Thomas R. Magee

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.

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.

Evidence That Osteogenic Progenitor Cells in the Human Tunica Albuginea May Originate from Stem Cells: Implications for Peyronie Disease

Abstract Tissue ossification in Peyronie disease (commonly known as Peyronies disease [PD]), a localized fibrotic lesion within the tunica albuginea (TA) of the penis, may result from osteogenic differentiation of fibroblasts, myofibroblasts, and/or adult stem cells in the TA, and may be triggered by chronic inflammation, oxidative stress, and profibrotic factors like transforming growth factor beta 1 (TGFB1). In this study, we have investigated whether cultures of cells from normal TA and PD plaques undergo osteogenesis, express markers for stem cells, and originate other cell lineages via processes modulated by TGFB1. We found that TA and PD cells in osteogenic medium (OM) expressed osteogenic markers, alkaline phosphatase, and osteopontin and underwent calcification. PD cells, but not TA cells, formed foci in soft agar that were positive for alkaline phosphatase and calcification and expressed the mRNAs for osteoblast-specific factors pleiotrophin and periostin and bone morphogenic protein 2. Both cultures expressed stem cell marker CD34 antigen but not protein tyrosine phosphatase, receptor type c. TA and PD cells expressed smooth-muscle cell markers smoothelin and transgelin. None of the cultures underwent adipogenesis in adipogenic medium. Incubation with TGFB1 increased osteogenesis and myofibroblast differentiation and reduced CD34 antigen expression in both cultures. TA and PD cells modulated the differentiation of the multipotent C3H 10T(1/2) cells in dual cultures, into osteoblasts and myofibroblasts. In conclusion, both TA and PD cultures contain cells, presumably stem cells, that undergo osteogenic and myofibroblast differentiation, and may induce these processes by paracrine interactions. This may explain progression of fibrosis in the PD plaque and its eventual calcification.

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.

Expression of penile neuronal nitric oxide synthase variants in the rat and mouse penile nerves

Abstract Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSα, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSß, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSα and β are expressed in the rat penis and are located in the nerves and whether the β form persists in the potent nNOS knock-out mouse (nNOS△△). A PnNOS antibody against the insert common to both PnNOSα and β detected the expected 155-kDa protein in PnNOSα-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSα mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSβ mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS△△ mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the β form. In conclusion, both PnNOSα and β are expressed in the rat penis at all ages and are located in the nerves. The β form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS△△ mouse occurs through transcription of the β mRNA, and this may explain the retention of erectile function when the expression of nNOSα is disrupted.

Gene expression profiles in the Peyronie’s disease plaque

OBJECTIVES To provide molecular insight into the pathophysiology of Peyronies disease (PD), a preliminary profile of differential gene expression between the PD plaque and control tunica albuginea was obtained with DNA microarrays. METHODS Seven PD plaques and five control tunica albugineas were studied. cDNA specimens were prepared from RNA isolated from one calcified PD plaque and one control tissue and hybridized with the Clontech Atlas 1.2 Array. Another set of plaque and control RNA samples was hybridized with the Affymetrix GeneChip. Relative changes of greater than 2.0 defined up-regulation and down-regulation, respectively. RNA from the remaining tissues was used to determine, by reverse transcriptase-polymerase chain reaction and Western blot analysis, the expression of selected individual genes. RESULTS Some of up-regulated genes in the PD plaque detected by the Clontech assay were pleiotrophin, monocyte chemotactic protein 1, and early growth response protein, which are involved in osteoblast recruitment, inflammation, and fibroblast proliferation, respectively. Ubiquitin and Id-2, which are involved in tissue remodeling, were down-regulated. The Affymetrix DNA chips identified the up-regulation of elastase (involved in elastic fiber degradation) and the myofibroblast markers alpha and gamma-smooth muscle actin, desmin, and others, as well as the down-regulation of collagenase IV and transforming growth factor-beta modulators. Four of the five genes selected for reverse transcriptase-polymerase chain reaction and Western blotting confirmed the DNA microarray results. CONCLUSIONS In the PD tissue, the genes involved in collagen synthesis, myofibroblast differentiation, tissue remodeling, inflammation, ossification, and proteolysis are up-regulated, and the genes that inhibit some of these processes and collagenase are down-regulated.

Profibrotic Role of Myostatin in Peyronie's Disease

INTRODUCTION The primary histologic finding in many urologic disorders, including Peyronies disease (PD), is fibrosis, mainly mediated by the transforming growth factor beta1 (TGFbeta1). AIM To determine whether another member of the TGFbeta family, myostatin, (i) is expressed in the human PD plaque and normal tunica albuginea (TA), their cell cultures, and the TGFbeta1-induced PD lesion in the rat model; (ii) is responsible for myofibroblast generation, collagen deposition, and plaque formation; and (iii) mediates the profibrotic effects of TGFbeta1 in PD. METHODS Human TA and PD tissue sections, and cell cultures from both tissues incubated with myostatin and TGFbeta1 were subjected to immunocytochemistry for myostatin and alpha-smooth muscle actin (ASMA). The cells were assayed by western blot, Real time-Polymerase chain reaction (RT-PCR), and ribonuclease protection. Myostatin cDNA and shRNA were injected, with or without TGFbeta1, in the rat penile TA, and plaque size was estimated by Masson. MAIN OUTCOME MEASURES Myostatin expression in the human TA, the PD plaque, and their cell cultures, and myostatin effects on the PD-like plaque in the rat. RESULTS A threefold overexpression of myostatin was found in the PD plaque as compared with the TA. In PD cells, myostatin expression was mainly in the myofibroblasts, and in the TA cells, it increased upon passage paralleling myofibroblast differentiation and was up-regulated by TGFbeta1. Myostatin or its cDNA construct increased the myofibroblast number and collagen in TA cells. Myostatin was detected in the TGFbeta1-induced PD-like plaque of the rat partly in the myofibroblasts, and in the TA. Myostatin cDNA injected in the TA induced a plaque and intensified the TGFbeta1 lesion, which was not reduced by myostatin shRNA. CONCLUSIONS Myostatin is overexpressed in the PD plaque, partly because of myofibroblast generation. Although myostatin induces a plaque in the rat TA, it does not appear to mediate the one triggered by TGFbeta1, thus suggesting that both proteins act concurrently and that therapy should target their common downstream effectors.