Effects Of Estrogen On Bone Marrow-Derived Endothelial Progenitor Cells-Mediated Cardiac Repair After Acute Infarction

Myocardial infarction has been a major cause of heart failure.Several studies demonstrated that premenopausal women with heart failure have a better prognosis than age-matched men.Whether endogenous sex hormones contribute to these differences in prognosis remains unknown.However,observational studies have demonstrated that postmenopausal women taking estrogen after a myocardial infarction have a lower incidence of heart failure.Furthermore,retrospective analysis of multicenter heart failure trials have shown that postmenopausal women taking estrogen have a better prognosis than women not on estrogen,supporting that estrogen may have beneficial effects on cardiovascular system.Hormone replacement therapy(HRT) in postmenopausal women was the accepted standard of care for many years until a randomized clinical trial showed that HRT actually increased the risk of cardiac events.Nevertheless,extensive experimental and epidemiological evidence indicates that estrogen has a protective role against cardiovascular system,which connects current studies about effects of estrogen on cardiovascular system with endothelial progenitor cells(EPCs) being a research focus for ten years.Experimental studies from several laboratories have reported that EPCs repair infarcted heart primarily through inducing neovascularization,resulting in enhancing perfusion,contributing to regeneration,confining extension of scar,inhibiting left ventricular remodeling and improving cardiac function.However,EPCs from patients with coronary artery disease and with risk factors for coronary artery disease show a lower number and impaired functions including migration,proliferation,homing, angiogenic activity and so on,compared with healthy volunteers.Accordingly,in an effort to enhance EPCs-mediated cardiovascular reparative benefits,current studies are focused on how to improve homing and neovascularization of EPCs.Stromal cell-derived factor-1αexclusively binds to CXCR4 and has CXCR4 as its only receptor.The SDF-1α/CXCR4 axis plays a pivotal role in homing and neovascularization of EPCs.Now,it is still not clear that whether estrogen mediates the homing and angiogenesis of EPCs by regulating SDF-1α/CXCR4 axis.Because of adverse reaction due to systemically applying estrogen and wide tissues and cells being targeted by estrogen,the effects and molecular mechanisms of estrogen on EPCs is hard to be clarified.Therefore,first,in this experiment,we investigated the effects and mechanisms of physiological estrogen on the migratory and angiogenic activity of bone marrow-derived endothelial progenitor cells(BM-EPCs) through establishing ovariectomized mouse models.Second,the effects and specific mechanisms of 17β-estradiol preconditioning in vitro on the migratory and angiogenic capacity of BM-EPCs were further illuminated.Finally,we studied the effects of 17β-estradiol preconditioning on homing of BM-EPCs in vivo and deciphered the effects and mechanisms of 17β-estradiol preconditioned BM-EPCs-mediated cardiac repair after acute myocardial infarction.PartⅠEffects of physiological estrogen on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cellsObjective To investigate the effects and mechanisms of physiological estrogen on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cells(BM-EPCs).Methods Six weeks female BALB/C mice were randomly divided into ovariectomized group,sham operative group and normal group.ELISA was taken to measure serum levels of estrogen in each group 1 and 4 weeks after observation.4 weeks later,we cultured and identified BM-EPCs,assessed migratory capacity of BM-EPCs of each group toward stromal cell-derived factor-1α(SDF-1α) with or without treatment of CXCR4 inhibitor AMD3100 by Transwell chamber,measured tube length of BM-EPCs of each group with or without AMD3100,and detected expression of CXCR4 by RT-PCR,fluorescence-activated cell sorting(FACS),and Western blotting.Results First,serum levels of estrogen in the ovariectomized group 1 and 4 weeks after ovariectomy were both significantly lower than sham operative group and normal group(23.09±4.01 pmol/L vs 894.53±71.98 pmol/L,23.09±4.01 pmol/L vs 867.52±77.08 pmol/L,respectively,after 1 week,P＜0.01;20.75±2.75 pmol/L vs 910.18±58.77 pmol/L,20.75±2.75 pmol/L vs 901.66±78.57 pmol/L,respectively, after 4 weeks,P＜0.01),but there were no difference between sham and normal group (P＞0.05).Second,the number of BM-EPCs migrated toward SDF-1αin ovariectomized group(102.67±7.02 per high power field) was significantly decreased than in sham operative group(172.00±9.17 per high power field;P＜0.01) and in normal group(174.67±10.41 per high power field;P＜0.01),respectively.However, there were no difference between sham and normal group(P＞0.05).After administration of AMD3100,the number of BM-EPCs migrated to SDF-1αin each group was significantly reduced(ovariectomized group,55.33±5.51 vs 102.67±7.02 per high power field,P＜0.01;sham group,57.00±4.58 vs 172.00±9.17 per high power field,P＜0.01;normal group,60.00±5.00 vs 174.67±10.41 per high power field,P＜0.01),but there was no difference between them(P＞0.05).Third,tube length of BM-EPCs in ovariectomized group(5386±405μm per power field) significantly decreased than in sham operative group(7768±466μm per power field; P＜0.01) and in normal group(7514±547μm per power field;P＜0.01),but there were no difference between sham group and normal group(P＞0.05).After treatment with AMD3100,the tube length of BM-EPCs in normal group was significantly impaired(3292±310μm with AMD3100 vs 7514±547μm per power field without AMD3100;P＜0.01).Finally,CXCR4 expression of BM-EPCs from ovariectomized group significantly decreased than sham operative group and normal group, respectively(P＜0.01).However,the difference was not shown between sham group and normal