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Modulation of Stem Cells Differentiation and Myostatin as an approach to Counteract fibrosis in Muscle Dystrophy and Regeneration after Injury
Abstract
We have completed in Year 3 the in vitro characterization of the three muscle derived stem cells (MDSC) isolated from the wild type (WT), myostatin knock out (Mst KO), and mdx mice. We had shown in Years 1 and 2 that the in vitro myogenic differentiation and myotube formation by MDSC was refractory to modulation by myostatin, follistatin, myostatin antibodies and shRNA, hormones, and nitric oxide, and specifically that the Mst KO and mdx MDSC were totally unable to form myotubes.. We have now found out: a) that this inability of the Mst KO and mdx MDSC to form myotubes in vitro occurs despite they can differentiate into myofibroblasts and neural and smooth muscle cells, albeit not into adipocytes, b) is not overcome even in dual paracrine and juxtacrine cultures with WT MDSC and with C2C12 myoblasts, and c) is not accompanied by the silencing of stem cell genes, like Sca 1 and Oct-4. MDSC from a myostatin+/dystrophin+ transgenic mouse expressing gfp under the Oct-4 promoter, morphologically resemble very small embryonic-like (VSEL) stem cells. Although the three MDSC cultures showed similar levels of expression for 98% of the 260 stem cell and myogenic genes assayed, the genetic inactivation of myostatin or dystrophin was associated with a dramatic down-regulation of Actc1, Acta1, Spp1, MyoD, and notch 2, critical genes for the onset of myogenesis. We also have defined novel antifibrotic strategies to apply concurrently with MDSC implantation into mdx mice during Year 4. The in vivo studies to determine the capacity of WT MDSC alone to repair notexin-injured diaphragm and gastrocnemius in old mdx mice are now ongoing.
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The octamer-binding transcription factor 4 gene encodes a nuclear protein (Oct4, also known as Pou5F1 and Oct3/4) that belongs to a family of transcription factors containing the POU DNA-binding domain. Expression can be detected in embryonic stem cells as well as in adult stem cells, such as bone marrow-derived mesenchymal stem cells. Expression of Oct4 is downregulated coincident with stem cell differentiation and loss of expression leading to differentiation. A role for maintaining pluripotency and self-renewal of embryonic stem cells is ascribed to Oct4 as a pluripotency marker. Results describing Oct4 expression in differentiated cells, including peripheral blood mononuclear cells (PBMCs), neonatal and adult stem cells, as well as cancer cells, must be interpreted with caution. In several publications, Oct4 has been ascribed a function in maintaining self-renewal of adult stem cells. In contrast, other publications reported Oct4 expression in human tumor cells. Here, we summarize the recent findings on Oct4 expression and present possibilities and reasons why several false positive results on Oct4 expression still occur in the recent literature. Also, simple solutions are provided to avoid these positive signals.
Duchenne muscular dystrophy is a recessive disease due to a mutation in the dystrophin gene. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with an antagonist, follistatin. Three different approaches were studied to overexpress follistatin in the muscles of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx mice were generated; second, a follistatin plasmid was electroporated in mdx muscles, and finally, follistatin was induced in mdx mice muscles by a treatment with a histone deacetylase inhibitor. The three approaches improved the success of the myoblast transplantation. Moreover, fiber hypertrophy was also observed in all muscles, demonstrating that myostatin inhibition by follistatin is a good method to improve myoblast transplantation and muscle function. Myostatin inhibition by follistatin in combination with myoblast transplantation is thus a promising novel therapeutic approach for the treatment of muscle wasting in diseases such as Duchenne muscular dystrophy.
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.
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.
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).
This supports mechanisms for the renoprotective effects of pioglitazone in diabetes additional to glycemic control.
M- and N-cadherin are members of the Ca(2+)-dependent cell-cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from -2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix-loop-helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.
Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.
Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose-dependent manner.
Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.
Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.
The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.
Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic
peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating
this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted
by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential.
cGK I+/− mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK
I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy
and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoAA188 in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent
with the notion that RhoAA188 escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoAA188 became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA
as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated
with vascular malfunction, predisposes individuals to vascular fibrosis.
Follistatin is a functional antagonist of several members of the TGF-beta family of secreted signaling factors, including myostatin, the most powerful inhibitor of muscle growth characterized to date. Myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. To assess the potential benefits of follistatin in treating muscle degenerative diseases, we examined the expression of myostatin and follistatin in Mdx mice, a model for Duchenne muscular dystrophy, and in B10 mice as a control. Our results demonstrated a temporary and coincident expression of follistatin and myostatin in both mouse strains, but this expression was significantly higher in Mdx mice than in B10 mice. The maximum expression of follistatin and myostatin in the presence of restoring necrotic muscle was detected 4 weeks after birth in Mdx mice. Interestingly, during the stage of complete regeneration, the absence of myostatin and follistatin proteins and a marked decrease in the expression of both genes were observed 9 weeks after birth in both mouse strains. These findings suggest that follistatin not only blocks myostatin but also allows other activators to function in muscle development, emphasizing that follistatin could be a very potent molecule in combating muscle loss during dystrophies and muscle ageing, disuse, or denervation.
We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)-mediated angiogenesis in MDSC transplantation-based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.
Examination of embryonic myogenesis of two distinct, but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3(-/-)) establishes for the first time that key elements of the pathology of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 1C (LGMD-1c) originate in the disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdx mutants, which lack a functional form of dystrophin, than in cav-3(-/-) mutants, which lack the Cav3 gene that encodes the protein caveolin-3; this finding is consistent with the milder phenotype of LGMD-1c, a condition caused by mutations in Cav3, and the earlier [embryonic day (E)9.5] expression of dystrophin. Myogenesis is severely disrupted in mdx embryos, which display developmental delays; myotube morphology and displacement defects; and aberrant stem cell behaviour. In addition, the caveolin-3 protein is elevated in mdx embryos. Both cav-3(-/-) and mdx mutants (from E15.5 and E11.5, respectively) exhibit hyperproliferation and apoptosis of Myf5-positive embryonic myoblasts; attrition of Pax7-positive myoblasts in situ; and depletion of total Pax7 protein in late gestation. Furthermore, both cav-3(-/-) and mdx mutants have cardiac defects. In cav-3(-/-) mutants, there is a more restricted phenotype comprising hypaxial muscle defects, an excess of malformed hypertrophic myotubes, a twofold increase in myonuclei, and reduced fast myosin heavy chain (FMyHC) content. Several mdx mutant embryo pathologies, including myotube hypotrophy, reduced myotube numbers and increased FMyHC, have reciprocity with cav-3(-/-) mutants. In double mutant (mdxcav-3(+/-)) embryos that are deficient in dystrophin (mdx) and heterozygous for caveolin-3 (cav-3(+/-)), whereby caveolin-3 is reduced to 50% of wild-type (WT) levels, these phenotypes are severely exacerbated: intercostal muscle fibre density is reduced by 71%, and Pax7-positive cells are depleted entirely from the lower limbs and severely attenuated elsewhere; these data suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels that are found in mdx embryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential for correct fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of muscular dystrophy and will be invaluable in establishing an earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols, leading to better clinical outcome for these patients.
