MicroRNA-34a regulates cardiac ageing and function.

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

JMJD8 Regulates Angiogenic Sprouting and Cellular Metabolism by Interacting With Pyruvate Kinase M2 in Endothelial Cells.

OBJECTIVE: Jumonji C (JmjC) domain-containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown. Here, we characterize the expression of JmjC domain-containing proteins after inducing endothelial differentiation of murine embryonic stem cells and study the function of JmjC domain-only proteins in endothelial cell (EC) functions. APPROACH AND RESULTS: We identified a large number of JmjC domain-containing proteins regulated by endothelial differentiation of murine embryonic stem cells. Among the family of JmjC domain-only proteins, Jmjd8 was significantly upregulated on endothelial differentiation. Knockdown of Jmjd8 in ECs significantly decreased in vitro network formation and sprouting in the spheroid assay. JMJD8 is exclusively detectable in the cytoplasm, excluding a function as a histone-modifying enzyme. Mass spectrometry analysis revealed JMJD8-interacting proteins with known functions in cellular metabolism like pyruvate kinase M2. Accordingly, knockdown of pyruvate kinase M2 in human umbilical vein ECs decreased endothelial sprouting in the spheroid assay. Knockdown of JMJD8 caused a reduction of EC metabolism as measured by Seahorse Bioscience extracellular flux analysis. Conversely, overexpression of JMJD8 enhanced cellular oxygen consumption rate of ECs, reflecting an increased mitochondrial respiration. CONCLUSIONS: Jmjd8 is upregulated during endothelial differentiation and regulates endothelial sprouting and metabolism by interacting with pyruvate kinase M2.

LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity.

LEOPARD syndrome (multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafness; LS), also called Noonan syndrome with multiple lentigines (NSML), is a rare autosomal dominant disorder associating various developmental defects, notably cardiopathies, dysmorphism, and short stature. It is mainly caused by mutations of the PTPN11 gene that catalytically inactivate the tyrosine phosphatase SHP2 (Src-homology 2 domain-containing phosphatase 2). Besides its pleiotropic roles during development, SHP2 plays key functions in energetic metabolism regulation. However, the metabolic outcomes of LS mutations have never been examined. Therefore, we performed an extensive metabolic exploration of an original LS mouse model, expressing the T468M mutation of SHP2, frequently borne by LS patients. Our results reveal that, besides expected symptoms, LS animals display a strong reduction of adiposity and resistance to diet-induced obesity, associated with overall better metabolic profile. We provide evidence that LS mutant expression impairs adipogenesis, triggers energy expenditure, and enhances insulin signaling, three features that can contribute to the lean phenotype of LS mice. Interestingly, chronic treatment of LS mice with low doses of MEK inhibitor, but not rapamycin, resulted in weight and adiposity gains. Importantly, preliminary data in a French cohort of LS patients suggests that most of them have lower-than-average body mass index, associated, for tested patients, with reduced adiposity. Altogether, these findings unravel previously unidentified characteristics for LS, which could represent a metabolic benefit for patients, but may also participate to the development or worsening of some traits of the disease. Beyond LS, they also highlight a protective role of SHP2 global LS-mimicking modulation toward the development of obesity and associated disorders.

Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth hormone-induced ERK hyperactivation, which contributes to short stature.

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.

Role of the microRNA-17-92 cluster in the endothelial differentiation of stem cells.

MicroRNAs (miRs) are small non-coding RNAs that recently emerged as potent regulators of gene expression. The members of the miR-17-92 cluster have been shown to control endothelial cell functions and neovascularization; however, the regulation and function of the cluster in endothelial cell lineage commitment has not been explored. This project aimed to test the role of the miR-17-92 cluster during endothelial differentiation. We demonstrate that miR-17, miR-18, miR-19 and miR-20 are increased upon the induction of endothelial cell differentiation of murine embryonic stem cells or induced pluripotent stem cells. In contrast, miR-92a and the primary miR-17-92 transcript were downregulated. The inhibition of each individual miR of the cluster by cholesterol-modified antagomirs did not affect endothelial marker gene expression. Moreover, the combination of all antagomirs had no effect. These findings illustrate that although the miR-17-92 cluster regulates vascular integrity and angiogenesis, none of the members has a significant impact on the endothelial differentiation of pluripotent stem cells.

Smooth muscle cell differentiation from human bone marrow: variations in cell type specific markers and Id gene expression in a new model of cell culture.

