The Experimental Study On Adipose Tissue-Derived Stem Cell For Diabetic Erectile Dysfunction

Background:Erectile dysfunction(ED)associated with diabetes mellitus is a worldwide problem,since epidemiological studies have reported that the incidence of ED in diabetic patients is three-fold higher than in non-diabetic persons.ED often occurs 10-15 years earlier and is more severe than that of non-diabetic men.Diabetic ED(DED)is multifactorial,including aetiology,advancing age,duration of diabetes,poor glycaemic control,hypertension,hyperlipidaemia,sedentary lifestyle,smoking,and the presence of other diabetic complications.Therefore,the proposed mechanisms of DED are related to vasculopathy,neuropathy,visceral adiposity,insulin resistance,and hypogonadism.Efficacy of phosphodiesterase type 5 inhibitors(PDE-5i),which are generally considered to be the firstline therapy for ED,is lower in diabetic men with ED compared with that in the general population.Recent studies have focused on cellular therapy of DED,and each cell type has been shown to be effective in an animal study.However,the exact mechanisms involved in stem cells improving erectile function are not clear.Besides,the therapeutic efficacy is positively correlated with the retention rate of ADSCs at the target site.Adipose-derived stem cells(ADSCs)have characteristics of rich in number,easily obtain,quickly proliferation in vitro and ability of access to autologous cell transplantation,which suggested the more advantages of this kind of stem cells in clinic application.Objectives:First,we aim to explore the feasibility of non-invasively monitoring intracavernous(IC)injection of ADSCs in rat corpus cavernosum using in vivo magnetic resonance(MR)imaging.Next,we explore the effect of magnetic targeting ADSCs on improving the erectile function of streptozocin(STZ)-induced diabetic rats using an external magnetic field.Then,we analyzed the possible mechanisms involved inADSCs improving erectile function.Methods:1)ADSCs were isolated from para-testicular fat of adult rats.Cell surface antigens of ADSCs(passage 3)were determined by flow cytometry analysis.To determine the multipotent differentiation capacity of ADSCs,osteogenic and adipogenic differentiation were performed in induction medium for 2 weeks.Loading of superparamagnetic iron oxide nanoparticles(SPIONs)into ADSCs and the labelling efficiency were confirmed by Prussian blue staining.In vitro toxicity experiments including trypan blue exclusion and an MTT assay were performed at 24 h after SPION labelling to assess cell viability and proliferation.A 1.3-T magnet was placed directly under the dish for 24 h,and then cell condensation by magnetic capturing was visually examined by Prussian blue staining.We performed in vitro and in vivo MR imaging of ADSCs using a horizontal bore 7.0-T scanner.2)Type-1 diabetes was induced by a single intraperitoneal injected with STZ.These diabetic rats were treated with phosphate-buffered saline(PBS)(DED,n=8),ADSCs labelled with SPIONs(ADSCs,n=10)and SPION-labelled ADSCs with magnetic field application(M-ADSCs,n=10).After transplantation,erectile function was evaluated by the intracavernous pressure(ICP)response to electric stimulation of the cavernous nerve.Smooth muscle and endothelium in corpus cavernosum were assessed using immunohistochemistry.Masson's trichrome staining was used to evaluate the ratio between the smooth muscle and the collagen which reflected the fibrosis in the corpus cavernosum.Immunofluorescence was used to assess whether SPION-labelled ADSCs moved toward the magnetic field applied at the injection site.3)Immunofluorescent staining was used to assess whether ADSCs could differentiate into endothelial or smooth muscle cells in the corpus cavernosum.Vascular endothelial growth factor(VEGF)in corpus cavernosum was assessed using immunohistochemistry.ADSCs derived exosomes(ADSC-Exo)was isolated by ultracentrifugation from the ADSCs culture medium.The transmission electron microscope and western blot were used to identify the characteristic of ADSC-Exo.ADSC-Exo was co-cultured with human umbilical vein endothelial