Mingcan Yang

The use of BDNF to enhance the patency rate of small-diameter tissue-engineered blood vessels through stem cell homing mechanisms.

The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.

The promotion of endothelial progenitor cells recruitment by nerve growth factors in tissue-engineered blood vessels.

Endothelial progenitor cells (EPCs) mobilization and homing are critical to the development of an anti-thrombosis and anti-stenosis tissue-engineered blood vessel. The growth and activation of blood vessels are supported by nerves. We investigated whether nerve growth factors (NGF) can promote EPCs mobilization and endothelialization of tissue-engineered blood vessels. In vitro, NGF promoted EPCs to form more colonies, stimulated human EPCs to differentiate into endothelial cells, and significantly enhanced EPCs migration. Flow cytometric analysis revealed that NGF treatment increased the number of EPCs in the peripheral circulation of C57BL/6 mice. Furthermore, the treatment of human EPCs with NGF facilitated their homing into wire-injured carotid arteries after injection into mice. Decellularized rat blood vessel matrix was incubated with EDC cross-linked collagen and bound to NGF protein using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldit-hio) propionate (SPDP). The NGF-bound tissue-engineered blood vessel was implanted into rat carotid artery for 1 week and 1 month. NGF-bound blood vessels possessed significantly higher levels of endothelialization and patency than controls did. These results demonstrated that NGF can markedly increase EPCs mobilization and homing to vascular grafts. Neurotrophic factors such as NGF have a therapeutic potential for the construction of tissue-engineered blood vessels in vivo.

Neurotrophin-3 Accelerates Wound Healing in Diabetic Mice by Promoting a Paracrine Response in Mesenchymal Stem Cells

Angiogenesis is a major obstacle for wound healing in patients with diabetic foot wounds. Mesenchymal stem cells (MSCs) have an important function in wound repair, and neurotrophin-3 (NT-3) can promote nerve regeneration and angiogenesis. We investigated the effect of NT-3 on accelerating wound healing in the diabetic foot by improving human bone marrow MSC (hMSC) activation. In vitro, NT-3 significantly promoted VEGF, NGF, and BDNF secretion in hMSCs. NT-3 improved activation of the hMSC conditioned medium, promoted human umbilical vein endothelial cell (HUVEC) proliferation and migration, and significantly improved the closure rate of HUVEC scratches. In addition, we produced nanofiber mesh biological tissue materials through the electrospinning technique using polylactic acid, mixed silk, and collagen. The hMSCs stimulated by NT-3 were implanted into the material. Compared with the control group, the NT-3-stimulated hMSCs in the biological tissue material significantly promoted angiogenesis in the feet of diabetic C57BL/6J mice and accelerated diabetic foot wound healing. These results suggest that NT-3 significantly promotes hMSC secretion of VEGF, NGF, and other vasoactive factors and that it accelerates wound healing by inducing angiogenesis through improved activation of vascular endothelial cells. The hMSCs stimulated by NT-3 can produce materials that accelerate wound healing in the diabetic foot and other ischemic ulcers.

The effect of heme oxygenase-1 complexed with collagen on MSC performance in the treatment of diabetic ischemic ulcer

Diabetic ischemic ulcer is an intractable diabetic complication. Bone marrow mesenchymal stem cells (BMSCs) have great potential in variety of tissue repair. In fact, poor cell viability and tolerance limit their ability for tissue repair. In addition, it is difficult for stem cells to home and locate to the lesion. In this study, we explore whether over-expression of heme oxygenase-1 (HO-1) in BMSCs complexed with collagen play an important role in treatment of diabetic ischemic ulcers. In vitro, over-expression of HO-1 promoted the proliferation and paracrine activity of BMSCs and the conditioned medium of BMSCs accelerated HUVECs migration and proliferation. These processes were closely related to Akt signaling pathway and were not dependent on Erk signaling pathway. In vivo, in order to make BMSCs directly act on the wound, we choose a solid collagen as a carrier, BMSCs were planted into it, ischemic wounds of diabetic mice were covered with the complex of BMSCs and collagen. The results indicate that the complex of HO-1-overexpressing BMSCs and collagen biomaterials can significantly promote angiogenesis and wound healing. These preclinical findings open new perspectives for the treatment of diabetic foot ulcers.

Adenosine accelerates the healing of diabetic ischemic ulcers by improving autophagy of endothelial progenitor cells grown on a biomaterial.

Endothelial progenitor cells (EPCs) seeded on biomaterials can effectively promote diabetic ischemic wound healing. However, the function of transplanted EPCs is negatively affected by a high-glucose and ischemic microenvironment. Our experiments showed that EPC autophagy was inhibited and mitochondrial membrane potential (MMP) was increased in diabetic patients, while adenosine treatment decreased the energy requirements and increased the autophagy levels of EPCs. In animal experiments, we transplanted a biomaterial seeded with EPCs onto the surface of diabetic wounds and found that adenosine-stimulated EPCs effectively promoted wound healing. Increased microvascular genesis and survival of the transplanted cells were also observed in the adenosine-stimulated groups. Interestingly, our study showed that adenosine increased the autophagy of the transplanted EPCs seeded onto the biomaterial and maintained EPC survival at 48 and 96 hours. Moreover, we observed that adenosine induced EPC differentiation through increasing the level of autophagy. In conclusion, our study indicated that adenosine-stimulated EPCs seeded onto a biomaterial significantly improved wound healing in diabetic mice; mechanistically, adenosine might maintain EPC survival and differentiation by increasing high glucose-inhibited EPC autophagy and maintaining cellular energy metabolism.

