Valentina Sala

High Levels of Cre Expression in Neuronal Progenitors Cause Defects in Brain Development Leading to Microencephaly and Hydrocephaly

Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D’Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreERT2 (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreERT2 recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreERT2 transgenic lines after tamoxifen mediated-CreERT2 translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreERT2 oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.

Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.12116.001

Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy.

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) – surprisingly – autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.

Novel therapy for myocardial infarction: can HGF/Met be beneficial?

Myocardial infarction (MI) is a leading cause of hospitalization worldwide. A recently developed strategy to improve the management of MI is based on the use of growth factors which are able to enhance the intrinsic capacity of the heart to repair itself or regenerate after damage. Among others, hepatocyte growth factor (HGF) has been proposed as a modulator of cardiac repair of damage due to the pleiotropic effects elicited by Met receptor stimulation. In this review we describe the mechanistic basis for autocrine and paracrine protection of HGF in the injured heart. We also analyse the role of HGF/Met in stem cell maintenance and in stem cell therapies for MI. Finally, we summarize the most significant results on the use of HGF in experimental models of heart injury and discuss the potential of the molecule for treating ischaemic heart disease in humans.

Signaling to cardiac hypertrophy: insights from human and mouse RASopathies

Cardiac hypertrophy is the heart’s response to a variety of extrinsic and intrinsic stimuli, some of which might finally lead up to a maladaptive state. An integral part of the pathogenesis of the hypertrophic cardiomyopathy disease (HCM) is the activation of the rat sarcoma (RAS)/RAF/MEK (mitogen-activated protein kinase kinase)/MAPK (mitogen-activated protein kinase) cascade. Therefore, the molecular signaling involving RAS has been the subject of intense research efforts, particularly after the identification of the RASopathies. These constitute a class of developmental disorders caused by germline mutations affecting proteins contributing to the RAS pathway. Among other phenotypic features, a subset of these syndromes is characterized by HCM, prompting researchers and clinicians to delve into the chief signaling constituents of cardiac hypertrophy. In this review, we summarize current advances in the knowledge of the molecular signaling events involved in the pathogenesis of cardiac hypertrophy through work completed on patients and on genetically manipulated animals with HCM and RASopathies. Important insights are drawn from the recognition of parallels between cardiac hypertrophy and cancer. Future research promises to further elucidate the complex molecular interactions responsible for cardiac hypertrophy, possibly pointing the way for the identification of new specific targets for the treatment of HCM.

MicroRNAs in myocardial ischemia: identifying new targets and tools for treating heart disease. New frontiers for miR-medicine

MicroRNAs (miRNAs) are natural, single-stranded, small RNA molecules which subtly control gene expression. Several studies indicate that specific miRNAs can regulate heart function both in development and disease. Despite prevention programs and new therapeutic agents, cardiovascular disease remains the main cause of death in developed countries. The elevated number of heart failure episodes is mostly due to myocardial infarction (MI). An increasing number of studies have been carried out reporting changes in miRNAs gene expression and exploring their role in MI and heart failure. In this review, we furnish a critical analysis of where the frontier of knowledge has arrived in the fields of basic and translational research on miRNAs in cardiac ischemia. We first summarize the basal information on miRNA biology and regulation, especially concentrating on the feedback loops which control cardiac-enriched miRNAs. A focus on the role of miRNAs in the pathogenesis of myocardial ischemia and in the attenuation of injury is presented. Particular attention is given to cardiomyocyte death (apoptosis and necrosis), fibrosis, neovascularization, and heart failure. Then, we address the potential of miR-diagnosis (miRNAs as disease biomarkers) and miR-drugs (miRNAs as therapeutic targets) for cardiac ischemia and heart failure. Finally, we evaluate the use of miRNAs in the emerging field of regenerative medicine.

Gene expression profiling of HGF/Met activation in neonatal mouse heart

Hepatocyte Growth Factor (HGF) controls growth and differentiation in different cell types, including cardiac cells. However, its downstream effectors are poorly understood. To investigate the transcriptional targets of HGF, we analyzed the hearts of neonatal mice with cardiomyocyte-specific HGF overexpression with whole genome DNA microarrays. When comparing HGF expressing versus control hearts, we found a total of 249 transcripts with significant gene expression changes (210 upregulated and 39 downregulated). Gene Ontology (GO) annotation analysis revealed that the transcripts modulated by HGF were enriched for metabolic functions including: protein translation, vesicle-mediated transport, regulation of transcription, regulation of muscle development. Using an automated literature meta-analysis approach, we obtained a co-occurrence network oriented to the positive regulatory role of Myc and Notch1 in controlling some of the genes which are downstream to HGF. GO analysis of this network returned genes involved in the regulation of heart development. HGF positively controls MyocD, an activator of cardiac gene expression, and Hdac5, an inhibitor of cardiac growth. These results may unveil a new role of HGF in the modulation of signaling pathways implicated in the activation or repression of cardiomyogenesis.

Digoxin and ouabain induce the efflux of cholesterol via liver X receptor signalling and the synthesis of ATP in cardiomyocytes

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition of Na(+)/K(+)-ATPase. We showed previously that in human hepatoma cells, digoxin and ouabain increase the rate of the mevalonate cascade and therefore have Na(+)/K(+)-ATPase-independent effects. In the present study we found that they increase the expression and activity of 3-hydroxy-3 methylglutaryl-CoA reductase and the synthesis of cholesterol in cardiomyocytes, their main target cells. Surprisingly this did not promote intracellular cholesterol accumulation. The glycosides activated the liver X receptor transcription factor and increased the expression of ABCA1 (ATP-binding cassette protein A1) transporter, which mediates the efflux of cholesterol and its delivery to apolipoprotein A-I. By increasing the synthesis of ubiquinone, another derivative of the mevalonate cascade, digoxin and ouabain simultaneously enhanced the rate of electron transport in the mitochondrial respiratory chain and the synthesis of ATP. Mice treated with digoxin showed lower cholesterol and higher ubiquinone content in their hearts, and a small increase in their serum HDL (high-density lipoprotein) cholesterol. The results of the present study suggest that cardioactive glycosides may have a role in the reverse transport of cholesterol and in the energy metabolism of cardiomyocytes.

Activated Met signalling in the developing mouse heart leads to cardiac disease.

BACKGROUND The Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine involved in many physiological processes, including skeletal muscle, placenta and liver development. Little is known about its role and that of Met tyrosine kinase receptor in cardiac development. METHODOLOGY/PRINCIPAL FINDINGS In this study, we generated two transgenic mice with cardiac-specific, tetracycline-suppressible expression of either Hepatocyte Growth Factor (HGF) or the constitutively activated Tpr-Met kinase to explore: i) the effect of stimulation of the endogenous Met receptor by autocrine production of HGF and ii) the consequence of sustained activation of Met signalling in the heart. We first showed that Met is present in the neonatal cardiomyocytes and is responsive to exogenous HGF. Exogenous HGF starting from prenatal stage enhanced cardiac proliferation and reduced sarcomeric proteins and Connexin43 (Cx43) in newborn mice. As adults, these transgenics developed systolic contractile dysfunction. Conversely, prenatal Tpr-Met expression was lethal after birth. Inducing Tpr-Met expression during postnatal life caused early-onset heart failure, characterized by decreased Cx43, upregulation of fetal genes and hypertrophy. CONCLUSIONS/SIGNIFICANCE Taken together, our data show that excessive activation of the HGF/Met system in development may result in cardiac damage and suggest that Met signalling may be implicated in the pathogenesis of cardiac disease.

A mouse model for spatial and temporal expression of HGF in the heart

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia–reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.