Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence.

Human stem cell therapy for type 2 diabetes/obesity (T2D/O) complications is performedwith stem cell autografts, exposed to the noxious T2D/O milieu, often with suboptimal results.We showed in the Obese Zucker (OZ) rat model of T2D/O that when their muscle-derived stemcells (MDSC) were from long-term T2D/O male rats, their repair ecacy for erectile dysfunctionwas impaired and were imprinted with abnormal gene- and miR-global transcriptional signatures(GTS). The damage was reproduced in vitro by short-term exposure of normal MDSC to dyslipidemicserum, causing altered miR-GTS, fat infiltration, apoptosis, impaired scratch healing, and myostatinoverexpression. Similar in vitro alterations occurred with their normal counterparts (ZF4-SC) fromthe T2D/O rat model for female stress urinary incontinence, and with ZL4-SC from non-T2D/O leanfemale rats. In the current work we studied the in vitro eects of cholesterol and Na palmitate aslipid factors on ZF4-SC and ZL4-SC. A damage partially resembling the one caused by the femaledyslipidemic serum was found, but diering between both lipid factors, so that each one appears tocontribute specifically to the stem cell damaging eects of dyslipidemic serum in vitro and T2D/Oin vivo, irrespective of gender. These results also confirm the miR-GTS biomarker value forMDSC damage.

Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence Are Damaged by In Vitro Exposure to its Dyslipidemic Serum, Predicting Inadequate Repair Capacity In Vivo.

Female stress urinary incontinence (FSUI) is prevalent in women with type 2 diabetes/obesity (T2D/O), and treatment is not optimal. Autograph stem cell therapy surprisingly has poor efficacy. In the male rat model of T2D/O, it was demonstrated that epigenetic changes, triggered by long-term exposure to the dyslipidemic milieu, led to abnormal global transcriptional signatures (GTS) of genes and microRNAs (miR), and impaired the repair capacity of muscle-derived stem cells (MDSC). This was mimicked in vitro by treatment of MDSC with dyslipidemic serum or lipid factors. The current study aimed to predict whether these changes also occur in stem cells from female 12 weeks old T2D/O rats, a model of FSUI. MDSCs from T2D/O (ZF4-SC) and normal female rats (ZL4-SC) were treated in vitro with either dyslipidemic serum (ZFS) from late T2D/O 24 weeks old female Zucker fatty (ZF) rats, or normal serum (ZLS) from 24 weeks old female Zucker lean (ZL) rats, for 4 days and subjected to assays for fat deposition, apoptosis, scratch closing, myostatin, interleukin-6, and miR-GTS. The dyslipidemic ZFS affected both female stem cells more severely than in the male MDSC, with some gender-specific differences in miR-GTS. The changes in miR-GTS and myostatin/interleukin-6 balance may predict in vivo noxious effects of the T2D/O milieu that might impair autograft stem cell (SC) therapy for FSUI, but this requires future studies.

Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture.

BACKGROUND: Previous work showed that muscle-derived stem cells (MDSCs) exposed long-term to the milieu of uncontrolled type 2 diabetes (UC-T2D) in male obese Zucker (OZ) rats, were unable to correct the associated erectile dysfunction and the underlying histopathology when implanted into the corpora cavernosa, and were also imprinted with a noxious gene global transcriptional signature (gene-GTS), suggesting that this may interfere with their use as autografts in stem cell therapy. AIM: To ascertain the respective contributions of dyslipidemia and hyperglycemia to this MDSC damage, clarify its mechanism, and design a bioassay to identify the damaged stem cells. METHODS: Early diabetes MDSCs and late diabetes MDSCs were respectively isolated from nearly normal young OZ rats and moderately hyperglycemic and severely dyslipidemic/obese aged rats with erectile dysfunction. Monolayer cultures of early diabetic MDSCs were incubated 4 days in DMEM/10% fetal calf serum + or - aged OZ or lean Zucker serum from non-diabetic lean Zucker rats (0.5-5%) or with soluble palmitic acid (PA) (0.5-2 mM), cholesterol (CHOL) (50-400 mg/dL), or glucose (10-25 mM). MAIN OUTCOME MEASURE: Fat infiltration was estimated by Oil red O, apoptosis by TUNEL, protein expression by Western blots, and gene-GTS and microRNA (miR)-GTS were determined in these stem cells' RNA. RESULTS: Aged OZ serum caused fat infiltration, apoptosis, myostatin overexpression, and impaired differentiation. Some of these changes, and also a proliferation decrease occurred with PA and CHOL. The gene-GTS changes by OZ serum did not resemble the in vivo changes, but some occurred with PA and CHOL. The miR-GTS changes by OZ serum, PA, and CHOL resembled most of the in vivo changes. Hyperglycemia did not replicate most alterations. CLINICAL IMPLICATIONS: MDSCs may be damaged in long-term UC-T2D/obese patients and be ineffective in autologous human stem cell therapy, which may be prevented by excluding the damaged MDSCs. STRENGTH & LIMITATIONS: The in vitro test of MDSCs is innovative and fast to define dyslipidemic factors inducing stem cell damage, its mechanism, prevention, and counteraction. Confirmation is required in other T2D/obesity rat models and stem cells (including human), as well as miR-GTS biomarker validation as a stem cell damage biomarker. CONCLUSION: Serum from long-term UC-T2D/obese rats or dyslipidemic factors induces a noxious phenotype and miR-GTS on normal MDSCs, which may lead in vivo to the repair inefficacy of late diabetic MDSCs. This suggests that autograft therapy with MDSCs in long-term UT-T2D obese patients may be ineffective, albeit this may be predictable by prior stem cell miR-GTS tests. Masouminia M, Gelfand R, Kovanecz I, et al. Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture. J Sex Med 2018;15:1678-1697.

