Effects Of VEGF Pre-conditioning On The Therapeutic Effects Of Bone Marrow Mononuclear Cells Transplantation In A Rat Model Of Chronic Cerebral Hypoperfusion Funded By NSFC

Chronic cerebral hypoperfusion(CCH)plays an important role in the pathogenesis and pathologic process of vascular dementia(VD)and Alzheimer disease(AD).The morbidity of VD and AD is rising year by year in China because of the accelerating aging population.VD and AD account for over 85%dementia in China and has become a serious medical and social problem.Exploring the therapeutic targets for CCH may provide promising new methods for the VD and AD treatment.Bone marrow mononuclear cells(BMMNCs)is a bunch of mononuclear cells that locate in bone marrow.BMMNCs contain many therapeutic cells such as stem/progenitor cells and immunocyte cells.Intravenously BMMNCs transplantation has been proved to be effective in acute ischemic stroke and has enter phaseⅡclinical research.My previous research has also demonstrated that intravenously BMMNCs transplantation can migrate into the ischemic brain of CCH rat model and play its neuroprotective roles via angiogenesis,illustrating that BMMNCs may be a new method for the CCH treatment.However,the efficiency of intravenously BMMNCs transplantation is very low.After the transplantation,most BMMNCs are blocked in the periphery organs,and only a small part of the BMMNCs can migrate into the ischemic brain.Blood brain barrier(BBB)permeability is vital for the BMMNCs migration towards the brain,and properly increasing the BBB permeability may accelerate more BMMNCs to migrate to the brain and enhance the therapeutic effects of intravenously BMMNCs transplantation in CCH rats.Vascular endothelial growth factor(VEGF)is a well-known neuroprotective cytokine which is widely expressed in the brain.VEGF can increase the BBB permeability,which is a fundamental process for the VEGF-induced neurogenesis.Previous research has indicated that proper dosage of exogenous VEGF can increase the BBB permeability without affecting the brain structure and function.In this study,firstly,we would explore the different dosage of VEGF pre-conditioning on the BBB permeability of CCH rats,aimed to find the proper dosage of VEGF-preconditioning;Secondly,we would explore the effects of VEGF pre-conditioning on the bio-distribution of BMMNCs after intravenously transplantation in CCH rat model,to determine if VEGF pre-conditioning can accelerate the brain migration of BMMNCs,or its effects on the periphery distribution/immunoregulation of BMMNCs;Finally,we would explore the effects of VEGF pre-conditioning on the therapeutic effects of BMMNCs transplantation in CCH rat model by assessing the angiogenesis,neurogenesis,neural degeneration and neurologic functions.The goal of this study is to find an effective way to enhance the therapeutic effects of BMMNCs transplantation in CCH.PartⅠEffect of VEGF pre-conditioning on the BBB permeability of CCH ratsObjective:To explore different dosage of VEGF pre-conditioning on the BBB permeability of CCH rats.Methods:We established CCH model by applying classic bilateral common artery occlusion(2-vessel occlusion,2VO)methods in SD rats.In this part,rats were randomly divided into three groups:sham rats treated with vehicle(vehicle),2VO rats treated with vehicle(2VO),2VO rats treated with recombinant rat VEGF(VEGF).The VEGF group were divided into five sub-groups and treated different dosage of VEGF(0.4μg/kg,0.6μg/kg,0.8μg/kg,1μg/kg and 1.2μg/kg).The VEGF or normal saline were injected via intracerebroventricular injection(icv).We assessed the brain edema and neurologic functional changes in the rats of different groups by using dry-wet weight method and Morris Water Maze(MWM)test.The highest dosage of VEGF that did not affect brain water content and neurologic function were chosen as the proper dosage of VEGF pre-conditioning in this study.Then we assessed the BBB permeability in rats of different groups by detecting changes of tight junction proteins ZO-1 and claudin-5,ZO-1-positive vessels,ALB staining and Evans blue leakage in the ischemic brain by using Western blot analysis,Immunofluorescence staining and spectrophotometric method.Results:Compared with the 0.4μg/kg,0.6μg/kg and 0.8μg/kg VEGF sub-groups,the brain edema and neurologic function were significantly worsened in the 1μg/kg and 1.2μg/kg VEGF group,the differences were statistically significant(P<0.05).Based on this finding,we selected the 0.8μg/kg as the target dosage of VEGF pre-conditioning