Abstract 157: Secretion of Cyclophilin A by Glutathione Peroxidase-1 Deficient Smooth Muscle Cells has a Paracrine Effect on Normal Smooth Muscle Cells to Activate NF-kB and Increase Cell Growth
In patients with coronary artery disease, a low level of erythrocyte glutathione peroxidase-1 (GPx) activity is associated with increased risk of cardiovascular events. The immunophilin cyclophilin A (CYP) is secreted by smooth muscle cells (SMC) in response to oxidative stress. We hypothesized that reduction in GPx results in hydrogen peroxide (H2O2)-dependent secretion of CYP, resulting in activation of normal SMC. Studies were performed in three different isolations of SMC cultured from aorta of GPx null and littermate control mice. As compared to control SMC, GPx null SMC had increased intracellular and extracellular H2O2 levels, as measured by DCFH and Amplex Red fluorescence. After 24 hours of culture in 0.1% serum, CYP levels were 3.6-fold greater in the conditioned media (CM) of GPx null SMC as compared to CM of control SMC, measured by relative densitometry of Western blots (p<0.05, n=7). Addition of H2O2 (0.1 mM) increased CYP in CM of control SMC, whereas N-acetylcysteine (10 mM) decreased CYP secretion by GPx null SMC. We examined activation of the transcription factor NF-κB in normal SMC using a luciferase reporter assay. CM from GPx null SMC increased NF-κB activation in normal SMC by 45% (p<0.05, n=4) and this response was partially inhibited by the CYP inhibitor cyclosporine (2.5 μg/ml) and by the flavoenzyme inhibitor diphenylene iodonium (10 μM). CYP (10 nM) increased NF-κB activation in control cells by 25% (p<0.05, n=7). In growth studies, CM from GPx null SMC increased cell number by 100% after 48 hours, as compared to CM from normal SMC (p<0.05, n=5), and this increase in growth was inhibited by cyclosporine. Addition of CYP to SMC did not increase expression of the NAPDH oxidase subunits Nox1or Nox4. In summary, reduction in GPx increases H2O2 levels, resulting in SMC secretion of CYP. Through a paracrine effect, CYP activates NF-kB and increases SMC proliferation. Reduction in cellular GPx activity may contribute to progression of atherosclerosis via a mechanism involving secretion of CYP and activation of SMC.