Dolores Vernet

Chronic daily tadalafil prevents the corporal fibrosis and veno-occlusive dysfunction that occurs after cavernosal nerve resection

Associate Editor

Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie's fibrotic plaque and in its rat model.

The myofibroblast shares phenotypic features of both fibroblasts and smooth muscle cells. It plays a critical role in collagen deposition and wound healing and disappears by apoptosis when the wound is closed. Its abnormal persistence leads to hypertrophic scar formation and other fibrotic conditions. Myofibroblasts are present in the fibrotic plaque of the tunica albuginea (TA) of the penis in men with Peyronies disease (PD), a localized fibrosis that is accompanied by a spontaneous induction of the inducible nitric oxide synthase (iNOS), also observed in the TGFbeta1-elicited, PD-like lesion in the rat model. iNOS expression counteracts fibrosis, by producing nitric oxide (NO) that reduces collagen deposition in part by neutralization of profibrotic reactive oxygen species. In this study we investigated whether fibroblast differentiation into myofibroblasts is enhanced in the human and rat PD-like plaque and in cultures of human tissue fibroblasts. We also examined whether NO reduces this cell differentiation and collagen synthesis. The myofibroblast content in the fibroblast population was measured by quantitative immunohistochemistry as the ratio between alpha-smooth muscle actin (ASMA; myofibroblast marker) and vimentin (general fibroblast marker) levels. We found that myofibroblast content was considerably increased in the human and TGFbeta1-induced rat plaques as compared to control TA. Inhibition of iNOS activity by chronic administration of L-iminoethyl-L-lysine to rats with TGFbeta1-induced TA lesion increased myofibroblast abundance and collagen I synthesis measured in plaque and TA homogenates from animals injected with a collagen I promoter construct driving the expression of beta-galactosidase. Fibroblast differentiation into myofibroblasts occurred with passage in the cell cultures from the human PD plaque, but was minimal in cultures from the TA. Induction of iNOS in PD and TA cultures with a cytokine cocktail and a NO donor, S-nitroso-N-acetyl penicillamine (SNAP), was detected by immunohistochemistry. Both treatments reduced the total number of cells and the number of ASMA positive cells, whereas only SNAP decreased collagen I immunostaining. These results support the hypotheses that myofibroblasts play a role in the development of the PD plaque and that the antifibrotic effects of NO may be mediated at least in part by the reduction of myofibroblast abundance and lead to a reduction in collagen I synthesis.

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass

Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass.

Long-Term Continuous Treatment with Sildenafil Ameliorates Aging-Related Erectile Dysfunction and the Underlying Corporal Fibrosis in the Rat

Abstract Aging-related erectile dysfunction is characterized by a loss of smooth muscle cells (SMCs) and fibrosis in the corpora cavernosa, and functionally by corporal veno-occlusive dysfunction (CVOD). Phosphodiesterase 5 (PDE5A) inhibitors, in part via upregulating inducible nitric oxide synthase (NOS2A), have antifibrotic properties in penile tissues. We aimed to determine whether in the aged rat the chronic long-term treatment with sildenafil ameliorates corporal SMC loss and fibrosis, stimulates NOS2A induction, and corrects the associated CVOD. Aged male rats (20 mo old) received sildenafil in their drinking water (20 mg/kg per day) or plain water for 45 days, and untreated young rats (5 mo old) served as controls (n = 8 per group). CVOD was assessed by dynamic infusion cavernosometry (DIC). Collagen:SMC (Masson trichrome) and collagen III:I (picrosirius red) ratios, SMC content (alpha-smooth muscle actin [ACTA2]), cell proliferation (proliferating nuclear antigen [PCNA]), apoptotic death (TUNEL), and NOS2A induction were measured by histochemistry and immunohistochemistry followed by quantitative image analysis. Collagen content was determined by hydroxyproline assay, and transforming growth factor beta-1 (TGFB1); xanthine oxidoreductase (XDH); ACTA2; NOS2A; and the Rho kinase inhibitor protein tyrosine phosphatase, nonreceptor type 11 (PTPN11), and activator, VAV, were measured by quantitative Western blot. In the aged rats treated with sildenafil, the erectile response by DIC was normalized, and the corporal SMC:collagen ratio and SMC number were increased. In addition, sildenafil reduced the corporal collagen content without affecting the collagen III:I ratio, increased the PCNA:apoptosis ratio, and stimulated NOS2A induction, although there was no effect on XDH, TGFB1, PTPN11, or VAV levels. These data show that long-term PDE5A treatment corrected CVOD in the aged rat and partially reversed the aging-related fibrosis and loss of SMC in the corpora cavernosa without affecting TGFB1 or PTPN11 levels, which are markers of oxidative stress. It may be speculated that similar effects may be achieved with this paradigm in men.

