Gaby Nolazco

Effects of long-term vardenafil treatment on the development of fibrotic plaques in a rat model of Peyronie's disease

To determine whether the phosphodiesterase‐5 (PDE5) inhibitor, vardenafil, given orally and in different regimens, has a similar effect to that of the PDE5 inhibitor sildenafil, which prevented the development of a Peyronies disease (PD)‐like plaque formation induced by injecting transforming growth factor β1 (TGF‐β1) into the tunica albuginea of the rat.

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.

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.

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.

Early onset of fibrosis within the arterial media in a rat model of type 2 diabetes mellitus with erectile dysfunction.

To determine, in the obese Zucker fa/fa rat (OZR), whether the loss in smooth muscle cells (SMCs) as well as the increase in fibrosis that occurs within the corpora cavernosa accompanying corporal veno‐occlusive dysfunction (CVOD), also occurs within the media of the arterial tree.

Separate or Combined Treatments with Daily Sildenafil, Molsidomine, or Muscle‐Derived Stem Cells Prevent Erectile Dysfunction in a Rat Model of Cavernosal Nerve Damage

INTRODUCTION Long-term daily administration of phosphodiesterase type 5 (PDE5) inhibitors in the rat prevents or reverses corporal veno-occlusive dysfunction (CVOD) and smooth muscle cell (CSMC) loss and fibrosis, in both aging and bilateral cavernosal nerve resection (BCNR) models for erectile dysfunction. In the aging rat model, corporal implantation of skeletal muscle-derived stem cells (MDSC) reverses CVOD. Nitric oxide (NO) and cyclic guanosine monophosphate can modulate stem cell lineage. AIM To investigate in the BCNR model the effects of sildenafil at lower doses, alone or in combination with MDSC or the NO donor molsidomine, on CVOD and the underlying corporal histopathology. MAIN OUTCOMES MEASURES CVOD, histological, and biochemical markers in rat corporal tissue. Methods.  Rats subjected to BCNR were maintained for 45 days either untreated, or received sildenafil in the water or retrolingually at 10, 2.5, and 1.25 mg/kg/day (medium, low, and very low doses), or intraperitoneal molsidomine, or MDSC implantation into the corpora cavernosa separately or in combination. Cavernosometry evaluated CVOD. Histopathology was assessed on penile sections by Masson trichrome, immunohistochemistry for α-smooth muscle actin (ASMA), or immunofluorescence for neuronal nitric oxide synthase (nNOS)/neurofilament 70, and in fresh tissue by Western blot for various markers and picrosirius red for collagen. RESULTS All treatments normalized erectile function (drop rate), and most increased the CSMC/collagen ratio and ASMA expression in corporal tissue sections, and reduced collagen content in the penile shaft. MDSC also increased nNOS and brain-derived neurotrophic factor. The combination treatment was not superior to MDSC or sildenafil given alone, and upregulated PDE5. CONCLUSIONS Lowering the dose of a continuous long-term sildenafil administration still maintained the prevention of CVOD in the BCNR rat previously observed, but it was less effective on the underlying histopathology. As in the aging rat model, MDSC also counteracted CVOD, but supplementation with very low-dose sildenafil did not improve the outcome.

Ageing-related corpora veno-occlusive dysfunction in the rat is ameliorated by pioglitazone

To determine whether ageing‐related changes in the penile corpora cavernosa, namely corporal veno‐occlusive dysfunction (CVOD), loss of smooth muscle cells (SMCs), and excessive collagen deposition, can be ameliorated by the peroxisome proliferator‐activated receptor γ (PPARγ) agonist pioglitazone, in a rat model of ageing as we have shown in a rat model of type 2 diabetes.

Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

IntroductionStimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD).MethodsTo examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis.ResultsSurprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration.ConclusionsAlthough WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs.

