Somitogenesis: From somite to skeletal muscle.

Myogenesis is controlled by an elaborate system of extrinsic and intrinsic regulatory mechanisms in all development stages. The aim of this review is to provide an overview of the different stages of myogenesis and muscle differentiation in mammals, starting from somitogenesis and analysis of the different portions that constitute the mature somite. Particular attention was paid to regulatory genes, in addition to mesodermal stem cells, which represent the earliest elements of myogenesis. Finally, the crucial role of growth factors, molecules of vital importance in contractile regulation, hormones and their function in skeletal muscle differentiation, growth and metabolism, and the role played by central nervous system, are discussed.

Urokinase-type plasminogen activator (uPA) stimulates triglyceride synthesis in Huh7 hepatoma cells via p38-dependent upregulation of DGAT2.

OBJECTIVE: The liver is the central organ of fatty acid and triglyceride metabolism. Oxidation and synthesis of fatty acids and triglycerides is under the control of peroxisome-proliferator-activated receptors (PPAR) α. Impairment of these receptors' function contributes to the accumulation of triglycerides in the liver resulting in non-alcoholic fatty liver disease. Urokinase-type plasminogen activator (uPA) was shown to regulate gene expression in the liver involving PPARγ transcriptional activity. In this study we questioned whether uPA modulates triglyceride metabolism in the liver, and investigated the mechanisms involved in the observed processes. METHODS AND RESULTS: Huh7 hepatoma cells were incubated with increasing concentrations of uPA for 24 h uPA dose-dependently increased the cellular triglyceride mass, and this effect resulted from increased de novo triglyceride synthesis mediated by the enzyme diglyceride acyltransferase 2 (DGAT2). Also, the amount of free fatty acids was highly up regulated by uPA through activation of the transcription factor SREBP-1. Chemical activation of PPARα further increased uPA-stimulated triglyceride synthesis, whereas inhibition of p38, an upstream activator of PPARα, completely abolished the stimulatory effect of uPA on both triglyceride synthesis and DGAT2 upregulation. The effect of uPA on triglyceride synthesis in Huh7 cells was mediated via binding to its receptor, the uPAR. In vivo studies in uPAR(-/-) mice demonstrated that no lipid droplets were observed in their livers compared to C57BL/6 mice and the triglyceride levels were significantly lower. CONCLUSION: This study presents a new biological function of the uPA/uPAR system in the metabolism of triglycerides and might present a new target for an early therapeutic intervention for NAFLD.

ADAM-10 could mediate cleavage of N-cadherin promoting apoptosis in human atherosclerotic lesions leading to vulnerable plaque: a morphological and immunohistochemical study.

Atherosclerosis remains a major cause of mortality. Whereas the histopathological progression of atherosclerotic lesions is well documented, much less is known about the development of unstable or vulnerable plaque, which can rupture leading to thrombus, luminal occlusion and infarct. Apoptosis in the fibrous cap, which is rich in vascular smooth muscle cells (VSMCs) and macrophages, and its subsequent weakening or erosion seems to be an important regulator of plaque stability. The aim of our study was to improve our knowledge on the biological mechanisms that cause plaque instability in order to develop new therapies to maintain atherosclerotic plaque stability and avoid its rupture. In our study, we collected surgical specimens from atherosclerotic plaques in the right or left internal carotid artery of 62 patients with evident clinical symptoms. Histopathology and histochemistry were performed on wax-embedded sections. Immunohistochemical localization of caspase-3, N-cadherin and ADAM-10 was undertaken in order to highlight links between apoptosis, as expressed by caspase-3 immunostaining, and possible roles of N-cadherin, a cell-cell junction protein in VSMCs and macrophages that provides a pro-survival signal reducing apoptosis, and ADAM-10, a "disintegrin and metalloproteases" that is able to cleave N-cadherin in glioblastomas. Our results showed that when apoptosis, expressed by caspase-3 immunostaining, increased in the fibrous cap, rich in VSMCs and macrophages, the expression of N-cadherin decreased. The decreased N-cadherin expression, in turn, was linked to increased ADAM-10 expression. This study shows that apoptotic events are probably involved in the vulnerability of atherosclerotic plaque.

Mineralocorticoid receptor blockade inhibits accelerated atherosclerosis induced by a low sodium diet in apolipoprotein E-deficient mice.

