The Effect Of VEGF-induced Connexin 43 On EPCs Proliferation,Migration And Repair Of Injured Vessels

1.Background and purposesVessel injured diseases are serious problems which lead to myocardial infarction and stroke.The endothelial cells,arranged in the innermost of the vessel wall,which play an important role in maintaining the anatomical and physiological functions of blood vessels.Various pathological factors can damage the endothelial cells,destroy the integrity of endothelium,and lead to atheroscerosis.Injury of endothelial cell is the important pathophysiological basis of occurrence and development of vascular injured diseases.However,mature endothelial cells are terminal differentiated cells with low proliferation capacity,which limits its capability of vascular injury repair.Endothelial progenitor cells(EPCs),which are derived from bone marrow and spleen,are a kind of progenitor cells that can differentiate into endothelial cells.A number of previous studies have confirmed that when the blood vessels are injured,EPCs can be mobilized into the peripheral blood,home to the area of vascular injury,proliferate and differentiate into endothelial cells,and participate in the repair of vascular injury.Vascular endothelial growth factor(VEGF)is a key vascular growth factors which can enhance the differentiation,proliferation,and other functions of EPCs in vitro.Futhermore,increasing the local expression of VEGF of injured blood vessels can promote EPCs homing to injured site,differentiating into endothelial cells and also inhibit the proliferation of smooth muscle cells.However the specific mechanism is not fully clear.Connexin(Cx)is the basic unit of the Gap junction(GJ).Six connexins consist of hallow channel called connexons,and connexons of adjacent cells form gap junction channels(GJs)which mediate the exchange of materials and energy between adjacent cells.In the 20 kinds of Connexins which have been found,Cx43 is widely distributed in blood vessels and plays an important role in maintaining the physiological function of blood vessels,and is closely related to the occurrence and development of atherosclerosis and other vessel injured diseases.Further studies notice that Cx43 can mediate adjacent EPCs to form gap junctions.our aims are as follows: 1.To observe whether VEGF promote the proliferation and migration of EPCs by increasing the expression of Cx43.2.To evaluate whether the Cx43 participates into the VEGF-mediated repair of injured vessels.2.Methods2.1 Culture and characterization of EPCsMonocyotes(MNCs)were isolated from rats' spleen tissues,and cultured in medium.Inverted phase contrast microscope was applied to observe cell morphological characteristics.On 7 days after isolation,FITC-UEA-I binding and Di I-ac-LDL intake were performed.VEGFR-2 and CD34 were carried out to identify the cells.2.2 Expression and location of Cx43 in EPCsEPCs were incubated with the primary antibody of Cx43 and fluorescently-labeled second antibody,and the expression and location of Cx43 was observed by laser scanning confocal microscope(LSCM).2.3 The expression of Cx43 stimulated by different concentration of VEGF and effect of interfered si RNA of Cx43 detected by western blotEPCs were stimulated by VEGF(0?10?50ng/m L)and western blot was performed to evaluate Cx43 expression;EPCs were treated with saline?None si RNA and Cx43 interfered si RNA respectively,and western blot was performed to observe the effect of si RNA.2.4 The effect of VEGF-induced Cx43 on gap junction of adjacent EPCs detected by fluorescence redistribution after photobleaching(FRAP)Cell experiment was divided into 3 groups:(1)control group(2)VEGF group(3)VEGF+si Cx43 group.The recovery rate of fluoresecnce was used to evaluate gap junction of EPCs.2.5 The effect of VEGF-induced Cx43 on proliferation and migration of EPCsAfter EPCs were synchronized with serum-free media,proliferation of EPCs was evaluated by CCK8 and the migration of EPCs was evaluated by transwell.2.6 VEGF-induced Cx43 participates in the repair of injured blood vessels in vivo2.6.1 Eight days after splenectomy,rats were implemented with carotid artery injury.Dio-perchlorate labelled EPCs(2×10~6)were injected through tail veins 24 hours after carotid artery injury.EPCs had been disposed according to different groups.To evaluate the difference of EPCs' homing in different group,rats were killed after three days,and frozen sections of target vessels were made to observe under inverted fluorescent microscope.2.6.2 Seven days after carotid artery injury,evans blue was injected to blood vessels,and target vessels were taken to observe the re-endothelialization after 15 miniutes;HE staining was performed to observe the intima hyperplasia