Hugo H. Davila

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.

Fibrin as an inducer of fibrosis in the tunica albuginea of the rat: a new animal model of Peyronie's disease

To investigate the role of fibrin in inducing fibrosis in the tunica albuginea (TA) of the rat penis, to develop a new animal model for Peyronies disease (PD).

Gene Transfer of Inducible Nitric Oxide Synthase Complementary DNA Regresses the Fibrotic Plaque in an Animal Model of Peyronie's Disease

Abstract The goal of the present study was to investigate the antifibrotic role of inducible nitric oxide synthase (iNOS) in Peyronies disease (PD) by determining whether a plasmid expressing iNOS (piNOS) injected into a PD-like plaque can induce regression of the plaque. A PD-like plaque was induced with fibrin in the penile tunica albuginea of mice and then injected with a luciferase-expressing plasmid (pLuc), either alone or with piNOS, following luciferase expression in vivo by bioluminescence imaging. Rats were treated with either piNOS, an empty control plasmid (pC), or saline. Other groups were treated with pC or piNOS, in the absence of fibrin. Tissue sections were stained for collagen, transforming growth factor (TGF) β1, and plasminogen-activator inhibitor (PAI-1) as profibrotic factors; copper-zinc superoxide dismutase (CuZn SOD) as scavenger of reactive oxygen species (ROS); and nitrotyrosine to detect nitric oxide reaction with ROS. Quantitative image analysis was applied. Both iNOS and xanthine oxido-reductase (XOR; oxidative stress) were estimated by Western blot analysis. Luciferase reporter expression was restricted to the penis, peaked at 3 days after injection, but continued for at least 3 wk. In rats receiving piNOS, iNOS expression also peaked at 3 days, but expression decreased at the end of treatment, when a considerable reduction of plaque size occurred. Protein nitrotyrosine, XOR, and CuZn SOD increased, and TGFβ1 and PAI-1 decreased. The piNOS gene transfer regressed the PD plaque and expression of profibrotic factors, supporting the view that endogenous iNOS induction in PD is defense mechanism by the tissue against fibrosis.

Antisense and short hairpin RNA (shRNA) constructs targeting PIN (Protein Inhibitor of NOS) ameliorate aging-related erectile dysfunction in the rat.

INTRODUCTION Over-expression of penile neuronal nitric oxide synthase (PnNOS) from a plasmid ameliorates aging-related erectile dysfunction (ED), whereas over-expression of the protein inhibitor of NOS (PIN), that binds to nNOS, increases ED. AIM To improve this form of gene therapy for ED by comparing the electrical field response of short hairpin RNA (shRNA) for PIN with that of antisense PIN RNA. MAIN OUTCOME MEASURE Both shRNA and antisense RNA gene therapy vectors increased intracavernosal pressure in aged rats. METHODS PIN small interfering RNA (siRNA), and plasmid constructs for cytomegalovirus promoter plasmid vector (pCMV-PIN), pCMV-PIN antisense RNA, pSilencer2.1-U6-PIN-shRNA; and pSilencer2.1-U6-randomer-shRNA were prepared and validated by transfection into HEK293 cells, determining the effects on PIN expression by Western blot. Plasmid constructs were then injected, followed by electroporation, into the penile corpora cavernosa of aged (20-month-old) Fisher 344 rats and, 1 month later, the erectile response was measured by intracavernosal pressure increase following electrical field stimulation (EFS) of the cavernosal nerve. PIN was estimated in penile tissue by Western blot and real-time reverse transcriptase-polymerase chain reaction. Cyclic guanosine monophosphate (cGMP) measurements were conducted by competitive enzyme immunoassay (EIA). Immunohistofluorescence detected PIN in corporal tissue sections. RESULTS In cell culture, PIN siRNA and plasmid-expressed pU6-PIN-shRNA effectively reduced PIN expression from pCMV-PIN. pSilencer2.1-U6-PIN-shRNA corrected the impaired erectile response to EFS in aged rats and raised it above the value for young rats, more efficiently than pCMV-PIN antisense RNA. PIN mRNA expression in the penis was decreased by >70% by the shRNA but remained unaffected by the antisense RNA, whereas PIN protein expression was reduced in both cases, particularly in the dorsal nerve. PIN antisense increased cGMP concentration in treated tissue by twofold. CONCLUSION pSilencer2.1-U6-PIN-shRNA gene therapy was more effective than the antisense PIN mRNA in ameliorating ED in the aged rat, thereby suggesting that PIN is indeed a physiological inhibitor of nNOS and nitrergic neurotransmission in the penis.

Protein Inhibitor of Nitric Oxide Synthase (NOS) and the N-Methyl-d-Aspartate Receptor Are Expressed in the Rat and Mouse Penile Nerves and Colocalize with Penile Neuronal NOS

Abstract Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-d-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS−/− mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

ANALYSIS OF THE PSA RESPONSE AFTER TESTOSTERONE SUPPLEMENATATION IN PATIENTS WHO HAVE PREVIOUSLY RECEIVED MANAGEMENT FOR THEIR LOCALIZED PROSTATE CANCER

1247 ANALYSIS OF THE PSA RESPONSE AFTER TESTOSTERONE SUPPLEMENATATION IN PATIENTS WHO HAVE PREVIOUSLY RECEIVED MANAGEMENT FOR THEIR LOCALIZED PROSTATE CANCER Hugo H Davila*, Charles N Arison, Mary K Hall, Raoul Salup, Jorge L Lockhart, Rafael E Carrion. Tampa, FL. INTRODUCTION AND OBJECTIVE: 60 years ago, Huggins et al. demonstrated that suppression of testosterone (T) levels caused regression of prostate cancer (Pca). However, despite decades of research there is no compelling evidence that that T has a causative role in Pca. Our aim was to investigate the impact of testosterone supplementation (TS) in patients having undergone radical prostatectomy (RP) antigen (PSA) was closely monitored after commencing TS. METHODS: We retrospectively studied 20 men with hypogonadal symptoms or low T levels who underwent TS with either injections 200mg/q2w (n=8) or transdermal-gel 5g qd (n=12). These patients had previously undergone an open (n=8) or laparoscopic (n=6) RP (group A), or external bean radiation therapy (EBRT) (n=6) (group B) undetectable PSA and normal digital rectal exam (DRE) post therapy and before starting TS. T and PSA levels were evaluated before and after TS. RESULTS: When we compared group A/B, the Mean Age was 69/66, mean time after Pca therapy was 74/57 months, and mean follow up time after TS was 12/9 months. Moreover, the Gleason score was 6.2/5, body mass index (BMI) was 28/35 (p 0.05) and 630 vs 834 (p>0.05). Using the same time points, the PSA level was 0.1 vs 0.2 (OR 0.14, 95% CI -3.33-3.47, p>0.05) and 0.1 vs 0.1 (OR 0.22, 95% CI -2.94-3.17, p>0.05) CONCLUSIONS: TS by T injection or transdermal-gel is effective in improving T level and hypogonadal symptoms in men follow