Maintenance of the contractile phenotype in corpus cavernosum smooth muscle cells by Myocardin gene therapy ameliorates erectile dysfunction in bilateral cavernous nerve injury rats.

The pathophysiology of erectile dysfunction post radical prostatectomy is not clearly clarified, and the low efficacy of traditional PDE5i treatment remains a major complaint in contemporary practice. This study aimed to demonstrate phenotypic modulation in bilateral cavernous nerve injury (BCNI) rats within 7 days, and subsequently validate gene therapy with Myocardin (Mycod) by maintaining a contractile phenotype in corpus cavernosum smooth muscle cells. Initially, 36 male rats were randomly divided into BCNI and negative control (NC) groups for histological and phenotypic molecular measurements at 3, 5, and 7 days. Afterwards, an additional 30 rats received a single intra-cavernous injection of 50 μL PBS, Ad-Myocd (1 × 1011  pfu/ml) or Ad-vector for 10 animals each, namely the NC+PBS, BCNI+Ad-Myocd, and BCNI+Ad-vector groups. Finally, the validity and mechanism of Myocd transfection was explored at 21 days in vivo and 48 h in vitro. Western blotting showed canonical declines in Myocd, α-SMA, and Calponin expression, as well as elevated Osteopontin (OPN) expression, before corporeal morphological and SM-to-collagen ratio changes at day 5 after injury. Overexpression of Myocd maintained the contractile phenotype of corpus cavernosum smooth muscle cells, ameliorated bilateral cavernous nerve injury rat erectile dysfunction, as well as promoted cell contractility and suppressed proliferative capacity. Simultaneously, confocal imaging revealed up-regulation and co-localization of serum response factor in gene-transferred cells. In conclusion, our study is the first to investigate corpus cavernosum smooth muscle cells phenotypes in the early stages of cavernous injury model rats, and Myocd reversed phenotypic modulation by activating serum response factor. The experimental results demonstrated the validity of gene therapy for erectile dysfunction.

Zhang HB ，Wang ZQ ，Chen FZ ，Ding W ，Liu WB ，Chen ZR ，He SH ，Wei AY ... -  《-》

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction.

Wei AY ，He SH ，Zhao JF ，liu Y ，Liu Y ，Hu YW ，Zhang T ，Wu ZY ... -  《-》

Myocardin restores erectile function in diabetic rats: phenotypic modulation of corpus cavernosum smooth muscle cells.

This study aimed to investigate whether gene transfer of myocardin to the penis of diabetic rats can modulate corpus cavernosum smooth muscle (CCSM) cells phenotype and restore erectile function. Five normal control rats, and 22 diabetic rats were randomly divided into four groups: rats transfected with adCMV-myocardin (N = 6), treated with empty vector (N = 6), injected with medium (N = 5), and sham-operated rats (N = 5). The erectile response was measured 7 days after transfection. The percent of smooth muscle and the expressions of SMα-actin, smooth muscle myosin heavy chain (SMMHC), calponin were evaluated. The increases in intracorporal pressure(ICP)/mean arterial pressure and total ICP in response to nerve stimulation in the adCMV-myocardin treated rats were significantly greater than those in the empty vector (P < 0.001 and P < 0.001), medium only (P < 0.001 and P < 0.001), and sham-operated rats (P < 0.001 and P < 0.001). The suppressed expressions of SMα-actin, SMMHC and calponin were completely restored, and the amount of smooth muscle in diabetic rats were not restored after treatment. It is concluded that myocardin ameliorated erectile responses in diabetic rats mainly via promoting phenotypic modulation of CCSM cells from a proliferative to a contractile state.

He S ，Zhang T ，Liu Y ，Liu L ，Zhang H ，Chen F ，Wei A ... -  《-》

Administration of H(2)S improves erectile dysfunction by inhibiting phenotypic modulation of corpus cavernosum smooth muscle in bilateral cavernous nerve injury rats.