Stromal cells follow a vascular smooth muscle differentiation pathway. However, cell culture models performed from human bone marrow do not allow the obtention of a large proportion of highly differentiated smooth muscle cells (SMC) and their differentiation pathways remain unclear. We have characterized a new model of SMC differentiation from human bone marrow stromal cells by using different factors (bFGF, EGF, insulin and BMP-4). A relative homogeneous population of differentiated SMC was reproducibly obtained in short-term culture with high expression of SMC markers. Id gene expression was investigated and showed that (1) Id2 mRNA expression was upregulated during SMC differentiation without change of Id1 mRNA and (2) Id1 gene expression highly increased concomitantly with a decrease of SMC markers while Id2 mRNA was slightly modulated. Our data suggested that Id genes are potentially implicated in the differentiation pathway of human SMC from bone marrow.

Differential expression of two TEF-1 (TEAD) genes during Xenopus laevis development and in response to inducing factors.

Transcription enhancer factors 1 (TEF-1 or TEAD) make a highly conserved family of eukaryotic DNA binding proteins that activate not only viral regulatory elements but muscle specific genes and are involved in several developmental processes. In this study, we report the identification and the expression pattern of NTEF-1 (TEAD1) and DTEF-1 (TEAD3), two members of this family in Xenopus laevis. Both X. laevis NTEF-1 (XNTEF-1 or XTEAD1) and DTEF-1 (XDTEF-1 or XTEAD3) possess a 72 amino acid TEA domain characteristic of TEF-1 proteins. XNTEF-1 is a 426 amino acid protein that has 96% identity with the avian or the mammalian NTEF-1 proteins while XDTEF-1 is a 433 amino acid protein with 77 to 80% identity with the avian and mammalian DTEF-1 sequences respectively. Temporal expression analysis by RT-PCR indicated that the two genes are expressed maternally and throughout embryonic development. In the adult, the two genes are broadly expressed although they showed differences of expression between tissues. Spatial expression analysis by whole mount in situ hybridization showed that the XNTEF-1 and XDTEF-1 mRNAS were predominantly detected in eye, embryonic brain, somites and heart. In animal cap assay, the two genes are activated by bFGF but are differently regulated by BMP4, and the muscle regulatory factor Mef2d.

Glitazones inhibit human monoamine oxidase but their anti-inflammatory actions are not mediated by VAP-1/semicarbazide-sensitive amine oxidase inhibition.

Glitazones are peroxisome proliferator-activated receptor gamma (PPARγ) agonists widely used as antidiabetic drugs also known as thiazolidinediones. Most of them exert other effects such as anti-inflammatory actions via mechanisms supposed to be independent from PPARγ activation (e.g., decreased plasma monocyte chemoattractant protein-1 (MCP-1) levels). Recently, pioglitazone has been shown to inhibit the B form of monoamine oxidase (MAO) in mouse, while rosiglitazone and troglitazone were described as non-covalent inhibitors of both human MAO A and MAO B. Since molecules interacting with MAO might also inhibit semicarbazide-sensitive amine oxidase (SSAO), known as vascular adhesion protein-1 (VAP-1), and since VAP-1/SSAO inhibitors exhibit anti-inflammatory activity, our aim was to elucidate whether VAP-1/SSAO inhibition could be a mechanism involved in the anti-inflammatory behaviour of glitazones. To this aim, MAO and SSAO activities were measured in human subcutaneous adipose tissue biopsies obtained from overweight women undergoing plastic surgery. The production of hydrogen peroxide, an end-product of amine oxidase activity, was determined in tissue homogenates using a fluorometric method. The oxidation of 1 mM tyramine was inhibited by pargyline and almost resistant to semicarbazide, therefore predominantly MAO-dependent. Rosiglitazone was more potent than pioglitazone in inhibiting tyramine oxidation. By contrast, benzylamine oxidation was only abolished by semicarbazide: hence SSAO-mediated. Pioglitazone hampered SSAO activity only when tested at 1 mM while rosiglitazone was inefficient. However, rosiglitazone exhibited anti-inflammatory activity in human adipocytes by limiting MCP-1 expression. Our observations rule out any involvement of VAP-1/SSAO inhibition and subsequent limitation of leukocyte extravasation in the anti-inflammatory action of glitazones.

Involvement of autotaxin/lysophosphatidic acid signaling in obesity and impaired glucose homeostasis.