Skeletal muscle alpha-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac alpha-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.
Cardiac fibrosis is a hallmark of cardiovascular remodeling associated with hypertension. The purpose of this study was to explore the effect and mechanism of soluble guanylate cyclase (sGC) stimulator BAY 41-2272, leading to intracellular cyclic guanosine monophosphate (cGMP) elevation, on the remodeling process induced by pressure overload. Seven-week-old male Wistar rats with hypertension induced by suprarenal aortic constriction (AC) were treated orally with 2 mg kg(-1) day(-1) of BAY 41-2272 for 14 days. BAY 41-2272 had no effects on blood pressure, but decreased AC-induced collagen accumulation in the left ventricle (LV), inhibiting the number of myofibroblasts and gene expressions of transforming growth factor-beta1 and type 1 collagen. In addition, the antifibrotic action of BAY 41-2272 was accompanied by reducing AC-induced angiotensin-converting enzyme (ACE) mRNA and its enzymatic activity, and angiotensin II concentration in LV. In cultured cardiac fibroblasts, BAY 41-2272 inhibited ACE synthesis and myofibroblast transformation, accompanied by elevating the intracellular cGMP concentration. These results suggest that sGC stimulator BAY 41-2272 might be effective to reduce fibrosis in hypertensive heart disease by attenuating angiotensin II generation through myofibroblast transformation.
Myostatin is a negative regulator of skeletal muscle size, previously shown to inhibit muscle cell differentiation. Myostatin requires both Smad2 and Smad3 downstream of the activin receptor II (ActRII)/activin receptor-like kinase (ALK) receptor complex. Other transforming growth factor-beta (TGF-beta)-like molecules can also block differentiation, including TGF-beta(1), growth differentiation factor 11 (GDF-11), activins, bone morphogenetic protein 2 (BMP-2) and BMP-7. Myostatin inhibits activation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using small interfering RNA to regulatory-associated protein of mTOR (RAPTOR), a component of TOR signaling complex 1 (TORC1), increases myostatin-induced phosphorylation of Smad2, establishing a myostatin signaling-amplification role for blockade of Akt. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. Inhibition of TORC2, via rapamycin-insensitive companion of mTOR (RICTOR), is sufficient to inhibit differentiation on its own. Furthermore, myostatin decreases the diameter of postdifferentiated myotubes. However, rather than causing upregulation of the E3 ubiquitin ligases muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." In vivo, inhibition of myostatin increases muscle creatine kinase activity, coincident with an increase in muscle size, demonstrating that this in vitro differentiation measure is also upregulated in vivo.
Muscle satellite cells are responsible for the postnatal growth and robust regeneration capacity of adult skeletal muscle. A subset of satellite cells purified from adult skeletal muscle is capable of repopulating the satellite cell pool, suggesting that it has direct therapeutic potential for treating degenerative muscle disease. Satellite cells uniformly express the transcription factor Pax7, and Pax7 is required for satellite cell viability and to give rise to myogenic precursors that express the basic helix-loop-helic (bHLH) transcription factors Myf5 and MyoD. Pax7 activates expression of target genes such as Myf5 and MyoD through recruitment of the Wdr5/Ash2L/MLL2 histone methyltransferase complex. Extensive genetic analysis has revealed that Myf5 and MyoD are required for myogenic determination, whereas myogenin and MRF4 have roles in terminal differentiation. Using a Myf5-Cre knockin allele and an R26R-YFP Cre reporter, we observed that in vivo about 10% of satellite cells only express Pax7 and have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells give rise to Pax7(+)/Myf5(+) satellite cells through basal-apical asymmetric cell divisions. Therefore, satellite cells in skeletal muscle are a heterogeneous population composed of satellite stem cells (Pax7(+)/Myf5(-)) and satellite myogenic cells (Pax7(+)/Myf5(+)). Evidence is accumulating that indicates that satellite stem cells represent a true stem cell reservoir, and targeting mechanisms that regulate their function represents an important therapeutic strategy for the treatment of neuromuscular disease.
Recent evidence suggests that treatment of type 2 diabetes with thiazolidinediones [peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists], ameliorates glomerulosclerosis and tubulointerstitial fibrosis in the rat kidney. In the current work, we have investigated whether these drugs, and specifically pioglitazone (PGT), act by preventing fibrosis and kidney dysfunction mainly through antioxidant and anti-inflammatory effects, independently of glycaemic control.
Male 2- to 3-month-old obese Zucker fa/fa (OZR) and ZDF fa/fa rats (ZDFR), and their control the lean Zucker rat (LZR), were used. Diabetic rats were given either a low dose (0.6 mg/kg/day) or a high dose (12 mg/ kg/day) of PGT in the chow for 2 or 4-5 months. Glycaemia, blood pressure, creatinine clearance and proteinuria were determined, and the underlying histopathology was defined with markers of fibrosis, glomerular damage, oxidative stress and inflammation by immunohistochemistry/ quantitative image analysis in tissue sections, and western blots and ad hoc assays in fresh tissue.
PGT at low doses given for 4-5 months considerably reduced blood pressure, proteinuria and creatinine clearance. This was associated with amelioration of renal tissue damage and fibrosis, evidenced by the glomerulosclerosis, tubulointerstitial fibrosis, tubular atrophy and podocyte injury indexes, and of oxidative stress and inflammation, as shown by the decrease in the respective markers, although glycaemia remained high and obesity was not affected.
