| Background：Coronary atherosclerotic heart disease (CAD) is one of the most common heartdiseases that are serious damage to human health world wide now, which remains a leadingcause of morbidity and mortality. Therefore, how to effectively prevent and treat coronaryheart disease has become an important territory aim to cardiovascular disease researchpresently. Despite rapid development in pharmaco-therapy, percutaneous coronaryinterventions(PCI) treatment and coronary artery bypass grafting (CABG), there areapproximately10%～37%of CAD patients who are not available to any type of coronaryinterventions because of some special lesions of coronary artery and the limit of anatomicalfactors. Further more, some patients merely accept the incomplete revascularizationtreatment. Thus leading to severe myocardial ischemia accompany with decrease of cardiacfunction. The disabling and fatality rate of patients are extremely high. However,therapeutic angiogenesis may induce collateral growth according to the gene, protein, andcellular level technology. Finally, angiogenesis leading to the formation of collateral vesselswith blood supply. Currently, therapeutic angiogenesis has become a therapy option forpatients with advanced CAD with unable to revascularization for example PCI or CABG.Therapeutic angiogenesis may improve the symptoms of CAD through increasing the bloodsupply of ischemic myocardium. As is known to all, cardiac micro-vascular endothelialcells (CMECs) is an especially cell type derive to coronary micro-vascular, which play akey role in the process of angiogenesis.NF-e2p45-related factor2(Nrf2) belongs to the Cap’n’collar (CNC) family of basic leucine zipper (bZip) transcription factor, which plays an important role in the process ofresistance to oxidative stress. The activation of Nrf2can lead to much downstreamantioxidant gene expression for example heme oxygenase-1(HO-1). Presently, foreignstudy has been confirmed that Nrf2may promote the increases of deep capillary numberand the sprouting of vascular endothelial cell. Nrf2also plays important role incardio-protection, such as fighting against pathological cardiac remodeling, protectionmyocardium from ischemia/reperfusion injury, delay atherosclerotic lesions and so on.However, the effects of Nrf2on CAD infarction/ischemia myocardial angiogenesis andunderlying mechanisms are not fully understood. At the same time, research found thatendogenous activation of Nrf2is limited in infarction/ischemia myocardial region. Theangiogenesis effect not only present a time-dependent phenomenon, but also the expressionlevels of promote angiogenesis gene have corresponding requirements. Therefore, all kindsof interference factors for promote myocardial Nrf2high expression can promotemyocardial repair by increase the therapeutic angiogenesis.In conclusion, using primary rat cardiac micro-vascular endothelial cells as the objectof study, we examined whether Nrf2promotes cell angiogenesis in vitro induced byhypoxia with the method of Nrf2gene transfection. At the same time, we discussed whetherthe effect of Nrf2promotes cell angiogenesis in vitro through regulation of HO-1expression.Methods：Part I. Isolation, culture and identification of CMECs Effects of hypoxia ornormoxia on the Nrf2and HO-1transcriptional and protein expressions in CMECs.Rat cardiac micro-vascular endothelial cells were obtained from the myocardial tissueof adult healthy male Sprague-Dawley (SD) rats using tissue block method. IsolatedCMECs were cultured in Dulbecco’s modified eagle’s medium (DMEM) at37°C in ahumidified atmosphere. The identity of CMECs was verified by the immunocytochemistrystaining with Factor VIII. Cells from passages3-4were used in all experiments. CMECswere divided into two groups: control group and various short period time of hypoxiagroups, various short period time of hypoxia groups including①1hr hypoxiagroup,②2hr hypoxia group,③4hr hypoxiagroup,④12hr hypoxiagroup, and⑤24hr hypoxia group. After cells were incubated at37°C in1%O2,5%CO2,94%N2for indicated time,real-time Reverse trans-cription-polymerase chain reaction (RT-PCR) and western blotwere used for detecting the Nrf2and HO-1transcriptional and protein expressions inCMECs. Meanwhile, CMECs were divided into control group and various long period timeof hypoxia groups, various long period time of hypoxia groups including①12hr hypoxiagroup,②24hr hypoxiagroup,③48hr hypoxiagroup, and④72hr hypoxiagroup. Aftercells were incubated at37°C in1%O2,5%CO2,94%N2for indicated time, real-timeRT-PCR and western blot analysis were used for detecting the Nrf2and HO-1transcriptional and protein expressions in CMECs. Through above experiment, wedetermined whether hypoxia actived Nrf2accompany by corresponding changes of HO-1level.Part II. The effects of transcription factor Nrf2on the migration and tube formation inCMECs under hypoxia conditions and regulation of downstream target genes HO-1.After purchase of shRNA-Nrf2or shRNA-control was transfected into the CMECsrespectively, RT-PCR and western blot were used for detecting the Nrf2mRNA and proteinexpressions in CMECs aimed to assure whether successfully interference. And then CMECswere divided into:①c ontrol group,②t ransfection with shRNA-control group, and③transfection with shRNA-Nrf2group. After cells were incubated for12h, the migration andtube formation of CMECs were determined by transwell chamber assay and Matrigel assayrespectively. RT-PCR