Aging related erectile dysfunction-potential mechanism to halt or delay its onset.

Erectile dysfunction (ED) will visit every man at some time in his life. The age at when that knock on the door is heard is totally dependent on one's genetics as well as other extrinsic factors. Unlike guests who come for a visit and then leave, once ED shows up it tends to hang around forever. To add insult to injury, the longer ED hangs around, the worse it will get. It is estimated that by the time a man is in his 40's, he has about a 40% chance of having some form of ED and this prevalence increases about 10% per decade thereafter. This suggests that the aging related process that leads to ED begins early in life. It turns out that the most common cause of ED, regardless of the patient's age, is due to a problem with the vascular system of the penis. However, this specific aging related vascular problem is not caused by arterial disease but due to a dysfunction and/or loss of the corporal smooth muscle cells (SMC), the main constituent of the corporal sinusoids. As one gets older, these SMC continue to degrade and disappear. When approximately 15% of these cells have been impacted, it results in an inability of the corporal tissue to retain and/or prevent the blood from "leaking" out of the corporal sinusoids into the systemic veins. However, the corporal SMC themselves begin to combat this aging process by expressing the inducible nitric oxide synthase (iNOS) enzyme to make nitric oxide (NO) in an attempt to quench the high intracellular oxidative stress responsible for the SMC apoptosis. When this iNOS pathway is then pharmacologically upregulated, reversal of these aging related changes in the corpora with correction of the venous leakage is observed. Since we believe that aging related ED is pathologically the same disorder as essential hypertension, the development of a therapeutic regimen that can halt, delay or possibly reverse the cellular processes that lead to aging related ED should also be applicable to those patients diagnosed with essential hypertension.

Long-term exposure of MCF-7 breast cancer cells to ethanol stimulates oncogenic features.

Alcohol consumption is a risk factor for breast cancer. Little is known regarding the mechanism, although it is assumed that acetaldehyde or estrogen mediated pathways play a role. We previously showed that long-term exposure to 2.5 mM ethanol (blood alcohol ~0.012%) of MCF-12A, a human normal epithelial breast cell line, induced epithelial mesenchymal transition (EMT) and oncogenic transformation. In this study, we investigated in the human breast cancer cell line MCF-7, whether a similar exposure to ethanol at concentrations ranging up to peak blood levels in heavy drinkers would increase malignant progression. Short-term (1-week) incubation to ethanol at as low as 1-5 mM (corresponding to blood alcohol concentration of ~0.0048-0.024%) upregulated the stem cell related proteins Oct4 and Nanog, but they were reduced after exposure at 25 mM. Long-term (4-week) exposure to 25 mM ethanol upregulated the Oct4 and Nanog proteins, as well as the malignancy marker Ceacam6. DNA microarray analysis in cells exposed for 1 week showed upregulated expression of metallothionein genes, particularly MT1X. Long-term exposure upregulated expression of some malignancy related genes (STEAP4, SERPINA3, SAMD9, GDF15, KRT15, ITGB6, TP63, and PGR, as well as the CEACAM, interferon related, and HLA gene families). Some of these findings were validated by RT-PCR. A similar treatment also modulated numerous microRNAs (miRs) including one regulator of Oct4 as well as miRs involved in oncogenesis and/or malignancy, with only a few estrogen-induced miRs. Long-term 25 mM ethanol also induced a 5.6-fold upregulation of anchorage-independent growth, an indicator of malignant-like features. Exposure to acetaldehyde resulted in little or no effect comparable to that of ethanol. The previously shown alcohol induction of oncogenic transformation of normal breast cells is now complemented by the current results suggesting alcohol's potential involvement in malignant progression of breast cancer.

