Regulating Energy Metabolism Of Stem Cells And Construction Of Small-diameter Tissue Engineering Blood Vessels In Situ

In 2014, more than 80 million American adults have several types of cardiovascular diseases. Cardiovascular diseases were the major factors causing death over the past three decades in China. Although the treatments of drugs and endovascular intervention surgeries decreased the mortality rate of cardiovascular diseases, more and more patients still need the vessel replacements or bypass surgeries at last. All these surgeries need vessel substitutes. Besides, there are huge clinical demands of vessel substitutes in cancer, trauma, the vessels caused by inflammation, hemodialysis and aortic dissection. Vascular substitutes can be got from autologous vessels or artificial vessels in clinical. The main autologous blood vessels contain radial artery, internal mammary artery and saphenous vein. There are two disadvantages in autologous vessels: 1. the quality of vessels in patients can not be guaranteed and the number of autologous blood vessels was limited; 2. the local traumas after the surgery for getting donor blood vessels would affect the life quality of patients. The artificial blood vessels using in clinical contain polyethylene terephthalate(PET) and expanded polytetrafluoroethylene(ePTFE). The using of PET or ePTFE leads to several complications such as intimal hyperplasia, calcification, thrombosis, infection, lack of growth potential for pediatric applications. These synthetic materials only are successfully used for the replacement of large diameter blood vessels(ID >6 mm). However, there are no artificial vascular grafts can be used in the replacements of small diameter blood vessels(ID < 6 mm) in clinical. Therefore, researches and applications of small diameter tissue engineering blood vessels(TEBVs) are imminent.Past researches indicated that there are unacceptable patency rates in small diameter blood vessels(ID < 6 mm) for long term. The risk factors that influence the patency rates of small diameter vessel grafts are acute thrombus formation and intimal hyperplasia. The vessel grafts were exposed to complex environment after transplantation, keep in contact with blood directly, which were different from other kinds of pipeline grafts, and easy to forming thrombosis after transplantation. The prevention of acute thrombosis is important to keep vessel grafts open in the acute phase. The pathological vessel smooth muscle proliferation and the abnormal deposition of extracellular matrix can lead to intimal hyperplasia, which is the main cause of vessel grafts blockage. The endothelium exists on the vessels surface, which can inhibit the thrombus formation, intimal hyperplasia, and play key roles in maintaining the homeostasis of vessels. The endothelium is important to the vessel grafts too. The earlier endothelialization of vessel grafts can cause the lower possibility of thrombosis. Besides, the endothelialization of vessel grafts also can inhibit the intimal hyperplasia in vessel grafts. So, the promoting of early endothelialization is the key to keep vessel grafts open.Recently researches indicated the in situ TEBVs were a type of hopeful vessel grafts. In these TEBVs, the no cells seeded but surface modified scaffolds were implanted directly and realized recellularization by recruiting the host cells in vivo. The implantations of in situ TEBVs avoid the limits correlated to the in vitro cellularization of scaffolds, specifically the choicing and expansion of cells. The endothelialization of in situ TEBVs concerned to following mechanism: endothelial Cells(ECs) from the neighboring vessel migrate over the anastomosis site of the TEBVs, and endothelial progenitor cells(EPCs) homing to the TEBVs and differentiate into ECs. The migration distance of ECs from the neighboring vessel is limited, so the endothelialization of middle piece in TEBVs is finished by the migration, adhesion and differentiation of EPCs. EPCs can mobilize and move towards artificial vascular inner surfaces lacking ECs at early transplantation stages, after which they can differentiate and form ECs or secret grow factors, leading to endothelialization of vessel grafts. So how to make the EPCs homing to the surface of TEBVs faster and performing their functions better need us to research.The researches focused on the metabolism of stem cells had been increasing steadily. Researches showed that different states of stem cells(quiescent state, differentiation or proliferation) had different metabolic states, and the switch of metabolic states in stem cells caused by changing of environment would lead to the switch of cell states. Increased the metabolism of stem cells can improve the functions of them. So, we hypothesized that the enhancement of EPCs metabolism will leading to better functions of them, which further contribute the endothelialization of TEBVs. Adenosine monophosphate activated protein kinase(AMPK) is a key molecule to regulate energy metabolism. AMPK is activated when the intracellular AMP/ ATP ratio increases, which activates downstream molecules, inhibits many synthetic processes, and promotes energy-producing catabolic processes. Stress, hungry, exercises, and metformin can active AMPK pathway. 