The Combination of High-Fat Diet and Oral Marijuana Promotes the Development of Fibrosis in the Mouse Corpora Cavernosa.

INTRODUCTION: The link between cannabis use and erectile dysfunction remains unclear. Moreover, the effect of cannabis in tandem with current Western dietary habits is an area in male sexual health that has yet to be explored. This study seeks to investigate the impact of diet and cannabis on penile health in an animal model. AIM: To determine the effects of diet and oral cannabis extract on fibrosis and oxidative stress within the corpora cavernosa of mice. METHODS: This is a pilot animal study in which groups of 2-month old C57BL/6J male mice were fed a normal chow diet (NCD) or high-fat diet (HFD) daily and treated with or without either MJ or THC extract for 2 months. After euthanization, mouse penises were isolated and processed for immunohistochemical studies to determine: (i) smooth muscle cell to collagen content, (ii) myofibroblast proliferation, and (iii) anti-oxidative activity. MAIN OUTCOME MEASURES: Quantitative assessment of immunohistochemical markers of fibrosis and oxidative stress within the corpora cavernosa of mice fed a high-fat diet in combination with either oral marijuana (MJ) or Δ-9-tetrahydrocannabinol extract (THC). RESULTS: The combination of HFD with MJ resulted in: (i) a decrease in the smooth/collagen ratio in the corpora cavernosa, (ii) an increase in alpha-smooth muscle actin expression in the tunica albuginea compatible with myofibroblast proliferation, and (iii) a decrease in heme oxygenase 1 expression indicating an increase in oxidative stress. Significant histological changes were not observed in the HFD + THC group. CONCLUSIONS: HFD combined with oral MJ extract led to structural alterations in erectile tissue that are associated with accelerated corporal fibrosis. However, the addition of THC to the diet did not exacerbate histological changes within the corpora. Further studies are warranted to elucidate the discrepant effects between MJ and THC in order to optimize the therapeutic potential of cannabis and minimize its adverse effects on penile health. S Nguyen, M Mangubat, S Eleswarapu, et al. The Combination of High-Fat Diet and Oral Marijuana Promotes the Development of Fibrosis in the Mouse Corpora Cavernosa. Sex Med 2021;9:100312.

Prevention of Osteoporosis in the Ovariectomized Rat by Oral Administration of a Nutraceutical Combination That Stimulates Nitric Oxide Production.

Osteoporosis represents an imbalance between bone formation and bone resorption. As a result of low estrogen levels, it is markedly prevalent during menopause, thus making such patients susceptible to fractures. Both bone formation and resorption are modulated by nitric oxide (NO). Currently, there are no risk-free pharmaceutical prevention therapies for osteoporosis. COMB-4, a nutraceutical combination of Paullinia cupana, Muira puama, ginger, and L-citrulline, known to activate the NO-cGMP pathway, was reported to accelerate fracture healing in the rat. To determine whether COMB-4 could be effective in preventing menopausal osteoporosis, it was compared to estradiol (E2) in an ovariectomized (OVX) rat osteoporosis model. Nine-month-old female Sprague Dawley rats were divided into SHAM, OVX, OVX+E2, and OVX+COMB-4. After 100 days of treatment, bone mineral density (BMD) and bone mineral content (BMC) were measured by DXA scan. TRAP staining was performed in the femur and lumbar vertebrae. TRACP 5b and osteocalcin levels were assayed in the serum. MC3T3-E1 cells were differentiated into osteoblasts and treated with COMB-4 for one week in order to evaluate calcium deposition by Alizarin staining, cGMP production by ELISA, and upregulation of the nitric oxide synthase (NOS) enzymes by RT-PCR. OVX resulted in a decrease in BMD, BMC, and serum osteocalcin and an increase in serum TRACP 5b. Except for an increase in BMC with COMB-4, both E2 and COMB-4 reverted all bone and serum markers, as well as the number of osteoclasts in the vertebrae, to SHAM levels. Incubation of MC3T3-E1 cells with COMB-4 demonstrated an increase in the three NOS isoforms, cGMP, and calcium deposition. COMB-4 increased BMD in OVX rats by inhibiting bone resorption and increasing calcium deposition presumably via activation of the NO-cGMP pathway. It remains to be determined whether COMB-4 could be a potential nutraceutical therapy for the prevention of premenopausal bone loss.

