Tiziana Crepaldi | Università degli Studi di Torino (original) (raw)

Papers by Tiziana Crepaldi

La Ricerca in clinica e in laboratorio

Biochemical Journal, 2012

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition... more 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.

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sar... more 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.

Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor... more Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor. The HGF/Met pathway has a prominent role in cardiovascular remodelling after tissue injury. The present review provides a synopsis of the cellular and molecular mechanisms underlying the effects of HGF/Met in the heart and blood vessels. In vivo, HGF/Met function is particularly important for the protection of the heart in response to both acute and chronic insults, including ischaemic injury and doxorubicin-induced cardiotoxicity. Accordingly, conditional deletion of Met in cardiomyocytes results in impaired organ defence against oxidative stress. After ischaemic injury, activation of Met provides strong anti-apoptotic stimuli for cardiomyocytes through PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) cascades. Recently, we found that HGF/Met is also important for autophagy regulation in cardiomyocytes via the mTOR (mammalian target of rapamycin) pathway. HGF/Met induces proliferation and migration of endothelial cells through Rac1 (Ras-related C3 botulinum toxin substrate 1) activation. In fibroblasts, HGF/Met antagonizes the actions of TGFβ1 (transforming growth factor β1) and AngII (angiotensin II), thus preventing fibrosis. Moreover, HGF/Met influences the inflammatory response of macrophages and the immune response of dendritic cells, indicating its protective function against atherosclerotic and autoimmune diseases. The HGF/Met axis also plays an important role in regulating self-renewal and myocardial regeneration through the enhancement of cardiac progenitor cells. HGF/Met has beneficial effects against myocardial infarction and endothelial dysfunction: the cellular and molecular mechanisms underlying repair function in the heart and blood vessels are common and include pro-angiogenic, anti-inflammatory and anti-fibrotic actions. Thus administration of HGF or HGF mimetics may represent a promising therapeutic agent for the treatment of both coronary and peripheral artery disease.

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a pla... more Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of Hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake, and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with an altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the pre-clinical level.

Hepatocyte growth factor, also known as scatter factor, is a pleiotropic cytokine, which stimulat... more Hepatocyte growth factor, also known as scatter factor, is a pleiotropic cytokine, which stimulates cell motility, invasion, proliferation, survival and morphogenesis, and induces the expression of specific genes by activating its receptor tyrosine kinase. In this work we have isolated, characterized and used as agonists two monoclonal antibodies (mAbs) directed against the extracellular domain of HGF receptor to investigate the requirements for receptor activation and for the different biological responses. The two mAbs display similar affinities, react with epitopes different from the hepatocyte growth factor binding site, and behave as either full or partial agonists. The full agonist mAb (DO-24) triggers all the biological effects elicited by hepatocyte growth factor, namely motility, proliferation, cell survival, invasion, tubulogenesis and angiogenesis. The partial agonist mAb (DN-30) induces only motility. Only the full agonist mAb is able to induce and sustain the expression of urokinase-type plasminogen activator receptor for prolonged periods of time, while both mAbs up-regulate the constitutive expression of urokinasetype plasminogen activator. Both mAbs activate receptor phosphorylation, which, being strictly dependent on mAb bivalence, requires receptor dimerization. Since simple receptor dimerization is not sufficient to trigger full biological responses, we propose that the region on the b chain of the receptor recognized by the full agonist mAb is crucial for optimal receptor activation.

