The Molecular Control Of S100A4 In Apoptosis Of Cardiovascular Endothelial Cells On Oxidative Stress By Exhaustion Exercise

A lot of oxygen free radicals being produced from human body that carrying out exhaustion exercise could induce apoptosis of cardiovascular endothelial cells. Among the factors that induce endothelial cells injury, oxidative stress plays a key role in inducing cardiovascular diseases. S100 proteins that belong to the family of calcium-binding proteins are a group of calcium-binding proteins that share similar structure and function and low molecular weight. They have distribution specificity of tissue. And different S100 protein has different biological activity and intracellular location and mode of expression. They could form homodimer or heterodimer and play an important role in cell proliferation, cell differentiation, muscle contraction, genetic expression, secretion and apoptosis. Their disfunction also could induce different diseases. S100A4 is a member of the family of S100 proteins, which mainly is centered on oncology area. As a promoted tumor metastasis factor, it is involved in development or growth or prognosis of tumor. Because S100A4 can not only express in cardiovascular endothelial cells, but also promote angiogenesis, we guess that it maybe have key protective effect on cardiovascular system injury. Recently the study revealed that in the course of proliferation of tumor cells , S100A4 binding to the c-terminal area of P53 could affect subcellular localization and transcriptional activity of P53 and apoptosis process of cells. As a potential protective factor of heart injury, the control mechanism of S100A4 on apoptosis of cardiovascular endothelial cells by exhaustion exercise remains elusive. Whether S100A4 is involved in the control of cardiovascular endothelial cell apoptosis and how to affect the apoptosis way of P53 is still to testify for further study. P53 is an important mediate factor of cell apoptosis. S100A4 can bind to the control region of C-terminal of wild type P53 and affect its function. In endothelial cell apoptosis way for exhaustion exercise oxidative stress injury, whether S100A4 affects in-out nucleolus or transcriptional activity of P53 and control apoptosis remains elusive. To explore the change law of S100A4 in cardiovascular endothelial cells injury by exhaustion exercise, we have established both male Wistar rats animal model of cardiovascular endothelial cells injury by exhaustion exercise and cultured vascular endothelial cells oxidative stress injury for H2O2. To construct prokaryotic expression vector for expressing S100A4 protein and establish purification technology of S100A4, the aim of the study is to establish a new way for protecting cardiovascular endothelial cells and to develop a fresh idea for developing a kind of (chemical or pharmaceutical) preparation to observe the effect of S100A4 on apoptosis of cardiovascular endothelial cells induced by H2O2 and to elucidate the control mechanism of S100A4 on apoptosis in vitro .The following is the mainly results:1 The expression and distribution of S100A4 and p53 in oxidative stress cardiovascular tissue induced by exhaustion exerciseMale Wistar rats were divided into 2 groups: the control group(CN) and the exhaustion exercise group (EI). After 10 days of loading-increasing treadmill running, the rats in both CN and EI were immediately decapitated for colleting blood sample and cardiovascular tissue sample. The results showed: after exhaustion exercise, lactate dehydrogenase(LDH) activity(72.13±3.68U/L,P<0.01) of rat blood in EI increased significantly as compared with CN(18.50±2.98U/L) ,but NO content of EI declined by 25.15% (P<0.01) when compared with that of CN, which revealed that exhaustion exercise could induce cardiovascular injury, especially vascular endothelial cell(VEC) injury. On the same condition that malondialdehyde(MDA) content increased by 1.90 fold,but superoxide dismutase(SOD) activity declined by 26.11% when compared with that of CN,which showed that an increasing number of oxygen free radicals is a mainly reason that induces oxidative stress injury of cardiovascular endothelial cells.HE results showed the structure of cardiovascular tissue in CN group is integrity and no pathological changes. And the myocardium and endothelial cells of EI group emerged vacuolar degeneration or interstitial edema et al. Electron microscope results revealed :mitochondria swell,vacuolization ,nucleolus enrich, marginalization,apoptotic body in vascular endothelial cells. According to the results of biochemical index of blood and pathological index, exhaustion exercise not only leads to oxidative stress injury of cardiovascular tissue, but also induces apoptosis of rat cardiovascular endothelial cell.Western-blot results proved that the expression of wild-type P53 in CN group was little and the expression of wild-type P53 in EI group increased . And the expression of S100A4 in CN group was little and the expression of S100A4 of EI group increased . Immunohistochemistry results also showed that the expression of both S100A4 and wild-type P53 was distributed in both myocardial cells and endothelial cells after exhaustion exercise ; but the expression of S100A4 incresed by 1.68 fold and the expression of wild-type P53 increased by 1.63 fold.2 Sequence Optimization , High-level Expression , purification and characterization of S100A4 proteinAccording to the natural S100A4 DNA sequence, the primers were designed based on E.coli codon preference and 6×His tag was introduced. The optimization sequence was amplified by PCR. The BBP sequence was inserted in pEasy-T3 to construct the recombinant cloning vector pEasy-S100A4. After determined by sequence analysis, the S100A4 gene was cloned into expression vector pBV-220 to construct the recombinant expression vector pBV220- S100A4, and was over-expressed in E.coli DH5α. The recombinant protein about 11.5 KDa was showed by SDS-PAGE and was S100A4 protein determined by Western-blot. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein(purity coefficient above 95% ).It laid the foundation for further research about the control mechanism of S100A4 in apoptosis of endothelial cells by oxidative stress injury.3 The control of S100A4 on apoptosis of vascular endothelial cells induced by oxidative stress injuryIn the vitro experimental models, the cultured vascular endothelial cells were stimulated by H2O2. The results demonstrated that H2O2 could lead to oxidative stress injury of the cultured vascular endothelial cells and decline the cell survival rate(28.67%) of vascular endothelial cells and induce apoptosis of vascular endothelial cells. Exogenous S100A4 plays an insignificant role in anti-oxidative stress injury and increasing the cell survival rate of vascular endothelial cells and decreasing apoptosis of vascular endothelial cells. Western-blot results suggested: the level on of both S100A4 and P53 increased after oxidative stress injury of vascular endothelial cells induced by H2O2; exogenous S100A4 increased the kytoplasm level on of P53 in endothelial cells and decreased the nuclear level of P53 in endothelial cells; Different dose of exogenous S100A4 declined the different nuclear level of P53 in endothelial cells. We concluded that the control of S100A4 on apoptosis of endothelial cells induced by oxidative stress injury depended on that S100A4 restrained P53 into cell nucleus and declined the effect of P53 on promoting apoptosis of cells.Take together, these data support that the oxidative stress injury and apoptosis of rat cardiovascular cells was induced by exhaustion exercises. These findings suggested that the expression of both S100A4 and P53 increased in oxidative stress cardiovascular tissue induced by exhaustion exercise. We constructed the recombinant expression vector pBV220- S100A4. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein (purity coefficient above 95%). The cell model of apoptosis induced by oxidative stress was established. It is showed that oxidative stress could lead to increasing level of expression of both S100A4 and P53. S100A4 played an important role in anti-oxidative stress endothelial cells injury and promoting survival rate of endothelial cells and decreasing apoptosis of cells. These findings suggested that exogenous S100A4 could affect the distribution of P53 in oxidative stress injury endothelial cells via increasing accumulation of intracytoplasmic P53 and decreasing the intranuclear level of P53. With the dose of S100A4 increasing ,the effect of S100A4 on P53 was proved more significantly, which revealed that S100A4 was able to restrain P53 into cell nucleus. Therefore, we guess that the ability of S100A4 to restrain P53 into cell nucleus perhaps was the one of control mechanism of apoptosis in endothelial cell injury induced by oxidative stress.