These results indicate that low doses of PGT ameliorate renal fibrosis and preserve renal function in this animal model of metabolic syndrome, independently of glycaemic control or effects on body weight.
Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.
Gene-targeted therapies, such as adeno-associated viral vector (AAV)-mediated gene therapy and cell-mediated therapy using myogenic stem cells, are hopeful molecular strategies for muscular dystrophy. In addition, drug therapies based on the pathophysiology of muscular dystrophy patients are desirable. Multidisciplinary approaches to drug design would offer promising therapeutic strategies. Myostatin, a member of the transforming growth factor-beta superfamily, is predominantly produced by skeletal muscle and negatively regulates the growth and differentiation of cells of the skeletal muscle lineage. Myostatin inhibition would increase the skeletal muscle mass and prevent muscle degeneration, regardless of the type of muscular dystrophy. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Although follistatin possesses potent myostatin-inhibiting activity, it works as an efficient inhibitor of activins. Unlike myostatin, activins regulate the growth and differentiation of nearly all cell types, including cells of the gonads, pituitary gland and skeletal muscle. We have developed a myostatin-specific inhibitor derived from follistatin, designated FS I-I. Transgenic mice expressing this myostatin-inhibiting peptide under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. mdx mice were crossed with FS I-I transgenic mice and any improvement of the pathological signs was investigated. The resulting mdx/FS I-I mice exhibited increased skeletal muscle mass and reduced cell infiltration in muscles. Muscle strength was also recovered in mdx/FS I-I mice. Our data indicate that myostatin inhibition by this follistatin-derived peptide has therapeutic potential for muscular dystrophy.
The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-beta1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-beta1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene-2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing approximately 60-70% myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited alpha-SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of G(i/o) inhibition, and a sustained increase in extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on alpha-SMA and Smad2 phosphorylation, while the NO inhibitor, L-nitroarginine methyl ester (hydrochloride) (L-NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS-NO-cGMP-dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF-beta1-mediated renal myofibroblast differentiation and collagen production.
Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8weeks were randomly allocated to the following 6 groups: WT (n= 11), wild-type animals without treatment; WT+ l-NAME (n= 14), wild-type animals receiving l-NAME, an inhibitor of NO synthase; WT+ l-NAME+ LU (n= 13), wild-type animals receiving l-NAME and LU302872, a dual ETA/ETB-receptor antagonist; ET1tg (n= 10), ET-1 transgenic mice; ET1tg+ l-NAME (n= 13); and ET1tg+ l-NAME+ LU (n= 13). After 6weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation,as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after l-NAME treatment. The combined ETA/ETB-receptor antagonist LU302872 abolished inflammation and interstitial fibrosis in l-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study l-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.
Muscle contusions are the most common muscle injuries in sports medicine. Although these injuries are capable of healing, incomplete functional recovery often occurs.
Suramin enhances muscle healing by both stimulating muscle regeneration and preventing fibrosis in contused skeletal muscle.
Controlled laboratory study.
In vitro: Myoblasts (C2C12 cells) and muscle-derived stem cells (MDSCs) were cultured with suramin, and the potential of suramin to induce their differentiation was evaluated. Furthermore, MDSCs were cocultured with suramin and myostatin (MSTN) to monitor the capability of suramin to neutralize the effect of MSTN. In vivo: Varying concentrations of suramin were injected in the tibialis anterior muscle of mice 2 weeks after muscle contusion injury. Muscle regeneration and scar tissue formation were evaluated by histologic analysis and functional recovery was measured by physiologic testing
In vitro: Suramin stimulated the differentiation of myoblasts and MDSCs in a dose-dependent manner. Moreover, suramin neutralized the inhibitory effect of MSTN on MDSC differentiation. In vivo: Suramin treatment significantly promoted muscle regeneration, decreased fibrosis formation, reduced myostatin expression in injured muscle, and increased muscle strength after contusion injury.
Intramuscular injection of suramin after a contusion injury improved overall skeletal muscle healing. Suramin enhanced myoblast and MDSC differentiation and neutralized MSTN's negative effect on myogenic differentiation in vitro, which suggests a possible mechanism for the beneficial effects that this pharmacologic agent exhibits in vivo.
These findings could contribute to the development of biological treatments to aid in muscle healing after experiencing a muscle injury.
INTRODUCTION AND OBJECTIVE: The repair of vaginal tissue after vaginal vault prolapse or for correcting genital abnormalities is based on surgical techniques with graft materials that are under constant development. However, in contrast to other approaches in female pelvic reconstruction, stem cells have not yet been tested for this purpose in the vagina. We have previously reported that MDSC differentiate into smooth muscle cells (SMC) in the rat penile corpora cavernosa and correct erectile function. Here, we have investigated in a rat model whether: a) MDSC mounted on SIS scaffolds generate in vitro SMC and other cell types; b) express specific markers applicable to their detection upon implantation into the vagina after hysterectomy and partial vaginectomy ; and c) stimulate vaginal tissue repair.
METHODS: MDSC were prepared from mouse skeletal muscle by the pre-plating procedure, grown on monolayer, or polymeric scaffolds on porcine SIS, under 2.5% or 20% fetal bovine serum with or without TGF1 (5ng/ml), and tested for differentiation by immunocytochemistry (ICC) and quantitative western blot and real time PCR. Putative MDSC markers were screened by DNA microarrays (SuperArray) followed by RT/PCR, ICC, and western blot. DMEM on SIS scaffolds were labeled with DAPI and implanted on the vagina of rats that underwent hysterectomy, using as controls intact rats and hysterectomized rats implanted or not with SIS scaffolds without MDSC. Rats were immunosuppressed with tacrolimus and sacrificed after 4 or 8 weeks. Dual immunofluorescence was used to detect MDSC differentiation and hematoxylin/eosin was used for histology.
RESULTS: MDSC, both on monolayers and scaffolds, differentiated in vitro into SMC, as determined by actin (ASMA), calponin, and smoothelin. MDSC expressed Oct-4 and
myoglobin, in addition to myst 4, Nanog, Notch 3, Wnt 1, CD63, and muscle creatine kinase, as potential markers. Both DAPI fuorescence and myoglobin antibody detected MDSC implanted in the vagina, and dual DAPI/ASMA indicated their conversion to SMC at 4 weeks. Vaginal
tissue repair was stimulated by MDSC on SIS, as compared to SIS
alone, by differentiating into SMC and restoring normal histology at 4
and 8 weeks.