Exogenous l-ARGININE does not stimulate production OF NO or cGMP within the rat corporal smooth muscle cells in culture.

BACKGROUND AND AIM: Nitric oxide (NO) is the intracellular chemical responsible for initiating a penile erection. Despite conflicting clinical data, it continues to be publicized and promoted that orally administered l-arginine, the putative substrate for NO, enhances the erectile response presumably by stimulating NO production by the corporal tissues resulting in an increase in cGMP production. To shed light on this issue, an in vitro study was conducted to explore the effect of direct exogenous administration of l-arginine as well as its precursor and metabolite, l-citrulline, on the NO-cGMP pathway within the cavernosal smooth muscle (CSM) cell. MATERIALS AND METHODS: CSM cells obtained from 8 to 10 week old Sprague-Dawley rats were grown in Dulbecco media with 20% fetal calf serum and then incubated with or without l-arginine (L-ARG) or l-citrulline (L-CIT) in a time course and dose-response manner. Sildenafil (0.4 mM), IBMX (1 mM), l-NAME (3 µM), ODQ (5 µM) and Deta Nonoate (10 µM) were used as either inhibitors or stimulators of the NO-cGMP pathway. mRNA and protein were extracted and used for the determination of the phosphodiesterase 5 (PDE5). PDE5 activity was determined by luminometry. cGMP content was determined by ELISA. Nitrite formation, an indicator of NO production, was measured in the cell culture media by a colorimetric assay. The cationic (CAT-1) and neutral (SNAT-1) amino acid transporters for L-ARG and L-CIT, respectively, were determined by Western blot. RESULTS: When compared to untreated CSM cells, incubation with 0.25-4.0 mM of L-ARG or 0.3-4.8 mM of L-CIT anywhere between 3 and 24 h did not result in any additional nitrite or cGMP production. The addition of l-NAME, IBMX or ODQ to these L-ARG and L-CIT treated cells did not alter these results. L-CIT but not L-ARG increased PDE5 mRNA and protein content as well as the activity of the PDE5 enzyme. Both CAT-1 and SNAT-1 were expressed in the CSM cells. CONCLUSIONS: This in vitro study demonstrates that exogenous administration of L-ARG or L-CIT failed to stimulate production of either NO or cGMP by the corporal CSM cells. A re-evaluation of the presumptive role of the exogenous administration of L-ARG in improving the synthesis of NO at least at the level of the CSM cells appears warranted.

Effect of ginger, Paullinia cupana, muira puama and l- citrulline, singly or in combination, on modulation of the inducible nitric oxide- NO-cGMP pathway in rat penile smooth muscle cells.