INTRODUCTION: A low-sodium diet (LSD) was shown to increase both angiotensin II (AngII) and aldosterone levels, and to accelerate atherosclerosis in apolipoprotein E-deficient (E0) mice. The aim of the present study was to examine whether accelerated atherosclerosis in E0 mice fed a LSD is mediated by aldosterone, using the mineralocorticoid receptor blocker, eplerenone (Epl). METHODS AND RESULTS: Mice were divided into three groups: normal diet (ND), LSD and LSD treated with Epl at 100 mg/kg per day (LSD+Epl) for 10 weeks. LSD significantly enhanced plasma renin and aldosterone levels, which were further increased in mice fed LSD+Epl. The aortic lesion area increased three-fold with LSD, while LSD+Epl significantly reduced the lesion area to values similar to ND. Serum and peritoneal macrophages obtained from LSD-fed mice exhibited pro-atherogenic properties including increased inflammation, oxidation and cholesterol accumulation, which were inhibited in mice fed LSD+Epl. In a J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, Epl was shown to have a direct anti-inflammatory effect. CONCLUSION: In E0 mice, Epl inhibited LSD-accelerated atherosclerosis, despite the elevation of renin and aldosterone levels. It is therefore suggested that the atherogenic action of LSD could be mediated, at least in part, by activation of the mineralocorticoid receptor. In addition, eplerenone may have direct anti-inflammatory actions.

The effects of aldosterone on diet-induced fatty liver formation in male C57BL/6 mice: comparison of adrenalectomy and mineralocorticoid receptor blocker.

OBJECTIVE: Obesity, diabetes, fatty liver, and hypertension are major determinants of the metabolic syndrome. The effects of aldosterone and mineralocorticoid receptor blockers on fatty liver are largely unknown. The aim of the present study was to evaluate the relationships between aldosterone and the development of fatty liver. MATERIALS AND METHODS: In our experiments, we performed adrenalectomy (ADX) or administered 100 mg/kg/day eplerenone, a specific mineralocorticoid receptor blocker, to male C57BL/6 mice fed with a 60% fat diet for 20 weeks. RESULTS: High-fat diet led to metabolic syndrome as indicated by increased body weight, elevated systolic blood pressure, impaired glucose tolerance, elevated insulin levels, and development of fatty liver. A marked reduction of aldosterone by ADX or blockade of aldosterone interaction with its receptor by eplerenone, which increased serum aldosterone considerably, resulted in reduced blood pressure, and reduced serum insulin and levels of triglycerides. However, differential effects were found on reduction of blood glucose to normal levels, which was observed only in ADX. Neither ADX nor eplerenone affected fatty liver formation or body weight. In cultured hepatocytes, triglyceride loading induced by high glucose, oleic acid or very low density lipoprotein was not affected by aldosterone, spironolactone or eplerenone. CONCLUSION: Our results suggest that whereas aldosterone might be involved in some of the diet-induced insulin and glucose metabolic effects, it played no role in the development of fatty liver.

Eplerenone reduced lesion size in early but not advanced atherosclerosis in apolipoprotein E-deficient mice.

The beneficial effects of eplerenone, a specific mineralocorticoid receptor blocker, were previously demonstrated in early atherosclerosis (ATS). The aim of the present study was to evaluate the effect of eplerenone in advanced versus early ATS. Apolipoprotein E knockout mice aged 16 or 32 weeks were randomly divided into eplerenone (100 mg·kg·d) or vehicle treatment for 14 weeks. Eplerenone reduced atherosclerotic lesion size by 51% only in early ATS. In peritoneal macrophages obtained from these mice, eplerenone reduced messenger RNA expression of pro-inflammatory markers, interleukin 6, tumor necrosis factor α, monocyte chemotactic protein 1, and increased anti-inflammatory marker arginase 1 to a greater extent in early compared with advanced ATS. These changes correspond to macrophage polarization toward alternative inflammatory phenotype. Messenger RNA expression of the mineralocorticoid receptor and aldosterone synthase were also reduced by eplerenone to a greater extent in early ATS, and these might increase the sensitivity of macrophages to mineralocorticoid blockade in early ATS. The results of the present study point to the benefits of early initiation of treatment with eplerenone in reducing experimental ATS.

FAD286, an aldosterone synthase inhibitor, reduced atherosclerosis and inflammation in apolipoprotein E-deficient mice.