Translational Perspective on the Role of Testosterone in Sexual Function and Dysfunction.

INTRODUCTION: The biological importance of testosterone is generally accepted by the medical community; however, controversy focuses on its relevance to sexual function and the sexual response, and our understanding of the extent of its role in this area is evolving. AIM: To provide scientific evidence examining the role of testosterone at the cellular and molecular levels as it pertains to normal erectile physiology and the development of erectile dysfunction and to assist in guiding successful therapeutic interventions for androgen-dependent sexual dysfunction. METHODS: In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current basic science literature examining the role of testosterone in sexual function and dysfunction. RESULTS: Testosterone plays an important role in sexual function through multiple processes: physiologic (stimulates activity of nitric oxide synthase), developmental (establishes and maintains the structural and functional integrity of the penis), neural (development, maintenance, function, and plasticity of the cavernous nerve and pelvic ganglia), therapeutically for dysfunctional regulation (beneficial effect on aging, diabetes, and prostatectomy), and phosphodiesterase type 5 inhibition (testosterone supplement to counteract phosphodiesterase type 5 inhibitor resistance). CONCLUSION: Despite controversies concerning testosterone with regard to sexual function, basic science studies provide incontrovertible evidence for a significant role of testosterone in sexual function and suggest that properly administered testosterone therapy is potentially advantageous for treating male sexual dysfunction.

Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0-2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

Implanted Muscle-Derived Stem Cells Ameliorate Erectile Dysfunction in a Rat Model of Type 2 Diabetes, but Their Repair Capacity Is Impaired by Their Prior Exposure to the Diabetic Milieu.

INTRODUCTION: Muscle-derived stem cells (MDSCs) and other SCs implanted into the penile corpora cavernosa ameliorate erectile dysfunction in type 1 diabetic rat models by replenishing lost corporal smooth muscle cells (SMCs) and decreasing fibrosis. However, there are no conclusive data from models of type 2 diabetes (T2D) and obesity. AIM: To determine whether MDSCs from obese Zucker (OZ) rats with T2D at an early stage of diabetes (early diabetic SCs isolated and cultured in low-glucose medium [ED-SCs]) counteract corporal veno-occlusive dysfunction and corporal SMC loss or lipo-fibrosis when implanted in OZ rats at a late stage of diabetes and whether MDSCs from these OZ rats with late diabetes (late diabetic SCs isolated and cultured in high-glucose medium [LD-SC]) differ from ED-SCs in gene transcriptional phenotype and repair capacity. METHODS: ED-SCs and LD-SCs were compared by DNA microarray assays, and ED-SCs were incubated in vitro under high-glucose conditions (ED-HG-SC). These three MDSC types were injected into the corpora cavernosa of OZ rats with late diabetes (OZ/ED, OZ/LD, and OZ/ED-HG rats, respectively). Untreated OZ and non-diabetic lean Zucker rats functioned as controls. Two months later, rats were subjected to cavernosometry and the penile shaft and corporal tissues were subjected to histopathology and DNA microarray assays. MAIN OUTCOME MEASURES: In vivo erectile dysfunction assessment by Dynamic Infusion Cavernosometry followed by histopathology marker analysis of the penile tissues. RESULTS: Implanted ED-SCs and ED-HG-SCs improved corporal veno-occlusive dysfunction, counteracted corporal decreases in the ratio of SMCs to collagen and fat infiltration in rats with long-term T2D, and upregulated neuronal and endothelial nitric oxide. LD-SCs acquired an inflammatory, pro-fibrotic, oxidative, and dyslipidemic transcriptional phenotype and failed to repair the corporal tissue. CONCLUSION: MDSCs from pre-diabetic rats injected into the corpora cavernosa of rats with long-term T2D improve corporal veno-occlusive dysfunction and the underlying histopathology. In contrast, MDSCs from rats with long-term uncontrolled T2D are imprinted by the hyperglycemic and dyslipidemic milieu with a noxious phenotype associated with an impaired tissue repair capacity. SCs affected by diabetes could lack tissue repair efficacy as autografts and should be reprogrammed in vitro or substituted by SCs from allogenic non-diabetic sources.

