Satyesh K. Sinha

Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ β-Catenin and Follistatin/Transforming Growth Factor-β Signaling Pathways

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

Decreased prevalence of diabetes in marijuana users: cross-sectional data from the National Health and Nutrition Examination Survey (NHANES) III

Objective To determine the association between diabetes mellitus (DM) and marijuana use. Design Cross-sectional study. Setting Data from the National Health and Nutrition Examination Survey (NHANES III, 1988–1994) conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention. Participants The study included participants of the NHANES III, a nationally representative sample of the US population. The total analytic sample was 10 896 adults. The study included four groups (n=10 896): non-marijuana users (61.0%), past marijuana users (30.7%), light (one to four times/month) (5.0%) and heavy (more than five times/month) current marijuana users (3.3%). DM was defined based on self-report or abnormal glycaemic parameters. We analysed data related to demographics, body mass index, smoking status, alcohol use, total serum cholesterol, high-density lipoprotein, triglyceride, serum 25-hydroxy vitamin D, plasma haemoglobin A1c, fasting plasma glucose level and the serum levels of C reactive protein and four additional inflammatory markers as related to marijuana use. Main outcome measures OR for DM associated with marijuana use adjusted for potential confounding variables (ie, odds of DM in marijuana users compared with non-marijuana users). Results Marijuana users had a lower age-adjusted prevalence of DM compared to non-marijuana users (OR 0.42, 95% CI 0.33 to 0.55; p<0.0001). The prevalence of elevated C reactive protein (>0.5 mg/dl) was significantly higher (p<0.0001) among non-marijuana users (18.9%) than among past (12.7%) or current light (15.8%) or heavy (9.2%) users. In a robust multivariate model controlling for socio-demographic factors, laboratory values and comorbidity, the lower odds of DM among marijuana users was significant (adjusted OR 0.36, 95% CI 0.24 to 0.55; p<0.0001). Conclusions Marijuana use was independently associated with a lower prevalence of DM. Further studies are needed to show a direct effect of marijuana on DM.

Using Stereologic Techniques for Podocyte Counting in the Mouse: Shifting the Paradigm

Background: The podocyte serves the important function of maintaining the glomerular filtration barrier, and many studies report a decrease in podocyte number relative to the development of proteinuric states. However, there is significant inconsistency in the number of podocytes counted, possibly due to different counting methods. We previously counted the three glomerular cell types in the mouse kidney and showed that the fractionator/disector method is a close approximation of the exhaustive count or the gold standard method. In this study, we compared the commonly used model-based approach with the design-based approach to count podocytes in the db/m and db/db mouse and illustrate that the design-based approach, which uses the fractionator/disector method, provides an accurate determination of podocyte number. Methods: In the design-based approach, toluidine blue-stained 1-µm-thick sections from glutaraldehyde perfusion-fixed kidneys were used (n = 15) with the fractionator/disector method. In the model-based approach, WT-1-immunolabeled podocyte nuclei in 3- to 4-µm-thick formalin-fixed paraffin-embedded sections of the same kidneys were counted with the Weibel-Gomez method. Glomerular volume was determined for each method. Results: We discovered that the fractionator/disector method counted 89 ± 10 podocytes compared to the Weibel-Gomez method, which counted 137 ± 38 podocytes and overestimated podocyte number by 54% (p < 0.05). In addition, glomerular volume (231 ± 52 × 103 vs. 192 ± 64 × 103 µm3) was significantly underestimated by 17% (p < 0.0002). Moreover, the model-based approach was more time consuming. Conclusion: We conclude that the fractionator/disector method offers an unbiased and efficient determination of podocyte counts.

Association of Race/Ethnicity, Inflammation, and Albuminuria in Patients With Diabetes and Early Chronic Kidney Disease