Phenotypic modulation of Corpus Cavernosum Smooth Muscle Cells (CCSMCs) is an important step in the development and progression of bilateral cavernous nerve injury induced erectile dysfunction (BCNI-ED). To investigate the effect of exogenous hydrogen sulfide (H2S) on the phenotypic modulation of CCSMCs in BCNI-ED rats, a total of 18 male Sprague-Dawley rats were equally divided into 3 groups, including sham-operated (Sham) group, BCNI group and BCNI treated with NaHS (BCNI + NaHS) group. The treated group received intraperitoneal injection of NaHS (100 μmol kg-1day-1) for 4 weeks starting day 1 postoperatively. Erectile function was measured by the ratio of intracavernous pressure (ICP)/mean arterial pressure (MAP), and relevant tissues were harvested for Immunohistochemistry, Hematoxylin and eosin (H&E), Masson's trichrome staining, H2S fluorescent probe WSP-1 and Western blot. The primary CCSMCs were isolated and pretreatment with NaHS before exposed to PDGF-BB (platelet-derived growth factor). Relative expression mRNA and protein of phenotypic biomarkers, RhoA, ROCK-1 and cell cycle proteins were detected. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST) and H2S levels in penile tissue was significantly decreased in the BCNI group compared with the Sham group. Compared with the BCNI group, administration of NaHS significantly increased the ratio of ICP/MAP, ratio of smooth muscle to collagen, expressions of a-SMA, calponin and decreased the expression of OPN, collagen-I, RhoA, ROCK1 in the penile tissue. PDGF-BB-treated CCSMCs exhibited higher expression of OPN, RhoA, ROCK1, and lower α-SMA, calponin, which were attenuated by NaHS pretreatment. NaHS suppressed RhoA/ROCK activity and decreased the expression of CDK2, Cyclin E1, while increased the expression of P27kip1 induced by PDGF-BB in CCSMCs. Taken together, this study indicated that exogenous H2S inhibited the phenotypic modulation of CCSMCs by suppressing RhoA/ROCK1 signaling and affecting its downstream factor, CDK2, Cyclin E1, P27kip1, thereby improved BCNI rat erectile function.

Qinyu-Zeng ，Shuhua-He ，Fengzhi-Chen ，Li-Wang ，Liren-Zhong ，Jialiang-Hui ，Wei-Ding ，Junhong-Fan ，Haibo-Zhang ，Anyang-Wei ... -  《-》

COX-2-10aa-PGIS gene therapy improves erectile function in rats after cavernous nerve injury.

Erectile dysfunction (ED) is a very common complication after radical prostatectomy. COX-2-10aa-PGIS is a newly engineered protein with COX-2 and prostacyclin synthase activities that converts arachidonic acid directly to prostacyclin (prostaglandin I2 [PGI2]). PGI2 is a potent smooth muscle relaxant. The purpose of this study was to explore the effect and mechanism of COX-2-10aa-PGIS gene therapy in penile rehabilitation. Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley rats was used to mimic radical prostatectomy-induced ED. Sprague-Dawley rats were randomly assigned into four groups: 1. sham surgery; 2. BCNC; 3. BCNC + null control recombinant adenovirus intracavernous injection; and 4. BCNC + Ad-COX2-10aa-PGIS intracavernous injection. Twenty-eight days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the mean arterial pressure (MAP) was monitored. At the end of the measurement, the penis was harvested and processed for (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (α-SMA), and transforming growth factor beta-1 (TGF-β1); (ii) Masson's trichrome stain for smooth muscle/collagen ratios; (iii) Western blot of eNOS, α-SMA, TGF-β1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. Erectile function was evaluated by ICP/MAP. Smooth muscle and endothelium functions in corpora cavernosum were assessed by Masson's trichrome stain, immunohistochemistry, and Western blot. Apoptosis was identified by TUNEL assay. The results were the following: 1. COX2-10aa-PGIS gene therapy improved erectile function (82%, compared with control) in the BCNC rat model; 2. COX2-10aa-PGIS gene therapy increased eNOS (121%) and α-SMA (118%) expression and decreased TGF-β1 (45%) expression; 3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury (64%); and 4. COX2-10aa-PGIS gene therapy improved smooth muscle/collagen ratios (81%). Our data demonstrated that COX2-10aa-PGIS improved erectile function after cavernous nerve injury through antifibrotic and anti-apoptotic mechanisms.

Lin H ，Yuan J ，Ruan KH ，Yang W ，Zhang J ，Dai Y ，Wang R ... -  《-》