Ischaemic heart disease is the main cause of death in western countries. Cardiac tissue is primar... more Ischaemic heart disease is the main cause of death in western countries. Cardiac tissue is primarily damaged by cardiomyocyte cell death triggered by low oxygen supply to the heart (hypoxia). The current therapeutic approach is coronary angioplastic intervention or thrombolytic treatments to resume blood flow in the ischaemic heart. Unfortunately, reperfusion itself causes a burst of ROS production responsible for cardiomyocyte death and myocardial dysfunction. Indeed, the majority of patients surviving to acute myocardial infarction undergoes progressive heart failure, with 50% mortality at five years from diagnosis. Apoptosis of cardiomyocytes is dangerous both during ischaemia and reperfusion. In line with this concept, we have shown that treatment of H9c2 cardiomyoblasts with cobalt chloride (CoCl2), a chemical mimetic of hypoxia, induces caspase-dependent apoptosis. Unexpectedly, we found that 3-methyladenine, an inhibitor of autophagy initiation, partially prevents CoCl2-mediated cell death, indicating that also autophagy contributes to cardiomyoblast death. Consistently, we found an increase in the autophagic flux in dying cells. Mechanistically, we have shown that CoCl2 upregulates Redd1, Bnip3 and phospho-AMPK proteins and causes inhibition of mTOR, the main negative regulator of autophagy. In light of these observations, it is important to discover new therapeutic tools displaying a dual prosurvival mechanism. To this aim, we have analyzed the cardioprotective action of HGF/Met axis in hypoxic injury. To activate Met signaling we have used either the HGF ligand or two different monoclonal antibodies (mAbs) directed against the extracellular moiety of Met receptor. Owing a divalent structure, the two mAbs can dimerize and activate Met receptor, thus displaying agonist activity. Hypoxic injury was fully prevented by either HGF or Met agonist mAbs through both anti-apoptotic and anti-autophagic functions. By pharmacological inhibition we showed that activation of mTOR is the protective signaling downstream to Met, being involved in the anti-autophagic effect. In conclusion, HGF or Met agonist mAbs promote cell survival by negative dual regulation of apoptotic and autophagic cell death and represent promising new therapeutic tools to manage cardiac diseases.

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurviva... more 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 (CoCl 2), 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 CoCl 2. 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.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents a... more Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyro-sine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hyper-trophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracel-lular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

Developmental Dynamics, 2004

The Pax3 and c-met genes are necessary for the development of tongue, diaphragm, and limb muscles... more The Pax3 and c-met genes are necessary for the development of tongue, diaphragm, and limb muscles. These hypaxial muscles derive from precursors that migrate out of the ventrolateral lip of the somites at occipital, cervical, and limb levels. In this work, we re-examined primary myogenesis in c-met signaling mutants using a skeletal muscle-specific lacZ transgene (Mlc3f-nlacZ-2E). This strategy allowed us to identify precisely the shoulder, limb, tongue, and dermal muscles that need Met for development and to confirm that the morphological structure of epaxial and body wall muscles was normal, even in the most severe c-met mutant. Surprisingly, however, X-gal staining showed that, in this mutant, hyoid arch-derived facial muscles were either reduced or absent, thus revealing that Met also contributes to the development of muscles in the head. Developmental Dynamics 231:582-591, 2004.

Molecular medicine (Cambridge, Mass.), 2012

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli, ... more 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...

Experientia, 2011

Myocardial infarction (MI) is a leading cause of hospitalization worldwide. A recently developed ... more 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

Molecular Biology of the Cell, 2007

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimu... more Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. Inhere we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

La Ricerca in clinica e in laboratorio

A decreased expression of major histocompatibility complex (MHC) class I antigens is a common fea... more A decreased expression of major histocompatibility complex (MHC) class I antigens is a common feature of many experimental and human tumors and can often be correlated with malignancy grade. In fact, reduction of class I antigens is associated in most tumors with an enhanced ability to elude immune surveillance. Loss of HLA-A,B,C antigens ranges from a decrease in the percentage of A,B,C-positive cells to selective loss of particular antigens and total loss of class I molecule expression. In man, this has been documented in melanomas, carcinomas, lymphomas, neuroblastoma and acute leukemias. The reduction in membrane antigens is generally associated with a parallel fall in immunoprecipitable intracellular proteins and the corresponding mRNAs in the absence of structural changes in the coding genes. The literature concerning the above mentioned topics is reviewed and discussed.

Biomedicines, 2014

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor (Met) play important roles in myo... more Hepatocyte growth factor (HGF) and its tyrosine kinase receptor (Met) play important roles in myocardial function both in physiological and pathological situations. In the developing heart, HGF influences cardiomyocyte proliferation and differentiation. In the adult, HGF/Met signaling controls heart homeostasis and prevents oxidative stress in normal cardiomyocytes. Thus, the possible cardiotoxicity of current Met-targeted anti-cancer therapies has to be taken in consideration. In the injured heart, HGF plays important roles in cardioprotection by promoting: (1) prosurvival (anti-apoptotic and anti-autophagic) effects in cardiomyocytes, (2) angiogenesis, (3) inhibition of fibrosis, (4) anti-inflammatory and immunomodulatory signals, and (5) regeneration through activation of cardiac stem cells. Furthermore, we discuss the putative role of elevated HGF as prognostic marker of severity in patients with cardiac diseases. Finally, we examine the potential of HGF-based molecules as new therapeutic tools for the treatment of cardiac diseases.

