The Effect And Mechanism Of Estrogen On Endothelial Progenitor Cells Of Patients With Cerebral Hemorrhage Associated With Hypertension

Background and Purpose:Stroke is one of the three major diseases threatening human health and the leading cause of death and disabilities in many countries and districts.Primary cerebral hemorrhage refers to non-traumatic hemorrhage of brain parenchyma,the proportion of cerebral hemorrhage(CH)is about 30%of total stroke. The mortality of CH during acute stage is about 30-40%,which is the highest among all kinds of cerebrovascular diseases.So many studies focused on CH and many discoveries about the pathogenesis,treatment, especially neurogenesis after stroke emerged.Studies showed evidences of neurogenesis related to neural stem cell after stroke but about 80%of new neurons died within 2 to 6 weeks after onset of stroke.The increasing evidences indicated that neurogensis after stroke is limited by inadequate angiogenesis/vasculogensis,which provide nutrition and proper microenviroment that facilitate new neurons' survival and growth.Since Asahara et al.reported that a subtype of hematopoietic progenitor cells from adults in 1997,namely "endothelial progenitor cells"(EPCs),showed endothelial cell feature,and play an important role in re-endothelialization during the neovascularization of ischemic organs.The patients with stroke are always associated with ageing and hypertension.Hypertension and ageing may decrease the mobilization, migration and proliferative capacity of EPCs.EPCs of patients with hypertension and old age are easy to senescence.While increasing evidences show that the number,mobilization,migration and proliferative capacity of EPCs may be improved in acute period of ischemia diseases (such as ischemia diseases of heart,cerebral and limbs),heart surgery, and organ injury.This suggests that stressing of organ can augment the number and ability of EPCs.Ischemia is believed to upregulate VEGF,which in turn are released to the circulation and induce mobilization of progenitor cells from the bone marrow.EPCs may act similar to monocytes/macrophages,which can increase arteriogenesis by providing cytokines and growth factors. Indeed,EPCs cultivated from different sources showed a marked expression of growth factors such as VEGF,HGF,and IGF-1.The release of growth factors in turn may influence the classical process of angiogenesis,namely the proliferation and migration as well as survival of mature endothelial cells.VEGF is a known chemokine critical for angiogenesis in adults.Intramuscular or intramyocardial VEGF gene transfer has been shown to increase EPC numbers in patients with limb ischemia or inoperable coronary disease.But it is unknown about EPCs functional activity change of old patients with CH associated with hypertension(CH-EH).And it's underlying mechanism needs further research.EPCs remain extremely rare in adult peripheral blood(0.01%of mononuclear cells,under steady state conditions).And studies showed that the number of incorporated cells with an endothelial phenotype into injury or ischemic tissues is generally quite low.It is necessary to seek drugs or methods to augment the number and function of EPCs. Fortunately,studies showed that estrogen,erythropoietin,statins, angiotensin converting enzyme inhibitors can increase the number and functions of EPCs.Imanishi's study demonstrated that 17β-estradiol can improve the secretion of VEGF by EPCs,and delay the onset of senescence of EPCs in healthy human.PI3-K blockers could attenuate the effects of estrogen on EPCs differentiation and senescence and permitted the conclusion that PI3K/Akt signaling pathway is a possible regulator of EPCs proliferation.The PI3-kinase enzymes are widely expressed and play crucial roles in many biological responses including cell survival and proliferation.However,Imanishi's study also indicated that understan ding of the relationship of these factors to hypertension and its vascular sequelae must await further developments in clinical expertise. It is unknown that the effect of estrogen to EPCs of patients with CH-EH. So we aimed to investigate the effect of estrogen on EPCs of patients with CH-EH and explore the underlying molecular mechanism.Methods:1.Study Subjects:Each of patients in control group and CH-EH group had a hypertension history(more than 5 years),hadn't the history of diseases and drugs treatment that maybe affect EPCs.The CH diseases that not associated with hypertension must be excluded.The CH-EH patients were enrolled between 1-3 days after onset of stroke.The volumes of hemorrhage were 20-30ml.2.Isolation and cultivation of EPCs:Peripheral blood(15 ml) was drawn by venipuncture,Mononuclear cells were isolated by density gradient method using Ficoll-Paque Plus.Cells were cultured with M199 medium,which contains 20%fetal-calf.The culture media were changed every 3 days beginning at day 4.3.Characterization of EPCs:At day 7,EPCs were incubated with Dil- acLDL and FITC-UEA-I,Samples were examined with laser scanning con-focal microscope.Only cells exhibiting double positive were identified as EPCs.4.Groups of experiment:Groups of the first experiment include hypertension group and CH-EH group.EPCs were cultured with the medium as described above.Groups of the second experiment include: 1).CH-EH EPCs group,2).CH-EH EPCs treated with 17β-estradiol10nmol/L group,3).CH-EH EPCs treated with 17β-estradiol100nmol/L group,4).CH-EH EPCs treated with 17β-estradiol 1μmol/L group,5).CH-EH EPCs treated with 17β-estradiol1μmol/L + LY294002 group.Every group of EPCs was cultured with the similar medium as above but also included different concentration of 