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase.

Abstract Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and β-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-β-gal) and “gutless” AdV (AdV-CMV-PnNOS; AdV-CMV-β-gal) vectors, and injected into the penis of adult (β-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of β-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-β-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 106 viral particles (vp) of AdV-CMV-β-gal, and with 107 vp β-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (107 vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.

Aging-Related Expression of Inducible Nitric Oxide Synthase and Markers of Tissue Damage in the Rat Penis

Abstract Erectile dysfunction in the aging male results in part from the loss of compliance of the corpora cavernosal smooth muscle due to the progressive replacement of smooth muscle cells by collagen fibers. We have examined the hypothesis that a spontaneous local induction of inducible nitric oxide synthase (iNOS) expression and the subsequent peroxynitrite formation occurs in the penis during aging and that this process is accompanied by a stimulation of smooth muscle apoptosis and collagen deposition. The penile shaft and crura were excised from young (3–5 mo old) and old (24–30 mo old) rats, with or without perfusion with 4% formalin. Fresh tissue was used for iNOS and proteasome 2C mRNA determinations by reverse transcription polymerase chain reaction assay, ubiquitin mRNA by Northern blot, and iNOS protein by Western blot. Penile sections from perfused animals were embedded in paraffin and immunostained with antibodies against iNOS and nitrotyrosine, submitted to the TUNEL assay for apoptosis, or stained for collagen, followed by image analysis quantitation. A 4.1-fold increase in iNOS mRNA was observed in the old versus young tissues, paralleled by a 4.9-fold increase in iNOS protein. The proteolysis marker, ubiquitin, was increased 1.9-fold, whereas a related gene, proteasome 2c, was not significantly affected. iNOS immunostaining was increased 3.6-fold in the penile smooth muscle of the old rats as compared with the young rats. The peroxynitrite indicator nitrotyrosine was increased by 1.6-fold, accompanied by a 3.6-fold increase in apoptotic cells and a 2.0-fold increase in collagen fibers in the old penis. In conclusion, aging in the penis is accompanied by an induction of iNOS and peroxynitrite formation that may lead to the observed increase in apoptosis and proteolysis and may counteract a higher rate of collagen deposition in the old penis.

Effect of muscle-derived stem cells on the restoration of corpora cavernosa smooth muscle and erectile function in the aged rat

To determine whether skeletal muscle‐derived stem cells (MDSCs) convert into smooth muscle cells (SMCs) both in vitro and in vivo, and in so doing ameliorate the erectile dysfunction (ED) of aged rats, and whether endogenous stem cells are present in the rat corpora cavernosa.