Cells(HUVECs).Whether ADSC-Exo could be incorporated into HUVECs was assessed by immunofluorescent staining.Transwell and an MTT assay were performed after co-cultured with ADSC-Exo to assess HUVECs migration and proliferation.Tube formation assay was performed to assess in-vitro proangiogenic property of ADSC-Exo.To explore the underlying mechanism of ADSC-Exo on endothelial cell proliferation and angiogenesis process,we also performed novel and functional miRNA sequencing in ADSC-Exo and ADSCs.STZ induced diabetic rays were randomly divided into three groups:diabetic group(DM,n=4),10 ?g ADSC-Exo treated group(10 ?g Exo,n=6),100 ADSC-Exo treated group(100 ?g Exo,n=6).Four weeks after IC injection of ADSC-Exo,ICP was assessed to evaluate the erectile function.Immunofluorescent staining was used to track PKH-67 labeled ADSC-Exo in penile tissue.Results:1)Flow cytometric analysis showed that most ADSCs expressed typical MSC markers,including CD90(100.0%)and CD44(98.9%),and had low expression of hematopoietic markers CD45(2.13%)and CD34(0.05%).The presence of adipogenic or osteogenic induced ADSCs(stained with Oil Red O or Alizarin Red Solution,respectively),further demonstrate the multipotential ability of primary cultured ADSCs.The SPIONs-labelled cells displayed the typical blue colour.When incubated with SPIONs(50 ?g ml-1),almost every ADSC showed blue-stained deposits.SPION labelling did not affect the proliferation and survival rate of ADSCs compared with that of unlabelled cells.When a magnetic field(1.3 T)was applied to the bottom of the cell culture dish,a clear accumulation of SPION-labelled ADSCs was observed at the edge of the magnet,suggesting that the external magnetic field attracted SPION-labelled ADSCs.A dramatically decreased signal intensity was observed in the SPION-labeled ADSCs compared with in distilled water,agarose gel and unlabeled ADSCs.On follow-up serial T2-weighted MR imaging,the hypointense signal intensity faded over time,and disappeared completely over the course of 1 week after ADSC transplantation.2)After ADSCs transplantation,erectile function was restored significantly compared with diabetic controls.ADSCs transplantion increased significantly the content of smooth muscle and endothelium in corpus cavernosum.Collagen deposition was decreased significantly in the ADSCs treated rats compared with diabetic control group,which reflected the fibrosis was significantly decreased The number of Ed U+ADSCs in the corpus cavernosum of ADSCs treated rats was significantly increased with magnetic field application compared with no application of the magnetic field.The M-ADSC group exhibited more improvement in erectile function and organizational structure than the ADSC group.3)EdU+ADSCs were negative for vWF and ?-SMA,indicating that ADSCs did not differentiate into endothelial or smooth muscle cells.VEGF expression in the corpus cavernosum was significantly increased in both ADSC-treated groups compared with the DED group.ADSC-Exo could be incorporated into HUVECs,and could improve the migration and proliferation of HUVECs.Besides,ADSC-Exo exhibited in-vitro proangiogenic property.Three proangiogenic microRNAs including miR-126,miR-130a,miR-132 and an antifibrotic microRNA family miR-let7b and miR-let7c were detected in the cellular and exosome lysates.After ADSC-Exo transplantation,erectile function was improved significantly compared with diabetic controls.However,there were no PKH-67 labeled ADSC-Exo within corpus cavernosum by immunofluorescent staining at 4 weeks after injection.Conclusions:MRI can trace transplanted ADSCs in the short term in animals in the context of erectile dysfunction.IC injection of ADSCs is effective on improving erectile function in diabetic rats.Magnetic targeting of ADSCs contributed to long-term cell retention in the corpus cavernosum and improved the erectile function of diabetic rats compared with ADSC injection alone.The paracrine effect of ADSCs appeared to play the major role in functional and structural recovery.ADSC-Exo therapy is expected to become an effective approach for diabetes-associated ED therapy.