CONCLUSIONS: MDSC/SIS implants stimulate vaginal tissue
repair in the rat, and autologous MDSC may constitute a promissory
approach for treatment of vaginal prolapse.
Source of Funding: Institutional funds, Division of Female
Pelvic Medicine and Reconstructive Surgery, Harbor-UCLA Medical
Center, and Department of Defense PC061300
ABSTRACT Introduction. The primary histologic finding in many urologic disorders, including Peyronie's disease (PD), is fibrosis, mainly mediated by the transforming growth factor β1 (TGFβ1). Aim. To determine whether another member of the TGFβ family, myostatin, (i) is expressed in the human PD plaque and normal tunica albuginea (TA), their cell cultures, and the TGFβ1-induced PD lesion in the rat model; (ii) is responsible for myofibroblast generation, collagen deposition, and plaque formation; and (iii) mediates the profibrotic effects of TGFβ1 in PD. Methods. Human TA and PD tissue sections, and cell cultures from both tissues incubated with myostatin and TGFβ1 were subjected to immunocytochemistry for myostatin and α-smooth muscle actin (ASMA). The cells were assayed by western blot, Real time-Polymerase chain reaction (RT-PCR), and ribonuclease protection. Myostatin cDNA and shRNA were injected, with or without TGFβ1, in the rat penile TA, and plaque size was estimated by Masson. Main Outcome Measures. Myostatin expression in the human TA, the PD plaque, and their cell cultures, and myostatin effects on the PD-like plaque in the rat. Results. A threefold overexpression of myostatin was found in the PD plaque as compared with the TA. In PD cells, myostatin expression was mainly in the myofibroblasts, and in the TA cells, it increased upon passage paralleling myofibroblast differentiation and was up-regulated by TGFβ1. Myostatin or its cDNA construct increased the myofibroblast number and collagen in TA cells. Myostatin was detected in the TGFβ1-induced PD-like plaque of the rat partly in the myofibroblasts, and in the TA. Myostatin cDNA injected in the TA induced a plaque and intensified the TGFβ1 lesion, which was not reduced by myostatin shRNA. Conclusions. Myostatin is overexpressed in the PD plaque, partly because of myofibroblast generation. Although myostatin induces a plaque in the rat TA, it does not appear to mediate the one triggered by TGFβ1, thus suggesting that both proteins act concurrently and that therapy should target their common downstream effectors. Cantini LP, Ferrini MG, Vernet D, Magee TR, Qian A, Gelfand RA, Rajfer J, and Gonzalez-Cadavid NF. Profibrotic role of myostatin in Peyronie's disease. J Sex Med 2008;5:1607–1622.
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, TGFβ1 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 TGFβ1 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, TGFβ1, or XOR. In the iNOS KO mouse the hyperglycemia-induced alterations were exacerbated, with additional increases in oxidative stress and TGFβ1. 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.
Research on the molecular and cellular pathophysiology of Peyronie’s disease (PD) and the potential implications for the discovery
of novel therapeutic targets for this prevalent condition has lagged considerably behind the advances in surgical techniques.
However, two animal models have been generated based on the injection in the tunica albuginea of the rat of either transforming
growth factor-β1 (TGF-β1) or fibrin to induce PD-like plaques and on the extensive characterization of fibroblast cell cultures from the human tunica
albuginea and the PD plaque. These in vivo and in vitro models of PD not only replicate key molecular pathways and cell differentiation
processes operating in most other types of tissue fibrosis, but also are excellent tools to test and validate new antifibrotic
approaches of wider applicability. The fibrogenic roles of TGF-β1, oxidative stress, fibrin, plasminogen activator inhibitor 1, myofibroblast differentiation, and others have started to be
elucidated in the context of the PD plaque, and stem cells have been found in the tunica albuginea that may be involved in
plaque calcification and ectopic osteogenesis. In addition, endogenous antifibrotic pathways have been found in the PD plaque,
mainly the inducible nitric oxide synthase/nitric oxide/cyclic guanosine monophosphate cascade, antioxidant enzymes, anti-TGF-β1 factors, and others, that may maintain the plaque in cellular and molecular turnover, particularly in terms of collagen deposition/breakdown
and myofibroblast differentiation/apoptosis. Both the modulation of these defense mechanisms and some antifibrotic agents
utilized in other conditions are starting to be tested therapeutically in human PD or its models or are potential targets
for experimental research.
The aim of this study was to investigate the relationship between history of selected diseases, genital traumas, and Peyronie’s disease. A hospital-based case-control study was conducted at the Andrologic and Surgical Outpatient Units of the Policlinico Gemelli, Rome, where 134 men with Peyronie’s disease and 134 male controls were interviewed. The association between Peyronie’s disease and selected characteristics was estimated by means of odds ratios (OR) and 95% confidence intervals (CI). Patients who underwent invasive procedures on the penis (i.e., urethral catheterization, cystoscopy, and transurethral prostatectomy) had a 16-fold increased risk for Peyronie’s disease (OR = 16.1, 95% CI: 1.8–142), while a nearly three-fold increase was observed among patients who had genital and/or perineal traumatisms (95% CI: 1.0–7.1). A history of urethritis, uricacidemia, and lipoma was also significantly associated with an increased risk for Peyronie’s disease. Twenty-one percent of the cases and none of the controls were affected by Dupuytren’s contracture, and 4% of cases and none of the controls reported familial history for Peyronie’s disease. The frequency of inflammatory or fibromatous lesions of the genital tract of the partner was significantly higher in men with Peyronie’s disease than among controls. These results were consistent when performing a stratified analysis according to the type of controls (i.e., controls affected by urologic or by digestive conditions) to rule out the potential effect of recall bias. The findings of the study lend support to clinical reports stressing the importance of genital traumatisms and genetic conditions in the development of Peyronie’s disease.