group(P＞0.05).Conclusion Physiological estrogen improves migratory and angiogenic capacity of BM-EPCs by up-regulating functional CXCR4 expression.PartⅡIn vitro experimental study of estrogen preconditioning for enhancing the migratory and angiogenic activity of bone marrow-derived endothelial progenitor cellsObjective To investigate the effects and mechanisms of estrogen preconditioning in vitro on the migratory and angiogenic capacity of bone marrow-derived endothelial progenitor cells(BM-EPCs).Methods 6 weeks aged BALB/C mice were ovariectomized and 4 weeks later, BM-EPCs were cultured and identified from ovariectomized BALB/C mice tibia and femur.After 48 hours coculture with 0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L 17β-estradiol,or,corresponding concentration of 17β-estradiol and estrogen receptors antagonist ICI182 780,BM-EPCs migratory activity toward stromal cell-derived factor-1αwere assessed by transwell chamber with or without treatment of CXCR4 inhibitor AMD3100,angiogenic capacity of BM-EPCs was evaluated by measuring tube length also with or without administration of AMD3100,and CXCR4 expression of BM-EPCs were detected by RT-PCR,FACS,and Western blotting.Results Migratory activity and CXCR4 expression of BM-EPCs were increased by 17β-estradiol in a dose-dependent manner(P＜0.05),however,these effects were completely blocked by ICI182 780(P＞0.05).17β-estradiol prominently enhances angiogenic capacity of BM-EPCs which was fully blocked by ICI182 780 as well. After administration of AMD3100,both migratory and angiogenic activity of BM-EPCs preconditioned with 17β-estradiol were significantly impaired(P＜0.05).Conclusion Estrogen enhances migratory and angiogenic activity of BM-EPCs by up-regulating functional CXCR4 expression via estrogen receptors pathway.PartⅢIn vivo experimental study of estrogen preconditioned bone marrow-derived endothelial progenitor cells transplanted for enhancing recovery after acute myocardial infarctionObjective To investigate the effects of estrogen preconditioning on homing and treatment of bone marrow-derived endothelial progenitor cells(BM-EPCs) transplanted for infarcted myocardium.Methods Female BALB/C mice aged six weeks were ovariectomized.Four weeks later,acute myocardial infarction models were established by ligating the anterior descending(LAD) branch of the left coronary artery.RT-PCR and immunohistochemistry were taken to detect the expression of stromal cell-derived factor-1αin myocardial infarcted area just before myocardial infarction and 1,3,14, 21days after myocardial infaction.3 Days after myocardial infarction,mice were randomly divided into three groups,and each group received 17β-estradiol preconditioned BM-EPCs,non-17β-estradiol preconditioned BM-EPCs,and saline as control through tail vein,respectively.4 and 11 days after BM-EPCs transplantation, MRI trafficking and Prussian staining were used to quantify BM-EPCs homed to infarcted myocardium.25 Days after BM-EPCs transplantation,transthoracic echocardiography was performed to measure left ventricular systolic(LVDs) and diastolic(LVDd) dimensions,fractional shortening(FS) and ejection fraction(EF).At the same time,immunohistochemistry and Masson's trichrome staining were used to assess the capillary density and the average ratio of fibrosis area to total left ventricular area,respectively.Results(1) SDF-1αexpression in infarcted myocardium area showed a higher level just 1 day after myocardial infarction,reached the highest level 3 days after myocardial infarction,and still maintained at a higher level 7 days after myocardial infarction(P＜0.01).However,14 and 21 days after myocardial infarction,expression of SDF-1αsignificantly decreased and had no statistic significance compared with in normal myocardium(P＞0.05).(2) Quantity of homed BM-EPCs in infarcted myocardium of 17β-estradiol preconditioned group is larger than of non-17β-estradiol preconditioned group(P＜0.01).(3) 25 Days after transplantation,echocardiography revealed less ventricular dilation in 17β-estradiol preconditioned group versus controlled group(LVDs:3.09±0.05 vs 3.27±0.10 mm,P＜0.05;LVDd,4.18±0.07 vs 4.3±0.05 mm,P＜0.05),but there were no significance between non-17β-estradiol preconditioned group versus controlled group(LVDs:3.18±0.07 vs 3.27±0.10 mm,P＞0.05;LVDd:4.24±0.06 vs 4.31±0.05 mm,P＞0.05).LV function was also significantly better in 17β-estradiol preconditioned group versus controlled group(FS, %:33±3.8 vs 26±3.2,P＜0.05),however,LV function of non-17β-estradiol preconditioned group was not significantly improved compared with of control(FS, %:28±4.7 vs 26±3.2,P＞0.05).Although EF value of 17β-estradiol and non-17β-estradiol preconditioned group increased 4.72%and 3.29%than of controlled group,respectively,there were no significance between them(P＞0.05).(4) Capillary density 25days after BM-EPCs transplantation in infarcted myocardium of 17β-estradiol preconditioned group was significantly greater than of controlled group (1428±214/mm~2 vs 1070±168/mm~2,P＜0.05).However,capillary density was similar between non-17β-estradiol preconditioned group and controlled group (1214±157/mm~2 vs 1070±168/mm~2,P＞0.05).(5) 25 Days after BM-EPCs transplantation,the area of left ventricular fibrosis was significantly less in 17β-estradiol preconditioned group than in controlled group(8.8±4.9%vs 49.0±4.6%, P＜0.05).Nevertheless,the area of left ventricular fibrosis of non-17β-estradiol preconditioned group had no significance compared with control(41.6±5.2%vs 49.0±4.6%,P＞0.05).Conclusions(1) During 1 week after acute myocardial infarction is optimal period for BM-EPCs transplantation for treating ischemic heart disease for SDF-1αexpression in infarcted myocardium showing a higher level.(2) estrogen preconditioning enhances homing of BM-EPCs which contributes to neovascularization,attenuating left ventricular remodeling,and improving cardiac function.