| 1. Background and Objective:Atherosclerosis(AS), a non-inflammation, retrogression and hyperplastic artery disease, induces artery thickening and lumina stenosis and, at last, leads to myocardial infarction, cerebral thrombosis or necrosis of limbs. Besides people's health, AS also leads to consumption of medical resources which has been a serious burden for many country, society and individual. The study of AS pathogenesy, prevention and therapy, which not only have considerable academic significance but enormous economic and social effects,will play important roles in the future.As a complicated disease,"impairment and resultant"is a popular pathogenesy of AS and supported by most scholars. This theory presumes that endothelial cells, which exposed to blood, are easily impaired by stress of blood flow, oxidative stress, medicine and immuno-complex deposit. Damage and denudation of endothelial cells will cause increase of intima permeability, mononuclear macrophage aggregation and smooth muscule cells proliferation. However, rapidly re-endothelialization can maintain endothelium integrity and block the progress of AS.At present, it has been an important subject for acceleration of re-endothelialization after blood vessel injury in AS related fields. Endothelial Progenitor Cells (EPCs), derived from haemopoietic stem celss, are precursor cells of blood vessel endothelial cells. EPCs can be obtained from bone marrow, peripheral blood and spleen. Stem cell and endothelial cell antigens are all expressed by EPCs. EPCs can be mobilized from bone marrow to peripheral blood, migrate and adhere into injured location, proliferate and differentiate into endothelial cells. It is reported that EPCs mobilization and transplantation can promote recovery of injured vessel.Estrogen plays an important role in generation and development of cardiovascular diseases. It is significant to clarify the effect and mechanism of estrogen on EPCs and recovery of injured artery.2. Methods:2.1 EPCs culture and characterization Briefly, total mononuclear cells (MNCs) were isolated from bone marrow or spleen by density gradient centrifugation with Ficoll separating solution. Cells were plated on culture dishes coated with human fibronectin and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal-calf serum (VEGF 50ng/ml, b-FGF 5ng/mL, EGF 10ng/mL).Cell growth and morphology were evaluated. To observe the function of EPCs, FITC-ulex europaeus agglutinin(UEA-I) binding and DiI-LDL intake are performed; To identify the cell phaenotype, immunofluorescence of CD31,eNOS and fluorescence activated cell sorting(FACS) of Sca-1,VEGFR-2 were carried out. Transmission electron microscope was used to determine EPCs ultramicrostructure.2.2 Effect of estrogen on EPCs biological function After EPCs were synchronized with phenol red and FBS-free media, 17β-estradiol was added to get the final concentration of 0μmol/L, 0.01μmol/L, 0.1μmol/L, 1μmol/L. EPCs differentiation, migration, proliferation, adhesion assay, apoptosis and angiogenesis were evaluated by MTT analysis, modified-Boyden, cell counting and FACS.2.3 Contribution of ERs/PI3K/Akt signal pathway on estrogen-induced EPCs migration and proliferationExpressions of ERs were confirmed by reverse transcription and polymerase chain reaction (RT-PCR), immunofluorescence and westernblot. To examine possible roles of ERs and PI3K pathway on EPCs migration and proliferation, the effect of ERs antagonist-ICI182780 and PI3K pharmacological inhibitor-LY294002 were tested. To indentify the role of Akt, Phosphorylation of Akt (Ser407) was detected by westernblot after treatment of E2, E2+ICI182780 and E2+LY294002.2.4 In vivo experiment of estrogen on recovery of injured vessel2.4.1 Injury of carotid