The Effect Of Inflammation Resolution And Nerve Reconstruction On Long-term Patency Of Small-diameter Tissue Engineering Blood Vessels

Cardiovascular disease is the biggest threat to human health.Ischemic heart disease is at the top of the list of cardiovascular diseases and kills 7.3 million people every year around the world.The demand for small-diameter tissue engineering blood vessels(TEBVs < 6 mm)is growing because of coronary artery bypass grafting(CABG),hemodialysis or peripheral vascular occlusion and so on.However,there has been no report on the application of small-diameter TEBVs in clinical products.The main reason is that small-diameter TEBVs are prone to five problems after transplantation: acute thrombosis,endometrial hyperplasia of the anastomotic site,aneurysm formation and infection of aneurysm,progression of atherosclerosis and calcification.Therefore,the construction of small-diameter TEBVs that can achieve long-term patency is still a difficult problem in the field as well as a global challenge.The inflammatory reaction caused by small-diameter TEBVs is responsible for vascular thrombosis and hyperplasia of endometrium,and the accepted solution is currently to promote engineering blood vessel's endothelium.Endothelial progenitor cells(EPCs)are targeted cells to promote the endothelialization of TEBVs in body,inducing its homing and differentiation into endothelial cells is a strategy of TEBVs construction in vivo.Studies have shown that acute inflammatory reactions can promote the occurrence of tissue regeneration and mobilize the EPCs in bone marrow,and the timely inflammation resolution is necessary for the survival of homing stem cells.Therefore,timely promotion of inflammation resolution may play an important role in the endothelialization of TEBVs and the maintenance of its long-term patency.Studies have found that the neural protuberant orientation factor 1(netrin-1)is expressed in natural vascular endothelial cells,which can react with UNC5 B and DCC receptors to promote angiogenesis,and have anti-inflammatory effect.We intend to construct small-diameter TEBVs modified by netrin-1 in a layered self-assembly way,and study its role and mechanism in regulating macrophage phenotype and inflammation resolution of small-diameter TEBVs through a large number of in vivo and in vitro experiments.Small-diameter TEBVs modified by netrin-1 promoted inflammation resolution in time and completed endothelialization successfully,which maintained patency for 14 months after implantation in normal rats.However,after implantation in diabetic rats,the rate of vascular calcification in TEBVs prone to atherosclerosis is extremely high due to the disorder of lipid metabolism,which directly affects the patency and function of transplanted blood vessels.Small-diameter TEBVs is a useful method for the treatment of small and medium-sized vascular diseases in diabetes.Therefore,how to prevent TEBVs vascular calcification and promote long-term patency under high glucose is the key problem to be solved in the treatment of diabetic vascular diseases by TEBVs transplantation.In autologous environment,besides the capillaries,almost all of the human body blood vessels are innervated by the sympathetic vasoconstrictor fiber,In our previous study,we found that the nerves growth was positively correlated with the smooth muscle reconstruction of the TEBVs,but the mechanism was still unclear.Therefore,in-depth study on the role of nerve reconstruction in small-diameter TEBVs has great importance.Therefore,based on above scientific questions,we conducted in-depth studies on the significance of inflammation resolution and nerve reconstruction on TEBVs construction on four aspects,which can be anti-thrombotic,anti-hypercalcification and maintaining long-term patency.1.The effect of netrin-1 on promoting inflammation resolution of small-diameter TEBVs and its mechanism studiesWe cross-linked chitosan nanoparticles packaging netrin-1 in surface of TEBVs using layers by layers method,which made TEBVs with good mechanical strength and long stable functional molecules slow-release ability to solve the inflammation and thrombosis problem.The effect of netrin-1 on phenotypic transformation of macrophages was measured in vitro by flow-cytometry and immunofluorescence,and the supernatant inflammatory factors were further detected to verify the conversion efficiency.In vivo,the inflammatory cells infiltration in situ and the effect of netrin-1 on inflammation resolution were detected by immunofluorescence.It was found that netrin-1 acted on the A2 b receptor to activate the PPAR? signaling pathway in macrophages,thus reprogramming them into an anti-inflammatory type with strong expression of CD163.After the netrin-1-modified TEBVs were transplanted into rats,it was found that netrin-1 could promote the infiltrating macrophages transform into anti-inflammatory type in situ at 3-7 days after vascular implantation,and then flow out of the intima