We have isolated and cultured human primordial germ cells (PGCs) from early embryos. The PGCs expressed embryonic germ (EG) cell-specific surface markers, including Oct4 and Nanos. We derived a cell population from these PGCs that we termed embryoid body-derived (EBD) cells. EBD cells can be extensively expanded in vitro for more than 50 passages and express lineage markers from all three primary germ layers. The myogenic potential of the EBD cells was examined both in vitro and in vivo.In vitro, the EBD cells can be induced to form multinucleated myotubes, which express late skeletal muscle-specific markers, including MHC and dystrophin, when exposed to human galectin-1. In vivo, the EBD cells gave rise to all the myogenic lineages, including the skeletal muscle stem cells known as satellite cells. Strikingly, these cells were able to partially restore degenerated muscles in the SCID/mdx mouse, an animal model of the Duchenne’s muscular dystrophy. These results indicate the EBD cells may be a promising source of myogenic stem cells for cell-based therapies for muscle degenerative disorders.
Peyronie's disease is a fibrotic disorder of the tunica albuginea of the penis resulting in varying degrees of penile curvature and sexual dysfunction. Diagnosis of the disorder is made by detailed sexual history and focused physical examination. A wide range of medical treatments has been employed to treat the disorder. Verapamil treatment administered by intraplaque injection is supported by generally consistent randomized controlled trials (RCT). Some RCT evidence exists to support the use of colchicine, potaba, L-carnitine and liposomal superoxide dismutase. Future studies of medical efficacy should focus on improving RCT design, implementation and results reporting to improve the quality of evidence generated by each study. The surgical treatment of Peyronie's disease is a viable and recommended alternative for men with compromised sexual function due to severe curvature or lesions causing penile instability. The choice of corrective procedure should be tailored to each patient after a detailed evaluation of disease severity and sexual function.
Increased TGF-beta1 protein expression in tunica albuginea has been found to be associated with Peyronie's disease. The present study is designed to investigate whether surgical trauma induces TGF-beta up-regulation and histological changes in rat penis.
Thirty-two adult male Sprague-Dawley rats were divided into two groups. The first group (n=24) underwent incision and suture repair of the tunica albuginea of the penis. The second group (n=8) received sham surgery (incision of the penile skin and underlying fascia) as the control group. The trauma-induced group was divided into four subgroups in which the rats were euthanized at 6 hours (n=6), 1 day (n=6), 3 days (n=6), and 8 weeks (n=6). Two sham-operated (control) animals were also euthanized at each of the above time points. All tunical tissues from the trauma-induced and sham-operated rats were collected and examined histologically using Trichrome and Hart elastic fiber stain. Electron microscopy was used to study the ultrastructural changes of both trauma induced and control specimens. Western blotting technique was performed to study TGF-beta protein expression in both experimental and sham-operated groups.
Tissue edema and hemorrhage between collagen bundles are noted in the experimental groups after 6 hours, 1 day and 3 days. At 8 weeks the most prominent changes observed were inflammatory cellular infiltration and disorganization of the collagen bundles. In the control group the tunica albuginea retains normal wavy regular appearance in all rats. This histological analysis is similar to the reported description of histological features of the acute phase of Peyronie's disease. Electron microscopy showed packed collagen bundles in the trauma-induced group with normal appearing elastic fibers. No abnormal change was detected in the control group. Immunoblot results revealed remarkable TGF-beta1 protein expression in 1, 5, 3, and 0 rats of trauma induced subgroups after 6 hours, 1 day, 3 days, and 8 weeks respectively. No TGF-beta1 protein expression in any rats in the control group was detected. No significant TGF-beta2 or TGF-beta3 protein expression was observed either in the trauma induced group or in the control group.
Trauma can induce histological changes similar to the acute phase of Peyronie's disease but not the overt picture of the chronic phase of Peyronie's disease. It can also result in an early but transient up-regulation of TGF-beta1 protein expression in the rat penis. We conclude that surgical incisional trauma does not result in Peyronie's disease-like changes in the tunica.
Ischemia-reperfusion (IR)-induced nephrotoxicity is associated with proteinuria. There are reports on the involvement of inducible nitric oxide synthase (iNOS) in proteinuria in conjunction with renal disease. This study was designed to investigate the effect of N6-(1-iminoethyl)-L-lysine hydrochloride (L-Nil), a selective inhibitor of iNOS, to prevent proteinuria in IR injury.
Ischemia was induced by 40-minute clamping of the renal arteries followed by 6-hour reperfusion. Rats were administered either L-Nil (3 mg/kg intravenous bolus followed by infusion of 1 mg/kg/h) or saline. To monitor glomerular and tubular functional changes before and after treatment, we measured blood urea nitrogen, plasma creatinine, and urinary N-acetyl-beta-D-glucosaminidase activity. Total protein (TP), albumin, and low- (LMW) and high-molecular-weight (HMW) protein excretion rates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of urine samples. Kidney ultrastructure was examined through a transmission electron microscope (TEM).
IR resulted in significant LMW and HMW proteinuria. L-Nil significantly prevented the IR-induced increases in TP, albumin, and alpha1-microglobulin excretion. TEM showed loss of microvilli of the proximal tubule cells, injured mitochondria, and foamy changes in the structure of nuclear and cytoplasm in IR group. L-Nil reduced IR-mediated renal ultrastructural changes and tubular proteinuria.
This study suggested possible differences in the mechanism(s) of nephrotoxicity induced by iNOS in the glomeruli and tubular cells. The types of proteins excreted in the urine should be considered in the treatment strategy. In conclusion, this study suggested the involvement of iNOS in IR-induced tubular proteinuria.
Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.
Radical prostatectomy (RP) remains the standard treatment for men with clinically localized prostate cancer, despite the range of alternative treatment modalities. Even with significant advances in surgical technique and superb results for cancer control and preservation of urinary function, erectile dysfunction (ED) following RP is a common complication. This is mainly attributed to temporary cavernous nerve damage (neuropraxia) resulting in penile hypoxia, smooth muscle apoptosis, fibrosis and veno-occlusive dysfunction. One of the most promising new approaches is the concept of early penile rehabilitation, which is thought to prevent ED after RP by countering post-RP pathophysiological changes during the period of neural recovery. Various treatments, such as vacuum constriction devices, intraurethral and intracorporal alprostadil, and phosphodiesterase type 5 (PDE5) inhibitors, might serve to facilitate recovery of erectile function. PDE5 inhibitors are considered as the first-line treatment for early penile rehabilitation, with superior erectile function outcomes compared to placebo. Definitive conclusions regarding the success of penile rehabilitation cannot be drawn at this time because of differences in study design, data acquisition, and definitions of potency. Continued prospective, rigorous study is needed to develop and bring forward this important field and to establish the best evidence basis for counseling and treating patients suffering from ED after RP.