after 14 days.3.Results3.1 FITC-UEA-I and Di I-ac-LDL double staining rate was 92.00±2.23%(n=5).CD34 and VEGFR-2 expression positive rate in cultured cells were 89.67±1.39%(n=3)and 92.07±1.35%(n=3)respectively,which indicated these cells were EPCs.3.2 Cx43 green fluorescence could be detected in cytoplasm and cell membrane.3.3 WB indicated the relaitive expression of Cx43 in EPCs treated with 10ng/m L VEGF was higher than 0ng/m L VEGF(1.42±0.47 vs 1.00±0.31,p<0.05)(n=5),the relative expression of Cx43 in EPCs treated with 50ng/m L VEGF was higher than 0ng/m L VEGF(2.07±0.49 vs 1.00±0.31,p<0.05)(n=5),and the relative expression of Cx43 in EPCs treated with 50ng/m L VEGF was highter than 10ng/m L VEGF group(2.07±0.49 vs 1.42±0.47 p<0.05)(n=5);The transfection efficiency of si Cx43 was 62.95±7.71%(si Cx43 vs NS,0.38±0.13 vs 1.00±0.14,p<0.05)(n=3).3.4 FRAP test showed fluorescent substances could transfer between adjacent EPCs through gap junction.The fluorescent recovery rate of adjacent EPCs were obviously higher than isolated EPCs(adjacent cells in control vs isolated cells in control,25.85±4.00% vs 11.66±1.62%,p<0.01),(adjacent cells in VEGF vs isolated cells in VEGF,65.65±6.24% vs 14.23±1.77%,p < 0.01),(adjacent cells in VEGF+si Cx43 vs isolated cells in VEGF+si Cx43 in VEGF+si Cx43,31.31±1.53% vs 14.83±7.48%,p<0.01)(n=6);VEGF could promote the Cx43-induced gap juction of adjacent EPCs(VEGF vs control,65.65±6.24% vs 25.85±4.00%,p<0.01)(n=6);When the expression of Cx43 was inhibited,the gap junction of adjacent EPCs decreased(VEGF vs VEGF+si Cx43,65.65±6.24% vs 31.31±1.53%,p<0.01)(n=6).3.5 Influence of VEGF-induced Cx43 on proliferation and migration of EPCs3.5.1 Effect of VEGF-induced Cx43 on EPCs proliferationOptical desity(OD)was used to reflect the proliferation of cells.CCK8 analysis showed proliferation of EPCs increased in VEGF group(VEGF vs control,1.73±0.07 vs 0.88±0.08,p<0.01)(n=6).When the expression of Cx43 was inhibited,the proliferation of EPCs decreased(VEGF vs VEGF+si Cx43,1.73±0.07 vs 1.37±0.11,p<0.01)(n=6).3.5.2 Effect of VEGF-induced Cx43 on EPCs migrationMigration of EPCs increased in VEGF group(VEGF vs control,128.22±3.59 vs 84.14±4.58,p<0.01)(n=12).When the expression of Cx43 was inhibited,the migration of EPCs decreased(VEGF vs VEGF+si Cx43,128.22±3.59 vs 104.00±3.92,p<0.05)(n=12).3.6 VEGF-induced Cx43 participates in the repair of injured vessels in vivo3.6.1 The effect of Cx43 on the homing of EPCs in rats with carotid artery injury was detected by Fluorescence labeling.No green fluorescence was detected on the intima of the saline group and Some green fluorescence-labeled EPCs can be observed on the intima of the other three groups under Fluorescent microscope.Homing EPCs increased in VEGF-EPCs group(VEGF-EPCs vs EPCs,15.67±2.08 vs 5.00±2.00,p<0.01)(n=3)(×100);When the expression of Cx43 was inhibited,the number of homing EPCs decreased(VEGF-EPCs vs VEGF-si Cx43-EPCs,15.67±2.08 vs 10.67±2.08,p<0.05)(n=3)(×100).3.6.2 Evans blue was injected into tail veins 7 days after carotid artery injury.The rate of re-endothelialization was calculated to evaluate the condition of re-endothelialization according to experimental methods.Transplanted EPCs could promote the re-endothelialization of injured area(EPCs vs saline,18.39±1.83% vs 5.51±1.18%,p<0.01)(n=3);Re-endothelialization of injured area increased in VEGF-EPCs group(VEGF-EPCs vs EPCs,52.66±3.78% vs 18.39±1.83%,p<0.01)(n=3);When the expression of Cx43 was inhibited,re-endothelialization reduced(VEGF-EPCs vs VEGF-si Cx43-EPCs,52.66±3.78% vs 29.58±2.44%,p<0.01)(n=3).3.6.3 HE staining was performed 14 days after the injury of carotid artery,the ratio of I/M detected by softwale was used to evaluate the effect of Cx43 on the intimal hyperplasia of injured vessels.It was shown that the transplanted EPCs could inhibit intimal hyperplasia(EPCs vs saline,0.714±0.024 vs 0.835±0.025,p < 0.01)(n=3),intimal hyperplasia is inhibited in VEGF group(VEGF-EPCs vs EPCs,0.278±0.018 vs 0.714±0.024,p<0.01)(n=3),and when the expression of Cx43 was inhibited,intimal hyperplasia increased(VEGF-EPCs vs VEGF-si Cx43-EPCs,0.278±0.018 vs 0.402±0.116,p<0.01)(n=3).4.Conclusion4.1 Spleen MNCs can differentiate into EPCs in specific condition.4.2 Cx43 mainly express in cytoplasm and cell membrane.4.3 VEGF increases the expression of Cx43 and si RNA inhibits the expression of Cx43 effectively.4.4 VEGF can promote the gap juction by increasing the expression of Cx43.4.5 VEGF can promote the proliferation and migration of the EPCs by increasing the expression of Cx43.4.6 VEGF can promote EPCs homing to injured area,promote re-endothelialization of injuered vessels,and inhibit intimal hyperplasia by increasing the expression of Cx43.