Basic Science Evidence for the Link Between Erectile Dysfunction and Cardiometabolic Dysfunction.

INTRODUCTION: Although clinical evidence supports an association between cardiovascular/metabolic diseases (CVMD) and erectile dysfunction (ED), scientific evidence for this link is incompletely elucidated. AIM: This study aims to provide scientific evidence for the link between CVMD and ED. METHODS: In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current literature on basic scientific support for a mechanistic link between ED and CVMD, and deficiencies in this regard with a critical assessment of current preclinical models of disease. RESULTS: A link exists between ED and CVMD on several grounds: the endothelium (endothelium-derived nitric oxide and oxidative stress imbalance); smooth muscle (SM) (SM abundance and altered molecular regulation of SM contractility); autonomic innervation (autonomic neuropathy and decreased neuronal-derived nitric oxide); hormones (impaired testosterone release and actions); and metabolics (hyperlipidemia, advanced glycation end product formation). CONCLUSION: Basic science evidence supports the link between ED and CVMD. The Committee also highlighted gaps in knowledge and provided recommendations for guiding further scientific study defining this risk relationship. This endeavor serves to develop novel strategic directions for therapeutic interventions.

The transcriptional signatures of cells from the human Peyronie's disease plaque and the ability of these cells to generate a plaque in a rat model suggest potential therapeutic targets.

INTRODUCTION: The success of medical therapies for Peyronie's disease (PD) has not been optimal, possibly because many of them went directly to clinical application without sufficient preclinical scientific research. Previous studies revealed cellular and molecular pathways involved in the formation of the PD plaque and in particular the role of the myofibroblast. AIMS: The current work aimed to determine under normal and fibrotic conditions what differentiates PD cells from tunica albuginea (TA) and corpora cavernosa (CC) cells by defining their global transcriptional signatures and testing in vivo whether PD cells can generate a PD-like plaque. METHODS: Human TA, PD, and CC cells were grown with transforming growth factor beta 1 (TGFß1; TA+, PD+, CC+) or without it (TA-, PD-, CC-) and assayed by (i) immunofluorescence, Western blot and RT-PCR for myofibroblast, smooth muscle cell and stem cell markers; (ii) collagen content; and (iii) DNA microarray analysis. The ability of PD+ cells to induce a PD-like plaque in an immuno-suppressed rat model was assessed by Masson trichrome and Picrosirius Red stainings. MAIN OUTCOMES MEASURES: Fibroproliferative features of PD cells and identification of related key genes as novel targets to reduce plaque size. RESULTS: Upon TGFß1stimulation, collagen levels were increased by myofibroblasts in the PD+ but not in the CC+ cells. The transcriptional signature of the PD- cells identified fibroproliferative, myogenic (myofibroblasts), inflammatory, and collagen turnover genes that differentiate them from TA- or CC- cells and respond to TGFß1 with a PD+ fibrotic phenotype, by upregulation of IGF-1, ACTG2, MYF5, ACTC1, PSTN, COL III, MMP3, and others. The PD+ cells injected into the TA of the rat induce a PD-like plaque. CONCLUSIONS: This suggests a novel combination therapy to eliminate a PD plaque by targeting the identified genes to (i) improve collagenase action by stimulating endogenous metalloproteinases specific to key collagen types and (ii) counteract fibromatosis by inhibiting myofibroblast generation, proliferation, and/or apoptosis.

Chronic high dose intraperitoneal bisphenol A (BPA) induces substantial histological and gene expression alterations in rat penile tissue without impairing erectile function.