17β-estradiol.EPCs treated with 17β-estradiol1μmol/L + LY294002 group were firstly cultured with LY294002 before culturing with 1μmol/L 17β-estradiol.5.Numbers assay of EPCs colonies and EPCs:The numbers of EPCs and EPCs colonies were determined by counting 5 random high-power(×200)microscope fields per subject at day 7.After the numbers of EPC and EPC colony were counted,cells were assayed or harvested for further study.6.Adhesion assay of EPCs:1×10~5cells/ml EPCs of every group were replanted onto fibronectin-coated culture dishes and incubated at 37℃for 30 minutes.Adherent cells were counted 5 random high-power (×200)microscope fields per subject.7.Migration assay of EPCs:2×10~4cells/ml EPCs of every group resuspended in 50ul M199 medium include different concentration of 17β-estradiol were placed in the upper chamber of a modified Boyden chamber.25ul M199 and human recombinant VEGF(50ng/ml)were placed in the lower compartment of the chamber.After 24h incubation at 37℃,cells migrating into the lower chamber were counted.8.Proliferation assay of EPCs:2×10~3 cell/ml EPCs of every group were replanted 96-well culture dishes,MTT Cell Proliferation and Cytotoxicity Assay Kit were applied to assay proliferation of EPCs.The OD value was measured at 570nm.9.Measurement of VEGF protein in serum and the culture medium:The VEGF protein content of serum of patients and culture medium(at day 7)were assayed by using VEGF ELISA Kit.The OD value was measured at 450nm.10.VEGF mRNA and protein expression assay of EPCs:Total RNA of EPCs in every group was extracted by using Trizol RNA Extraction Kit,and proteins of EPCs in these groups were also extracted. Semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR)analysis was performed to assay VEGF mRNA.VEGF protein was assessed by Western blotting.11.Senescence-associatedβ-galactosidase activity assay: After the first medium changing,the EPCs of different groups were cultured with different medium which included different concentration of 17β-estradiol.EPCs at day 14 were harvested and senescenceassociatedβ-galactosidase activity was measured by using Senescenceβ-Galactosidase Staining Kit.Senescence cells among per 100 cells were counted 5 random high-power(×200)microscope fields per subject. Results:1.Morphological features of EPCs:Culture of total peripheral blood mononuclear cell resulted in the emergence of characteristic spindle-shaped cells and colonies consisting of peripheral spindle-shaped cells emanating from round central cells within 72 hours of culture in CH-EH group,bands or vessels like structure formed by layers of EPCs can be found in some fields.In hypertension group,the colonies of EPCs appeared at day 5,relative later to that of CH-EH.After intervention of different concentration of estrogen,the number and the size of EPCs colonies increased significantly.2.Characterization of EPCs:These cells could be shown to uptake Dil- acLDL and FITC-UEA-I,and exhibiting double-positive. According the results of morphological features and double-positive cells, the cells that we cultured were EPCs.3.Number assay of EPCs colonies and EPCs,adhesion, migration and proliferation of EPCs:Compared with hypertension group,the number of EPCs and EPCs colonies,adhesive migrating and proliferative faculty of EPCs in CH-EH group significantly increased (P＜0.01 or P＜0.05).After treated with different concentration 17β-estradiol,Compared with CH-EH group,except CH-EH EPCs treated with 17β-estradiol10nmol/L group's colonies counts,the number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in different concentration groups significantly increased (P＜0.01),and dose-dependently improved(P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).4.Measurement of VEGF protein in serum and the culture medium,VEGF mRNA and VEGF protein expression assay of EPCs: Compared with hypertension group,the content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased in CH group(P＜0.01).After treated with different concentration 17β-estradiol,Compared with CH-EH group,the content of VEGF protein in culture medium,VEGF mRNA and protein expression of EPCs in different concentration groups significantly increased(P＜0.01),and dose-dependently improved(P＜0.01) with a peak at 1umol/l concentration(P＜0.01).5.Senescence-associatedβ-galactosidase activity assay: Compared with hypertension group,the number of senescence EPCs in CH-EH group had no significant difference(P＞0.05).After treated with different concentration 17β-estradiol,compared with CH-EH group,the number of senescence EPCs in different concentration groups significantly decreased(P＜0.01),and dose-dependently improved (P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on senescence EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).Conclusions:1.The number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in acute period of CH-EH significantly increased.The content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased.This may be the mechanism of acute cerebral hemorrhage augment EPCs numbers and function.The destiny of EPCs easy to senescence was not changed compared with EPCs of patients with hypertension.2.Estrogen can increase the numbers and function of EPCs of patients with CH-EH in vitro,and this effect is dose-dependence. Estrogen can improve VEGF mRNA and protein expression and VEGF secretion of EPCs,and reduce EPCs senescence.The effect is dose-dependent.These may be mechanisms of estrogen augment numbers and function of EPCs of patients with CH-EH.3.The role of Estrogen augments number and function of EPCs, reduces EPCs senescence of patients with CH-EH in vitro is related to PI3K/Akt signaling pathway.