Autotaxin (ATX) is a secreted lysophospholipase D involved in synthesis of lysophosphatidic acid (LPA), a phospholipid growth factor acting via specific receptors (LPA1R to LPA6R) and involved in several pathologies including obesity. ATX is secreted by adipocytes and contributes to circulating LPA. ATX expression is up-regulated in obese patients and mice in relationship with insulin resistance and impaired glucose tolerance. LPA1R is the most abundant subtype in adipose tissue. Its expression is higher in non-adipocyte cells than in adipocytes and is not altered in obesity. ATX increases and LPA1R decreases while preadipocytes differentiate into adipocytes (adipogenesis). LPA inhibits adipogenesis through down-regulation of the pro-adipogenic transcription factor PPARγ2. Adipocyte-specific knockout (FATX-KO) mice or mice treated with the LPAR antagonist Ki16425 gain more weight and accumulate more adipose tissue than wild type or control mice fed a high fat diet (HFD). These observations suggest that LPA (via LPA1R) exerts a tonic inhibitory effect on adipose tissue expansion that could, at least in part, result from the anti-adipogenic activity of LPA. A possible negative impact of LPA on insulin-sensitivity might also be considered. Despite being more sensitive to nutritional obesity, FATX-KO and Ki16425-treated mice fed a HFD show improved glucose tolerance when compared to wild type mice. Moreover, exogenously injected LPA acutely impairs glucose tolerance and insulin secretion. These observations show that LPA exerts a tonic deleterious impact on glucose homeostasis. In conclusion, ATX and LPA1R represent potential interesting pharmacological targets for the treatment of obesity-associated metabolic diseases.

Resveratrol directly affects in vitro lipolysis and glucose transport in human fat cells.

Resveratrol is a naturally occurring polyphenol found in many dietary sources and red wine. Recognized as a cancer chemoprevention agent, an anti-inflammatory factor and an antioxidant molecule, resveratrol has been proposed as a potential anti-obesity compound and to be beneficial in diabetes. Most of the studies demonstrating the anti-adipogenic action of resveratrol were performed as long-term treatments on cultured preadipocytes. The aim of this study was to analyse the acute effects of resveratrol on glucose uptake and lipolysis in human mature adipocytes. Samples of subcutaneous abdominal adipose tissue were obtained from overweight humans and immediately digested by liberase. Fat cells were incubated (from 45 min to 4 h) with resveratrol 1 µM-1 mM. Then, glycerol release or hexose uptake was determined. Regarding lipolysis, the significant effects of resveratrol were found at 100 µM, consisting in a facilitation of isoprenaline stimulation and an impairment of insulin antilipolytic action. At 1 and 10 µM, resveratrol only tended to limit glucose uptake. Resveratrol 100 µM did not change basal glucose uptake but impaired its activation by insulin or by benzylamine. This inhibition was not found with other antioxidants. Such impairment of glucose uptake activation in fat cells may led to a reduced availability of glycerol phosphate and then to a decreased triacylglycerol assembly. Therefore, resveratrol increased triacylglycerol breakdown triggered by ß-adrenergic activation and impaired lipogenesis. Consequently, our data indicate that resveratrol can be considered as limiting fat accumulation in human fat cells and further support its use for the mitigation of obesity.

Influence of secreted factors from human adipose tissue on glucose utilization and proinflammatory reaction.

The objective of the present study was to characterize the nature of the autocrine/paracrine signal within human adipose tissue that may alter glucose metabolism and the inflammatory status in adipocytes. We prepared a conditioned medium from abdominal dermolipectomies in the absence (CM) or the presence (CMBSA) of bovine serum albumin (BSA), and we tested the influence of CM and CMBSA on glucose transport, maximal insulin response, and the expression of inflammation marker genes in differentiated human SGBS adipocytes. We found that CMBSA increased basal and reduced insulin-stimulated glucose incorporation along with a reduced mRNA level of the glucose transport GLUT4, and an increased expression of GLUT1. These effects were associated with a potent upregulation in the mRNA level of the proinflammatory cytokines IL-6 and MCP-1. These regulations were strongly attenuated in the absence of BSA during the preparation of CM, or after BSA depletion of CM, and were attributed to water-soluble molecules rather than lipids. Finally, fractionation of CMBSA by isoelectric focusing showed that part of its bioactivity could be reproduced with proteins with pHi ranging from 6.6 to 7.6. In conclusion, our results demonstrate that the production by human adipose tissue of autocrine/paracrine neutral proteins is able to increase the inflammatory status of the adipocytes and to deteriorate their glucose metabolism and maximal insulin response, and their release is greatly amplified by the presence of albumin.

Comparative functional analysis of ZFP36 genes during Xenopus development.

