VEGF Regulates Macrophage Polarization To Improve The In Vivo Performance Of Decellularized Small Diameter Vascular Grafts

Background:Coronary heart disease seriously endangers human health.Coronary artery bypass grafting is one of the main modalities for the treatment of coronary artery disease,and autologous vessels are the gold standard,but are available in limited quantities.Therefore,there is an urgent clinical need for small-diameter(<6 mm)artificial vessels,and unfortunately,no satisfactory products are available at this time.Currently,small-diameter vascular materials perform poorly after implantation,often with problems such as neointimal hyperplasia,thrombosis,aneurysm formation,and rejection reactions ultimately leading to vascular graft failure.Tissue-engineered vascular grafts(TEVG)are a promising vascular alternative.Allogeneic decellularized matrix vascular grafts have good biocompatibility and mechanical properties and can be used as materials for constructing small-diameter TEVGs,but they contain allogeneic antigens that may stimulate adverse immune responses in vivo.Thus,adequate elimination of the immunogenicity of heterogeneous vessels is the basis for the construction of TEVG.Neointimal hyperplasia can lead to early stenosis and occlusion of small diameter TEVG,while the degree of vascular regeneration and remodeling determines the long-term patency of small diameter TEVG.Macrophages are early response cells after implantation of biomaterials and can be classified into M1 and M2 types.M2 type macrophages can release anti-inflammatory factors,reduce TEVG neointimal hyperplasia and promote tissue regeneration.Recent studies have found that vascular endothelial growth factor(VEGF)can regulate macrophage M2 polarization.Therefore,in this study VEGF will be used to regulate macrophage M2 polarization to improve the in vivo performance of decellularized small diameter vascular grafts(neointimal hyperplasia,vascular regeneration and remodeling).Objective:1.To investigate the effect of different decellularization protocols on the immunogenicity of bovine internal mammary arteries and to prepare low immunogenicity decellularized bovine internal mammary arteries,which will provide a suitable source of material for the construction of small caliber TEVG;2.To evaluate the effect of VEGF on macrophage phenotype by cellular assay;Effect of VEGF on macrophage phenotype and inflammatory factor expression after decellularized bovine internal mammary arteries implantation in vivo will be evaluated by establishing subcutaneous implantation and infrarenal abdominal aortic replacement models in New Zealand white rabbits;3.The effect and mechanism of VEGF on neointimal hyperplasia,vascular regeneration and remodeling after decellularized bovine internal mammary arteries implantation will be explored by establishing a New Zealand white rabbit infrarenal abdominal aortic replacement model.Methods:1% SD,1% SDS,1% Triton X-100,0.5% SDS+0.5% Triton X-100,0.5% SDS+0.5% SD,0.5% SD+0.5% Triton X-100 were used to decellularize the bovine internal mammary artery.The decellularized internal mammary arteries were examined for α-Gal content,MHC-Ⅰcontent,non-specific antigen content,DNA content,HE staining,DAPI staining,EVG staining,Masson staining,collagen content,elastin content,scanning electron microscope,mechanical properties,cytotoxicity test.Rat subcutaneous implant models were established,graft-specific antibodies,subcutaneous implant fibrous capsule thickness and macrophage and lymphocyte counts were measured.RAW264.7 cells were induced by VEGF in vitro,and RAW264.7cell viability was detected by CCK-8,cell phenotypic changes were detected by flow cytometry,and m RNA expression of related inflammatory factors was detected by Real time PCR.New Zealand white rabbit subcutaneous implantation model and infrarenal abdominal aortic replacement model were established,and decellularized bovine internal mammary arteries were implanted.rhVEGF-165 was delivered to the vascular grafts by adventitial administration,and the observation time points: 7 days and 28 days.The thickness of the neointima and the thickness of the fibrous capsule of the vascular grafts were measured,and the number of M0(CD68),M1(CD86)and M2(CD206)macrophages and the expression of pro-inflammatory factors(IL-1)and anti-inflammatory factors(IL-10)in the grafts were detected by immunofluorescence staining.New Zealand white rabbit infrarenal abdominal aortic replacement model was established,and VEGF was delivered to the vascular grafts by adventitial administration,and the observation time points: 7 days,28 days and 90 days.HE staining,EVG staining,Masson staining,Sirius red staining,immunofluorescence staining,mechanical properties,collagen content,and elastin content detection