INTRODUCTION: COMP-4 is a natural compound-based dietary supplement consisting of the combination of ginger, Paullinia cupana, muira puama and l-citrulline, which when given long-term has been shown in the aged rat to a) upregulate iNOS in the penile smooth muscle cells (SMC), b) reverse the corporal SMC apoptosis and fibrosis associated with corporal veno-occlusive dysfunction (CVOD), and c) improve resulting erectile function. To elucidate the mechanism of how COMP-4 and its individual components modulate the iNOS-cGMP pathway, an in vitro study was conducted using a rat corporal primary SMC culture to determine its effect on NOS, soluble guanylate cyclase (sGC), cGMP and the phosphodiesterase 5 enzyme (PDE5). MATERIALS AND METHODS: Primary SMC cultures using the explant technique were initiated by cutting small pieces of corporal tissue from 8 week old Sprague-Dawley rats. The SMC were grown in Dulbecco media with 20% fetal calf serum. The SMC were then incubated with or without COMP-4 (0.69 mg/ml) or its ingredients alone (ginger: 0.225 mg/ml; muira puama, Paullinia cupana and l-citrulline each at 0.9 mg/ml) for up to 24 h mRNA and protein were extracted and used for the determination of NOS, sGC and PDE5 content. cGMP content was determined by ELISA. L-NIL (4 µM) was used as an inhibitor of iNOS activity. RESULTS: Compared to the control values, COMP-4 upregulated expression of cGMP by 85%, induced a 42 fold increase in sGC as well as a 15 fold increase in both iNOS protein and mRNA content while it decreased both PDE5 mRNA and protein content each by about 50%. L-NIL completely inhibited the effect of COMP-4 on cGMP production. When compared with each of the individual four components of COMP-4, it appears that COMP-4 itself had the most profound effect in modulating each one the specific steps within the iNOS-cGMP pathway. CONCLUSIONS: This in vitro study demonstrates that COMP-4 is capable of activating the endogenous cellular iNOS-cGMP pathway within the CSM cells, which is theorized to be responsible for reducing the fibrosis and apoptosis as well as the CVOD observed in the aging rat penis. Further studies will be necessary in order to determine whether supplementation of COMP-4 on a daily basis may be beneficial in halting or reversing this aging related erectile dysfunction in the clinical setting.

Vitamin D induces myogenic differentiation in skeletal muscle derived stem cells.

Skeletal muscle wasting is a serious disorder associated with health conditions such as aging, chronic kidney disease and AIDS. Vitamin D is most widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance. Recently, vitamin D supplementation has been shown to improve muscle performance and reduce the risk of falls in vitamin D deficient older adults. However, little is known of the underlying molecular mechanism(s) or the role it plays in myogenic differentiation. We examined the effect of 1,25-D3 on myogenic cell differentiation in skeletal muscle derived stem cells. Primary cultures of skeletal muscle satellite cells were isolated from the tibialis anterior, soleus and gastrocnemius muscles of 8-week-old C57/BL6 male mice and then treated with 1,25-D3 The efficiency of satellite cells isolation determined by PAX7+ cells was 81%, and they expressed VDR. Incubation of satellite cells with 1,25-D3 induces increased expression of: (i) MYOD, (ii) MYOG, (iii) MYC2, (iv) skeletal muscle fast troponin I and T, (v) MYH1, (vi) IGF1 and 2, (vii) FGF1 and 2, (viii) BMP4, (ix) MMP9 and (x) FST. It also promotes myotube formation and decreases the expression of MSTN. In conclusion, 1,25-D3 promoted a robust myogenic effect on satellite cells responsible for the regeneration of muscle after injury or muscle waste. This study provides a mechanistic justification for vitamin D supplementation in conditions characterized by loss of muscle mass and also in vitamin D deficient older adults with reduced muscle mass and strength, and increased risk of falls.

Vitamin D and Cardiac Differentiation.

Calcitriol (1,25-dihydroxycholecalciferol or 1,25-D3) is the hormonally active metabolite of vitamin D. Experimental studies of vitamin D receptors and 1,25-D3 establish calcitriol to be a critical regulator of the structure and function of the heart. Clinical studies link vitamin D deficiency with cardiovascular disease (CVD). Emerging evidence demonstrates that calcitriol is highly involved in CVD-related signaling pathways, particularly the Wnt signaling pathway. Addition of 1,25-D3 to cardiomyocyte cells and examination of its effects on cardiomyocytes and mainly Wnt11 signaling allowed the specific characterization of the role of calcitriol in cardiac differentiation. 1,25-D3 is demonstrated to: (i) inhibit cell proliferation without promoting apoptosis; (ii) decrease expression of genes related to the regulation of the cell cycle; (iii) promote formation of cardiomyotubes; (iv) induce expression of casein kinase-1-α1, a negative regulator of the canonical Wnt signaling pathway; and (v) increase expression of noncanonical Wnt11, which has been recognized to induce cardiac differentiation during embryonic development and in adult cells. Thus, it appears that vitamin D promotes cardiac differentiation through negative modulation of the canonical Wnt signaling pathway and upregulation of noncanonical Wnt11 expression. Future work to elucidate the role(s) of vitamin D in cardiovascular disorders will hopefully lead to improvement and potentially prevention of CVD, including abnormal cardiac differentiation in settings such as postinfarction cardiac remodeling.