OBJECTIVE African Americans (AAs) and Hispanics have higher diabetes and end-stage renal disease but similar or lower early chronic kidney disease (CKD) compared with whites. Inflammation plays a critical role in the pathogenesis of diabetes-related CKD. We postulated that in contrast to the general population, AAs and Hispanics have a higher prevalence of early diabetic CKD and systemic inflammatory markers compared with whites. RESEARCH DESIGN AND METHODS We analyzed the National Health and Nutrition Examination Survey 1999–2008 of 2,310 diabetic patients aged ≥20 years with fasting plasma glucose (FPG) ≥126 mg/dL. We performed multiple linear regression among patients with early CKD (urinary albumin excretion [UAE] ≥30 μg/mL and estimated glomerular filtration rate ≥60 mL/min/1.73 m2) to test the relationship between UAE and C-reactive protein (CRP) by race/ethnicity, adjusting for demographics, diabetes duration, FPG, hemoglobin A1c, uric acid, white blood cell count, medication use, cardiovascular disease, and related parameters. RESULTS In patients with diabetes, the prevalence of early CKD was greater among Hispanics and AAs than whites (P < 0.0001). AAs had higher adjusted odds ratio (AOR) for CRP ≥0.2 mg/dL (AOR 1.81 [95% CI 1.19–2.78]), and Hispanics had higher AOR for UAE ≥30 μg/mL (AOR 1.65 [1.07–2.54]). In a regression model adjusted for confounding variables, there was a significant association between UAE and CRP in the mid-CRP tertile (CRP 0.20–0.56 mg/dL, P = 0.001) and highest CRP tertile (CRP ≥0.57 mg/dL, P = 0.01) for Hispanics, but only in the mid-CRP tertile (P = 0.04) for AAs, compared with whites. CONCLUSIONS AAs and Hispanics with diabetes have a higher prevalence of early CKD compared with whites, which is significantly associated with UAE and/or CRP.

Role of γ melanocyte-stimulating hormone–renal melanocortin 3 receptor system in blood pressure regulation in salt-resistant and salt-sensitive rats

Melanocortin 3 receptor (MC3-R) has high affinity and specificity to gamma melanocyte-stimulating hormone (gammaMSH), a natriuretic peptide involved in regulation of blood pressure (BP) and sodium excretion. Recent studies showing increased MC3-R expression and elevated plasma gammaMSH in normal rats fed a high-salt diet support the role of this system in sodium homeostasis. We hypothesized that dysregulation of MC3-R response to dietary salt may contribute to salt retention and BP elevation in salt-sensitive hypertension. We examined renal MC3-R expression, plasma gammaMSH concentration, and response to MC3-R agonist and antagonist in Dahl salt-sensitive (DSS) and Dahl salt-resistant (DSR) rats fed high-salt (8%) or low-salt (0.07%) diets for 3 weeks. Consumption of high-salt diet significantly increased BP in the DSS but not the DSR group. High-salt diet led to a 5-fold increase in plasma gammaMSH and a 2-fold increase in renal MC3-R in DSR rats. Plasma gammaMSH and renal MC3-R abundance in DSS rats were maximally elevated on low-salt diet and remained unchanged on high-salt diet. Administration of MC3-R agonist melanotan II significantly lowered BP and raised fractional Na excretion in the DSR but not the DSS rats consuming high-salt diet. In contrast, MC3-R antagonist SHU9119 significantly raised BP and lowered fractional Na excretion in both groups. Thus, the data suggest that gammaMSH-renal MC3-R pathway is activated and appears to be biologically functional in the DSS rats.

17β-estradiol inhibits oxidized low-density lipoprotein-induced increase in matrix metalloproteinase-9 expression in human macrophages.

Beneficial effects of estrogen have been attributed to improved lipid profiles and to direct effects on the arterial wall. Macrophage-derived matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where they may contribute to plaque disruption. We have shown that oxidized low-density lipoprotein (Ox-LDL) increases matrix metalloproteinase-9 (MMP-9) expression in macrophages (Mφ). In this study, we tested the hypothesis that 17β-estradiol regulates basal and Ox-LDL-induced expression of MMPs and their tissue inhibitor (TIMPs) in human Mφ. Peripheral blood mononuclear cells isolated from normal human subjects were cultured for 7 days to transform into Mφ. On day 7, Mφ were starved with serum-free medium for 16 hours and then treated with 17β-estradiol and/or progesterone (PROG) in the presence or absence Ox-LDL for 24 hours. Levels and activity of MMP-2 and MMP-9 and levels of TIMP-1 and TIMP-2 were determined. After exposure to Ox-LDL, MMP-9 expression increased by 60% and TIMP-1 expression decreased by 29% (P < 0.05 and P < 0.05, respectively, compared to control), whereas TIMP-2 expression was unchanged. 17β-estradiol reduced the levels of Ox-LDL-induced MMP-9 protein as measured by Western blot (P < 0.05; n = 5) and Ox-LDL-induced MMP-9 activity (P < 0.05; n = 5) as measured by gelatin zymography. Conclusively, estradiol abolished Ox-LDL-stimulated increase in the levels of macrophage-derived MMP-9 protein and activity in human Mφ. This effect was reversed by TAM but not by PROG. These data suggest that at least part of the protective effect of estrogen occurs by attenuation of Ox-LDL alterations in MMP-9 expression.

