Human Telomerase Reverse Transcriptase-modified Endothelial Progenitor Cells Restore Erectile Function Of Streptozotocin-induced Diabetic Rats

Objective:To construct and identify the recombinant lentiviral vector overexpressing human telomerase reverse transcriptase (hTERT) gene.Methods:The hTERT gene was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). The vector GV341 and the amplified product were digested with Agel and Nhel enzymes before the vector and RT-PCR fragment were ligated by DNA ligase. Then the ligated product was transformed into DH5a cells. The positive clones were picked up by means of polymerase chain reaction (PCR). For further identification, sequence analysis and blasting with database of GenBank were carried out. The lentiviral vector system had three parts before packaging, including the GV341 vector, the pHelper 1.0 vector and the pHelper 2.0 vector. These vectors were cotransfected into 293T cells in serum-free medium. Then the medium was collected and concentrated before virus titer was assessed.Results:The results of sequence analysis revealed that the sequence of hTERT gene in GV431 vector was completely correct. After drug screening, the concentrated virus titer was2×108 TU/ml.Conclusions:The recombinant lentiviral vector overexpressing hTERT gene was successfully constructed.Objective:To study the expression level of exogenous hTERT gene in rat EPCs and the effect of hTERT overexpression on rat EPCs after rat EPCs were transfected with recombinant lentiviral vector overexpressing hTERT gene.Methods:Rat EPCs were primary isolated by density gradient centrifugation. Cellular immunofluorescence staining was adopted to analyze the expression of surface markers such as CD31, CD34, CD 133 and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot was used to detect eNOS expression in rat EPCs. To determine the multipotent differentiation capacity of rat EPCs, the experiments of adipogenic and myogenic differentiation were conducted with induction medium. Rat EPCs were transfected with the recombinant leniviral vector or negative control lentiviral vector to generate EPCs-hTERT or EPCs-control. EPCs-hTERT and EPCs-control were selected in medium containing puromycin. Expression of hTERT and rat telomerase reverse transcriptase (rTERT) was detected using Western blot, Real-time PCR and cellular immunofluorescence staining. Telomeric repeat amplification protocol (TRAP) was used to assess telomerase activity of EPCs-hTERT. For further study, we detected proliferation capacity of EPCs-hTERT with the method of cell counting kit-8 (CCK-8) assay and 5-Ethynyl-2’-deoxyuridine (EdU) assay. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay used to detect intracelluar reactive oxygen species (ROS). Then, we detected the survival of EPCs-hTERT exposed to oxidative stress induced by hydrogen peroxide by means of CCK-8 assay.Results:We successfully isolated and cultured rat EPCs in vitro expressing CD31 (96.8±0.5%), CD34 (97.2±1.4%), CD133 (92.8±5.3%) and VEGFR-2 (91.5±3.7%). eNOS was also expressed in EPCs. Adipogenic and myogenic cells were positively stained with Oil Red O and a-smooth muscle actin (a-SMA). After transfection with lentiviral vector, mRNA and protein expression of hTERT was significantly increased in EPCs-hTERT compared with EPCs and EPCs-control, while the expression of rTERT did not change. Results of cellular immunofluorescence staining revealed that hTERT was expressed in cytoplasm of EPCs-hTERT. Telomrase activity of EPCs-hTERT was significantly increased. Proliferation capacity and survial cells in oxidative stress of EPCs-hTERT was significantly upregulated while intracellular ROS level was downregulated.Conclusions:We successfully isolated and cultured rat EPCs, and EPCs-hTERT showed increased telomerase activity, proliferation capacity and anti-oxidative stress activity, but decreased intracellular ROS level.Objective:To evaluated the possibility and mechanism of diabetic ED treatment with EPCs genetically modified with hTERT.Methods:Diabetes was induced via an intraperitoneal injection of streptozotocin. Diabetic ED rats were selected using the apomorphine (APO) test after 8 weeks. Thirty male rats were devided into five groups:normal control group, diabetic ED rats (diabetic ED group), diabetic ED rats treated with EPCs (EPCs group), diabetic ED rats treated with EPCs-control (EPC-control group) and diabetic ED rats treated with EPCs-hTERT (EPCs-hTERT group). Intracavernosal pressure (ICP) was measured using electrical stimulation for each group 2 weeks after EPCs injections. ICP/mean artery pressure (MAP) ratio was calculated to evaluate erectile function of every group. The amount of retained EPCs and the smooth muscle content in penile tissues were assessed in vivo by means of immunofluorescence staining. Masson’s trichrome staining was performed to determine collagen content in penile tissues. The degree of apoptosis was assessed using TUNEL assay. The protein expression of Bcl-2, Bax, transforming growth factor-β1 (TGF-(31)/Smad2/3 pathway were assessed using western blotting. The expression of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and phospho-eNOS (p-eNOS, ser1177) and nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) concentrations were detected.Results:The ICP/MAP ratio induced by electrical stimulation was markedly increased in the EPCs-hTERT group compared with the EPCs and EPCs-control group. Immunofluorescence demonstrated increased cell survival in penile tissues after EPCs-hTERT implantation. The smooth muscle content was increased in penile tissues of the EPCs-hTERT group. The smooth muscle/collagen ratio was markedly increased in the EPCs-hTERT group. TGF-β1/p-Smad pathway expression decreased significantly in DMED rats after EPCs-hTERT treatment. The degree of apoptosis in penile tissues of the EPCs-hTERT group was considerably reduced, as demonstrated by the decreased Bax/Bcl-2 ratio. eNOS, p-eNOS and nNOS expression and NO, cGMP concentrations increased significantly in the EPCs-hTERT group.Conclusions:EPCs-hTERT injection was an effective treatment for diabetic ED. More retained cells in penile tissues to alleviate penile fibrosis, decrease apoptosis level and increase NO generation is the possible mechanism of the treatment.