artery was preformed in ovariectomized mice. One and three days later, mobilization of EPCs was evaluated by FACS as double positive of Sca-1/VEGFR-2. Evens blue was injected and area of reendothelization was calculated after 7 days;2.4.2 EPCs tracing: After 14 days of splenectomy and MNCs culture, 1×106 autologous spleen-derived EPCs were labeled with DAPI and transplanted through tail vein. Cell tracing and evaluation of reendothelization were performed on the injured sites. Neointima was observed by H.E staining.3. Results:3.1 Culture and Characterization of EPCs3.1.1 Morphology and growth of EPCsAt first day, cultured MNCs were round and lucency, with enough photonasty. Four days later, cells number increased and cell body stretched. Oval-shaped or spindle like cells appeared which accompanied with some cell process. At 7th day, number of spindle cells augmented. Cell cluster and clones appeared. At 14th day, cells connected with each other and lead to some chord or reticulate, blood capillary-shaped structure. After 21 days culture, cells mixed together, resulting in cobble-stone morphology.3.1.2 Ultramicrostructure of EPCsEPCs were about 15-20um. Pseudopods were found on the cellular membrane. Some cytoplasmic organoids were wraped up by tegument which were staff-shaped and generated some parallel tubiform (Weibel-palade body). At same time, many phagocytic vesicles, chondriosomes and endocytoplasmic reticulums were found too.3.1.3 EPCs phaenotype analysisRed fluorescence were found which indicated positive signal of CD31(89.9±6.16%, n=4),eNOS(92.4±1.77%, n=4) in EPCs,control showed no staining. DAPI nuclear staining confirmed that fluorescence were mainly in plasm. After 7 days of culture, fluorescence activated cell sorting showed expression of Sca-1 38.8±9.09% (n=5), VEGFR-2 55.9±17.18% (n=6).3.1.4 EPCs functional identificationAfter cellular staining of DiI-acLDL and FITC-UEA-I, red (acLDL-DiI intake) and green (FITC-UEA-I binding) fluorescence cells were found. Confocal microscopy indicated 87.2±14.2% EPCs were double positive (yellow, n=5) which were considered as differentiating EPCs.3.2 Effects of estrogen on EPCs biological function3.2.1 Effects of estrogen on EPCs migration The number of migrated EPCs increased in estrogen group (0.01μmol/L E2 vs control, 42.1±8.15 vs 19.7±6.16, P<0.01), (0.1μmol/L E2 vs control, 41.5±4.56 vs 19.7±6.16, P<0.01), (1μmol/L E2 vs control, 55.3±7.89 vs 19.7±6.16, P<0.01) (n=12).3.2.2 Effects of estrogen on EPCs proliferation MTT analysis showed absorbance increased in estrogen groups (0.01μmol/L E2 vs control, 0.41±0.04 vs 0.38±0.06, P<0.05), (0.1μmol/L E2 vs control, 0.46±0.06 vs 0.38±0.06, P<0.01), (1μmol/L E2 vs control, 0.52±0.05 vs 0.38±0.06, P<0.01) (n=30). The increase in proliferation activity was confirmed by manual counting of EPCs(×105/well) ,(0.01μmol/L E2 vs control, 5.94±0.29 vs 5.74±0.16,P<0.05), (0.1μmol/L E2 vs control, 6.14±0.32 vs 5.74±0.16,P<0.01); (1μmol/L E2 vs control, 6.19±0.33 vs 5.74±0.16,P<0.01) (n=30).3.2.3 Effects of estrogen on EPCs apoptosisThe number of apoptosis EPCs reduced in estrogen group (0.01μmol/L E2 vs control, 0.27±0.03 vs 0.29±0.02, P>0.05), (0.1μmol/L E2 vs control, 0.24±0.01 vs 0.29±0.02, P<0.05), (1μmol/L E2 vs control, 0.21±0.02 vs 0.29±0.02, P<0.01) (n=3).3.2.4 Effects of estrogen on EPCs adhesion The number of EPCs increased in estrogen group (0.01μmol/L E2 vs control, 38.7±6.94 vs 35.7±4.12, P<0.05); (0.1μmol/L E2 vs control, 42.2±5.05 vs 35.7±4.12, P<0.01); (1μmol/L E2 vs control, 47.9±5.97 vs 35.7±4.12, P<0.01), (n=30).3.2.5 Effects of estrogen on EPCs-angiogenesisThe value of estrogen group increased (0.01μmol/L E2 vs control, 2.9±0.7 vs 2.1±0.59, P<0.01), (0.1μmol/L E2 vs control, 4.0±0.66 vs 