INTRODUCTION: Bisphenol A (BPA), released from plastics and dental sealants, is a suspected endocrine disruptor and reproductive toxicant. In occupationally exposed workers, BPA has been associated with erectile dysfunction (ED). AIMS: To determine whether long-term exposure to high doses of BPA in the rat affects serum levels of testosterone (T) and estradiol (E2), and induces corporal histopathology and resultant ED. METHODS: Young rats were injected intraperitoneal (IP) injection daily with BPA at 25 mg/kg/day or vehicle (n = 8/group). Erectile function was measured at 3 months by cavernosometry and electrical field stimulation (EFS). BPA was assayed in serum, urine, and penile tissue, and serum T and E2 were determined. Quantitative Masson trichrome, terminal deoxynucleotidyl transferase dUTP nick end labeling, Oil Red O, immunohistochemistry for calponin, α-smooth muscle actin, and Oct 4 were applied to penile tissue sections. Protein markers were assessed by Western blots and 2-D minigels, and RNA by DNA microarrays. MAIN OUTCOME MEASURES: Erectile function, histological, and biochemical markers in corporal tissue. RESULTS: In the BPA-treated rats, total and free BPA levels were increased in the serum, urine, and penile tissue while serum T and E2 levels were reduced. In addition, the corpora cavernosa demonstrated a reduction in smooth muscle (SM) content, SM/collagen ratio, together with an increase in myofibroblasts, fat deposits, and apoptosis, but no significant change in collagen content or stem cells (nuclear/perinuclear Oct 4). In the penile shaft, BPA induced a downregulation of Nanog (stem cells), neuronal nitric oxide synthase (nitrergic terminals), and vascular endothelial growth factor (angiogenesis), with genes related to SM tone and cytoskeleton upregulated 5- to 50-fold, accompanied by changes in the multiple protein profile. However, both cavernosometry and EFS were unaltered by BPA. CONCLUSIONS: While rats treated chronically with a high IP dose of BPA developed hypogonadism and a corporal histo- and molecular-pathology usually associated with ED, no changes were detected in erectile function as measured by EFS and cavernosometry. Further studies using alternate routes of BPA administration with various doses and length of exposure are needed to expand these findings.

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

INTRODUCTION: Stimulating 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). METHODS: To 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. RESULTS: Surprisingly, 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. CONCLUSIONS: Although 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.

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.

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

BACKGROUND: Previous 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. METHODS: MI 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. RESULTS: As 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. CONCLUSIONS: Long-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.

Amelioration of diabetes-induced cavernosal fibrosis by antioxidant and anti-transforming growth factor-ß1 therapies in inducible nitric oxide synthase-deficient mice.

OBJECTIVE: • To investigate whether sustained long-term separate treatments of diabetic inducible nitric oxide synthase knockout (iNOSKo) mice with allopurinol, an antioxidant inhibiting xanthine oxidoreductase, decorin, a transforming growth factor-ß1 (TGFß1) -binding antagonist, and molsidomine, a long-life nitric oxide donor, prevent the processes of diabetes-induced cavernosal fibrosis. MATERIALS AND METHODS: • Eight week old male iNOS knock out (iNOSKo) mice were made diabetic by injecting 150 mg/kg B.W Streptozotocin (1P) with were either left untreated or treated with the oral antioxidant allopurinol (40 mg/kg/day), or decoin (50 mg, 1P, twice), as an anti-TGFß1 agent (n = 8/group). • Glycemia and oxidative stress markers were determined in blood and urine. • Paraffin-embedded tissue sections from the penile shaft were subjected to Masson trichrome staining for the smooth muscle (smc)/collagen ratio, and imunostaining for smc content, profibrotic factors, oxidative stress, cell replication and cell death markers followed by quantitative image analysis. RESULTS: • Eight-week treatment with either allopurinol or decorin counteracted the decrease in smooth muscle cells and the increase in apoptosis and local oxidative stress within the corpora tissue. • Decorin but not allopurinol increased the smooth muscle cell/collagen ratio, whereas allopurinol but not decorin inhibited systemic oxidative stress. • Molsidomine was effective in reducing both local and systemic oxidative stress, but did not prevent corporal fibrosis. CONCLUSION: • Both allopurinol and decorin appear as promising approaches either as a single or a combined pharmacological modality for protecting the diabetic corpora from undergoing apoptosis and fibrosis although their functional effects still need to be defined.

Antifibrotic effects of pioglitazone at low doses on the diabetic rat kidney are associated with the improvement of markers of cell turnover, tubular and endothelial integrity, and angiogenesis.