Treatment with a combination of ginger, L-citrulline, muira puama and Paullinia cupana can reverse the progression of corporal smooth muscle loss, fibrosis and veno-occlusive dysfunction in the aging rat.

AIMS: Aging associated erectile dysfunction is characterized within the corpora by a progressive apoptosis of the smooth muscle cells and their replacement by collagen. Nitric oxide from iNOS has been shown to inhibit these histological changes in the corpora while PDE5 inhibitors as well as certain nutraceuticals such as ginger, paullinia cupana, muira puama and L-citrulline are known to enhance the effects of NO. We evaluated whether the daily oral administration for 2 months with a combination of ginger, paullinia cupana, muira puama and L-citrulline (COMP-4) can effectively delay the ongoing corporal fibrosis, smooth muscle cell apoptosis and cavernosal veno-occlusive dysfunction (CVOD) seen in middle aged rats similar to that seen with tadalafil. METHODS: 10 Month old Fisher 344 rats were treated or not for two months with COMP-4, tadalafil or a combination of tadalafil plus COMP-4. CVOD was determined by dynamic infusion cavernosometry. Penile sections of the corpora cavernosa were subjected to Masson trichrome staining to evaluate fibrosis and immunohistochemistry for desmin as a marker of smooth muscle content and inducible nitric oxide synthase (iNOS) followed by image analysis. Oxidative stress levels were determined by GSH/GSSG ratio in whole blood. RESULTS: a decline in the non-treated rat's erectile function is evident by 10-12 months of age and is accompanied by a decrease in the corporal smooth muscle content determined by desmin expression and an increase in corporal fibrosis. The daily treatment for two months with COMP-4 reverses this process by reducing systemic oxidative stress and increasing desmin and iNOS expression, similar to that seen with tadalafil or the combination of COMP-4 plus tadalafil. CONCLUSION: An oral combination of ginger, muira puama, Paullinia cupana and L-citrulline seems to be as effective as daily PDE5 inhibitor therapy in either delaying or reversing the onset of the histological and functional characteristics of aging related erectile dysfunction.

Sildenafil attenuates inflammation and oxidative stress in pelvic ganglia neurons after bilateral cavernosal nerve damage.

Erectile dysfunction is a common complication for patients undergoing surgeries for prostate, bladder, and colorectal cancers, due to damage of the nerves associated with the major pelvic ganglia (MPG). Functional re-innervation of target organs depends on the capacity of the neurons to survive and switch towards a regenerative phenotype. PDE5 inhibitors (PDE5i) have been successfully used in promoting the recovery of erectile function after cavernosal nerve damage (BCNR) by up-regulating the expression of neurotrophic factors in MPG. However, little is known about the effects of PDE5i on markers of neuronal damage and oxidative stress after BCNR. This study aimed to investigate the changes in gene and protein expression profiles of inflammatory, anti-inflammatory cytokines and oxidative stress related-pathways in MPG neurons after BCNR and subsequent treatment with sildenafil. Our results showed that BCNR in Fisher-344 rats promoted up-regulation of cytokines (interleukin- 1 (IL-1) ß, IL-6, IL-10, transforming growth factor ß 1 (TGFß1), and oxidative stress factors (Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), TNF receptor superfamily member 5 (CD40) that were normalized by sildenafil treatment given in the drinking water. In summary, PDE5i can attenuate the production of damaging factors and can up-regulate the expression of beneficial factors in the MPG that may ameliorate neuropathic pain, promote neuroprotection, and favor nerve regeneration.

1,25-Vitamin D3 promotes cardiac differentiation through modulation of the WNT signaling pathway.

