Xuefeng Qiu

Combined Strategy of Mesenchymal Stem Cell Injection With Vascular Endothelial Growth Factor Gene Therapy for the Treatment of Diabetes-Associated Erectile Dysfunction

This study was designed to investigate the effect of vascular endothelial growth factor 164 adenovirus (Ad-VEGF(164))-transfected mesenchymal stem cells (MSC) on improving erectile function in diabetic rats. Forty-five male Sprague-Dawley rats were injected with streptozotocin to develop type 1 diabetes, whereas 10 served as normal controls. Diabetic rats were randomly divided into 3 groups: rats that underwent intracavernous injection with phosphate-buffered saline (DM+PBS), unmodified MSCs (DM+MSC), and Ad-VEGF(164)-transfected MSCs (DM+VMSC). Normal controls received intracavernous injection of PBS. Four weeks after injection, erectile function was measured by cavernous nerve electrostimulation. Penile tissue was harvested for histology and enzyme-linked immunoassay. Prior to injection, high expression of VEGF was confirmed in Ad-VEGF(164)-transfected MSCs by enzyme-linked immunoassay. Four weeks after injection, the erectile function, as well as the content of smooth muscle and endothelium in corpus cavernosum increased significantly in the MSC-injected groups compared with the DM+PBS group. There was a significant improvement of erectile function, the content of smooth muscle and endothelium, and the VEGF concentration in the corpus cavernosum in the DM+VMSC group compared with the DM+MSC group. Our study validates the effect of intracavernous injection of MSCs for diabetes-associated erectile dysfunction in an animal model. The combined strategy of MSC injection with VEGF gene therapy-enhanced therapy of MSCs for the treatment of diabetes-associated erectile dysfunction.

Neurotrophic effect of bone marrow mesenchymal stem cells for erectile dysfunction in diabetic rats

It has been demonstrated that intracavernous injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) had beneficial effects on improving erectile function in type-1 diabetic rats. This study was designed to investigate the neurotrophic effect of BM-MSCs for type-1 diabetic rats. Streptozocin-induced type-1 diabetic rats were randomly divided into three groups: diabetic group, BM-MSCs-treated group and BM-MSCs-conditioned medium-treated group. At the 3d, 1 and 2w time points after BM-MSCs injection, three randomly selected rats in MSCs 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 evaluate the erectile function. Immunohistochemistry was used to track labelled BM-MSCs in penile tissue and to detect neuronal nitric oxide synthase (nNOS) and neurofilament (NF) positive fibres in penile dorsal nerve. 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. 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 by promoted nNOS and NF positive nerve fibres within penile dorsal nerve in type-1 diabetic rats. Histological data revealed a time-dependent decrease in the number of BM-MSCs in the corpus cavernosum following injection. Furthermore, the beneficial effect of BM-MSCs was partially repeated by BM-MSCs-conditioned medium. Intracavernous injection of BM-MSCs is effective in improving nerve regeneration in diabetic rats. Paracrine effects of BM-MSCs are probably involved in the improvement.

In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.

Identification and Characterization of the MicroRNA Profile in Aging Rats with Erectile Dysfunction

INTRODUCTION Aging-related erectile dysfunction (A-ED) is a neurovascular and refractory disorder with complicated pathophysiological mechanisms and a high prevalence. MicroRNAs (miRNAs), which modulate a variety of cell functions, may be involved in the pathophysiological processes of this disorder. AIM To investigate the miRNA profile in the corpus cavernosum (CC) of aging rats with ED, and to analyze the target genes and signaling pathways regulated by the dysregulated miRNAs. METHODS According to the apomorphine test, the experimental animals were divided into three groups: aging rats with ED (group AE), aging rats with normal erectile function (group AN), and young rats as normal controls (group YN). After the erectile functional test, CCs from each group were then collected for histological and molecular measurements. MAIN OUTCOME MEASURES Intracavernous pressure response to electric stimulation of the cavernous nerve was used to evaluate erectile function. Histological changes within CC were evaluated using immunofluorescent staining. GeneChip array was used to analyze the miRNA expression profiling. The miRNA profilings were further validated by real-time polymerase chain reaction. The TargetScan or DAIAN web platform and DAVID were used for bioinformatic analysis. RESULTS Accompanied with significantly decreased erectile function, the content of smooth muscle and endothelium within the CC of rats with A-ED was significantly decreased compared with both AN group and YN group. miR-1, miR-200a, miR-203, and miR-206 were found and validated up-regulating with above twofold change in AE group. According to the bioinformatics analysis, the four up-expressing miRNAs could regulate eNOS/NO/PKG and PGE1/PKA pathways through regulating 13 target genes. CONCLUSIONS Four miRNAs were found up-regulated significantly in the CC of rats with A-ED. The four miRNAs might play important roles in the development of A-ED by regulating the eNOS/NO/PKG and PGE1/PKA pathways despite lots of experiments still need to be validated.

MicroRNA-200a is up-regulated in aged rats with erectile dysfunction and could attenuate endothelial function via SIRT1 inhibition

MiR-200a was shown to be upregulated in the corpus cavernosum (CC) of rats with aging-related erectile dysfunction (A-ED) in our previous study. Among its target genes, SIRT1 was also reported as a protective factor in erectile function by our groups previously. Thus, miR-200a might attenuate the erectile function in A-ED via SIRT1 inhibition. In the present study, three animal groups were included: aged rats with ED (group AE, n = 8), aged rats with normal erectile function (group AN, n = 8), and young rats as normal controls (group YN, n = 8). CCs from each group were collected for histological and molecular measurements to validate the dysregulation of miR-200a and SIRT1. After that, the cavernous endothelial cells (CECs) from CC of aged rats with normal erectile function were transfected with miR-200a in vitro. Then the expression of SIRT1 and molecules within the eNOS/NO/PKG pathway were measured to investigate whether the transfection could imitate the attenuated process of erectile function in the aged. As a result, miR-200a was upregulated while the SIRT1, the levels of eNOS and cGMP were all downregulated in the CCs from AE group. After transfection in vitro, the miR-200a was upregulated while the SIRT1 and levels of eNOS and cGMP were obviously downregulated. Finally, based on the results of our previous study, we further verify that up-regulation of miR-200a could participate in the mechanisms of A-ED via SIRT1 inhibition, and mainly attenuate endothelial function via influencing the eNOS/NO/PKGpathway.

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

AB160. Intracavernous transplantation of bone marrow-derived mesenchymal stem cells restores erectile function of streptozocin-induced diabetic rats

Objective To explore the effects of transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) on improving erectile function of 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 labeled with Chloromethyl Benzamido derivatives of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (CM-DiI), and then transplanted into corporal cavernosum of STZ-induced diabetic rats. Four weeks after transplantation, all rats were analyzed for erectile function and penile histology. Results After BM-MSCs transplantation, the ICP/MAP ratio was increased significantly compared with diabetic controls. Content of smooth muscle and endothelium in corporal cavernosa of BM-MSCs transplanted rats was significantly increased compared to diabetic controls. Immunofluorescence analysis demonstrated that CM-DiI-labeled BM-MSCs could stay in corporal cavernosa for at least 4 weeks and some of them expressed von Willebrand Factor, CD31, calponin, or α-smooth muscle actin, cells markers for endothelial cells or smooth muscle cells, respectively. Conclusions Intracavernous transplantation of BM-MSCs had beneficial effects on erectile function of diabetic rats and increased the content of endothelium and smooth muscle in corporal cavernosum.