Liver, 2008

In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not ... more In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not lyse the hepatitis B virus deoxyribonucleic-acid-transfected human hepatoma cell line 2.2.15, but under the same experimental conditions they did lyse K562 cells. Peripheral blood mononuclear cells from 10 out of 16 patients with chronic active hepatitis B exhibited cytotoxic activity against 2.2.15 cells in the presence of a relatively reduced natural killer cell activity to the K562 cell target. Enhancement of the cytotoxic activity to 2.2.15 cells was statistically significant in the group of patients being treated with leukocyte alpha-interferon. The activity was not influenced by the degree of human leukocyte antigen type matching between effector and target, and was enhanced by depletion of T-cells and by in vitro interferon treatment. These results therefore support the concept of a natural killer-like cell activated by clinical administration of interferon in chronic active hepatitis B patients. This cell effector was lytic for the virus B negative HEP-G2 cells also. However, T-cells purified from a few patients failed to lyse the HEP-G2 while lysing the 2.2.15 target, thus indicating that a preferential recognition of the virus-infected target may be exerted by certain T-lymphocyte subsets. The use of the human leukocyte antigen type defined, highly differentiated, hepatitis B virus releasing 2.2.15 cell line as target for fresh lymphocytes in this cytolytic assay did not disclose cytolytic T-cells in an obvious way. Further manipulation of this system perhaps using T-cell clones may be the next step to exploit the investigative possibilities offered by the availability of the 2.2.15 cell target.

Transgenic research, 2013

Hepatocyte Growth Factor (HGF) controls growth and differentiation in different cell types, inclu... more 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 ne...

La Ricerca in clinica e in laboratorio

Biochemical Journal, 2012

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition... more 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.

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sar... more 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.

Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor... more Met tyrosine kinase receptor, also known as c-Met, is the HGF (hepatocyte growth factor) receptor. The HGF/Met pathway has a prominent role in cardiovascular remodelling after tissue injury. The present review provides a synopsis of the cellular and molecular mechanisms underlying the effects of HGF/Met in the heart and blood vessels. In vivo, HGF/Met function is particularly important for the protection of the heart in response to both acute and chronic insults, including ischaemic injury and doxorubicin-induced cardiotoxicity. Accordingly, conditional deletion of Met in cardiomyocytes results in impaired organ defence against oxidative stress. After ischaemic injury, activation of Met provides strong anti-apoptotic stimuli for cardiomyocytes through PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase) cascades. Recently, we found that HGF/Met is also important for autophagy regulation in cardiomyocytes via the mTOR (mammalian target of rapamycin) pathway. HGF/Met induces proliferation and migration of endothelial cells through Rac1 (Ras-related C3 botulinum toxin substrate 1) activation. In fibroblasts, HGF/Met antagonizes the actions of TGFβ1 (transforming growth factor β1) and AngII (angiotensin II), thus preventing fibrosis. Moreover, HGF/Met influences the inflammatory response of macrophages and the immune response of dendritic cells, indicating its protective function against atherosclerotic and autoimmune diseases. The HGF/Met axis also plays an important role in regulating self-renewal and myocardial regeneration through the enhancement of cardiac progenitor cells. HGF/Met has beneficial effects against myocardial infarction and endothelial dysfunction: the cellular and molecular mechanisms underlying repair function in the heart and blood vessels are common and include pro-angiogenic, anti-inflammatory and anti-fibrotic actions. Thus administration of HGF or HGF mimetics may represent a promising therapeutic agent for the treatment of both coronary and peripheral artery disease.

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a pla... more Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of Hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake, and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with an altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the pre-clinical level.