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Low levels of vitamin D are associated with high risk of myocardial infarction, even after controlling for factors associated with coronary artery disease. A growing body of evidence indicates that vitamin D plays an important role in CVD-related signaling pathways. However, little is known about the molecular mechanism by which vitamin D modulates heart development. The WNT signaling pathway plays a pivotal role in tissue development by controlling stem cell renewal, lineage selection and, even more importantly, heart development. In this study, we examined the role of 1,25-D3 (the active form of vitamin D) on cardiomyocyte proliferation, apoptosis, cell phenotype, cell cycle progression and differentiation into cardiomyotubes. We determined that the addition of 1,25-D3 to cardiomyocytes cells: i) inhibits cell proliferation without promoting apoptosis; ii) decreases expression of genes related to the regulation of the cell cycle; iii) promotes formation of cardiomyotubes; iv) induces the expression of casein kinase-1-α1, a negative regulator of the canonical WNT signaling pathway; and v) increases the expression of the noncanonical WNT11, which it has been demonstrated to induce cardiac differentiation during embryonic development and in adult cells. In conclusion, we postulate that vitamin D promotes cardiac differentiation through a negative modulation of the canonical WNT signaling pathway and by upregulating the expression of WNT11. These results indicate that vitamin D repletion to prevent and/or improve cardiovascular disorders that are linked with abnormal cardiac differentiation, such as post infarction cardiac remodeling, deserve further study.

1,25(OH)(2)vitamin D(3) enhances myogenic differentiation by modulating the expression of key angiogenic growth factors and angiogenic inhibitors in C(2)C(12) skeletal muscle cells.

Vitamin D is mostly recognized for its regulation of calcium homeostasis in relation to the intestine, kidney, and bone. Although clinical studies have linked vitamin D with increased muscle function and strength, little is known of its underlying molecular mechanism. We recently demonstrated that 1,25-D3 exerts a direct pro-myogenic effect on skeletal muscle cells; this has provoked our investigation of 1,25-D's effect on angiogenesis, a vital process for new capillary development and tissue repair. In this study, we examined the mechanism by which 1,25-D3 modulates key angiogenic growth factors and angiogenic inhibitors. C(2)C(12) myoblasts were incubated with 100 nM 1,25-D3 or placebo for 1, 4 and 10 days. At the end of the respective incubation time, total RNA was isolated for PCR arrays and for qRT-PCR. Total proteins were isolated for Western blots and proteome profiler arrays. The addition of 1,25-D3 to C(2)C(12) myoblasts increased VEGFa and FGF-1: two pro-angiogenic growth factors that promote neo-vascularization and tissue regeneration, and decreased FGF-2 and TIMP-3: two myogenic and/or angiogenic inhibitors. Our previous study demonstrated that 1,25-D3 altered IGF-I/II expression, consistent with the observed changes in VEGFa and FGF-2 expression. These results extend our previous findings and demonstrate the modulation of angiogenesis which may be an additional mechanism by which 1,25-D3 promotes myogenesis. This study supports the mechanistic rationale for assessing the administration of vitamin D and/or vitamin D analogs to treat select muscle disorders and may also provide an alternative solution for therapies that directly manipulate VEGF and FGF's to promote angiogenesis.

Sildenafil promotes neuroprotection of the pelvic ganglia neurones after bilateral cavernosal nerve resection in the rat.