BACKGROUND/AIMS: Pioglitazone and other thiazolidinediones are renoprotective in diabetic nephropathy at doses that normalize glycemia, presumably as a consequence of glycemic control. However, low doses of pioglitazone that did not normalize glycemia in rat models of type 2 diabetes prevented tubulointerstitial fibrosis and glomerulosclerosis through counteracting inflammation, oxidative stress, cell cycle arrest, and fibrosis. The current work tested whether this low-dose treatment also reduces other fibrosis and inflammation factors in the diabetic kidney and prevents tubular cell loss, endothelial damage, and abnormal angiogenesis. METHODS: ZDF fa/fa rats (ZDF) were fed for 4 months chow with 0.001% pioglitazone, and the untreated ZDF and the non-diabetic lean Zucker rats (LZR) received regular chow. Proteinuria, creatinine clearance, blood pressure, and renal quantitative histopathology markers were determined. RESULTS: Correction of renal function in ZDF by pioglitazone, occurring with a glycemia >250 mg/dl, was accompanied by normalization of the renal levels of connective tissue growth factor and fibronectin (fibrosis), TNF-α, interleukin-6 and MCP-1 (inflammation), megalin (tubular cells), the PCNA/caspase-3 ratio (positive cell turnover), VEGF (abnormal angiogenesis), and the ratio between eNOS and iNOS (endothelial dysfunction). CONCLUSION: This supports mechanisms for the renoprotective effects of pioglitazone in diabetes additional to glycemic control.

The genetic inactivation of inducible nitric oxide synthase (iNOS) intensifies fibrosis and oxidative stress in the penile corpora cavernosa in type 1 diabetes.

INTRODUCTION: Endogenously elicited inducible nitric oxide synthase (iNOS) induction counteracts fibrosis and oxidative stress in penile tissues in rat models of Peyronie's disease and erectile dysfunction. AIM: The current study aimed to determine whether the genetic blockade of iNOS expression in the iNOS knock out (iNOS KO) mouse intensifies fibrosis and oxidative stress in the penile corpora cavernosa, and this is exacerbated by streptozotocin (STZ)-induced diabetes and counteracted by insulin. MAIN OUTCOMES MEASURES: Quantitative assessment of histological and biochemical markers in mouse corporal tissue. METHODS: Male iNOS KO and wild type (WT) mice were left untreated or injected with STZ, with or without insulin treatment. At 8 weeks, glycemia, glucosuria, and proteinuria were determined, and corporal tissue sections were obtained and subjected to Masson trichrome staining for smooth muscle (SM)/collagen ratio, and immunostaining for α-smooth muscle actin (ASMA) for, SM content, proliferating cell nuclear antigen (PCNA) for cell replication, TGFß1 as profibrotic factor, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis, and xanthine oxidoreductase (XOR) for oxidative stress. Collagen was estimated by the hydroxyproline reaction. RESULTS: The corporal SM/collagen ratio and SM content were reduced, and collagen content increased in iNOS KO mice as compared with WT mice, but apoptosis was decreased and cell replication increased, whereas TGFß1 and XOR did not vary. Severe hyperglycemia caused in the WT a reduction of the corporal SM/collagen ratio and SM content and an increase in apoptosis without changes in PCNA, TGFß1, or XOR. In the iNOS KO mouse the hyperglycemia-induced alterations were exacerbated, with additional increases in oxidative stress and TGFß1. Insulin normalized glycemia and partially protected the SM in both the WT and the iNOS KO mice. CONCLUSIONS: The antifibrotic, antioxidative, and SM-protective roles of iNOS in the penile corpora cavernosa were confirmed in the iNOS KO/STZ mouse model. These findings support the importance of endogenously-elicited iNOS induction in protecting the penile corpora cavernosa from the pro-fibrotic effects of hyperglycemia.

Treatment of Peyronie's disease with PDE5 inhibitors: an antifibrotic strategy.

Peyronie's disease (PD) is a localized fibrotic condition of the tunica albuginea that is associated with risk factors for corpora cavernosa fibrosis (such as advanced age and diabetes) and Dupuytren contracture, another localized fibrotic process. Most of the current pharmacological treatments for PD are not based on antifibrotic approaches that have shown promising results in animal models and clinical efficacy in other fibrotic conditions, which may explain why they are generally unsuccessful. Evidence gathered in human specimens and animal models of PD have elucidated aspects of its etiology and histopathology, showing that overexpression of transforming growth factor beta1, plasminogen activator inhibitor 1, reactive oxygen species and other profibrotic factors, which are, in most cases, assumed to be induced by trauma to the tunica albuginea, leads to myofibroblast accumulation and excessive deposition of collagen. At the same time, a steady overexpression of inducible nitric oxide synthase, leading to increased nitric oxide and cGMP levels, seems to act as an endogenous antifibrotic mechanism. This process has also been reported in corporal and cardiovascular fibrosis, and has led to the demonstration that long-term continuous administration of phosphodiesterase type 5 inhibitors counteracts the development of a PD-like fibrotic plaque in a rat model, and later extended to the prevention of corporal fibrosis in animal models of erectile dysfunction.

