Studies Of Transplantation Of Endothelial Progenitor Cells Transfected By ENOS Gene On Vascular Endothelium Repair

Backgroud and objectiveCardiovascular disease is the leading cause of morbidity and mortality. Endothelial injury and dysfunction plays an important role in atherosclerotic physiological and pathophysiological processes. Vascular endothelial cells (ECs) not only provides a critical interface between the vessel itself and blood element, but also release a variety of bio-actively substance. In the normal state, vascular endothelium presents a nonadhesive surface to circulating leucocytes and platelets while helping to prevent the clotting of blood, regulate vascular tone through the release of nitric oxide production and vascular relaxation. The injury and apoptosis of ECs induced by cardiovascular risk factors damage vascular integrality. Macrophage adhesion and invasion, and smooth muscle cells migration and growth finally lead to stenosis of the targeted vessel. Accelerated reendothelialization effectively inhibit smooth muscle cells proliferation and neointima formation and therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis after angioplasty.Vascular endothelium repair need the proliferation, migration of neighboring mature ECs. However, mature ECs have lower proliferation rate and limited repair ability. Endothelial progenitor cells (EPCs) are the precursor of ECs, expressing surface antigens of both stem cells and endothelial cells. In culture, EPCs acquire both morphological and functional characteristics of endothelial cells. Bone-derived EPCs can be mobilized into peripheral blood and incorporate into sites of vascular injury, proliferate and differentiate into endothelial cells. These EPCs accelerate the regeneration of new endothelium.Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) is critical for the maintenance of cardiovascular homeostasis. NO exerts potent vasodilation, antiplatelet, anti-inflammatory, anti-proliferation, and cytoprotective effects on the vessel wall. Recent studies have revealed that eNOS play important role in EPCs mobilization, differentiation, and homing. Experimental studies suggest that something including VEGF, statins, estrogen, erythropoietin (EPO) can mobilize EPCs, promote reendothelialization and prevented neointima formation. They play such effects via a pathway involving upregulation eNOS. However, eNOS knockout or NOS inhibitor L-NAME have impaired mobilization of EPCs. We postulate that transplantation of EPCs overexpressing vasculoprotective genes such as eNOS may have therapeutic potential as a strategy to enhance the biological properties of reconstituted endothelium.Methods1. Identifying and amplifying retrovirus containing human eNOS gene by infecting 293 cells repeatly.2. EPCs culture and characterizationBone marrow mononuclear cells of adult rats were isolated by Ficoll gradient centrifugation. Cells were plated on culture dishes coated with human fibronectin and maintaine in M199 supplemented with 20% fetal-calf serum (VEGF 50ng/mL, b-FGF 5ng/mL, EGF 10ng/mL). cell growth and morphology were evaluated. Identify the cell phaenotype eNOS, vWF by immunofluorescence. To observe the function of EPCs, FITC-ulex europaeus agglutinin(UEA-I) binding and DiI-LDL intake are performed.3. effect of eNOS gene modified on EPCs biological functionPrimary cultured EPCs were infected with the retrovirus containing eNOS. Following up expression of GFP, we detected the infective efficiency, and the positive rate of GFP expression at 24h, 48h and 72h after retrovirus infection. RT-PCR and Western blot were used to detect the level of eNOS mRNA and protein in the infected EPCs to ensure eNOS overexpression as a result of retrovirus infection. EPCs proliferation and migration ability were observed and adhesion assay was performed.The concentration of NO and VEGF in culture medium were measured with method of nitrate reductase and ELISA.4. effect of eNOS gene modified EPCs on ECs and SMCs migration and proliferationIn the cell coculture system, eNOS gene modified EPCs were seeded in the lower chamber and SMCs or ECs in the upper chamber. Flow cytometry analysis and MTT were used to determine the effects of EPCs on the proliferation of SMCs or ECs. The number of SMCs or ECs migration was counted.5. effect of eNOS gene modified EPCs transplantation on vascular endothelium repair The carotid arteries intima injury model were made by balloon damage in SD rats. eNOS gene modified EPCs were injected by intravenous tail vein after induction of arterial injury. The injured segments were isolated 2 weeks after EPCs transplantation. RT-PCR and Western blot were used to detect the level of eNOS mRNA and protein in the injured artery. Thirty minutes before euthanasia, rat received Evans blue intravenously. The morphology of arterial intima and media was studied by optical microscopy and image analysing system. The distribution of GFP labeling EPCs were traced. Endothelium-dependent vasodilation response (EDVR) was determined by acetylcholine (Ach)–induced vasodilation response in isolated carotid arteries.Result1. Following repeatedly infecting 293 package cell line, high titer retrovirus particles yielded.2. EPCs culture and characterization 3-5 days later, cells number increased and cell body stretched. Oval-shaped or spindle like cells appeared which accompanied with some cell process. 