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats

It was recently reported in the rat that vardenafil given in a continuous long-term manner was successful in preventing smooth muscle fibrosis in the penile corpora cavernosa and corporal veno-occlusive dysfunction (CVOD) that occur following bilateral cavernosal nerve resection (BCNR), a model for human erectile dysfunction after radical prostatectomy. To expand on this finding and to determine whether this effect was common to other PDE5 inhibitors, and occurred in part by stimulation of the spontaneous induction of inducible nitric oxide synthase (iNOS, also known as NOS2), male Fischer 344 rats (N=10/group) were subjected to either BCNR or unilateral cavernosal nerve resection (UCNR) and treated with sildenafil (20 mg kg−1 day−1) in the drinking water daily for 45 days. Additional BCNR groups received L-NIL (6.7 mg kg−1 day−1) as inhibitor of iNOS activity, with or without concurrent sildenafil administration. It was determined that sildenafil, like vardenafil, (1) prevented the 30% decrease in the smooth muscle cell/collagen ratio, and the 3–4-fold increase in apoptosis and reduction in cell proliferation, and partially counteracted the increase in collagen, seen with both UCNR and BCNR; and (2) normalized the CVOD, measured by dynamic infusion cavernosometry, induced by both BCNR and UCNR. The long-term inhibition of iNOS activity exacerbated corporal fibrosis and CVOD in the BCNR rats, but sildenafil functional effects were not affected by L-NIL. These data suggest that the salutary effects of continuous long-term PDE5 inhibitors on erectile function post-cavernosal nerve resection involve their ability to prevent the alterations in corporal histology induced by cavernosal nerve damage, in a process apparently independent from endogenous iNOS induction.

Effects of Long-term Oral Administration of L-Arginine on the Rat Erectile Response

PURPOSE Nitric oxide (NO), the neurotransmitter responsible for mediating penile erection in the rat, is synthesized from L arginine by nitric oxide synthase (NOS) in a reaction blocked by L-NAME (N-omega-nitro-L-arginine methyl ester). To determine whether dietary supplementation of L-arginine can stimulate penile erection and whether ancillary pathways for penile erection may exist, a series of experiments were conducted in the Fischer 344 rat. MATERIALS AND METHODS Adult male (5 month old) and aged (20 month old) rats were fed L-arginine (2.25%) and L-NAME (0.7%) dissolved in tap water for 8 weeks. Animals (n = 6) underwent electrical field stimulation (EFS) of the cavernosal nerve to induce erection and both maximal intracavernosal pressure (MIP) and mean arterial pressure (MAP, mm. Hg +/- SEM) were measured. Tissue and serum levels of L-arginine were measured by an automated amino acid analyzer. Penile eNOS (endothelial) and nNOS (neuronal) content were measured by western blot densitometry. Total penile NOS enzyme activity was measured by the L-arginine to L-citrulline conversion assay. RESULTS The L-arginine fed animals demonstrated a significant increase in EFS-induced MIP when compared to the controls in both the adult (104 +/- 4 vs. 86 +/- 6, p = 0.04) and aged (87 +/- 5 vs. 66 +/- 4, p = 0.02) animals, without changes in MAP. L-NAME virtually abolished the MIP in adult rats (8 +/- 3, p < 0.0001), while increasing the MAP (186 +/- 8, p < 0.0001). Serum and penile tissue levels of L-arginine were increased by 64-148% in all groups compared to control animals. Penile eNOS and nNOS content remained unchanged in control and treated animals. Penile NOS activity was increased nearly 100% in the L-arginine treated groups vs. controls. CONCLUSIONS Long-term oral administration of supra-physiologic doses of L-arginine improves the erectile response in the aging rat. We postulate that L-arginine in the penis may be a substrate-limiting factor for NOS activity and that L-arginine may up-regulate penile NOS activity but not its expression. The blockade of penile erection by EFS with L NAME suggests that if ancillary corporeal vasodilator mechanisms develop, a basal level of NO synthesis is still required for activation and relaxation of the corporeal smooth muscle. These data support the possible use of dietary supplements for treatment of erectile dysfunction.

Pioglitazone prevents corporal veno-occlusive dysfunction in a rat model of type 2 diabetes mellitus.

To determine whether corporal veno‐occlusive dysfunction (CVOD), corporal smooth muscle (SM) loss, fibrosis and oxidative stress occur in a rat model of type 2 diabetes, and whether these are counteracted by pioglitazone, as pioglitazone is vasculoprotective, and corporal SM is an extension of arterial SM.