| Cardiovascular disease is the leading cause of death worldwide.Myocardial ischemia and hypoxia caused by vascular stenosis or obstruction are the primary causes of cardiovascular disease pathogenesis.Although there exist alimentary therapeutic,drug,and surgical treatments,coronary artery bypass grafting(CABG)is still considered to be the best choice for patients who need long-term vascular reconstruction.However,the source of the blood vessels is very limited.Therefore,the construction of small-caliber(<6 mm)tissue-engineered blood vessels(TEBVs)for clinical applications has excellent application prospects.However,currently,small-caliber TEBVs are facing problems of thrombosis and intimal hyperplasia after transplantation,resulting in a low patency rate of these transplanted blood vessels.Endothelialization of TEBVs can resist thrombosis effectively and inhibit intimal hyperplasia.EPCs are the type of precursor stem cell that can be mobilized from the bone marrow to the peripheral blood and can target specific sites to differentiate into endothelial cells,which are the main cells for endothelialization in TEBVs.What's more,the EPCs can also secrete many paracrine cytokines to promote endothelial cells growth and angiogenesis.Therefore,after TEBV transplantation,promoting the mobilization,homing,proliferation,and migration of EPCs to achieve rapid endothelialization is an effective method to promote the long-term patency of TEBVs.Icariin is one of the most abundant monomeric chemical constituents in the traditional Chinese herb Epimedium brevicornu Maxim.Structurally,icariin belongs to the 8-prenyl flavonol glycoside compound.Its pharmacological role is very extensive and includes anticancer and anti-inflammation properties,immune protection,neuroprotection,and cardiovascular protection.Various studies have shown that it has a very strong protective effect on cardiomyocytes and venous endothelial cells from harmful factors.Therefore,icariin is very promising for the treatment of cardiovascular diseases.?-cyclodextrin is a kind of molecule that has an excellent biocompatibility and a special hollow annular hydrophobic cavity.?-cyclodextrin can form a stable inclusion structure with hydrophobic drug molecules and can control the drug release.Moreover,due to the mechanism that is similar to the anticoagulation of heparin,sulfate-modified ?-cyclodextrin can bind to antithrombin due to its strong negative charge and can change the configuration of antithrombin to inactivate thrombin and the coagulation factors.Therefore,in this study,we constructed icariin-loaded ?-cyclodextrin sulfate and modified it to small-caliber TEBVs in order to make icariin-loaded ?-cyclodextrin sulfate modified small-caliber TEBVs exert anticoagulant effect and inhibit thrombus formationin the early stage of transplantation,providing a good environment for endothelialization of TEBVs.Then the slow release of Icariin can promote the migration,homing and proliferation of EPCs and promote endothelial cells migration,and make TEBVs complete endothelialization as soon as possible,correct the disorder,and maintain long-term patency.Methods1.The effects and mechanisms of Icariin on EPCs and RAVECs in vitroEPCs were isolated from spleens of rats and the optimal concentration of Icariin was determined by CCK-8 method.CCK-8 assay was also used to detect the proliferation of EPCs on the effect of Icariin and whether the pathway was associated with VEGFR2.Scratch experiments and transwell experiments were used to detect the migration of RAVECs and EPCs on the effect of Icariin and whether the pathway was related to VEGFR2.2.Preparation of inclusion complex of icariin and sulfated sodium salt of ?-cyclodextrin and its related characteristicsInfrared spectrometer was used to detect the stability of Icariin under different conditions.On this basis,the inclusion complex of icariin and sulfated sodium salt of ?-cyclodextrin was carried out.Infrared spectrum was used to detect wether the icariin was loaded into sulfated sodium salt of ?-cyclodextrin successfully.The appearance of inclusion complex of icariin and sulfated sodium salt of ?-cyclodextrin was observed using SEM.The APTT was tested to assess the anticoagulant effects.3.Construction of TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin and transplantationPreparation of decellularized TEBVs.Construct the TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin and use SEM to observe.The release of Icariin was detected.Three months after transplantation,the vascular patency was observed by micro-CT,and the grafts were taken out for HE,masson,immunofluorescence staining and SEM observation.Results1.We successfully isolated EPCs from rat spleens.CCK-8 results showed that the optimal concentration of Icariin for EPCs was 15 ?M,and at this optimum concentration,Icariin can promote EPCs proliferation via VEGFR2.Transwells and scratches results showed that Icariin can promote EPCs and RAVECs migration via VEGFR2.2.The results of infrared spectrum indicated that Icariin was stable and we successfully prepared the inclusion complex of icariin and sulfated sodium salt of ?-cyclodextrin.The SEM results showed that the inclusion of Icariin did not change the appearance of ?-cyclodextrin sulfate.The APTT results showed that sulfated sodium salt of ?-cyclodextrin has good anticoagulant effect.3.SEM results showed that we successfully constructed the TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin.The sustained-release experiments showed that Icariin could release from the small-diameter TEBVs steadily.The results of transplantation of TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin showed that the TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin maintained vascular patency and completed endothelialization well.ConclusionThe TEBVs modified by icariin-loaded sulfated sodium salt of ?-cyclodextrin can inhibit thrombus formation at the early stage of transplantation and provide a good environment for endothelialization of TEBVs.The slow release of Icariin promotes the homing,migration and proliferation of EPCs and promotes endothelial cells migration,which promotes endothelialization of TEBVs as soon as possible,corrects disorders,and achieves long-term patency. |
PDF Full Text Request