The Change Of Expression Of PDCD4 And DDR2 Gene In Peroxynitrite-Elicited Apoptotic Process In Human Cerebral Vascular Smooth Muscle Cells

Nitric oxide (NO) is an essential mediator of vascular function under physiological and pathophysiological conditions. NO signal transduction pathway not only sustains homoeostasis of vessel, but affects the initiation and development of vascular diseases as well. Peroxynitrite (ONOO") is the production of interreaction between NO and superoxide anion, which possesses strong oxidizing property. Previous researches relevant to the effect of ONOO- on cardiovascular system have mainly concentrated on its effect on the contraction or relaxation state of vascular smooth muscles. To date, few reports regarding the biological effect of ONOO- on human cerebral vascular smooth muscle cells (HCVSMCs), especially the induction of apoptosis, have appeared in literature. In this project, we selected PDCD4 and DDR2 as a breakthrough point to probe the molecular mechanism underlying the action of ONOO- on HCVSMCs.Programmed cell dead factor 4 (PDCD4) is a recently discovered tumor suppressor gene. Human PDCD4 gene is localized to chromosome 10q24. PDCD4 has attracted great interest as an inhibitor of tumor promoter induced neoplastic transformation and a specific inhibitor of cap-dependent mRNA translation in vitro and in vivo. Discoidin domain receptors (DDRs) are unusual receptor tyrosine kinases whose ligands are fibrilla collagen rather than growth factor-like peptides. They recognize and respond to fibrilla, while this integrins often mediate binding and attachment to components of the extracellular matrix such as collagen. It is evident that DDRs and fibrilla act in coordinated fashion to modulate cellular responses involving adhesion, spreading, locomotion, proliferation, survival and differentiation state. DDRs consist of two closely related receptor types. DDR1 is expressed mainly in epithelial cells, whereas, DDR2 is often found in mesenchymal cells. DDR2 regulates collagen turnover that is mediated by SMCs in atherosclerosis. Abnormality of structure or function in VSMCs is thought to be one of vascular disease pathogenesis.In our research, HCVSMCs cultured in vitro were subjected to different concentrations of ONOO-. Cell viability was measured by MTT assay. The rate of cell apoptosis was measured by flow cytometer. The morphological changes were assessed by acridine orange and Ho33342 and PI double staining to determine whether cell experienced apoptosis under the stress of ONOO". The expression of DDR2 at both mRNA and protein levels was examined by the means of RT-PCR and Western-blot simultaneously.The results showed that direct exposure of HCVSMCs to ONOO- was able to block the cell proliferation in a concentration-dependent manner, which is further revealed via triggering the apoptotic pathway. Under the stress by ONOO", the expression of PDCD4 gene was apparently increased at both mRNA and protein levels. The expression of DDR2 gene was finally decreased at both mRNA and protein levels.In conclusion, ONOO- can block proliferation of HCVSMCs, at least in part, via apoptotic pathway rather than necrotic pathway. PDCD4 and DDR2 are key mediators in ONOO- -induced apoptosis of HCVSMCs.