Research On The Proteomic Investigation And Regulation Of Neointima Development On Xenogeneic Small Diameter Vascular Graft

Background: There is a growing clinical demand for small-diameter tissue-engineered vascular grafts as materials for blood flow reconstruction in coronary and peripheral vascular disease,and as arteriovenous fistula vessels in dialysis access for end-stage renal disease.Many advances have been made in recent years in the investigation of small-diameter tissue-engineered vascular grafts,but in general they are far from clinical needs.The low rate of post-transplant vascular patency is mainly related to factors such as protein adsorption secondary to thrombosis,neointima formation,and incomplete endothelialization during the acute phase.Among them,neointima formation is the initiating problem of the series,and endothelialization is the key to solve the series.The formation and proliferation of neointima is an unavoidable pathophysiological process that causes narrowing of the graft lumen and,in more severe cases,luminal occlusion resulting in graft failure.For this reason,it is important to elucidate the mechanism of neointima formation in small-diameter artificial vessels and the subsequent techniques to be used to manage neointima formation and proliferation in small-diameter artificial vessels to prevent luminal stenosis and improve the long-term patency.Objectives:(1)To understand the process of neointima formation in small-diameter tissue-engineered vascular grafts in vivo,and the characteristics and changes at the cellular and protein levels in small-diameter artificial blood vessels;(2)To investigate the target mechanisms and methods for regulating neointima formation and proliferation in small-diameter blood vessels in vivo: anticoagulation,regulation of inflammatory response and promotion of beneficial regeneration;(3)To explore whether heparin nanoparticles loaded with anti-inflammatory factors in small-diameter tissue-engineered vascular grafts can effectively inhibit neointima formation and proliferation and promote regeneration.formation and proliferation and promote the possibility of regeneration.Methods: This study was divided into two major parts and two animal models: Part I: including the contents of Chapters 2 and 3.First,we used combined decontaminants and DNase to treat bovine internal mammary arteries to prepare xenogeneic small caliber decellularized vascular scaffolds;and verified the decellularization effect by detecting the DNA content of the decellularized vessels,improved the mechanical properties of decellularized bovine internal mammary arteries using photo-oxidative cross-linking,and implanted into rabbit abdominal aorta,and collect the neointima of the grafts at 1,4,7,14,21 and 28 days after surgery,and combine histological,proteomic and bioinformatics analysis to explore the process of neointima formation and proliferation in vivo in xenogeneic small caliber vessels,and its inner cell and protein level characteristics and changes to seek regulatory targets;Part II: includes the contents of Chapter 4,based on the results of Part I,optimizing The graft was implanted into the rat abdominal aorta,and the internal diameter,blood flow and morphological changes of the graft were continuously detected by Doppler ultrasound.The histological and PCR techniques were applied to observe the neointima remodeling,cellular infiltration and inflammatory factor expression in the graft on days 7 and 28,to investigate and verify the role and effects of combining anticoagulation and anti-inflammatory dual biological factors to regulate neointima formation in vivo.Results: Part I: Including the results in Chapters 2 and 3: Bovine internal mammary arteries can be treated with decellularization,cellular components can be removed sufficiently,biocompatibility is good,there is some degree of damage to the extracellular matrix,the biomechanical properties of the decellularized vessels can be complemented by cross-linking,and their suture tension strength and burst pressure are close to the properties of natural arteries.The results after New rabbit abdominal aortic xenografts revealed that there is an intrinsic regularity in the formation and evolution of neointima in xenograft small-caliber vascular grafts,associated with the processes of plasma protein adsorption and activation of the initial inflammatory response,neutrophil aggregation,natural regression,macrophage and stromal cell infiltration,and tissue remodeling;divided into 3 phases,acute instability: acute inflammation and thrombosis;early stability: Acute inflammatory cells recede and the structure of the neointima layer becomes stable;late stable remodeling phase: ECM and stromal layer remodeling.Combining proteomic and bioinformatics analysis,a total of 154 proteins were expressed in the neointima of the acute unstable phase and the neointima of the stable period,as well as in the plasma samples: dominating the cascade response of the coagulation and complement systems and the inflammatory response;and 104 upregulated differential proteins in the stable phase: mainly involved in the remodeling of the extracellular matrix.Part II: Results from Chapter 4: Successful optimization of the construction of xenogeneic small caliber vessels loaded with anticoagulant heparin and anti-inflammatory transforming growth factor complex cross-linked with good biocompatibility and biomechanical properties,slow release of low molecular heparin as well as TGF-β1;reduced infiltration of inflammatory type cells and stromal cells in the inner neointima of rat infrarenal abdominal aortic vessels after transplantation was found,and pro-inflammatory factors Expression of pro-inflammatory factors was reduced,while there was no excessive deposition of regenerated collagen and the luminal surface was covered by endothelial epithelial cells at a later stage,which attenuated the neointimal hyperplasia and provided excellent vascular patency.Conclusion: Decellularized small-diameter vascular scaffolds with good biocompatibility and biomechanical properties were successfully prepared by combined decellularization treatment method,and in combination with animal models,it was found that the short-term formation and proliferation process of neointima in unmodified xenogeneic small-diameter vessels mainly involved plasma proteins,coagulation,inflammatory response and stromal regenerative remodeling.Combined cross-linked decellularized small-diameter vessels loaded with chitosan heparin nanoparticles and transforming growth factor reduced inflammatory cell infiltration and excessive collagen deposition,regulated excessive proliferation of neointima,promoted stable neointima to facilitate endothelial regeneration,and improved the patency of small-diameter vessels.It provides a new theoretical basis for the preparation of tissue-engineered artificial small vessels and the regulation of neointima formation.The composite xenogeneic small-diameter vascular graft have good clinical application prospects.