5-Aminoimidazole-4- carboxamide 1-β-D-ribofuranoside(AICAR) is the agonist of AMPK. When ingested, AICAR can be phosphorylation and become the ZMP, the analogue of AMP. Then ZMP combined with AMPK and the AMPK pathway will be activated. AICAR had been approved for clinical application. So, we improved the metabolism of EPCs by treatment of AICAR, and then observe the changes of EPCs functions and patency rate of TEBVs.Therefore, we enhanced the metabolism of EPCs by AICAR treatment in vitro to clarify the impact of metabolism on cell functions. Then the AICAR modified TEBVs were constructed and applied in rat carotid artery transplantation model to verify whether AICAR can increase the endothelialization and patency rate of TEBVs by improving metabolism and functions of EPCs.Further, more than 387 million people had diabetes. The diabetic vascular diseases were serious complications caused by diabetes. The vessel grafts after transplantation in diabetes patients had a higher probability of blockage, because the cells participated in the regeneration of vessel grafts were damaged under hyperglycemia and hypertonic condition. So the vessel grafts applied in diabetes patients must have better biocompatibility and the function to resist hyperglycemia and hypertoni. It is unclear whether our TEBVs could be used in diabetes patients, and had higher patency rate. What the changing of stem cells participated in the endothelialization of TEBVs under hyperglycemia and hypertonic condition would happen need us to clarify too.Therefore, we changed the metabolism of EPCs under high glucose(HG) condition by treatment of AICAR, and observed the influence of metabolism on damaged functions. Then, AICAR modified TEBVs were used in diabetic models to verify our hypothesis. We considered that the research of TEBVs based on improving stem cell metabolism can provide new idea to the construction of vessel grafts in future.We conducted exhaustive researches listed as following.1. The effect of AICAR on the cells involved in the regeneration of TEBVs.First, we found AICAR can improve the metabolism of EPCs detected by rhodamine123, MitoTracker Green staining and intracellular ATP assay. Then metabolism enhanced EPCs had a better adhesion function detected by flow chamber assay. The migration and paracrine of EPCs were improved detected by ELISA and Transwell assay. We considered that enhanced metabolism of EPCs can improve the functions of them, which play important roles in the endothelialization of TEBVs.Besides, we observed the other cells participated in regeneration of TEBVs. The data showed that AICAR can inhibit platelet aggregation by inhibiting the secretion of 5-hydroxytry and thromboxane A2, and improve the migration of ECs near the anastomosis. We found AICAR can inhibit the proliferation and secretion, but improve metabolism and differentiation of vescular smooth muscle cells.2. The construction of AICAR modified TEBVs.To verify whether enhance EPCs by treating AICAR can increase the endothelialization and patency of TEBVs, we constructed AICAR modified TEBVs. The accellular rat carotid arteries were used in our experiment as blood vessel scaffold materials. The AICAR contained chitosan nanoparticles prepared using ion exchange method and collagen were mixed and coated on the scaffold materials to construct AICAR modified TEBVs. By release, mechanical, and biocompatibility experiments, we considered the AICAR modified TEBVs are suitable to the transplantation model. Then, we transplanted the TEBVs to the rat carotid arteries. After transplantation 3 months, the AICAR modified TEBVs had higher endothelialization and patency rate than them in control group. We considered that AICAR can increase the endothelialization and patency rate of TEBVs by improving the metabolism and functions of EPCs.3. AICAR improves the metabolism and functions of EPCs under HG condition.Further, we detected the metabolism and functions of EPCs under HG condition. The data showed that AICAR can improve the damaged metabolism of EPCs detected by staining of rhodamine123 and MitoTracker Green, oxygen consumption rate and intracellular ATP assay. Besides, we found the autophagy of EPCs was enhanced by treating AICAR, which was benefit to the cells damaged in diabetes. The enhanced diabetic EPCs had better migration and paracrine functions detected by ELISA and Transwell experiments. Above results indicated that ACIAR can reverse the damaged metabolism and functions of EPCs under HG condition, which would contribute to the endothelialization of TEBVs in diabetic models.4. The construction and application of AICAR modified TEBVs in diabetic models.EPCs play important roles in formation and repair of diabetic macrovascular and microvascular complication. To verify the functions of enhanced EPCs, we used EPCs to therapy mice lower limb ischemic ulcers. The results indicated that AICAR treated EPCs had better therapeutic effect rather than untreated EPCs. The living cell numbers, micro vessel density and ulcer healing time in AICAR group were better than them in control group, but the ulcers therapy by AICAR treated AMPKa2EPCs showed significant delay healing, and lower density of angiogenesis and less migration and survival of EPCs in the ulcer tissue. Then, we applied the AICAR modified TEBVs in the diabetic rat carotid arteries models. After transplantation 3 months, AICAR modified TEBVs still had high patency rate, although many vessel grafts which had good patency rate in normal transplantation failed in diabetic models. Besides, we found the delayed endothelialization and cellularization exist in diabetic model.In conclusion, by treatment of ACIAR, enhanced metabolism of EPCs improve the function of EPCs, and reversed the damages functions in diabetes models, further contributing to the endothelialization and patency rate of TEBVs.