Effect of brain-derived neurotrophic factor on mesenchymal stem cell-seeded electrospinning biomaterial for treating ischemic diabetic ulcers via milieu-dependent differentiation mechanism.

Great challenges in transplantation of mesenchymal stem cells (MSCs) for treating ischemic diabetic ulcers (IDUs) are to find a suitable carrier and create a beneficial microenvironment. Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, is considered angiogenic and neuroprotective. Given that IDUs are caused by vascular disease and peripheral neuropathy, we used BDNF as a stimulant, and intended to explore the role of new biomaterials complex with MSCs in wound healing. BDNF promoted the proliferation and migration of MSCs using MTT, transwell, and cell scratch assays. The activity of human umbilical vein endothelial cells (HUVECs) was also enhanced by the MSC-conditioned medium in the presence of BDNF, via a vascular endothelial growth factor-independent pathway. Since proliferated HUVECs in the BDNF group made the microenvironment more conducive to endothelial differentiation of MSCs, by establishing co-culture systems with the two cell types, endothelial cells derived from MSCs increased significantly. A new biomaterial made of polylactic acid, silk and collagen was used as the carrier dressing. After transplantation of the BDNF-stimulated MSC/biomaterial complex, the ulcers in hindlimb ischemic mice healed prominently. More blood vessel formation was observed in the wound tissue, and more MSCs were co-stained with some endothelial-specific markers such as cluster of differentiation (CD)31 and von Willebrand Factor (vWF) in the treatment group than in the control group. These results demonstrated that BDNF could improve microenvironment in the new biomaterial, and induce MSCs to differentiate into endothelial cells indirectly, thus accelerating ischemic ulcer healing.

Regulation of Cellular Response Pattern to Phosphorus Ion is a New Target for the Design of Tissue-Engineered Blood Vessel

Regulation of cellular response pattern to phosphorus ion (PI) is a new target for the design of tissue-engineered materials. Changing cellular response pattern to high PI can maintain monocyte/macrophage survival in TEBV and the signal of increasing PI can be converted by klotho to the adenosine signals through the regulation of energy metabolism in monocytes/macrophages.

5‐Aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside reduces intimal hyperplasia of tissue engineering blood vessel by inhibiting phenotype switch of vascular smooth muscle cell

Intimal hyperplasia (IH) is the cause of clinical failure in patients with vascular transplants and intravascular stents. The proliferation and phenotype switching of vascular smooth muscle cells (VSMCs) play important roles in IH. Inhibiting the proliferation of VSMCs and maintaining the differentiated phenotype of VSMCs is one way to reduce IH. In this article, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) was used in experiments after drug screening. We found that the metabolism, autophagy, and differentiation of VSMCs were enhanced which were important to the normal function of VSMCs, but the secretion of VSMCs was reduced after AICAR treatment. AICAR induces G1 phase arrest and inhibits the proliferation of VSMCs using the MTT and EdU assays and cell cycle analysis. Then, the rat carotid artery vessel transplantation model was used to evaluate the function of AICAR in vivo. AICAR-modified tissue-engineered blood vessels (TEBVs) had a higher patency rate and less IH than the control TEBVs. In conclusion, AICAR can improve the normal function of VSMCs by increasing the metabolism and autophagy of VSMCs but inhibit the proliferation, paracrine, and phenotypes switching of VSMCs, further contribute the reducing of IH in TEBVs.

Netrin-1 Promotes Inflammation Resolution to Achieve Endothelialization of Small-Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ

Abstract The transplant of small‐diameter tissue engineering blood vessels (small‐diameter TEBVs) (<6 mm) in vascular replacement therapy often fails because of early onset thrombosis and long‐standing chronic inflammation. The specific inflammation state involved in small‐diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small‐diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin‐1) is found present in endothelial cells of natural blood vessels and has anti‐inflammatory effects. This work generates netrin‐1‐modified small‐diameter TEBVs by using layer‐by‐layer self‐assembly to resolve the inflammation. The results show that netrin‐1 reprograms macrophages (MΦ) to assume an anti‐inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small‐diameter TEBVs modified by netrin‐1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small‐diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ‐derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

Construction of a small-caliber tissue-engineered blood vessel using icariin-loaded β-cyclodextrin sulfate for in situ anticoagulation and endothelialization

The rapid endothelialization of tissue-engineered blood vessels (TEBVs) can effectively prevent thrombosis and inhibit intimal hyperplasia. The traditional Chinese medicine ingredient icariin is highly promising for the treatment of cardiovascular diseases. β-cyclodextrin sulfate is a type of hollow molecule that has good biocompatibility and anticoagulation properties and exhibits a sustained release of icariin. We studied whether icariin-loaded β-cyclodextrin sulfate can promote the endothelialization of TEBVs. The experimental results showed that icariin could significantly promote the proliferation and migration of endothelial progenitor cells; at the same time, icariin could promote the migration of rat vascular endothelial cells (RAVECs). Subsequently, we used an electrostatic force to modify the surface of the TEBVs with icariin-loaded β-cyclodextrin sulfate, and these vessels were implanted into the rat common carotid artery. After 3 months, micro-CT results showed that the TEBVs modified using icariin-loaded β-cyclodextrin sulfate had a greater patency rate. Scanning electron microscopy (SEM) and CD31 immunofluorescence results showed a better degree of endothelialization. Taken together, icariin-loaded β-cyclodextrin sulfate can achieve anticoagulation and rapid endothelialization of TEBVs to ensure their long-term patency.