ZFP36 constitutes a small family of RNA binding proteins (formerly known as the TIS11 family) that target mRNA and promote their degradation. In mammals, ZFP36 proteins are encoded by four genes and, although they show similar activities in a cellular RNA destabilization assay, there is still a limited knowledge of their mRNA targets and it is not known whether or not they have redundant functions. In the present work, we have used the Xenopus embryo, a model system allowing gain- and loss-of-function studies, to investigate, whether individual ZFP36 proteins had distinct or redundant functions. We show that overexpression of individual amphibian zfp36 proteins leads to embryos having the same defects, with alteration in somites segmentation and pronephros formation. In these embryos, members of the Notch signalling pathway such as hairy2a or esr5 mRNA are down-regulated, suggesting common targets for the different proteins. We also show that mouse Zfp36 protein overexpression gives the same phenotype, indicating an evolutionary conserved property among ZFP36 vertebrate proteins. Morpholino oligonucleotide-induced loss-of-function leads to defects in pronephros formation, reduction in tubule size and duct coiling alterations for both zfp36 and zfp36l1, indicating no functional redundancy between these two genes. Given the conservation in gene structure and function between the amphibian and mammalian proteins and the conserved mechanisms for pronephros development, our study highlights a potential and hitherto unreported role of ZFP36 gene in kidney morphogenesis.

Depot-specific regulation of autotaxin with obesity in human adipose tissue.

Autotaxin (ATX) is a lysophospholipase D involved in synthesis of a bioactive mediator: lysophosphatidic. ATX is abundantly produced by adipocytes and exerts a negative action on adipose tissue expansion. In both mice and humans, ATX expression increases with obesity in association with insulin resistance. In the present study, fat depot-specific regulation of ATX was explored in human. ATX mRNA expression was quantified in visceral and subcutaneous adipose tissue in obese (BMI > 40 kg/m(2); n = 27) and non-obese patients (BMI < 25 kg/m(2); n = 10). Whatever the weight status of the patients is, ATX expression was always higher (1.3- to 6-fold) in subcutaneous than in visceral fat. Nevertheless, visceral fat ATX was significantly higher (42 %) in obese than in non-obese patients, whereas subcutaneous fat ATX remained unchanged. In obese patients, visceral fat ATX expression was positively correlated with diastolic arterial blood pressure (r = 0.67; P = 0.001). This correlation was not observed with subcutaneous fat ATX. Visceral fat ATX was mainly correlated with leptin (r = 0.60; P = 0.001), inducible nitric oxide synthase (r = 0.58; P = 0,007), and apelin receptor (r = 0.50; P = 0.007). These correlations were not observed with subcutaneous fat ATX. These results reveal that obesity-associated upregulation of human adipose tissue ATX is specific to the visceral fat depot.

Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs.

The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE(-/-) mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis.

Vestigial like gene family expression in Xenopus: common and divergent features with other vertebrates.

The Drosophila Vestigial and Scalloped proteins form heterodimers that control wing development and are involved in muscle differentiation. Four vestigial like genes have been described in mammals. Similar to the Drosophila vestigial gene, they encode a short conserved domain (TONDU) required for interaction with the mammalian paralogues of Drosophila Scalloped (i.e., TEAD proteins). We previously identified two TEAD genes in Xenopus laevis and we report here the expression of four distinct vestigial like genes in Xenopus (vgll1-4) that represent amphibian orthologs of the mammalian vestigial like genes. Vgll1 has a unique expression pattern which is restricted to epidermal cells, both in the embryo and in the adult. Vgll2 is expressed in the skeletal muscle lineage downstream of myogenic factors and in the embryonic brain similar to the avian and mammalian orthologues. Vgll3 expression is transient, identifies embryonic hindbrain rhombomere 2, and is negatively regulated by en2, but not by egr2. Vgll4 is mainly expressed in anterior neural structures. In summary, the four Xenopus vgll genes have unique/complex expression profiles and they are differently expressed during embryogenesis. Moreover, these amphibian vestigial like genes display distinct responses to the major signaling pathways (i.e., activin, FGF or BMP) that orchestrate pattern-formation during early development.

Induction and modulation of smooth muscle differentiation in Xenopus embryonic cells.

By comparison with skeletal or cardiac developmental programs, little is known regarding the specific factors that promote specification and differentiation of smooth muscle cells from pluripotent cells. We have analyzed the developmental expression of a subset of smooth muscle genes during Xenopus early development and showed that similar to mammals and avians, Xenopus smooth muscle myosin heavy chain (SM-MHC) is a highly specific marker of smooth muscle differentiation. Embryonic cells from animal pole explants of Xenopus blastula can be induced by basic fibroblast growth factor, Wnt, and bone morphogenetic protein signals to adopt the smooth muscle pathway. Explants from early embryos that contain neural crest cells can also differentiate into cells expressing smooth muscle genes. We examined the interplay of several transcription factors, that is SRF, myocardin, and GATA6, that induce the expression of SM-MHC in animal cap cells and found that myocardin-dependent expression of smooth muscle genes in animal cap cells is synergized by SRF but is strongly antagonized by GATA6.