were performed on vascular grafts.Vascular ring experiment was used to detect the contraction and relaxation function of vascular grafts.Immunofluorescence staining will be used to detect the number of smooth muscle cells(α-SMA,Calponin and SM-MHC),endothelial cells(CD31),M0(CD68),M1(CD86),M2(CD206)type macrophages and the expression of pro-inflammatory factors(IL-1,TNF-α)and anti-inflammatory factors(IL-4,IL-10)in the grafts.In vitro,the conditioned medium was collected after VEGF-induced polarization of RAW264.7 cells,and human vascular smooth muscle cells were stimulated with the conditioned medium,and human vascular smooth muscle cells were assayed for viability by CCK-8and migration ability by scratch assay.Results:All decellularization protocols were effective in removing antigenic molecules MHC I and α-Gal;1% SDS,0.5% SDS+0.5% SD,0.5%SDS+0.5% Triton decellularization protocols were effective in removing cell nuclear components;The 1% SDS and 0.5% SDS+0.5% SD decellularization protocols had the strongest ability to remove heterologous antigens,while the 1% Triton protocol is the weakest;The1% SDS decellularization protocols had the greatest damage to the extracellular matrix and biomechanical properties,while the remaining protocols had no significant effects;All decellularization protocols had no significant cytotoxic effects;1% SDS and 0.5% SDS+0.5% SD-treated vascular slices produced the lowest levels of graft-specific antibodies,the thinnest thickness of fibrous capsule,and the lowest numbers of macrophages and lymphocytes in rats.In cellular assays,rhVEGF-165 was not toxic to RAW 264.7macrophages at concentrations up to 50 ng/ml,and showed significant inhibition of cell growth at concentrations up to or above 100 ng/ml;flow cytometry analysis showed that different proportions of M2 macrophages were detected at different concentrations of rhVEGF-165 stimulation,with 50 ng/ml rhVEGF-165 treatment resulted in the highest proportion of M2;Real time PCR measured m RNA expression,and the expression levels of IL-1β and TNF-α were significantly reduced in rhVEGF-165-induced RAW 264.7 macrophages compared with LPS,while the expression levels of CD206 and IL-10 were significantly increased;in the New Zealand rabbit subcutaneous implantation model,compared with the control group,the fibrous capsule thickness decreased,the number of CD206-positive cells and the ratio of M2/M1 macrophages increased,and the expression of anti-inflammatory factors increased and the expression of pro-inflammatory factors decreased in the VEGF group;in the vascular replacement model,compared with the control group,the fibrous capsule thickness and neointima thickness decreased,the number of CD206-positive cells and the ratio of M2/M1 macrophages increased,and the expression of anti-inflammatory factors increased and expression of pro-inflammatory factors decreased.In the New Zealand white rabbit infrarenal abdominal aortic replacement model,at 90 d the vascular grafts in the control group appeared significantly dilated,and their inner diameter was larger than that of the VEGF group and the rabbit abdominal aorta;the vessel wall in the control group was thicker than that of the VEGF group;the number of smooth muscle cells and endothelial cells in the VEGF group was more than that of the control group;the vasoconstriction and diastolic functions in the VEGF group were better than those in the control group;The collagen content in the VEGF group was lower than that in the control group,while the elastin content was higher than that in the control group,and the Young’s modulus and maximum tensile strength were higher in the VEGF group than the control group;the number of CD206-positive cells and the ratio of M2/M1 macrophages increased in the VEGF group,the expression of anti-inflammatory factors increased and the expression of pro-inflammatory factors decreased,and CD206 was also highly expressed in the region of SM-MHC positive high expression at the same time;in cellular experiments,the VEGF-induced macrophage conditioned medium promoted proliferation and migration of human vascular smooth muscle cells.Conclusion:1.Bovine internal mammary arteries treated with 0.5% SDS+ 0.5%SD decellularization protocols have complete cellular component removal,low immunogenicity,and well-preserved extracellular matrix,which are ideal materials for constructing small diameter TEVG;2.VEGF regulates macrophage M2 polarization,inhibits inflammatory responses,attenuates neointimal hyperplasia,and promotes smooth muscle cell regeneration and vascular remodeling after decellularized bovine internal mammary arteries implantation in vivo.In conclusion,VEGF can improve the in vivo performance of decellularized bovine internal mammary arteries by regulating macrophage polarization.