was used for detecting the HO-1and VEGF mRNA expression levelsin CMECs. Western blot analysis and ELISA were used for detecting the HO-1and VEGFprotein expressions in CMECs respectively.Part III. The role of transcription factor Nrf2promote the migration and tube formationin CMECs under hypoxia conditions and related mechanism.After Len-Nrf2or Len-control was transfected into the CMECs respectively, Real-timeRT-PCR and western blot were used for detecting the Nrf2mRNA and protein expressionsin CMECs aimed to assure whether successfully interference. And then CMECs weredivided into:①control group,②transfection with Len-control group, and③transfectionwith Len-Nrf2group. Real-time RT-PCR and western blot analysis were used for detectingthe HO-1mRNA and protein expressions levels in CMECs. In the next experiments, the further research was used HO-1inhibitor Znpp todeterming the role of HO-1in Nrf2promote the migration and tube formation in CMECsunder hypoxia conditions. CMECs were divided into:①control group,②transfection withLen-control group,③transfection with Len-Nrf2group, and④transfection withLen-Nrf2+Znpp group. After cells were incubated for12h, the migration and tubeformation of CMECs were determined by transwell chamber assay and Matrigel assayrespectively.Results(1) Typical isolated CMECs using tissue block method exhibited “cobblestone”morphology. For further characterization, after culture for7days, cells were distinguishedby immunohistochemistry. The majority (﹥95%) of cells showed VIII factor positivereactions.(2) After short-term exposure to hypoxia (1h,2h,4h,12h, and24h), the Nrf2mRNAand protein expression levels were significantly upregulated beginning at4h compared tonormoxia-control. Compared to normoxia-control, the Nrf2mRNA expression levelincreased from0.99±0.01to2.29±0.06at4h (P<0.01), the Nrf2protein expression levelupregulated from0.28±0.03to0.42±0.02at4h (P<0.01). Nrf2gradually increased withinhypoxia24h in a time-dependent manner. Nrf2mRNA and protein expression levels weretemporarily upregulated compared to normoxia-control in long-term exposure to hypoxia(12h,24h,48h, and72h), the maximum expression of Nrf2was observed after48h(mRNA expression:0h:0.98±0.02,48h:5.55±0.05, protein expression:0h:0.27±0.02,48h:1.20±0.08, P<0.01), and then decreased thereafter. The same result was shown by themRNA and protein expressions of HO-1.(3) CMECs were transiently transfected with either shRNA-control or shRNA-Nrf2,Nrf2shRNA significantly inhibited Nrf2mRNA and nucleus protein expressions comparedto the shRNA-control (mRNA expression: from0.64±0.08to0.23±0.04, protein expression:from0.73±0.02to0.36±0.01, P<0.01). And transfection with shRNA-Nrf2significantlyinhibited migration and tube formation of CMECs to hypoxia12h at48h post-infectioncompared with the shRNA-control, the cell migration number and the tube formation lengthdecreased from61.4±2.9and (5.50±0.10)mm to38.8±3.7and (2.87±0.21) mm respectively (P<0.01).(4) Knockdown of Nrf2by shRNA markedly decreased HO-1mRNA and proteinexpression in CMECs to hypoxia12h at48h post-infection compared with theshRNA-control. The HO-1mRNA and protein expression decreased from0.61±0.03and0.70±0.04to0.21±0.04and0.47±0.07respectively (P<0.01). The same result was shownby the mRNA and protein expressions of VEGF（P<0.01）.(5) At a MOI of20, the transfection efficiencies of CMECs were91.9±1.3%(n=5),while CMECs activity and morphology were unaffected. CMECs were transientlytransfected with either Len-control or Len-Nrf2, Nrf2overexpression significantlyincreased Nrf2mRNA and nucleus protein expression levels compared with the Len-controlafter48h of transfection (mRNA expression: from0.96±0.01to4.40±0.05, proteinexpression: from0.33±0.03to0.77±0.03, P<0.01).(6) Nrf2overexpression markedly increased HO-1mRNA and protein expressions inCMECs to hypoxia12h at48h post-infection compared with the Len-control. Compared toLen-control, the HO-1mRNA expression increased from0.95±0.01to4.50±0.08at4h(P<0.01), the HO-1protein expression upregulated from0.42±0.02to1.03±0.03(P<0.01).Transfection with Len-Nrf2markedly increased migration and tube formation of CMECs inresponse to hypoxia12h compared with the Len–control (the cell migration number:80.0±3.8, the tube formation length:(62.8±3.5) mm, P<0.01). HO-1inhibitor Zincprotoporphyrin (Znpp) markedly inhibited the effects of increased migration and tubeformation by Nrf2overexpression compared with the Len-Nrf2(the cell migration number:from80.0±3.8to55.4±4.0, the tube formation length: from (62.8±3.5) mm to (49.6±2.7)mm, P<0.01).Conclusion(1) Hypoxia temporarily upregulated the mRNA and protein expression levels of Nrf2,accompanied by temporarily enhanced the mRNA and protein expression levels of HO-1inrat CMECS.(2) Knockdown of Nrf2by Lentiviral delivery of shRNA significantly inhibits themigration and tube formation in CMECs to hypoxia. Indicate Nrf2plays an important rolein the process of CMECs angiogenesis. (3) Nrf2knockdown sequentially downregulated HO-1and proangiogenic factorVEGF expression in CMECs to hypoxia.(4) Nrf2over-expressing may promote the migration and tube formation in CMECsunder hypoxic conditions.(5) HO-1is involved in Nrf2over-expressing promotes the migration and tubeformation in CMECs to hypoxia.(6) Nrf2may mediate angiogenesis of CMECs under hypoxic condition throughupregulating HO-1expressions. |
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