INTRODUCTION: Aldosterone is known to be involved in atherosclerosis and cardiovascular disease and blockade of its receptor was shown to improve cardiovascular function. It was, therefore, hypothesized that inhibition of aldosterone synthesis would also reduce atherosclerosis development. METHOD: To test this hypothesis, we examined the effect of FAD286 (FAD), an aldosterone synthase inhibitor, on the development of atherosclerosis in spontaneous atherosclerotic apolipoprotein E-deficient mice. Mice were divided into three treatment groups: normal diet, low-salt diet (LSD) and LSD treated with FAD at 30 mg/kg per day (LSD + FAD) for 10 weeks. RESULTS AND CONCLUSION: Histomorphometry of the aortas obtained from these mice showed that atherosclerotic lesion area increased by three-fold under LSD compared with normal diet and FAD significantly reduced lesion area to values similar to normal diet. Changes in atherosclerosis were paralleled by changes in the expression of the inflammation markers (C-reactive protein, monocyte chemotactic protein-1, interleukin-6, nuclear factor kappa B and intercellular adhesion molecule-1) in peritoneal macrophages obtained from these mice. Surprisingly, whereas LSD increased serum or urine aldosterone levels, FAD did not alter these levels when evaluated at the end of the study. In J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, FAD was shown to have a direct dose-dependent anti-inflammatory effect. In apolipoprotein E-deficient mice, FAD reduces atherosclerosis and inflammation. However, these actions appeared to be dissociated from its effect on inhibition of aldosterone synthesis.

Paraoxonase 1 deficiency in mice is associated with reduced steroid biosynthesis: effects on HDL binding, cholesteryl ester accumulation and scavenger receptor type BI expression.

OBJECTIVE: Selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis. METHODS AND RESULTS: PON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthesis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demonstrated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice. CONCLUSION: PON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.

Apolipoprotein E and its role in aging and survival.

The study of biological aging has seen spectacular progress in the last decade and markers are increasingly employed for understanding physiological processes that change with age. Recently, it has been demonstrated that apolipoprotein E (apoE) has a major impact on longevity, but its mechanisms are still not fully understood. ApoE-deficient (E(o)) mice have proved to be a very popular model for studying spontaneous hypercholesterolemia and the subsequent development of atherosclerotic lesions, but only limited data are available with regard to aging and aging changes. We used this murine model to better characterize the involvement of apoE in aging and to evaluate its role in the maintenance of normal organ morphology. Our results show that E(0) mice at different ages (6, 12, 20 weeks old) developed age-dependent morphological and biochemical alterations, including fibrosis (newly formed collagen), pro-inflammatory cytokine (IL-6 and iNOS), lipofuscin accumulation, and decrease of antioxidant enzymes (superoxide dismutase and catalase) in several organs (kidney, liver and heart). It is significant that the observed degenerative findings in E(0) mice at different ages (6, 12, 20 weeks old) were not identified in control mice (C57BL), at 6, 12 and 20 weeks of age. Consequently, since these mice showed enzymatic and structural alterations, normally linked to the age, such as increase of lipofuscin, pro-inflammatory cytokines and decrease of antioxidant enzymes, we can conclude that apoE is a useful player in studies of longevity and age-related diseases, such as inflammatory status and atherosclerosis that are known risk factors for functional decline and early mortality. Moreover, it is possible that apoE may also play a role in other pathological conditions including, for example, cancer, rheumatoid arthritis and macular degeneration.

Paraoxonase 2 attenuates macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1.

This study questioned the role of paraoxonase 2 (PON2) in attenuation of macrophage lipids accumulation. Mouse peritoneal macrophages (MPMs) harvested from PON2-deficient mice versus control C57BL/6 mice, look like foam cells and were larger in size and filled with lipid droplets. Macrophage triglyceride (but not cholesterol) content, biosynthesis rate, and microsomal acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) activity (not mRNA and protein) in PON2-deficient versus control MPM were all significantly increased by 4.6-, 3.6-, and 4.4-fold, respectively. Similarly, microsomal DGAT1 activity and cellular triglyceride content were significantly decreased in human PON2-transfected cells as well as upon incubation of PON2-deficient MPM with recombinant PON2. In all the above experimental systems, PON2 also decreased macrophage oxidative state. Incubation of PON2-deficient MPM with the free radicals generator 2,2'-amidinopropane hydrochloride increased cellular oxidative stress and DGAT1 activity by 2.2- and 3.4-fold, respectively, whereas incubation of microsomes from PON2-deficient MPM with superoxide dismutase decreased DGAT1 activity by 40%. We thus conclude that PON2 attenuates macrophage triglyceride accumulation and foam cell formation via inhibition of microsomal DGAT1 activity, which appears to be sensitive to oxidative state.