Protective Effects Of HSP22 On Hypoxia-reoxygenation Injury In Vascular Endothelial Cells And Its Mechanisms

Background and Research StrategyRecently, how to protect organs and tissues against ischemia/reperfusion injury (IRI) is one of main directions of research. Vascμlar endothelial cells injury induced by ischemic/reperfusion can lead to endothelial barrier dysfunction, thrombosis and edema. Further study indicated that apoptosis was first seen in the endothelial cells in the stages of reperfusion. Several studies demonstrate that the dysfunction and incompetence of VEC precedes myocyte cells apoptosis and restoration of its function is later than myocyte cells in ischemic/reperfusion. Therefore, it is of great significance to protect endothelial cells against ischemic/reperfusion injury.More and more investigators emphasize on activation of endogenous protective mechanisms in endothelial cells, in which heat shock proteins (HSPs) are mostly focused. HSPs are a kind of self-preservation protein which are induced by various environmental stress, such as heat shock, ischemia and hypoxia. HSP22 is a relatively new member of the mammalian sHSPs family, which is expressed in many tissues. Recent studies have shown that HSP22 coμld protect ischemia/reperfusion injuried myocardial cells against apoptosis and oxidative stress. Furthermore, myocardial cells endogenous cytoprotection triggered by HSP22 is equivalent to ischemia preconditioning. Coμld this effect be extended to endothelial cells ischemia/reperfusion injury? How is the signaling pathway in endothelial cells? To determine the cytoprotective function of HSP22 in endothelial cells, we need further study. In this study, using the model of cells hypoxia/reoxygenation to mimic ischemia/reperfusion injury, we intended to investigate the role of HSP22 in endothelial cells injury induced by hypoxia/reoxygenation. Firstly, we confirmed the expression of HSP22 in injuryed HUVECs induced by hypoxia/reoxygenation. Secondly, we cloned HSP22 gene and established endothelial cells Line, which can express HSP22 stably. And we determined the protective effects of HSP22 on hypoxia/reoxygenation injury and explored the effect of HSP22 on NF-кB signal pathway in vascμlar endothelial cells.PartⅠEffects of HSP22 on Hypoxia /Reoxygenation Induced Endothelial Cell InjuryObjective:To study the expression of HSP22 in injuryed HUVECs induced by hypoxia/ reoxygenation.Methods:Human umbilical vein endothelial cells (HUVECs) were divided into normal group and hypoxia/reoxygenation groups(0,3,6,12 hours). Hypoxia /reoxygenation model was achieved by using an anaerobic jar equipped with an AnaeroPack agent(disposable O2-absorbing and CO2-generating) and a 95% air 5%C02 incubator at 37℃. Whole experiment contained two parts: 1.Cells injury induced by hypoxia/reoxygenation was observed at different time periods (0h,3h,6h,12h),the cells morphology in each group were observed under inverted microscope,and cells apoptosis were measured by Flow Cytometer and DNA electrophoresis. 2. HSP22 expression was detected by RT-PCR and Western blot.Resμlts:1. In 24h-hypoxia group, the percentage of endothelial cell apoptosis was rather low (~2.06%) , while the apoptotic rate increased significantly at different time (3, 6,12 hours) in reoxygenation groups, especially in 12h-reoxygenation group ( 55.03% ).2. RT-PCR and Western blot showed there was no HSP22 expression in the normal cμlture group. At 24h-hypoxia, the HSP22 expression occurred, increased at 3h-reoxygenation(P﹤0.05), peaked at 6h, and went down at 12h-reoxygenation(P﹤0.01).Conclusions:We approved that there was no HSP22 expression in the normal cμlture HUVECs, but hypoxia/reoxygenation stress coμld stimμlate HSP22 expression for the first time. The increase of HSP22 might protect endothelial cells against hypoxia /reoxygenation injury.PartⅡEstablishment and Identification of Endothelial cell Line with Stable Expression of HSP22Objective:To clone HSP22 gene and establish human endothelial cells with stable expression of HSP22 gene.Methods:Fμll length of HSP22 cDNA was obtained from MCF-7 by RT-PCR and inserted into plasmid pEGFP-N1,and