Changes that occur in the skeletal muscle environment with the progress of muscular dystrophies may affect stem cell function and result in impaired muscle regeneration. It has previously been suggested that the success of stem cell transplantation could therefore be dependent both on the properties of the cell itself and on the host muscle environment. Here we engrafted young and mature adult mdx-nude mice, which are the genetic homolog of Duchenne muscular dystrophy, with a small number of satellite cells freshly isolated from young, normal donor mice. We found that the donor satellite cells contributed to muscle regeneration and self-renewal as efficiently within mature adult, as in young, dystrophic host muscle. Donor-derived satellite cells also contributed to robust regeneration after further injury, showing that they were functional despite the more advanced dystrophic muscle environment. These findings provide evidence that muscle tissue in a later stage of dystrophy may be effectively treated by stem cells.
There is considerable interindividual variability in motor function among patients with Duchenne muscular dystrophy (DMD); moreover, pathogenetic mechanisms of motor dysfunction in DMD are not understood. Using multiparametric analysis, we correlated initial histologic alterations in quadriceps muscle biopsies from 25 steroid therapy-free patients with DMD with 13 relevant clinical features assessed by a single clinical team during a long-term period (mean, >10 years). There was no residual muscle dystrophin by immunohistochemistry and Western blot analysis in the biopsies. Myofiber size, hypercontracted fibers, necrotic/basophilic fibers, endomysial and perimysial fibrosis, and fatty degeneration were assessed by morphometry. Endomysial fibrosis was the only myopathologic parameter that significantly correlated with poor motor outcome as assessed by quadriceps muscle strength, manual muscle testing of upper and lower limbs at 10 years, and age at ambulation loss (all p<0.002). Motor outcome and fibrosis did not correlate with genotype. Myofibers exhibited oxidative stress-induced protein alterations and became separated from capillaries by fibrosis that was associated with both increase of CD206+ alternatively activated macrophages and a relative decrease of CD56+ satellite cells (both p<0.0001). This study provides a strong rationale for antifibrotic therapeutic strategies in DMD and supports the view that alternatively activated macrophages that are known to inhibit myogenesis while promoting cellular collagen production play a key role in myofibrosis.
Satellite cells exist in postnatal muscle tissue and constitute the main source of muscle precursor cells for growth and repair. These cells carry out important roles for skeletal muscle formation postnatally during growth of muscle mass as well as damage-induced regenerative processes. Muscle regeneration supports muscle function in aging and has a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and extracellular matrix. Myostatin, a member of TGF-beta family, was mainly expressed in muscle tissue. Decorin, a member of the small leucine-rich proteoglycan gene family, is composed of a core protein and a dermatan/chondroitin sulfate chain. Recent studies have shown that decorin enhanced the proliferation and differentiation of myogenic cells by suppressing myostatin activity. Thus, decorin appears to be a new molecule in the myostatin signaling pathway and a promising target for treatment of progressive neuromuscular diseases. Therefore, in this study, we examined the localization of decorin as well as myostatin in a muscular dystrophy model in mdx mice and B10 Scott Snells mice as a control to elucidate the differences between decorin and myostatin messages as well as protein distribution. This study revealed increased expression of decorin protein as well as mRNA at the regenerative stage of mdx mice compared to early stages, while only weak expression of decorin was detected in the control mice. Our study contributes to identifying the relationship between decorin and myostatin as well as the development of a therapeutic strategy for progressive neuromuscular diseases.
Background and purpose:
Patients with diabetes mellitus exhibit generalized endothelial and cardiac dysfunction with decreased nitric oxide production. Elevated intracellular cyclic guanosine monophosphate (cGMP) levels contribute to an effective cardioprotection in different pathophysiological conditions. In this study, we investigated whether chronic treatment with the phosphodiesterase-5 inhibitor vardenafil could improve diabetic cardiovascular dysfunction by up-regulating the nitric oxide-cGMP pathway in the vessel wall and myocardium.
Experimental approach:
Diabetes was induced in young rats by a single intraperitoneal injection of streptozotocin (60 mg x kg(-1)). In the treatment group, vardenafil (10 mg x kg(-1) x day(-1)) was given orally for 8 weeks. Diabetic control animals received vehicle for the same time. Left ventricular pressure-volume relations were measured by using a microtip Millar pressure-volume conductance catheter, and indexes of contractility, such as the slope of end-systolic pressure-volume relationship (E(max)) and preload recruitable stroke work (PRSW), were calculated. In organ bath experiments for isometric tension with rings of isolated aortae, endothelium-dependent and independent vasorelaxation was investigated by using acetylcholine and sodium nitroprusside.
Key results:
When compared with the non-diabetic controls, diabetic rats showed increased myocardial and vascular transforming growth factor-beta1 expression, impaired left ventricular contractility (impairment of E(max) by 53%, PRSW by 40%; P < 0.05) and vascular dysfunction. Treatment with vardenafil resulted in higher cGMP levels, reduced transforming growth factor-beta1 expression, significantly improved cardiac function (improvement of E(max) by 95%, PRSW by 69%; P < 0.05) and greater vasorelaxation to acetylcholine and sodium nitroprusside in aortae from diabetic animals.
Conclusions and implications:
Our results demonstrate that impaired vascular cGMP signalling contributes to the development of diabetic vascular and cardiac dysfunction, which can be prevented by chronic phosphodiesterase-5 inhibition.