OBJECTIVE: To determine the gene expression profile of pelvic ganglia neurones after bilateral cavernosal nerve resection (BCNR) and subsequent treatment with sildenafil in relation to neurotrophic-related pathways. MATERIALS AND METHODS: Fisher rats aged 5 months were subjected to BCNR or sham operation and treated with or without sildenafil (20 mg/kg body-weight in drinking water) for 7 days. Total RNA isolated from pelvic ganglia was subjected to reverse transcription and then to quantitative reverse transcriptase-polymerase chain reaction (PCR) with the RAT-neurotrophic array. Results were corroborated by real-time PCR and western blotting. Another set of animals were injected with a fluorescent tracer at the base of the penis, 7 days before BCNR or sham operation, and were sacrificed 7 days after surgery. Sections of pelvic ganglia were used for immunohistochemistry with antibodies against neurturin, neuronal nitric oxide synthase, tyrosine hydroxylase and glial cell line-derived neurotrophic factor receptor α2. RESULTS: A down-regulation of the expression of neuronal nitric oxide synthase accompanied by changes in the level of cholinergic neurotrophic factors, such as neurturin and its receptor glial cell line-derived neurotrophic factor receptor α2, artemin, neurotrophin-4 and cilliary neurotrophic factor, was observed 7 days after BCNR in pelvic ganglia neurones. Treatment with sildenafil, starting immediately after surgery, reversed all these changes at a level similar to that in sham-operated animals. CONCLUSIONS: Sildenafil treatment promotes changes in the neurotrophic phenotype, leading to a regenerative state of pelvic ganglia neurones. The present study provides a justification for the use of phosphodiesterase 5 inhibitors as a neuroprotective agent after BCNR.

Vitamin D and cardiovascular disease: potential role in health disparities.

Cardiovascular disease (CVD), which includes coronary artery disease and stroke, is the leading cause of mortality in the nation. Excess CVD morbidity and premature mortality in the African American community is one of the most striking examples of racial/ ethnic disparities in health outcomes. African Americans also suffer from increased rates of hypovitaminosis D, which has emerged as an independent risk factor for all-cause and cardiovascular mortality. This overview examines the potential role of hypovitaminosis D as a contributor to racial and ethnic disparities in cardiovascular disease (CVD). We review the epidemiology of vitamin D and CVD in African Americans and the emerging biological roles of vitamin D in key CVD signaling pathways that may contribute to the epidemiological findings and provide the foundation for future therapeutic strategies for reducing health disparities.

1,25(OH)2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells.

Skeletal muscle wasting is an important public health problem associated with aging, chronic disease, cancer, kidney dialysis, and HIV/AIDS. 1,25-Dihydroxyvitamin D (1,25-D3), the active form of vitamin D, is widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance and for its calcemic effects on target organs, such as intestine, kidney, and parathyroid glands. Emerging evidence has shown that vitamin D administration improves muscle performance and reduces falls in vitamin D-deficient older adults. However, little is known of the underlying mechanism or the role 1,25-D3 plays in promoting myogenic differentiation at the cellular and/or molecular level. In this study, we examined the effect of 1,25-D3 on myoblast cell proliferation, progression, and differentiation into myotubes. C(2)C(12) myoblasts were treated with 1,25-D3 or placebo for 1, 3, 4, 7, and 10 d. Vitamin D receptor expression was analyzed by quantitative RT-PCR, Western blottings and immunofluorescence. Expression of muscle lineage, pro- and antimyogenic, and proliferation markers was assessed by immunocytochemistry, PCR arrays, quantitative RT-PCR, and Western blottings. Addition of 1,25-D3 to C(2)C(12) myoblasts 1) increased expression and nuclear translocation of the vitamin D receptor, 2) decreased cell proliferation, 3) decreased IGF-I expression, and 4) promoted myogenic differentiation by increasing IGF-II and follistatin expression and decreasing the expression of myostatin, the only known negative regulator of muscle mass, without changing growth differentiation factor 11 expression. This study identifies key vitamin D-related molecular pathways for muscle regulation and supports the rationale for vitamin D intervention studies in select muscle disorder conditions.

Sildenafil promotes smooth muscle preservation and ameliorates fibrosis through modulation of extracellular matrix and tissue growth factor gene expression after bilateral cavernosal nerve resection in the rat.