Multinucleated giant cells in atherosclerotic plaques of human carotid arteries: Identification of osteoclast-like cells and their specific proteins in artery wall

UNLABELLED The mechanism(s) mediating atherosclerotic calcification may be similar to those governing bone remodeling, and osteoblast-like cells have been observed in plaque. We tested the hypothesis that osteoclast-like cells (OLCs) also exist in atherosclerotic arteries. In 205 tissue blocks obtained from 21 patients undergoing carotid endarterectomy, we performed histopathologic analysis, histochemical staining for tartrate-resistant acid phosphatase (TRAP), and immunohistochemical analysis for osteoclast and macrophage antigens, including CD68, colony stimulating factor-1 receptor (CSF-1R), cathepsin K (cat-K), receptor activator of nuclear factor-κB (RANK), and osteoprotegerin (OPG). Lesions were classified according to the AHA system, and further grouped as calcified or non-calcified (with necrotic cores or suture granulomas). Multinucleated giant cells morphologically similar to osteoclasts were frequently seen, sometimes exhibited morphologic evidence of polarization, were closely associated with regions of calcification, fibrosis, or granulomatous tissue, and also appeared to be associated with neovascularization and regions of intraplaque hemorrhage. TRAP-positive cells often expressed the osteoclast-associated antigens cat-K, RANK, and OPG. Calcification typically occurred at the base of plaque or in necrotic cores in various morphologies, including a fine powdery pattern, a diffuse pattern of larger deposits near cholesterol clefts and necrotic centers, and nodular forms. Regions of frank ossification were rarely observed. CONCLUSION OLCs are frequently found in plaque, and co-localize with sub-regions of cholesterol deposition, mineralization, and necrotic and foreign debris. True bone tissue is rare in carotid plaque, although more common in other arteries. Our findings suggest that arterial OLCs might degrade mineral deposits, prevent formation of calcification or both and therefore counterbalance the activity of the osteoblast-like cells in atherosclerosis.

Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury

Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.

TESTOSTERONE REGULATION OF MUSCLE MASS: IN VITRO AND IN VIVO ROLE OF FOLLISTATIN.: 60

Testosterone (T) administration increases muscle mass and decreases fat mass in men. We have previously reported that T stimulates myogenic differentiation in mesenchymal multipotent C3H10T1/2 (10T1/2) cells through an androgen receptor (AR)-mediated pathway though interaction with β-catenin/TCF4 signaling. We found that an overexpression of full-length TCF4 led to an increase in MyoD and MHC II and follistatin (Fst) expression. Objectives To investigate (1) if the T-supplementation leads to Fst up-regulation in vivo and (2) if Fst-knockdown abrogates the myogenic effects in an in vitro differentiation model. Methods and Results In vivo: Castrated C57BL6J mice were sham treated and subcutaneously implanted with physiologic (1 cm) and supraphysiologic (2 cm) doses of T for 6 weeks. Body composition analysis was performed by DEXA scanning. Consistent with previous reports, castration resulted in decreased fat-free mass, total body weight, and weight of androgen-responsive levator ani (LA) muscle. In LA muscle, castration led to a decrease in Fst mRNA levels, which were restored by T supplementation in a dose-dependent manner. In vitro: To investigate the role of Fst in mediating myogenic effects of T treatment, incubation of 10T1/2 with recombinant Fst (0.4 μg/mL) led to a significant increase in key myogenic markers MyoD and MHC II (protein and mRNA) in the absence of T. In addition, treatment of these cells with anti-Fst antibody (1 μg/mL) and Fst siRNA abolished T-induced increase in MyoD and MHC II protein levels. Combined with in vivo data, in vitro our studies suggest that a T-induced increase in muscle mass may be mediated via induction of Fst. Conclusions Fst plays major role during T-induced in vitro and in vitro changes in muscle mass. As Fst in known to bind and antagonize myostatin (Mst), the most potent inhibitor of muscle mass, we believe that T levels may regulate endogenous Fst/Mst interaction and thereby regulate body composition. As the promyogenic effects of T are achieved with a concomitant increased risk of elevated prostate-specific antigen (PSA), we posit that the use of Fst with a low dose of T may have therapeutic implications similar to those achieved with high levels of T supplementation.