surface at 14 days.Thus,TEBVs achieved early inflammation resolution and provide good regeneration microenvironment for the homing stem cells.2.The effect of inflammation resolution on small-diameter TEBVs' endothelialization and its mechanism studiesThis part thoroughly studied the influence of macrophages,which play an important role in inflammation resolution,on the proliferation,migration and angiogenesis of EPCs.On this basis,the relationship between inflammation resolution and endothelialization of small-diameter TEBVs was studied in rats and the mechanism was explored.Through a lot of contrast experiment,we found an ultra-high speed centrifugal method with sucrose cushion for extracting M? exosomes,which cost low time and money and can obtain high purity exosomes.EPCs were treated by exosomes derived from distinct phenotypes of macrophages,and its effect was observed.The results found exosomes derived from pro-inflammatory type M? could enhance the EPC migration ability,but were harmful to the proliferation.Exosomes derived from anti-inflammatory type M? could obviously improve the EPC proliferation and tube formation ability.After implanted the netrin-1-modified small-diameter TEBVs in vivo,we found the endothelialization process was closely linked with the inflammation resolution.In the inflammation resolution group,the TEBVs achieved early endothelialization at 30 days and maintained long-term patency,which confirmed that timely promoting the inflammation resolution of the TEBVs was a good approach for early endothelialization.In clinical vascular transplantation,inflammation always induces thrombosis and obstruction of vascular grafts.Therefore,our study will provide a new perspective for the long-term survival of vascular grafts in the host,and may also provide an effective treatment for other inflammatory diseases.3.The effect of neuronal exosomes on vascular smooth muscle cell calcification under high glucose conditions and its mechanism studyThis section study found that neuronal exosomes have a significant effect on inhibiting VSMC phenotype transformation and oxidative stress under high glucose condition.We further made the mass spectrometry analysis of neuronal exosomes and found they contained considerable amounts of inhibiting oxidative stress protein prdx-1.Electron microscopy and BCA protein concentration measurements showed that the number of neuronal exosomes decreased significantly under high glucose condition,and immunofluorescence results showed that it is due to the inhibition of Rab35 activation which is responsible for vesicle transport.Through exogenous delivery of DENND1 A,we found that Rab35 was activated in neurons and the secretion function of exosomes was enhanced and restored.Later,the effects of neuronal exosomes on SMC were studied,and we found that neuronal exosomes could inhibit the ROS increase and protein damage in SMC under high glucose condition.Its mechanism may be that exosomes could inhibit the excessive opening of mitochondrial membrane channels and the increase of mitochondrial membrane potential in SMC under high glucose condition.Abnormal cell proliferation,migration and calcification induced by high glucose were significantly inhibited after adding neuronal exosomes into the supernatant of SMC,and the results of inhibitor group confirmed that this effect of exosomes was probably achieved through prdx-1.4.Construction of two stage response system on small-diameter TEBVs to induce nerve reconstructionIn order to protect the function of the netrin-1 and DENND1 A to promote nerve and the its regulating function in vivo,we designed and constructed a dual-layer responsive hydrogel encapsulation system for netrin-1 and DENND1 A factors based on the special local microenvironment of TEBVs in vivo.The results showed that the hydrogel could protect the factors from diffusion and degradation after implantation.After the local production of ROS and its reaction with thioether groups in hydrogel,the outer gel gradually hydrolyzed and released netrin-1,which effectively promoted the growth of nerve fibers and guided onto the TEBVs.Nerve grew into the adventitia of TEBVs in the experimental group at 14 days.The concentration of ATP released by nerve increased gradually after the nerve grew into the TEBVs,which could react with ssDNA of ATP adapter in the inner hydrogel.Then the inner hydrogel gradually hydrolyzed and released DENND1 A into the neuron,thus activated Rab35 of the neuron and promoted its exosome secretion under high glucose,which significantly reduced the calcium content and calcium deposition of TEBVs in diabetic rats,and increased the blood flow velocity of the carotid artery.This system effectively inhibited intimal hyperplasia and calcification caused by phenotype transformation of VSMCs and maintained long-term patency of TEBVsin diabetes mellitus.Our research provides a new strategy for clinical prevention,delay and treatment of diabetic vascular calcification.