotective effect of systemically administered 2MT on cardiac I/R injury by sparing energy metabolism with reversible hypothermia. Our results highlight the potential of drug-induced hypothermia therapy as an adjunct to coronary intervention in severe ischaemic heart disease.

Requirement of Cavin-2 for the expression and stability of IRß in adequate adipocyte differentiation.

OBJECTIVE: Adipogenesis plays an essential role in maintaining energy and hormonal balance. Cavin-2, one of the caveolae-related proteins, is abundant in adipocytes, the leading site of adipogenesis. However, the details of the roles of Cavin-2 in adipogenesis remain unknown. Here, we demonstrate the requirement of Cavin-2 for the expression and stability of IRß in adequate adipocyte differentiation. METHODS: Cavin-2 knockout (Cavin-2 KO) and wild-type (WT) mice were fed with a high-fat diet (HFD) for 8 weeks. We evaluated body weight, food intake, and several tissues. Glucose homeostasis was assessed by glucose and insulin tolerance tests. Insulin signaling in epididymal white adipose tissue (eWAT) was determined by Akt phosphorylation. In vitro study, we evaluated adipocyte differentiation, adipogenesis-related genes, and insulin signaling to clarify the relationship between Cavin-2 and adipogenesis under the manipulation of Cavin-2 expression. RESULTS: Caveolae structure decreased in eWAT of Cavin-2 KO mice and Cavin-2 knockdown 3T3-L1 cells. Cavin-2 enhanced the stability of insulin receptor (IR) through direct association at the plasma membrane in adipocytes, resulting in accelerated insulin/IR/Akt signaling-induced adipogenic gene expression in insulin-containing solution-stimulated 3T3-L1 adipocytes. IR-mediated Akt activation also enhanced Cavin-2 and IR expression. Cavin-2 knockout mice showed insulin resistance with dyslipidemia and pathological hypertrophic adipocytes after a HFD. CONCLUSIONS: Cavin-2 enhances IR stability through binding IR and regulates insulin signaling, promoting adequate adipocyte differentiation. Our findings highlight the pivotal role of Cavin-2 in adipogenesis and lipid metabolism, which may help to develop novel therapies for pathological obesity and adipogenic disorders.