The Promotion Of Endothelial Progenitor Cells Homing By Nerve Growth Factors In Constructing Tissue-engineered Blood Vessels In Vivo

Tissue-engineered blood vessels (TEBV) have extensive and important applications in blood vessel replacement therapies associated with vascular bypass or the repair of acute vascular injury. However, transplantation leads to high occlusion rates of small diameter (∠6mm) vascular grafts because of thrombosis and neointimal proliferation in small caliber vascular grafts that lack endothelium. Recently, artificial blood vessel and endovascular stent that were modified by anti-freezing methods can inhibit thrombosis in some degree, but the effect of long-time anti-freezing is not ideal sufficiently for the graft need to be used perpetually. Endothelial cells play a very important role in the process of anti-thrombosis, inhibiting platelet aggregation, secreting vascular active factor and preventing smooth muscle cells pathological proliferation. Endothelialization of the vascular graft can solve this problem because the formation of the endothelial cell layer of the vascular surface can prevent thrombosis and restenosis. Endothelial progenitor cells (EPCs) is a kind of precursor cell which can differentiate into endothelial cells. It has been proven that endothelialization of damaged vessels can be promoted by the mobilization, migration and differentiation of endothelial progenitor cells (EPCs). Following endothelial insult or ischemia, EPCs mobilize from the bone marrow and migrate to sites of damage where they differentiate into cells that replace the apoptotic endothelial cells. This process may promote vascular re-endothelialization and angiogenesis while reducing neointimal proliferation and thrombosis, as well as lowering the possibility of blood vessel stenosis. Because the vessels and nerves of living creatures are always concomitant and interdependent, their growth patterns are similar and they follow the same migration routes. Importantly, vascular activity and nutrition are regulated by nerves. Recent studies indicate that the nervous system plays an important role in the development of the embryonic cardiovascular system through paracrine activity involving neurotrophins. NGF, the most representative of the neurotrophic factors, is a multifunctional polypeptide that can combine with TrkA on endothelial cells to trigger proliferation and migration of endothelial cells, as well as increase the expression of adhesion molecules that promote angiogenesis. It is not clear, however, if NGF can promote EPCs mobilization, homing and endothelialization of TEBV in vivo. According to this, we investigated whether NGF can promote EPCs mobilization and endothelialization of tissue-engineered blood vessels. The main results and conclusions are as following:1.CD133+ progenitor cells were purified by positive selection with anti-CD133 microbeads and we conducted colony forming unit assays in methylcellulose semisolid medium. We found that NGF can promote EPC colony formation, but it does not act through the Akt pathway. RT-PCR showed that at the 14th day of EPC culture, EPCs treated with NGF expressed TrkA receptor but not the P75 receptor. Real-time PCR showed that NGF enhanced the expression of CD31 and VE-Caherin in EPCs, which indicated that NGF promoted EPCs to differentiate into endothelial cells.2.MTT assay showed that NGF can protect EPC viability and promoted their proliferation, but this effect is inhibited by the Akt inhibitor triciribine, demonstrating that NGF upregulates EPC proliferation through the Akt pathway. To determine if NGF can induce EPC migration, transwell assays were performed. Violet cells represented EPCs that migrated from the upper chamber into the low chamber. NGF can induce EPCs'migration and Akt plays a crucial role in NGF-induced migration of EPC.3.To confirm that NGF stimulates EPC mobilization from bone marrow into the peripheral circulation, C57BL/6 mice were injured by wire and treated with NGF for three days. Flow cytometric was used to determine the number of EPCs which were mobilizate from bone marrow to peripheral circulation. And the results showed that NGF not only increased the mobilization of EPCs in wire injury C57BL/6 mice, but also NGF synergized with VEGF to promote the mobilization of EPCs.4.To determine the effects of NGF to EPCs homing, CD133+ progenitor cells were purified by positive selection with anti-CD133 microbeads. Wire-induced injury to the mouse carotid artery causes complete removal of endothelium. NGF-treated EPCs and control EPCs were labeled with CM-Dil and calcein and injected into injured mice. The homing of transferred EPCs to sites of wire injury was observed by LSCM. The result showed NGF enhanced EPCs to homing to the site of injuried carotid artery.5.We used decellularized rat arteries incubated with collagen and cross-linked with EDC to generate TEBV. Then the arteries were treated by SPDP-conjugated NGF and transplanted into rat arteries. After one month, the open rate of NGF-bound TEBV is higher than control TEBV significantly and the degree of endothelialization in NGF-bound TEBV also increased.The above results suggested NGF can promote the proliferation, differentiation, migration and colony forming. Furthermore, NGF enhance the mobilization and homing of EPCs in C57BL/6 mice that treated by carotid artery wire injury. The open rate and endothelialization of NGF-bound TEBV increase significantly.