Study On The Role Of Gap Junctions On Vascular Injury Repair

The prevalence of vascular injury diseases such as atherosclerosis, hypertension and the vascular complication of diabetes are increasing, and become second major cause leading to death after tumor. The injury and dysfunction of endothelial cells (ECs) by mechanical removal and inflammation,which induces a cascade of proinflammatory events resulting in infiltration of monocytic cells and smooth muscle cells(SMCs) proliferation, is a initial event of pathogenesis of atherosclerosis and a major cause of adverse repair response to vascular injury. After ECs injured, under the action of many factors such as growth factors, cytokines, chemical factors, inflammatory mediators and shear stress, vascular SMCs migrate from media to intima, proliferate, synthesize and secrete extracellular matrix. These processes are pathophysiological mechanism of vascular injury adverse repair and neointimal formation, and are also primary elements involving in that vascular injury diseases develop and progress. To inhibit the migration and proliferation of vascular SMCs is of improtance to treat vascular injury diseases. However, knowledge about its mechanism of these processes is still poor. Therefore, to explore itself internal mechanism of migrating and proliferating and to find a measure of inhibiting its migration and proliferation will provide a new strategy for preventing and treating vascular injury diseases.The maintenance of vessel wall homeostasis requires cellular interaction and coordination of the behavior of individual cells. It is established that measures to promote the reendothelization of injured vessel could effectively inhibit the migration and proliferation of vascular smooth muscle, subsequently reduce neointimal formation, and finally prevent vascular injury diseases from developing or progressing. In the past, it had been thought that endothelial repair only depended on the regeneration and migration of ECs at the edge of injured endothelium. But recently, it was proved that the adjacent ECs could not regenerate or its regenerative ability was limited and could not effectively repair the injured endothelium. Hence, to study the mechanism of endothelial wound repair is very important for preventing and treating vascular injury diseases.Reendothelization of injured vessel could inhibit SMC migration and proliferation, subsequently reduce neointimal formation and promote the benign repair of injured vessel. But the mechanisms still remain unclear. It has been thought that ECs and SMCs could interact through bioactive substances in an autocrine and/or paracrine way. But it is found in vivo and in vitro that interaction between ECs and SMCs didn't disappear after blocking the pathway of autocrine and/or paracrine. It suggested that other pathway exist between two type cells besides autocrine and paracrine.Gap junctions play a key role in the maintenance of tissue homeostasis and the regulation of cellular growth, differentiation, and development. Recently, it was found that connexins is of very importance during the development and progression of vascular injury diseases. It was reported that upregulation of Cx43 protein was found in neointima of saphenous vein cultured in vitro and that the expression of Cx43 protein changed in the early stages of atherosclerosis. However, whether connexins act through the function dependent or independent on direct intercellular communication via gap junction remain unknown. And substances transferred between ECs and SMCs across gap junction are still unclear.ObjectivesIntercellular communication via gap junctions may play a key role on vascular injury repair. In this study, we attempted to explore the role of gap junctions on endothelial wound repair, the proliferation and phenotypic transition of SMCs and neointimal formation of vessel after balloon injury. Subsequently, we also tried to investigate if Ca2+ could transfer between ECs and SMCs across gap junctions during the processes of interaction of ECs and SMCs through a coculture system of ECs and SMCs. Lastly, we wanted to study a possible mechanism of Ca2+ transferred from ECs and SMCs across gap junctions would act on SMCs. And we hoped that this study could provide some experimental evidences for the strategy of preventing and