Stimulating vaginal repair in rats through skeletal muscle-derived stem cells seeded on small intestinal submucosal scaffolds.

OBJECTIVES: Grafts are used for vaginal repair after prolapse, but their use to carry stem cells to regenerate vaginal tissue has not been reported. In this study, we investigated whether 1) muscle-derived stem cells (MDSC) grown on small intestinal submucosa (SIS) generate smooth-muscle cells (SMC) in vitro and upon implantation in a rat model of vaginal defects; 2) express markers applicable to the in-vivo detection of vaginal endogenous stem cells; and 3) stimulate the repair of the vagina. METHODS: Mouse MDSC grown on monolayer, SIS, or polymeric mesh, were tested for cell differentiation by immunocytochemistry, Western blot and real-time polymerase chain reaction (PCR). Stem cell markers were screened by DNA microarrays followed by real-time PCR, immunocytochemistry, and Western blot. Rats that underwent hysterectomy and partial vaginectomy were left as such or implanted in the vagina with 4',6-Diamidino-2-Phenylindole (DAPI)-labeled MDSC on SIS, or SIS without MDSC, immunosuppressed, and killed at 2-8 weeks. Immunofluorescence, hematoxylin-eosin, and Masson trichrome were applied to tissue sections. RESULTS: Muscle-derived stem cell cultures on monolayer and on scaffolds differentiate into SMC, as shown by alpha-smooth muscle actin (ASMA), calponin, and smoothelin markers. Muscle-derived stem cells express embryonic stem cell markers Oct-4 and nanog. Dual DAPI/ASMA fluorescence indicated MDSC conversion to SMC. Muscle-derived stem cells/SIS stimulated vaginal tissue repair, including keratin-5 positive epithelium formation and prevented fibrosis at 4 and 8 weeks. Oct-4+ putative endogenous stem cells were identified. CONCLUSION: Muscle-derived stem cells/SIS implants stimulate vaginal tissue repair in the rat, thus autologous MDSC on scaffolds may be a promising approach for the treatment of vaginal repair.

Mechanisms of penile fibrosis.

INTRODUCTION: Penile fibrosis has been conceptually identified with the plaque that develops in the tunica albuginea in Peyronie's disease (PD), or with localized processes induced in the corpora cavernosa by ischemic or traumatic events. Recently, it has been proposed that a diffuse, progressive, and milder intracorporal fibrosis, which affects also the media of the penile arteries, is responsible for vasculogenic erectile dysfunction (ED) associated with aging, smoking, diabetes, hypertension, and post-radical prostatectomy. These processes differ in etiology, time course, target cells, and treatment, but have many features in common. AIM: To review the literature pertaining to fibrosis in the penis, related to PD and ED. METHODS: PubMed search for pertinent publications mainly during 2001-2008. RESULTS: This review focuses initially on PD and then deals with studies on ED in animal and cell culture models, discussing some of the pathophysiological similarities between tunical fibrosis in PD and corporal fibrosis in corporal veno-occlusive dysfunction (CVOD), and emerging therapeutic strategies. The role of profibrotic factors, the excessive deposit of collagen fibers and other extracellular matrix, the appearance of a synthetic cell phenotype in smooth muscle cells or the onset of a fibroblast-myofibroblast transition, and in the case of the corporal or penile arterial tissue the reduction of the smooth muscle cellular compartment, are discussed. This histopathology leads either to localized plaques or nodules in penile tissues, or to the diffuse fibrosis causing impairment of tissue compliance that underlies CVOD and arteriogenic ED. The antifibrotic role of the sustained stimulation of the nitric oxide/cyclic guanosine monophosphate pathway in the penis and its possible relevance to exogenous and endogenous stem cell differentiation is also briefly presented. CONCLUSIONS: Fibrotic processes in penile tissues share a similar cellular and molecular pathophysiology and common endogenous mechanisms of defense that have inspired novel pharmacological experimental approaches.