Experimental Research And Mechanism Of Bone Marrow Mesenchymal Stem Cells In The Treatment Of Diabetic Erectile Dysfunction Explore

Erectile dysfunction (ED) is a common complication of diabetes. The prevalence of ED in diabetic men is three times greater than in the non-diabetics. Type5phosphodiesterase inhibitor (PDE5I), as the first line therapy for ED, is the main therapy for diabetic ED. However, the effects of PDE5I for diabetic ED are lower due to the complicated mechanisms involved in the development of diabetic ED. It's necessary to develop a new strategy to treat diabetes associated ED. Bone marrow derived mesenchymal stem cells (BM-MSCs) are kind of adult stem cells in bone marrow. They can not only differentiate into several tissue cells but also secrete several kinds of cytokines which can improve tissue growth. It has been demonstrated that intracavernous injection of BM-MSCs had beneficial effects on improving erectile function in aging rats and cavernous nerve injured rats. However, the exact mechanisms involved in BM-MSCs improving erectile function are not clear. This project was designed to explore the feasibility of intracavernous injection of BM-MSCs on improving erectile function of type Ⅰ diabetic rats and the possible mechanisms.Part Ⅰ Intracavernous injection of BM-MSCs improves erectile function in diabetic ratsAim:To explore the effects of transplantation of bone marrow derived mesenchymal stem cells (BM-MSCs) on improving erectile function of in streptozocin (STZ) induced diabetic rats.Methods:Male Sprague Dawley rats were injected either with STZ to induce diabetes or with citrate buffer as controls. Rat BM-MSCs were harvested and transplanted into corporal cavernosum of STZ induced diabetic rats8weeks after establishment of diabetes. Four weeks after transplantation, all rats were analyzed for erectile function and penile histology. Erectile function was evaluated by the ratio between intracavernous pressure (ICP) and mean arterial pressure (MAP) during electrostimulation of cavernous nerve. Smooth muscle and endothelium in corpora cavernosum were assessed using immunohistochemistry. Masson's trichrome staining was used to evaluate the fibrosis in the corpus cavernosum.Results:After BM-MSCs transplantation, the ICP/MAP ratio was increased significantly compared with diabetic controls. Content of smooth muscle and endothelium in corporal cavernousa of BM-MSCs transplanted rats was significantly increased compared to diabetic controls. The fibrosis in the BM-MSCs treated rats was significantly decreased compared with diabetic control group.Conclusion:Intracavernous transplantation of BM-MSCs had beneficial effects on improving erectile function of diabetic rats and increasing the content of endothelium and smooth muscle in corporal cavernosum.Part Ⅱ Possible mechanisms involved in intracavernous injection of BM-MSCs improving erectile function in diabetic ratsAim:To explore the possible mechanisms involved in intracavernous injection of bone marrow derived mesenchymal stem cells (BM-MSCs) on improving erectile function of in streptozocin (STZ) induced diabetic rats.Methods:Streptozocin induced type-1diabetic rats were randomly divided into three groups: diabetic group, BM-MSCs treated group and BM-MSCs conditioned medium treated group. At the1d,3d,1w and2w time points after BM-MSCs injection,3randomly selected rats in BM-MSCs treated group were sacrificed and penile samples were harvested to detect BM-MSCs in penile tissue. Four weeks after intracavernous injection of BM-MSCs or BM-MSCs conditioned medium, intracavernous pressure (ICP) was assessed to evaluated the erectile function of experimental animals. Immunohistochemistry was used to track labeled BM-MSCs in penile tissue and to detect neuronal nitric oxide synthase (nNOS) and neurofilament (NF) positive fibers in penile dorsal nerve. Immunofluorescent staining was used to detect the differentiation of BM-MSCs in the corpus cavernosum. Enzyme lined immunosorbent assay (ELISA) was used to measure the concentrations of vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) in BM-MSCs conditioned medium.Results:BM-MSCs secreted detectable levels of VEGF, BDNF and NGF. Intracavernous injection of BM-MSCs improved erectile function in diabetic rats. The functional improvement was accompanied with promoted nNOS and NF positive nerve fibers within penile dorsal nerve in type-1diabetic rats. Histological data revealed a time-dependent decrease in the number of BM-MSCs in the corpus cavernosum following injection. Immunofluorescent staining results revealed that4weeks after injection, there were very limited BM-MSCs within corpus cavernosum. Some of the BM-MSCs expressed CD31and vWF which are specific antigens for endothelial cells, while some of them expressed α-SMA and calponin which are specific antigens for smooth muscle cells. Furthermore, the beneficial effects of BM-MSCs were partially repeated by BM-MSCs conditioned medium.Conclusion:Intracavernous injection of BM-MSCs is effective on improving nerve regeneration in diabetic rats. BM-MSCs injected within corpus cavernosum could differentiate into smooth muscle cell like and endothelial cell like cells. Paracrine effects of BM-MSCs are probably involved in the improvement.Part Ⅲ Combined strategy of bone marrow derived mesenchymal stem cells injection with VEGF gene therapy for the treatment of diabetes associated erectile dysfunctionAim:This study was designed to investigate the effects of VEGF164adenovirus (Ad-VEGF164) transfected BM-MSCs on improving erectile function in diabetic rats.Methods:Male Sprague-Dawley rats were injected with streptozotocin (STZ) to develop type Ⅰ diabetes. Diabetic rats were randomly divided into3groups:rats underwent intracavernous injection with PBS (DM+PBS), unmodified BM-MSCs (DM+MSC) and Ad-VEGF164transfected BM-MSCs (DM+VMSC). Normal controls received intracavernous injection of PBS (N+PBS). Four weeks after injection, erectile function was measured by cavernous nerve electrostimulation. Penile tissue was harvested for histology and ELISA.Results:Prior to injection, high expression of VEGF was confirmed in Ad-VEGF164transfected BM-MSCs by ELISA. Four weeks post-injection, the erectile function, the content of smooth muscle and endothelium in corpus cavernosum increased significantly in the BM-MSCs injected groups compared to the DM+PBS group. There was a significant improvement of erectile function, the content of smooth muscle and endothelium, and the VEGF concentration in corpus cavernosum in the DM+VMSC group compared with DM+MSC group.Conclusion:The combined strategy of BM-MSCs injection with VEGF gene therapy enhanced therapy of BM-MSCs for the treatment of diabetes associated erectile dysfunction.