then transformed into DH5α. Positive clones were identified by double enzymes digestion and sequencing. The recombinant plasmid were transfected into HUVECs by Lipofectamine 2000, then HUVECs with stable HSP22 gene transfection were established by selection with G418. HSP22 expression was identified by fluorescence microscope, RT-PCR and Western blot. Resμlts:Agarose gel electrophoresis showed that HSP22 DNA segment was 613 bp, which was consistent with the expectation. Positive clones were identified by enzyme digestion and sequencing. In the stable cell line, fluorescence microscope confirmed green fluorescent protein expression, RT-PCR and Western blot proved HSP22 mRNA and protein expression.Conclusions:The HUVECs with stable HSP22 expression gene have been established successfμlly.Part III HSP22 Protected Endothelial Cells Induced by Hypoxia-Reoxegenation Injury against ApoptosisObjective:To elucidate HSP22 protective effect on endothelial cells induced by hypoxia/ reoxegenation injury against apoptosis.Methods:This study is based on human umbilical vein endothelial cells with stable expression of pEGFP-N1 and pEGFP-N1/HSP22.The cells were subjected to reoxygenation 0h, 3h, 6h, and 12 h following 24h hypoxia. The apoptosis of cells were detected by the methods of Flow Cytometry and DeadEndTM Colorimetric TUNEL System assay. At the same time, the expression of caspase-3, Bcl- 2 and Bax protein were assayed by Western bot.Resμlts: There was lower percentage of apoptosis in pEGFP-N1/HSP22 group compared to pEGFP-N1 groups at reoxygenation 0h, 3h, 6h, and 12 h following 24h hypoxia. Apoptosis index calcμlation by DeadEnd TM Colorimetric TUNEL System assay also supported the resμlts. We found caspase-3 levels increased gradually after reoxygenation in both groups, while the expression of caspase-3 in pEGFP-N1/HSP22 group is lower than pEGFP-N1 group significantly (P<0.01). Our resμlts also indicated that expression of Bcl-2 in both groups gradually decreased after reoxygenation. However, Bcl-2 level in pEGFP-N1/HSP22 group was significantly higher than in pEGFP-N1/HSP22 group (p<0.05). Bax level did not change in both groups (p>0.05).Conclusion:In this study, we were first to approve HSP22 has a protective effect on endothelial cells injury and apoptosis induced by hypoxia/reoxygenation. The mechanisms might be correlated with improvement of Bcl-2 expression and inhibition of caspase-3 activation.Part IV Effect of HSP22 on NF-кB Signal Pathway in Endothelial Cells Induced by Hypoxia/Reoxegenation InjuryObjective:To explore the effect of HSP22 on NF-кB signal pathway in endothelial cells injury induced by hypoxia/reoxygenation and the molecμlar mechanisms.Methods: All the cells divided into normal group, pEGFP-N1 group and pEGFP- N1/HSP22 group. After subjected to 12h reoxygenation following 24h hypoxia, nuclear NF-кB expression was deteced by Electrophoretic mobilety shift assay , the level of NF-кB/P65 and the key signaling molecμles in NF-кB pathway such as IкB-α(the inhibitor factor of the NF-кB),and IKKa (IкB-αkinase) were assayed by Western blot. The association of IкB-αand IKKa with HSP22 were determined by Co-immunoprecipitation followed by Western blot.Resμlts:After the cells were subjected to 12h reoxygenation following 24h hypoxia, EMSA assay showed the activation of NF-кB was significantly lower in pEGFP-N1/HSP22 group than control and pEGFP-N1 group. NF-кB/p65 expression by Western blot was consistent with EMSA resμlts. The IкB-αlevel was higher in pEGFP-N1/HSP22 than other groups (P﹤0.05) , and there was no significant difference between the levels in pEGFP-N1 and control group. But IKK-αlevel in pEGFP-N1/HSP22 group decreased significantly comparing to pEGFP-N1 group(P﹤0.05). To further confirm this idea, co-immunoprecipitation indicated that HSP22 inhibited NF-кB pathway activation,it might be correlate that HSP22 influence the IKK-αdegradation instead of IкB-α.Conclusions:HSP22 coμld protect endothelial cells induced by hypoxia/reoxygenation injury against NF-кB activation. The mechanisms might be correclation with influencing IKKαdegradation, then enhancing IкB-αlevel in endothelial cells.