Hepatocyte growth factor, also known as scatter factor, is a pleiotropic cytokine, which stimulat... more Hepatocyte growth factor, also known as scatter factor, is a pleiotropic cytokine, which stimulates cell motility, invasion, proliferation, survival and morphogenesis, and induces the expression of specific genes by activating its receptor tyrosine kinase. In this work we have isolated, characterized and used as agonists two monoclonal antibodies (mAbs) directed against the extracellular domain of HGF receptor to investigate the requirements for receptor activation and for the different biological responses. The two mAbs display similar affinities, react with epitopes different from the hepatocyte growth factor binding site, and behave as either full or partial agonists. The full agonist mAb (DO-24) triggers all the biological effects elicited by hepatocyte growth factor, namely motility, proliferation, cell survival, invasion, tubulogenesis and angiogenesis. The partial agonist mAb (DN-30) induces only motility. Only the full agonist mAb is able to induce and sustain the expression of urokinase-type plasminogen activator receptor for prolonged periods of time, while both mAbs up-regulate the constitutive expression of urokinasetype plasminogen activator. Both mAbs activate receptor phosphorylation, which, being strictly dependent on mAb bivalence, requires receptor dimerization. Since simple receptor dimerization is not sufficient to trigger full biological responses, we propose that the region on the b chain of the receptor recognized by the full agonist mAb is crucial for optimal receptor activation.

Ischaemic heart disease is the main cause of death in western countries. Cardiac tissue is primar... more Ischaemic heart disease is the main cause of death in western countries. Cardiac tissue is primarily damaged by cardiomyocyte cell death triggered by low oxygen supply to the heart (hypoxia). The current therapeutic approach is coronary angioplastic intervention or thrombolytic treatments to resume blood flow in the ischaemic heart. Unfortunately, reperfusion itself causes a burst of ROS production responsible for cardiomyocyte death and myocardial dysfunction. Indeed, the majority of patients surviving to acute myocardial infarction undergoes progressive heart failure, with 50% mortality at five years from diagnosis. Apoptosis of cardiomyocytes is dangerous both during ischaemia and reperfusion. In line with this concept, we have shown that treatment of H9c2 cardiomyoblasts with cobalt chloride (CoCl2), a chemical mimetic of hypoxia, induces caspase-dependent apoptosis. Unexpectedly, we found that 3-methyladenine, an inhibitor of autophagy initiation, partially prevents CoCl2-mediated cell death, indicating that also autophagy contributes to cardiomyoblast death. Consistently, we found an increase in the autophagic flux in dying cells. Mechanistically, we have shown that CoCl2 upregulates Redd1, Bnip3 and phospho-AMPK proteins and causes inhibition of mTOR, the main negative regulator of autophagy. In light of these observations, it is important to discover new therapeutic tools displaying a dual prosurvival mechanism. To this aim, we have analyzed the cardioprotective action of HGF/Met axis in hypoxic injury. To activate Met signaling we have used either the HGF ligand or two different monoclonal antibodies (mAbs) directed against the extracellular moiety of Met receptor. Owing a divalent structure, the two mAbs can dimerize and activate Met receptor, thus displaying agonist activity. Hypoxic injury was fully prevented by either HGF or Met agonist mAbs through both anti-apoptotic and anti-autophagic functions. By pharmacological inhibition we showed that activation of mTOR is the protective signaling downstream to Met, being involved in the anti-autophagic effect. In conclusion, HGF or Met agonist mAbs promote cell survival by negative dual regulation of apoptotic and autophagic cell death and represent promising new therapeutic tools to manage cardiac diseases.

Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurviva... more 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 (CoCl 2), 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 CoCl 2. 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.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents a... more Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyro-sine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hyper-trophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracel-lular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

Developmental Dynamics, 2004

The Pax3 and c-met genes are necessary for the development of tongue, diaphragm, and limb muscles... more The Pax3 and c-met genes are necessary for the development of tongue, diaphragm, and limb muscles. These hypaxial muscles derive from precursors that migrate out of the ventrolateral lip of the somites at occipital, cervical, and limb levels. In this work, we re-examined primary myogenesis in c-met signaling mutants using a skeletal muscle-specific lacZ transgene (Mlc3f-nlacZ-2E). This strategy allowed us to identify precisely the shoulder, limb, tongue, and dermal muscles that need Met for development and to confirm that the morphological structure of epaxial and body wall muscles was normal, even in the most severe c-met mutant. Surprisingly, however, X-gal staining showed that, in this mutant, hyoid arch-derived facial muscles were either reduced or absent, thus revealing that Met also contributes to the development of muscles in the head. Developmental Dynamics 231:582-591, 2004.