Duchenne-Meryon muscular dystrophy (DMD) is the most common and lethal genetic muscle disease. Ameliorating muscle necrosis, inflammation, and fibrosis represents an important therapeutic approach for DMD. Imatinib, an antineoplastic agent, demonstrated antiinflammatory and antifibrotic effects in liver, kidney, lung, and skin of various animal models. This study tested antiinflammatory and antifibrotic effects of imatinib in mdx mice, a DMD mouse model. We treated mdx mice with intraperitoneal injections of imatinib at the peak of limb muscle inflammation and the onset of diaphragm fibrosis. Controls received PBS vehicle or were left untreated. Muscle necrosis, inflammation, fibrosis, and function were evaluated by measuring serum CK activity, endomysial CD45 immunoreactive inflammation area, endomysial collagen III deposition, and hind limb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was assessed by Western blot in diaphragm tissue and in primary cultures of peritoneal macrophages and skeletal muscle fibroblasts. Imatinib markedly reduced muscle necrosis, inflammation, and fibrosis, and significantly improved hind limb grip strength in mdx mice. Reduced clinical disease was accompanied by inhibition of c-abl and PDGFR phosphorylation and suppression of TNF-alpha and IL-1beta expression. Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis by inhibiting c-abl and PDGFR signaling pathways and downstream inflammatory cytokine and fibrotic gene expression.
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.
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.
The penis and aorta from both 7-month-old male diabetic OZR (5 months of diabetes) and aged-matched nondiabetic lean Zucker rats (LZR) rats were harvested (eight per group). The penis and aorta were subjected to histo- or immnohistochemistry, followed by quantitative image analysis (QIA) to determine the contents of SMC, collagen and the pro-fibrotic transforming growth factor (TGF)beta1. The turnover of SMCs was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) assays. Quantitative Western blots determined calponin (SMC marker) and PCNA, and hydroxyproline was used for collagen. In vitro relaxation of corporal strips was measured.
In vitro relaxation of corporal tissue from OZR was considerably less than in the LZR. In the media of the penile dorsal artery (PDA) of OZR, there was a considerable reduction in the SMC content and the SMC/collagen ratio, as well as an increase in apoptosis, but there were no changes in PCNA or TGFbeta1 expression, or in the intima-media/lumen ratio. In the aorta of the OZR, in contrast to the PDA, there was a reduction in PCNA as well as a more pronounced decrease in the SMC/collagen ratio, mainly from an increase in collagen, but there were no changes in TGFbeta1 or the wall/lumen morphometry. In the OZR, Western blots of aortic tissue confirmed the decrease in PCNA and a reduction in the SMC marker calponin.
These data show that 5 months after the onset of hyperglycaemia in the OZR, the rats develop both abnormal corporal SMC relaxation and a generalized fibrosis of the arterial media of both the large and small diameter vessels. It is possible that this pan-fibrosis of the media of the arterial system might contribute to the diabetes-related ED that occurs during this period in this rat model.
Peyronie's Disease (PD) has been studied for more than 260 years since Francois de la Peyronie's description in 1743. Based on the current literature, the prevalence of PD seems 3-9% with an average age of onset in the fifth life decade. Much effort has been spent on developing nonsurgical treatment options to cure or at least prevent disease progression.
The recent examination of drug trials for erectile dysfunction has led us to assess PD trial methodology more closely.
An Iinternet search on PubMed was performed using MeSH words PD, clinical trials, oral, transdermal, intralesional and shock wave therapy focusing on 26 representing studies published over the last 15 years. Mean Outcome Measures. A comprehensive review of the current literature on nonsurgical treatment options for PD was conducted to address methodological issues and challenges in PD trials highlighting trial design, patient population, and symptom and sign assessment.
The majority of the reviewed studies are underpowered and the heterogeneity in the methodological approach and patient assessment between the studies is one of the remarkable findings from our review. Studies should use a uniform means of defining the degree and type of penile deformity and a large enough cohort of patients should be studied for adequate study power. An ideally designed PD intervention trial should comprise: (i) a randomized, placebo-controlled design; (ii) with a PD patient set representative of the general PD population; and (iii) a comprehensive symptom and sign assessment before and at the end of treatment which includes an assessment of at least deformity, pain, and sexual function.
A number of challenges exist for the design of PD intervention trials and deciphering the data generated from them. The field would benefit greatly from a consensus statement or guidelines development on the design and conduct of such trials.
Despite its high prevalence and impact on the quality of life of patients, and that it is an excellent model for the study of fibrotic processes, Peyronie's disease (PD) is an orphan disease in biomedical research. The development of animal and cell culture models has advanced substantially the understanding of its molecular and cellular pathology and the proposal of new therapies.
To review the literature pertaining to the use of these models for the study of PD.
PubMed search conducted from the first report of an animal model for PD.
This model, based on the finding that transforming growth factor beta1 (TGF beta 1) is overexpressed in the PD plaque, consists on the injection of TGF beta 1 into the tunica albuginea of the rat. This leads to a PD-like plaque retaining many of the histological and biochemical features of human PD. Another rat model, based on the hypothesis that the PD plaque arises from trauma to the penis, causing fibrinogen extravasation that initiates as fibrin a fibrotic response, consists on injection of fibrin into the tunica. The cell culture model is based on the demonstration that myofibroblasts are abundant in the human PD plaque.
These models have: (i) clarified the role of microtrauma, myofibroblasts, and oxidative stress in plaque development; (ii) demonstrated that this tissue is under sustained turnover by fibrotic and antifibrotic mechanisms; (iii) showed the interplay of collagenolytic and fibrinolytic systems and their inhibitors; (iv) detected an endogenous antifibrotic process consisting of the expression of inducible nitric oxide synthase that counteracts oxidative stress, collagen synthesis, and myofibroblast generation; (v) characterized the antifibrotic effects of chronic treatment with phosphodiesterase type 5 (PDE5) inhibitors; (vi) discovered the cytogenetic instability of PD cells and alterations in their gene expression; and (vii) detected stem cells in the tunica albuginea with a potential role in fibrosis and ossification.
Corporal veno-occlusive dysfunction (CVOD), which usually is associated with a loss of smooth muscle cells (SMC) and an increase in fibrosis within the corpora cavernosa, can be induced by an injury to the cavernosal nerves. The corporal tissue expresses inducible nitric oxide synthase (iNOS), presumably as an antifibrotic and SMC-protective response.
We studied the temporal relationship in the corpora between the expression of iNOS, other histological and biochemical changes, and the development of CVOD, after bilateral cavernosal nerve resection (BCNR) in the rat.
