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

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

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

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

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

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

Effect of muscle-derived stem cells on the restoration of corpora cavernosa smooth muscle and erectile function in the aged rat.

OBJECTIVE: To determine whether skeletal muscle-derived stem cells (MDSCs) convert into smooth muscle cells (SMCs) both in vitro and in vivo, and in so doing ameliorate the erectile dysfunction (ED) of aged rats, and whether endogenous stem cells are present in the rat corpora cavernosa. MATERIALS AND METHODS: MDSCs were obtained from mouse muscle, and shown by immunocytochemistry for alpha-smooth muscle actin (alpha SMA) to originate in vitro in myofibroblasts and SMCs, discriminating SMCs by calponin 1 expression. In vivo these MDSCs, labelled with 4',6-diamidino-2-phenylindole, were implanted into the corpora cavernosa of young adult (5-month old) and aged (20-month old) rats for 2 and 4 weeks. Histological changes were assessed by immunohistochemistry and quantitative Western blot. Functional changes were determined by electrical field stimulation (EFS) of the cavernosal nerve. RESULTS: The exogenous cells replicated and converted into SMCs, as shown in corporal tissue sections by confocal immunofluorescence microscopy for proliferating cell nuclear antigen (PCNA), alpha SMA, and smoothelin, and also by Western blot for alpha SMA and PCNA. MDSC differentiation was confirmed by the activation of the alpha SMA promoter-linked beta-galactosidase in transfected cells, both in vitro and after implantation in the corpora. Putative endogenous stem cells were shown in corporal tissue sections and Western blots by detecting CD34 and a possible Sca1 variant. EFS showed that implanted MDSCs raised in aged rats the maximal intracavernosal pressure/mean arterial pressure levels above (2 weeks) or up to (4 weeks) those of young adult rats. CONCLUSIONS: MDSCs implanted into the corpora cavernosa of aged rats converted into SMCs and corrected ED, and endogenous cells expressing stem cell markers were also found in untreated tissue. This suggests that exogenous stem cell implantation and/or endogenous stem cell modulation might be viable therapeutic approaches for ageing-related ED.

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.