Molecular medicine (Cambridge, Mass.), 2012

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli, ... more 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...

Experientia, 2011

Myocardial infarction (MI) is a leading cause of hospitalization worldwide. A recently developed ... more 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

Molecular Biology of the Cell, 2007

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimu... more Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. Inhere we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

La Ricerca in clinica e in laboratorio

A decreased expression of major histocompatibility complex (MHC) class I antigens is a common fea... more A decreased expression of major histocompatibility complex (MHC) class I antigens is a common feature of many experimental and human tumors and can often be correlated with malignancy grade. In fact, reduction of class I antigens is associated in most tumors with an enhanced ability to elude immune surveillance. Loss of HLA-A,B,C antigens ranges from a decrease in the percentage of A,B,C-positive cells to selective loss of particular antigens and total loss of class I molecule expression. In man, this has been documented in melanomas, carcinomas, lymphomas, neuroblastoma and acute leukemias. The reduction in membrane antigens is generally associated with a parallel fall in immunoprecipitable intracellular proteins and the corresponding mRNAs in the absence of structural changes in the coding genes. The literature concerning the above mentioned topics is reviewed and discussed.

Biomedicines, 2014

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor (Met) play important roles in myo... more Hepatocyte growth factor (HGF) and its tyrosine kinase receptor (Met) play important roles in myocardial function both in physiological and pathological situations. In the developing heart, HGF influences cardiomyocyte proliferation and differentiation. In the adult, HGF/Met signaling controls heart homeostasis and prevents oxidative stress in normal cardiomyocytes. Thus, the possible cardiotoxicity of current Met-targeted anti-cancer therapies has to be taken in consideration. In the injured heart, HGF plays important roles in cardioprotection by promoting: (1) prosurvival (anti-apoptotic and anti-autophagic) effects in cardiomyocytes, (2) angiogenesis, (3) inhibition of fibrosis, (4) anti-inflammatory and immunomodulatory signals, and (5) regeneration through activation of cardiac stem cells. Furthermore, we discuss the putative role of elevated HGF as prognostic marker of severity in patients with cardiac diseases. Finally, we examine the potential of HGF-based molecules as new therapeutic tools for the treatment of cardiac diseases.

Liver, 2008

In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not ... more In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not lyse the hepatitis B virus deoxyribonucleic-acid-transfected human hepatoma cell line 2.2.15, but under the same experimental conditions they did lyse K562 cells. Peripheral blood mononuclear cells from 10 out of 16 patients with chronic active hepatitis B exhibited cytotoxic activity against 2.2.15 cells in the presence of a relatively reduced natural killer cell activity to the K562 cell target. Enhancement of the cytotoxic activity to 2.2.15 cells was statistically significant in the group of patients being treated with leukocyte alpha-interferon. The activity was not influenced by the degree of human leukocyte antigen type matching between effector and target, and was enhanced by depletion of T-cells and by in vitro interferon treatment. These results therefore support the concept of a natural killer-like cell activated by clinical administration of interferon in chronic active hepatitis B patients. This cell effector was lytic for the virus B negative HEP-G2 cells also. However, T-cells purified from a few patients failed to lyse the HEP-G2 while lysing the 2.2.15 target, thus indicating that a preferential recognition of the virus-infected target may be exerted by certain T-lymphocyte subsets. The use of the human leukocyte antigen type defined, highly differentiated, hepatitis B virus releasing 2.2.15 cell line as target for fresh lymphocytes in this cytolytic assay did not disclose cytolytic T-cells in an obvious way. Further manipulation of this system perhaps using T-cell clones may be the next step to exploit the investigative possibilities offered by the availability of the 2.2.15 cell target.

Transgenic research, 2013

Hepatocyte Growth Factor (HGF) controls growth and differentiation in different cell types, inclu... more 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 ne...