treating vascular injury diseases.Methods1. Role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair: Rat aortic ECs (RAECs) were cultured by explanted rat aortic wall tissue and were identified with cell immunofluorescence staining for vWF and transmission electron microscope. Cell immunofluorescence staining was applied to detect the expressions of connexin (Cx) 37, Cx40 and Cx47 in RAECs. Fluorescence redistribution after photobleaching (FRAP) was used to measure the communications between cells via gap junctions. Cell apoptosis was detected by Annexin V/PI staining combined with flow cytometry (FCM). The monolayer of cultured RAECs was scraped by a mechanical method and the endothelial wound healing rate was quantified by an analysis of the photographs taken every 24h after endothelial cell layers were wounded. Meanwhile, 18α-glycyrrhetinic acid (18α-GA), a specific blocker of gap junction, was administered to observe its effect on endothelial wound repair.2. Effect of the blocker of gap junction on phenotypic transition in cultured rat vascular SMCs: Rat aortic SMCs (RASMCs) were cultured by explanted rat aortic wall tissue and identified with cell immunofluorescence staining for SMα-actin. Cell immunofluorescence staining was applied to detect the expressions of connexin (Cx) 43 in RASMCs. FRAP was used to measure the communications between cells via gap junctions. MTT and RT-PCR were used to measure the proliferative capability of RASMCs and the expression of smooth muscle (SM)α-actin respectively. Meanwhile, 18α-glycyrrhetinic acid (18α-GA), a specific blocker of gap junction, was administered to observe its effect on the contents above.3. Role of gap junctions on the regulation of vascular tone and the repair of vascular wound: vascular rings from rat Carotid artery were made and used to compare to the changes of vascular response to NE or Ach with or without 18α-GA. The model of vascular injury was established with rat carotid balloon injury. And animals were administrated with intraperitoneal injections of carbenoxolone (3mg/kg.d) in carbenoxolone group or saline (2ml/d) in control group for 2weeks after carotid balloon injury. After 2 weeks, HE staining and DAPI-Evens blue double staining were applied to evaluate the neointimal formation of targeted vessels. And cell immunofluorescence staining and western blot were used to detect protein Cx43 expression on targeted vessels.4. To explore the transfer of Ca2+ between endothelia cells and SMCs across gap junctions and its implication: ECs and SMCs were cultured according to the protocols above. A coculture system was made by seeding ECs and SMCs on the both side of transwell insert with millipores respectively and observed with transmission electron microscope. Then, the proliferation of SMCs was evaluated by 3H-TdR incorporation. After loading both type cells with fluo-3 and pretreating ECs with ETB receptor blocker or SMCs with ETA(10-5M) and ETB (10-5M)receptor blocker, one type cells was stimulated with ET-1 and laser confocal scanning microscope was used to observe the change of immuofluorescence intensity in unstimulated other type cells. The effect of 18α-GA or heparin on above was also observed. After stimulating ECs according to above, the expression of c-fos and c-jun mRNA in unstimulated SMCs was detected with RT-PCR.Results1. Role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair: In cultured RAECs, immunofluorescence staining for vWF was positive and Weibel-Palade bodies were found. Cx37, Cx40 and Cx47 were all expressed in RAECs. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells (5.7±0.63 % vs. 82.26±1.68%,P<0.01). Compared with in control group, mean fluorescence recovery rate in 18α-GA group were significant lower (33.58±1.73% vs. 82.26±1.68%, P<0.01). Therefore, 18α-GA could inhibit dye transfer between conjugated cells. FCS also showed that 18α-GA at a concentration of 50μM could not increase the apoptosis of RAECs (2.044 vs. 2.156, P>0.05). The scrape widths of endothelium were similar in two groups at the time of wound. However, at 24h after endothelial wound, the scrape widths in 18α-GA group were significantly bigger than those in control group(237.38±20.40μm vs. 126.29±21.40μm, P<0.05)。