in this study.We found that rats in the VEGF(0.8μg/kg)group had significant lower ZO-1 and claudin-5 expressions,lower ZO-1-positive vessels and more ALB or Evans blue leakage in the brain than that of the vehicle or 2VO groups.Conclusion:0.8μg/kg VEGF pre-conditioning can significantly increase the BBB permeability of 2VO rats without inducing brain edema or worsening neurologic function.PartⅡEffects of VEGF pre-conditioning on the bio-distribution of BMMNCs in ischemic brian after intravenously transplantation in CCH rat modelObjective:To explore the effects of VEGF pre-conditioning(0.8μg/kg)on the bio-distribution of BMMNCs after intravenously transplantation in CCH rat model,to determine if VEGF pre-conditioning can accelerate the brain migration of BMMNCs,or its effects on the periphery distribution/immunoregulation of BMMNCs.Methods:We established CCH model by applying 2VO methods in SD rats.We collected the BMMNCs from the femur and ilium of e-GFP transgenic SD rats or normal SD rats by using gradient centrifugation method.The BMMNCs(1.2×10~7BMMNCs/kg)were transplanted into rats via tail vein injection.In this part,rats were randomly divided into six groups:sham rats treated with vehicle(sham+vehicle),sham rats treated with BMMNCs transplantation(sham+BMMNC),2VO rats treated with vehicle(2VO+vehicle),2VO rats treated with BMMNCs transplantation(2VO+BMMNC),2VO rats treated with recombinant rat VEGF(0.8μg/kg)and BMMNCs transplantation(2VO+VEGF+BMMNC)and 2VO rats treated with VEGFR inhibitor SU5416(4nM)and BMMNCs transplantation(2VO+SU5416+BMMNC).All rats were treated with icv injection(normal saline,VEGF or SU5416)and tail vein injection(DMEM or BMMNCs suspension).We detected the eGFP-positive BMMNCs in the brain and lung of rats in each group at 6 hour after cell transplantation,and detected the changes of IL-10、L-1βand TNF-αin serum by using ELISA test.Results:VEGF pre-conditioning(0.8μg/kg)significantly increased the brain migration of BMMNCs,and VEGFR inhibitor SU5416 significantly abolished this effect.VEGF pre-conditioning(0.8μg/kg)had no impact on the lung migration and serum changes of IL-10、L-1βand TNF-αlevels that induced by BMMNCs transplantation.Conclusion:VEGF pre-conditioning(0.8μg/kg)can significantly accelerate brain migration of BMMNCs after transplantation and had no effects on the lung migration or BMMNCs-induced periphery immunoregulation(IL-10、L-1βand TNF-αlevels).PartⅢEffects of VEGF pre-conditioning on the therapeutic effects of BMMNCs transplantation in CCH rat modelObjective:To explore the effects of VEGF pre-conditioning(0.8μg/kg)on the therapeutic effects of BMMNCs transplantation in CCH rat model by assessing the angiogenesis,neurogenesis,neural degeneration and neurologic functions.Methods:We established CCH rat model by applying 2VO methods and collected the BMMNCs from the femur and ilium of SD rats by using gradient centrifugation method.The BMMNCs(1.2×10~7 BMMNCs/kg)were transplanted into rats via tail vein injection.In this part,rats were randomly divided into six groups:sham rats treated with vehicle(sham+vehicle),sham rats treated with BMMNCs transplantation(sham+BMMNC),2VO rats treated with vehicle(2VO+vehicle),2VO rats treated with BMMNCs transplantation(2VO+BMMNC),2VO rats treated with recombinant rat VEGF(0.8μg/kg)and BMMNCs transplantation(2VO+VEGF+BMMNC)and2VO rats treated with VEGFR inhibitor SU5416(4nM)and BMMNCs transplantation(2VO+SU5416+BMMNC).All rats were treated with icv injection(normal saline,VEGF or SU5416)and tail vein injection(DMEM or BMMNCs suspension).We detected the neurologic function by using MWM test,angiogenesis(BrdU-positive vessels),neurogenesis(DCX-positive cells within SVZ)and neural apoptosis(FJB-positive cells)by using immunofluorescence analysis in rats of different groups to explore the effects of VEGF-preconditioning on the therapeutic effects of BMMNCs transplantation in CCH rat model.Results:VEGFpre-conditioning(0.8μg/kg)significantlyenhancedthe neuroprotective effects induced by BMMNCs transplantation;Compared with the rats from2VO+BMMNC,ratsin2VO+VEGF+BMMNCshowedincreased BrdU/Lectin-positive vessels,up-regulated DCX-positive cells within SVZ and less FJB-positive cells in the ischemic brain,the differences were statistically significant.SU5416 significantly abolished BMMNCs-induced angiogenesis,neurogenesis and neural protection in 2VO rats.Conclusion:VEGF pre-conditioning(0.8μg/kg)can significantly enhance the therapeutic effects of BMMNCs transplantation in CCH rat model.