2.1±0.59, P<0.01), (1μmol/L E2 vs control, 4.7±0.48 vs 2.1±0.59, P<0.01) (n=15).3.2.6 Effects of estrogen on EPCs differentiation The number of EPCs increased in estrogen group (0.01μmol/L E2 vs control, 138.9±9.58 vs 130.3±16.32, P>0.05), (0.1μmol/L E2 vs control, 154.3±11.83 vs 130.3±16.32,P<0.05), (1μmol/L E2 vs control, 218±39.78 vs 130.3±16.32,P<0.01) (n=12).3.3 Estrogen induced EPCs proliferation and migration by estrogen receptors and PI3K/Akt-dependent pathways3.3.1 Total cDNA of EPCs was amplificated by RT-PCR. PCR products, about 202bp and 245bp, were found on 1.5% agarose gel stained with ethidium bromide. Westernblot showed brown immuno-compound of 56KD and 66KD in 1, 2, 3, 4, 7 days MNCs, which are uniformity with expecting ERαand ERβmRNA and protein position. Immunohistochemical staining was used to detecte positive signal in EPCs plasm and nuclear permitting the conclusion of ERαand ERβexpression.3.3.2 In ICI182780(10μmol/L)+E2(1μmol/L),LY294002(10μmol/L)+E2(1μmol/L),E2(1μmol/L) and control group, migrated EPCs were 21.8±9.04,21.1±8.49,55.3±7.8,19.7±6.76(n=12). Proliferation analysis showed number of EPCs were 5.85±0.22×105,5.80±0.17×105,6.19±0.33×105,5.74±0.16×105. MTT showed OD value were 0.398±0.033,0.389±0.036,0.519±0.055,0.376±0.058(n=30).3.3.3 Phosphorylation Akt(Ser407) was induced by estrogen with different concentra- tion(0.01μmol/L,0.1μmol/L,1μmol/L) and 1μmol/L E2 at different time (5min, 15min, 30min, 60min). Compared to E2 group, ratio of phosphorylation Akt(Ser407)/Akt were reduced in ICI182780+E2 and LY294002+E2.3.4 Estrogen induced recovery of injured carotid artery3.4.1 Before carotid artery injury, EPCs number of peripheral blood were (variectomy+E2, 0.064±0.016%), (ovariectomy, 0.028±0.01%) and (non-ovariectomy, 0.063±0.022%) (n=11).One or three days after carotid artery injury, EPCs of peripheral blood were 0.42±0.135% (n=6), 1.47±0.38%(n=5) (ovariectomy + E2); 0.13±0.024% (n=6), 0.25±0.024% (n=6) (ovariectomy);0.43±0.16%(n=6),0.65±0.21%(n=4)(non-ovariectomy); 0.12±0.019% (n=6), 0.25±0.062% (n=6)(ovariectomy +E2+LY) and 0.12±0.019% (n=6),0.24±0.067% (n=6) (ovariectomy+E2+ICI).3.4.2 Area of re-endothelization were (ovariectomy, 28.33±13.49%, n=5) vs (ovariectomy+E2, 69.53±14.14%, n=5) vs (non-ovariectomy,83.11±7.94%, n=4 )(P<0.01).3.4.3 Bone marrow and spleen MNCs(n=4)showed different expression (58.9±2.72% vs 74.9±9.41%,P<0.05) of stem cell antigen-1(Sca-1)-a marker of mice hematopoietic stem cell. MNCs (1.48±0.29×107 MNCs/2 femurs and tibias, 4.32±0.83×107 MNCs/spleen, n=6) were cultured for 14 days to obtain EPCs (0.71±0.26×106/2 femurs and tibias; 1.29±0.32×10~6/spleen,n=4) for transplantation.3.4.4 Fourteen days after autologous spleen-derived EPCs transplantation, blue fluorescence cells were found in injured sites that were positive of CD31 too. Compared to normal mice, Re-endothelization increased (65.8±3.9% vs 40.3±1.9%, n=6, P<0.05) and neointima area reduced (0.34±0.045 vs 0.74±0.181, n=6, P<0.05).4. Conclusion4.1 EPCs are resident in bone marrow and spleen, which can differentiate towards endothelial cells in specific condition.4.2 Estrogen induces EPCs differentiation, migration, proliferation, adhesion, angiogenesis and inhibites apoptosis.4.3 ERαandβare expressed in mice EPCs, which are mainly located in nuclear and plasm. Migration and proliferation effects of estrogen on EPCs are ERs/PI3K/Akt signal pathway dependent.4.4 Estrogen can induce EPCs mobilization through ERs/PI3K pathway.4.5 EPCs can home to injured sites which are helpful to promote endothelium recovery of injured carotid artery.4.6 Autologous spleen-derived EPCs transplantation promotes endothelium recovery and inhibites neointima area in mice. |
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