INTRODUCTION: It has been shown that phosphodiesterase type 5 (PDE5) inhibitors preserve smooth muscle (SM) content and ameliorate the fibrotic degeneration normally seen in the corpora cavernosa after bilateral cavernosal nerve resection (BCNR). However, the downstream mechanisms by which these drugs protect the corpora cavernosa remain poorly understood. AIM: To provide insight into the mechanism, we aimed to determine the gene expression profile of angiogenesis-related pathways within the penile tissue after BCNR with or without continuous sildenafil (SIL) treatment. METHODS: Five-month-old Fisher rats were subjected to BCNR or sham operation and treated with or without SIL (20 mg/kg/BW drinking water) for 3 days or 45 days (N = 8 rats per group). Total RNAs isolated from the denuded penile shaft and prostate were subjected to reverse transcription and to angiogenesis real-time-polymerase chain reaction arrays (84 genes). Changes in protein expression of selected genes such as epiregulin (EREG) and connective tissue growth factor (CTGF) were corroborated by Western blot and immunohistochemistry. MAIN OUTCOMES MEASURES: Genes modulated by BCNR and SIL treatment. RESULTS: A decreased expression of genes related to SM growth factors such as EREG, platelet-derived growth factor (PDGF), extracellular matrix regulators such as metalloproteinases 3 and 9, endothelial growth factors, together with an upregulation of pro-fibrotic genes such as CTGF and transforming growth factor beta 2 were found at both time points after BCNR. SIL treatment reversed this process by upregulating endothelial and SM growth factors and downregulating pro-fibrotic factors. SIL did not affect the expression of EREG, VEGF, and PDGF in the ventral prostate of BCNR animals. CONCLUSIONS: SIL treatment after BCNR activates genes related to SM preservation and downregulates genes related to fibrosis in the corpora cavernosa. These results provide a mechanistic justification for the use of SIL and other PDE5 inhibitors as protective therapy against corporal SM loss and fibrosis after radical prostatectomy.

1,25(OH)2vitamin D3 inhibits cell proliferation by promoting cell cycle arrest without inducing apoptosis and modifies cell morphology of mesenchymal multipotent cells.

The vitamin D receptor (VDR) and its ligand 1,25D play an important role in regulating cell growth and cell fate. We examined the effect of 1,25D on cell morphology, cell proliferation, cell cycle progression and apoptosis on mesenchymal multipotent cells. Multipotent cells were treated with and without 1,25D in a time- and dose-dependent manner. Changes in cell morphology were evaluated by a green fluorescence fluorocrome. Cell proliferation was determined by the Formazan assay and PCNA antigen expression. The expression of genes related to the cell cycle was analyzed by DNA microarrays, RT(2)PCR arrays and western blots. Apoptosis was evaluated by TUNEL assay, and the expression of pro- and anti-apoptotic related genes by RT(2)PCR arrays and western blots. 1,25D inhibited cell proliferation, induced cell cycle arrest, and promoted accumulation of cells in G0/G1 phase without inducing apoptosis. An increase in cell size was associated with a decrease in the GTPase Rho and the atypical Rho family GTPase Rhou/Wrch-1 expression without inducing Wnt-1 expression. Survivin expression was also increased and may represent a novel 1,25D-mediated pathway regulating tissue injury and fibrosis. The data provide a mechanistic explanation for the anti-proliferative and anti-apoptotic properties of 1,25D in mesenchymal multipotent cells.

Vitamin D and the cardiovascular system.

Several epidemiologic and clinical studies have suggested that there is a strong association between hypovitaminosis D and cardiovascular disease (CVD). Hypovitaminosis D was reported as a risk factor for increased cardiovascular events among 1739 adult participants in the Framingham Offspring Study. Analysis of more than 13,000 adults in the Third National Health and Nutrition Examination Survey (NHANES III) showed that even though hypovitaminosis D is associated with an increased prevalence of CVD risk factors, its association with all-cause mortality is independent of these risk factors. Importantly, epidemiologic studies suggested that patients who had chronic kidney disease and were treated with activated vitamin D had a survival advantage when compared with those who did not receive treatment with these agents. Mechanistically, emerging data have linked vitamin D administration with improved cardiac function and reduced proteinuria, and hypovitaminosis D is associated with obesity, insulin resistance, and systemic inflammation. Preliminary studies suggested that activated vitamin D inhibits the proliferation of cardiomyoblasts by promoting cell-cycle arrest and enhances the formation of cardiomyotubes without inducing apoptosis. Activated vitamin D has also been shown to attenuate left ventricular dysfunction in animal models and humans. In summary, emerging studies suggest that hypovitaminosis D has emerged as an independent risk factor for all-cause and cardiovascular mortality, reinforcing its importance as a public health problem. There is a need to advance our understanding of the biologic pathways through which vitamin D affects cardiovascular health and to conduct prospective clinical interventions to define precisely the cardioprotective effects of nutritional vitamin D repletion.

