Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy

Abstract

Introduction. The application of gene therapy for a nonlife-threatening disease, such as erectile dysfunction (ED), requires a higher safety level and more efficacious systems for gene transfer. Aim. To establish a novel technique for gene expression in a rat model of hypercholesterolemic ED that uses the RTP801 promoter, a hypoxia-inducible promoter. Methods. Two-month-old male Sprague-Dawley rats were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. Main Outcome Measures. Cavernous expression of hypoxia-inducible factor (HIF)-1 alpha was evaluated by Western blot. After intracavernous injection of pSV-Luc or pRTP801-Luc, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. Results. HIF-1 alpha was up-regulated in the corpus cavernosum of hypercholesterolemic rats. Although pSV-Luc did not induce gene expression in either the control or the cholesterol group, pRTP801-Luc significantly induced gene expression in the cholesterol group and resulted in higher luciferase activity than did pSV-Luc up to 14 days after injection. Immunohistochemistry showed that the gene expression area was also greater in the pRTP801-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. This suggests that pRTP801-Luc exerts its effect mainly by inducing promoter activity under hypoxia, not by increasing the number of transfected cells. Conclusion. The RTP801 promoter-driven gene expression system increased gene expression in the corpus cavernosum tissue of rats with cholesterol-induced ED. This may be a useful system for the development of gene therapy in vasculogenic ED.