The times of the wound need to reach complete recover in 18α-GA group were significantly more than those in control group(4.2±0.2d vs.2.6±0.3d,P<0.05).2. Effect of the blocker of gap junction on phenotypic transition in cultured rat vascular SMCs: RASCMCs were cultured successfully with positive immunofluorescence staining for SMα-actin. At 3rd day after cultured, Cx43 was expressed in RASMCs. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells[(7.3±0.58 )% vs. ( 80.61±6.57 )%,P<0.01]. Compared to control group, mean fluorescence recovery rate in 18α-GA group were significant lower(41.43±7.62% vs. 80.61±6.57 % , P < 0.05). Therefore, 18α-GA could inhibit dye transfer between conjugate3.d cells. In this experiment, we found that 18α-GA couldn't induce the expression of Cx43 protein in RASMCs(0.85±0.06 vs 0.83±0.03,P>0.05), but 18α-GA could inhibit the proliferation of RASMCs(0.465±0.016 vs. 0.563±0.081,P<0.05 vs. control group) and promote the expression of SMα-actin in cultured RASMCs(1.384±0.13 vs.0.753±0.09,P<0.01 vs. control group).3. Role of gap junctions on the regulation of vascular tone and the repair of vascular wound: 18α-GA alone didn't cause significantly the response of vascular rings to systole or diastole. In control group, NE or Ach could induce vascular rings to contract or relax, but 18α-GA could inhibit the contraction or relaxation of vascular rings triggered by NE or Ach(NE: 0.60±0.03 vs. 0.21±0.04; Ach: 0.15±0.01 vs. 0.62±0.03; P<0.05). 2 weeks after carotid balloon injury, carbenoxolone could significantly reduce the neointimal formation and the stenosis of blood vessel lumen. When nuclei number of neointima was used to evaluate the neointimal formation, result suggested that nuclei number of neointima in carbenoxolone group was significantly lower than that in control group((89±28. 40 vs. 236±15.04,n=5,P<0.01). The expression of Cx43 protein in neointima was abundant. Western blot result also suggested that the expression of Cx 43 in carbenoxolone group was significantly lower than that in control group(0.93±0.06 vs. 0.38±0.11, n=3,P<0.01).4. To explore the transfer of Ca2+ between endothelia cells and SMCs across gap junctions and its implication: At first day after coculture, there was no significantly difference of 3H-TdR incorporation between EC/SMC and SMC/SMC(10900±1320 10430±1200, P>0.05). At second day, 3H-TdR incorporation in EC/SMC and SMC/SMC was 17200±1734 and 20900±1659 respectively. Although there were some difference, it didn't reach statistical standard. At third day, there was significantly difference on 3H-TdR incorporation between EC/SMC and SMC/SMC (25800±1984 vs. 33070±3569, P<0.05). after pretreating EC/SMC cocultured for 3d with 18α-GA for 24h, 3H-TdR incorporation was significantly higher compared to EC/SMC which was not pretreated((30500±2650l vs. 25800±1984,P<0.05). In the coculture system, [Ca2+]i in SMCs increased following stimulation of endothelia cells with ET-1, and 18α-GA could inhibit the increase of [Ca2+], but heparin not. [Ca2+]i in ECs also increased following stimulation of SMCs with ET-1, and 18α-GA inhibit the increase of [Ca2+]i and heparin could inhibit the increase of [Ca2+]i partly. The expression of c-fos and c-jun mRNA in the unstimulated SMCs increased following stimulation of endothelia cells with ET-1, compared with no stimulation(c-fos:0.957±0.051 vs. 0.682±0.038; c-jun:0.816±0.054 vs.0.467±0.032;P<0.01)。.Conclusions1. There are intercellular communications via gap junctions among conjugated RAECs and this kind of intercellular communication might regulate the process of endothelial wound repair.2. Intercellular communications via gap junctions could influence smooth muscle cell proliferation and phenotypic transition of SMCs from synthetic to contractile state.3. Gap junctions participate in maintaining and regulating vascular tone under the physiological condition, and promote the neointimal formation after vascular injury under the pathological condition. Hence, gap junctions play a key role on the development and progression of vascular injury diseases.4. Ca2+ can flow between ECs and SMCs across gap junction and is an important signal mediator coordinates the behavior of ECs and SMCs. Ca2+ flow from SMCs to ECs need the involvement of IP3, but which don't involve in Ca2+ flow from ECs to SMCs. Ca2+ flowing into SMCs from ECs via gap junctions increase the expression of c-fos and c-jun in SMCs.