Effects of sildenafil and/or muscle derived stem cells on myocardial infarction

BackgroundPrevious studies have shown that long-term oral daily PDE 5 inhibitors (PDE5i) counteract fibrosis, cell loss, and the resulting dysfunction in tissues of various rat organs and that implantation of skeletal muscle-derived stem cells (MDSC) exerts some of these effects. PDE5i and stem cells in combination were found to be more effective in non-MI cardiac repair than each treatment separately. We have now investigated whether sildenafil at lower doses and MDSC, alone or in combination are effective to attenuate LV remodeling after MI in rats.MethodsMI was induced in rats by ligature of the left anterior descending coronary artery. Treatment groups were: “Series A”: 1) untreated; 2) oral sildenafil 3 mg/kg/day from day 1; and “Series B”: intracardiac injection at day 7 of: 3) saline; 4) rat MDSC (106 cells); 5) as #4, with sildenafil as in #2. Before surgery, and at 1 and 4 weeks, the left ventricle ejection fraction (LVEF) was measured. LV sections were stained for collagen, myofibroblasts, apoptosis, cardiomyocytes, and iNOS, followed by quantitative image analysis. Western blots estimated angiogenesis and myofibroblast accumulation, as well as potential sildenafil tachyphylaxis by PDE 5 expression. Zymography estimated MMPs 2 and 9 in serum.ResultsAs compared to untreated MI rats, sildenafil improved LVEF, reduced collagen, myofibroblasts, and circulating MMPs, and increased cardiac troponin T. MDSC replicated most of these effects and stimulated cardiac angiogenesis. Concurrent MDSC/sildenafil counteracted cardiomyocyte and endothelial cells loss, but did not improve LVEF or angiogenesis, and upregulated PDE 5.ConclusionsLong-term oral sildenafil, or MDSC given separately, reduce the MI fibrotic scar and improve left ventricular function in this rat model. The failure of the treatment combination may be due to inducing overexpression of PDE5.

101 ANDROGEN MODULATION OF CELL LINEAGE COMMITMENT BY MUSCLE-DERIVED STEM CELLS.

Resident stem cells in the adult skeletal muscle are assumed to participate in normal tissue growth and in repair after injury by differentiating into committed myogenic precursors, presumably satellite cells. We have shown that androgens stimulate in vitro the myogenic differentiation of a mouse mesenchymal multipotent cell line (C3H-10T(1/2)) and inhibit its adipogenic differentiation and postulated that they would similarly affect the in vitro lineage commitment of mouse muscle-derived stem cells (MDSCs). To test this hypothesis, we isolated by the preplating procedure from the mouse hind limb muscles a MDSC cell population (pP6). This cell fraction comprised about 1% of the total muscle mononucleated cells and expressed the stem cell markers CD34 and Sca1+, but it was CD45-, as shown by immunocytochemistry and Western blot. The Sca1+ cells were further enriched by magnetic beads selection. Both populations of pP6 cells expressed androgen receptors and a low level of the early and intermediate myogenic markers, MyoD and myogenin, but not the late marker, myosin heavy chain II (MHC-II), as detected by Western blot. MyoD was more expressed in the other cell fractions, pP2 (mainly fibroblasts) and pP3-pP5 (rich in satellite cells). pP6 cells in adipogenic medium for 1 week differentiated into adipocytes, and in osteogenic medium for 2 and 4 weeks formed myofibroblasts and osteoblasts, as detected with Oil Red O, α-smooth muscle actin (ASMA), and alkaline phosphatase staining, respectively, and by Western blot for CBPα, ASMA, and alkaline phosphatase. In myogenic medium for 2 weeks, pP6 failed to form myotubes, intensify the MyoD and myogenin signals, or express MHC-II. However, when incubated with murine C2C12 myotubes on the top compartment of dual culture chambers the cells underwent intense myogenesis and led to a clear MHC-II signal. As predicted, testosterone (100 nM) and DHT (30 nM) increased myotube formation in dual cultures and reduced adipocyte number and stimulated osteogenesis in monocultures but did not affect fibrogenesis (myofibroblasts). Casodex and flutamide abolished these effects. In conclusion, we found that androgens do stimulate paracrinely induced myogenesis and inhibit adipogenesis in MDSC cultures, thus suggesting that a similar process may occur in vivo in the myofibers.