Rats underwent either BCNR or sham operation. Cavernosometry was performed 1, 3, 7, 15, 30, and 45 days (N = 8/groups) after surgery. Penile tissue sections were subjected to Masson trichrome staining for SMC and collagen, and immunodetection for alpha smooth muscle actin, iNOS, neuronal NOS (nNOS), endothelial NOS (eNOS), proliferating cell nuclear antigen (PCNA), and terminal transferase dUTP nick end labeling (TUNEL). Quantitative western blot analysis was done in homogenates.
Time course on the development of fibrosis and CVOD.
Following BCNR, CVOD was detectable 30 days later, and it became more pronounced by 45 days. In contrast, the SMC/collagen ratio in the BCNR corpora was reduced at 7 days and bottomed at 30 and 45 days, due in part to the reduction of SMC, presumably caused by an increase in apoptosis peaking at 3 days. PCNA also peaked at 3 days, but then decayed. nNOS was reduced early (3-7 days) and disappeared at 30 days, whereas eNOS was not affected. iNOS was induced at day 3, and steadily increased peaking at 30 days.
CVOD develops in the BCNR rat as a result of the early loss of corporal SMC by the neuropraxia-induced apoptosis, which the initial cell replication response cannot counteract, followed by fibrosis. The time course of iNOS induction supports the antifibrotic role of iNOS.
This study sought to test the efficacy of phosphodiesterase type 5A (PDE5A) inhibition for treating advanced hypertrophy/remodeling caused by pressure overload, and to elucidate cellular and molecular mechanisms for this response.
Sildenafil (SIL) inhibits cyclic guanosine monophosphate-specific PDE5A and can blunt the evolution of cardiac hypertrophy and dysfunction in mice subjected to pressure overload. Whether and how it ameliorates more established advanced disease and dysfunction is unknown.
Mice were subjected to transverse aortic constriction (TAC) for 3 weeks to establish hypertrophy/dilation, and subsequently treated with SIL (100 mg/kg/day) or placebo for 6 weeks of additional TAC.
The SIL arrested further progressive chamber dilation, dysfunction, fibrosis, and molecular remodeling, increasing myocardial protein kinase G activity. Isolated myocytes from TAC-SIL hearts showed greater sarcomere shortening and relaxation, and enhanced Ca(2+) transients and decay compared with nontreated TAC hearts. The SIL treatment restored gene and protein expression of sarcoplasmic reticulum Ca(2+) uptake adenosine triphosphatase (SERCA2a), phospholamban (PLB), and increased PLB phosphorylation (S16), consistent with improved calcium handling. The phosphatase calcineurin (Cn) and/or protein kinase C-alpha (PKCalpha) can both lower phosphorylated phospholamban and depress myocyte calcium cycling. The Cn expression and PKCalpha activation (outer membrane translocation) were enhanced by chronic TAC and reduced by SIL treatment. Expression of PKCdelta and PKCepsilon also increased with TAC but were unaltered by SIL treatment.
SIL treatment applied to well-established hypertrophic cardiac disease can prevent further cardiac and myocyte dysfunction and progressive remodeling. This is associated with improved calcium cycling, and reduction of Cn and PKCalpha activation may be important to this improvement.
Peyronie's disease (PD) is a wound-healing disorder in which a fibrotic plaque forms in the tunica albuginea layer of the penis. It clinically presents as any combination of penile pain, angulation, and erectile dysfunction. Recent studies indicate that PD has a prevalence of 3%-9% in adult men. Although the exact etiology has not been established, PD likely results from a predisposing genetic susceptibility combined with an inciting event such as microtrauma during intercourse. During the initial acute phase (6-18 months), the condition may progress, stabilize, or regress. For this reason authorities recommend a more conservative treatment approach, with a trial of oral and/or intralesional pharmacotherapy, before surgical reconstruction is considered. Oral therapies most commonly employed include tocopherol (vitamin E) and paraaminobenzoate (Potaba), with colchicine, tamoxifen, propoleum, and acetyl-L-carnitine being used less often. There are a limited number of long-term placebo-controlled studies with these oral agents, and for the most part, studies have failed to show a consistent beneficial effect. Intralesional injection therapy for PD is more commonly used as a first-line therapy. The current standard of care includes injection with interferon-alpha-2b, verapamil, or collagenase. Interferon-alpha-2b, in particular, has been documented in a large, multicenter, placebo-controlled study to show significant benefit over placebo in decreasing penile curvature, plaque size, penile pain, and plaque density. However, intralesional interferon is associated with posttreatment flu-like symptoms unless patients are premedicated with a nonsteroid anti-inflammatory agent. Other available therapies that have not consistently shown efficacy in placebo-controlled studies include corticosteroids, orgotein, radiation, and extracorporeal shockwave therapy. Surgery is considered when men with PD do not respond to conservative or medical therapy for approximately 1 year and cannot perform satisfactory sexual intercourse. Ongoing basic research in PD will likely identify future targets for medical exploitation.
Recent studies have challenged the conventional hypothesis that inflammation is the major player in the fibrosis cascade. Emerging evidence points to a critical role for interactions between tissue-resident and infiltrating, non-resident cells, in mediating fibrotic responses. Improved understanding of the biology of extracellular matrix (ECM) remodeling and the pathways that regulate assembly of the ECM and its interactions with growth factors/cytokines have led to the identification of new and attractive therapeutic targets. These include molecules that regulate fibrocytic cell infiltration, epithelial and myofibroblast differentiation, ECM synthesis and degradation. However, it is imperative that these new therapies be timed and compartmentalized to target the tissue of interest, as the dynamics of cellular differentiation and ECM remodeling may be different between organ systems. This review will summarize the current understanding of the mechanisms involved in the development of fibrosis, based on recent in vitro and in vivo studies, and comment on novel molecular pathways for drug discovery.
Progression of diabetic nephropathy (DN) is manifested by gradual scarring of both the renal glomerulus and tubulointerstitial region. Over the past several years, the general understanding of the pathogenic factors that lead to renal fibrosis in DN has expanded considerably. In this review, some of the important factors that appear to be involved in driving this fibrosing process are discussed, with special emphasis on newer findings and insights. It is now clear that multiple cell types in the kidney contribute to progressive fibrosis in DN. New concepts about bradykinin, TGF-beta and eNOS signaling as well as JAK/STAT activation and the central role of inflammation in both glomerular and tubulointerstitial fibrosis are discussed.