Vitamin D reduces the expression of collagen and key profibrotic factors by inducing an antifibrotic phenotype in mesenchymal multipotent cells.

Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5'-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0.1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.

Regulation of myogenic differentiation by androgens: cross talk between androgen receptor/ beta-catenin and follistatin/transforming growth factor-beta signaling pathways.

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts.

Tissue fibrosis, the excessive deposition of collagen/extracellular matrix combined with the reduction of the cell compartment, defines fibroproliferative diseases, a major cause of death and a public health burden. Key cellular processes in fibrosis include the generation of myofibroblasts from progenitor cells, and the activation or switch of already differentiated cells to a fibrotic synthetic phenotype. Myostatin, a negative regulator of skeletal muscle mass, is postulated to be involved in muscle fibrosis. We have examined whether myostatin affects the differentiation of a multipotent mesenchymal mouse cell line into myofibroblasts, and/or modulates the fibrotic phenotype and Smad expression of the cell population. In addition, we investigated the role of follistatin in this process. Incubation of cells with recombinant myostatin protein did not affect the proportion of myofibroblasts in the culture, but significantly upregulated the expression of fibrotic markers such as collagen and the key profibrotic factors transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor (PAI-1), as well as Smad3 and 4, and the pSmad2/3. An antifibrotic process evidenced by the upregulation of follistatin, Smad7, and matrix metalloproteinase 8 accompanied these changes. Follistatin inhibited TGF-beta1 induction by myostatin. Transfection with a cDNA expressing myostatin upregulated PAI-1, whereas an shRNA against myostatin blocked this effect. In conclusion, myostatin induced a fibrotic phenotype without significantly affecting differentiation into myofibroblasts. The concurrent endogenous antifibrotic reaction confirms the view that phenotypic switches in multipotent and differentiated cells may affect the progress or reversion of fibrosis, and that myostatin pharmacological inactivation may be a novel therapeutic target against fibrosis.

Alterations in myostatin expression are associated with changes in cardiac left ventricular mass but not ejection fraction in the mouse.

Myostatin (Mst) is a negative regulator of skeletal muscle in humans and animals. It is moderately expressed in the heart of sheep and cattle, increasing considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth. We determined whether Mst overexpression in the heart of transgenic mice reduces left ventricular size and function, and inhibits in vitro cardiomyocyte proliferation. Young transgenic mice overexpressing Mst in the heart (Mst transgenic mice (TG) under a muscle creatine kinase (MCK) promoter active in cardiac and skeletal muscle, and Mst knockout (Mst (-/-)) mice were used. Xiscan angiography revealed that the left ventricular ejection fraction did not differ between the Mst TG and the Mst (-/-) mice, when compared with their respective wild-type strains, despite the decrease in whole heart and left ventricular size in Mst TG mice, and their increase in Mst (-/-) animals. The expected changes in cardiac Mst were measured by RT-PCR and western blot. Mst and its receptor (ActRIIb) were detected by RT-PCR in rat H9c2 cardiomyocytes. Transfection of H9c2 with plasmids expressing Mst under muscle-specific creatine kinase promoter, or cytomegalovirus promoter, enhanced p21 and reduced cdk2 expression, when assessed by western blot. A decrease in cell number occurred by incubation with recombinant Mst (formazan assay), without affecting apoptosis or cardiomyocyte size. Anti-Mst antibody increased cardiomyocyte replication, whereas transfection with the Mst-expressing plasmids inhibited it. In conclusion, Mst does not affect cardiac systolic function in mice overexpressing or lacking the active protein, but it reduces cardiac mass and cardiomyocyte proliferation.