Cyclophilin A promotes atherosclerosis in part by inducing reactive oxygen species and promoting endothelial cell apoptosis and macrophage recruitment into lesions.

Objective Cyclophilin A (CyPA, encoded by Ppia) is a pro-inflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (AngII)-induced reactive oxygen species (ROS) production in the aortas of Apoe−/− mice. In this study we sought to evaluate the role of CyPA in AngII–induced cardiac hypertrophy. Methods and Results Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia−/− mice infused with AngII (1000 ng/min/kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe−/− mice. In contrast to Apoe−/− mice, Apoe−/− Ppia−/− mice exhibited significantly less AngII-induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. AngII-induced ROS production, cardiac fibroblast proliferation and migration were markedly decreased in Apoe−/− Ppia−/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusions CyPA is required for AngII-mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

ROS-dependent acetylation of CyPA is required for the generation of extracellular CyPA. Acetylated extracellular CyPA regulates VSMC and EC activation, suggesting that inhibition of acetylation of CyPA may prevent the pathogenesis of oxidative stress-related cardiovascular diseases.

Objectives-Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (Ϫ/Ϫ)) mice. OPG (Ϫ/Ϫ) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (Ϫ/Ϫ) mice. Methods and Results-Male OPG (Ϫ/Ϫ), OPG (ϩ/Ϫ), and OPG (ϩ/ϩ) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1␣,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (Ϫ/Ϫ) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs Key Words: osteoprotegerin Ⅲ alkaline phosphate Ⅲ vascular smooth muscle cells Ⅲ calcium deposits V ascular calcification, which is frequently observed in patients with end-stage renal disease, diabetes, aging, and osteoporosis, can also lead to cardiovascular diseases and even sudden death. 1-3 Until recently, vascular calcification was considered to be a passive process that occurred as a nonspecific response to tissue injury or necrosis. Now it is becoming increasingly clear that vascular calcification is an actively regulated process that resembles bone metabolism and involves alkaline phosphatase (ALP) and other bonerelated proteins. 4 -7 Osteoprotegerin (OPG) is abundantly produced by osteoblasts at the bone surface and inhibits osteoclast activity, working as a key regulator of bone homeostasis. 8,9 Since it has been reported that OPG (Ϫ/Ϫ) mice exhibit severe osteoporosis attributable to enhanced osteoclastogenesis, OPG is considered to be a protective factor in bone metabolism. 10,11 In the vasculature, the function of OPG is unknown because it is unclear whether vascular calcification takes place in OPG (Ϫ/Ϫ) mice or not. 10,11 Moreover, it was reported that the serum OPG level is associated with the presence and severity of coronary artery disease (CAD). 12 It remains to be clarified whether OPG is involved in the progression of CAD or whether the upregulation of serum OPG concentration is a compensatory mechanism. ALP is a crucial enzyme for initiating mineralization in bone and is present in systemic arteries, arterioles, and some capillaries. 13 It is possible that this enzyme plays a role in arterial calcification by the same mechanism of action as that in bone. 14 Activation of ALP in the arterial wall may result in enhanced vascular calcification.It is well known that either an elevated serum phosphate level or treatment with high doses of vitamin D induces vascular calcification in animal models as well as in humans. 15,16 In the present study, using OPG (Ϫ/Ϫ) mice, we established a mouse model in which arterial calcification can be quickly induced by treatment with a high phosphate diet plus 1␣,25-dihydroxyvitamin D3 (calcitriol) injection, and this model allowed us to perform detailed pathological and biochemical examinations at desired tim...

Objective Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl prolyl cis-trans isomerase (PPIase) activity, scaffold function and, significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Approach and Results Overexpression of CyPA in CyPA deficient VSMC (CyPA−/−VSMC) significantly increased AngII-stimulated ROS production. NADPH oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox overexpressing CyPA−/−VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA−/−VSMC compared to WT-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting for p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA−/−VSMC. Mechanistically, inhibition of p47phox phosphorylation and PX domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-PPIase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC suggesting that PPIase activity was required for their interaction. Conclusions These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton which enhances the translocation of the p47phox to the caveolae.