7-10 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 20 days culture, cells mixed together, resulting in cobble-stone morphology. EPCs expressed eNOS and vWF phaenotype. EPCs can bind FITC-UEA-I and intake acLDL. EPCs can release nitric oxide.3. effect of eNOS gene modified on EPCs biological function(1) EPCs transfection efficiency and eNOS mRNA and protein expression in EPCs after transfectionAfter pMSCV-eNOS and pMSCV-GFP transfection, the positive rate of GFP expression in EPCs was growing up in time-dependent manner. There was no more increase in the positive rate of GFP expression as the expression time exceeded 72h. 72h after infection, the percent of EPCs expressing GFP exceeded 90%. Findings with RT-PCR and Weatern blot showed that infection of retrovirus resulted in eNOS overexpression in EPCs.(2) effect of eNOS overexpression on EPCs proliferationMTT analysis showed absorbance increased in pMSCV-eNOS group ( pMSCV- eNOS group vs control group, 0.582±0.014 vs 0.357±0.009,P<0.01), (pMSCV- GFP group vs control group, 0.365±0.009 vs 0.357±0.009,P>0.05). Whereas NOS inhibitor L-NAME decreased the increase on EPCs proliferation (pMSCV-eNOS group vs L-NAME group, 0.582±0.014 vs 0.46±0.009,P<0.01) .(3) effect of eNOS overexpression on EPCs migrationThe number of migrated EPCs increased in pMSCV-eNOS group ( pMSCV- eNOS group vs control group, 20.50±1.87 vs 12.17±0.75, P<0.01), (pMSCV- GFP group vs control group, 12.33±1.03 vs 12.17±0.75, P>0.05). L-NAME decreased the increase on EPCs migration (pMSCV-eNOS group vs L-NAME group, 20.50±1.87 vs 15.50±1.05,P<0.01).(4) effect of eNOS overexpression on EPCs adhesionThe number of adhesion EPCs increased in pMSCV-eNOS group ( pMSCV- eNOS group vs control group, 42.00±1.41 vs 30.67±0.82,P<0.01), (pMSCV- GFP group vs control group, 30.67±0.82 vs 30.00±1.41,P>0.05). L-NAME decreased the increase on EPCs adhesion (pMSCV- eNOS group vs L-NAME group, 42.00±1.41 vs 34.83±1.17,P<0.01).(5) effect of eNOS overexpression on EPCs releasing NOThe concentration of NO in pMSCV-eNOS group was significantly higher than control groups (68.32±1.46 vs 35.13±1.28, P<0.01). L-NAME decreased the increase on NO (pMSCV- eNOS group vs L-NAME group, 42.00±1.41 vs 34.83±1.17, P<0.01).(6) effect of eNOS overexpression on EPCs releasing VEGFThe concentration of VEGF in pMSCV-eNOS group was significantly higher than control groups (203.47±16.26 vs 153.71±14.17, P<0.01). L-NAME decreased the increase on VEGF (pMSCV- eNOS group vs L-NAME group, 203.47±16.26 vs 174.96±14.50, P<0.01).4. effect of eNOS gene modified EPCs on ECs and SMCs migration and proliferationeNOS gene modified EPCs can promote ECs proliferation (eNOS-EPCs group vs control group, 0.482±0.009 vs 0.361±0.009,P<0.01 ) and migration (eNOS-EPCs group vs control group, 17±1 vs 11±1, P<0.01). Compared with the control group, the transition of ECs from G1 phase to S phase was promeoted in eNOS-EPCs group (eNOS-EPCs group vs control group, 62.3±2.32 vs 43.9±1.21, P<0.01). eNOS gene modified EPCs can inhibit SMCs proliferation (eNOS-EPCs group vs control group, 0.247±0.006 vs 0.350±0.004, P<0.01 ) and migration (eNOS-EPCs group vs control group, 11±1 vs 26±1, P<0.01). Compared with the control group, the transition of SMCs from G1 phase to S phase was inhibited in eNOS-EPCs group (eNOS-EPCs group vs control group, 8.61±0.74 vs 15.08±1.96, P<0.01).5. effect of eNOS gene modified EPCs transplantation on vascular endothelium repairThe injured segments were isolated 2 weeks after EPCs transplantation. Findings with RT-PCR and Western blot in injured artery showed that eNOS was overexpressed in eNOS-EPCs transplantation group. Immunohistochemistry detection showed that eNOS expression was significantly enhanced in eNOS-EPCs transplantation group. EPCs with GFP were found in injured sites. Re-endothelialization increased and the neointima formation relieved in EPCs transplantation group. EDVR increased significantly in EPCs transplantation group compared with injury group. Furthermore, the effect of EPCs was further enhanced by overexpression of eNOS.Conclusion1. Retrovirus could infected EPCs safety and high efficiency. After transfection, EPCs expressed eNOS mRNA and protein. eNOS gene modification can promoted EPCs proliferation, migration, adhesion and increased EPCs releasing NO and VEGF. NOS inhibitor L-NAME could inhibited the effect of eNOS gene modification on EPCs.2. eNOS gene modified EPCs could improve ECs proliferation and migration and inhibit SMCs proliferation and migration.3. EPCs transplantation could home into the vascular injury site . EPCs could inhibit neointima hyperplasia and accelerate reendothelialization of denuded vessel. And EPCs transplantation could improve significantly EDVR. Furthermore, the effects of EPCs were further enhanced by overexpression of eNOS, suggesting that transplantation of genetically modified EPCs may have potential as a therapeutic strategy for vasculoproliferative diseases.