Leonardo Elia

The knockout of miR-143 and -145 alters smooth muscle cell maintenance and vascular homeostasis in mice: Correlates with human disease

Mechanisms controlling vascular smooth muscle cell (VSMC) plasticity and renewal still remain to be elucidated completely. A class of small RNAs called microRNAs (miRs) regulate gene expression at the post-transcriptional level. Here, we show a critical role of the miR-143/145 cluster in SMC differentiation and vascular pathogenesis, also through the generation of a mouse model of miR-143 and -145 knockout (KO). We determined that the expression of miR-143 and -145 is decreased in acute and chronic vascular stress (transverse aortic constriction and in aortas of the ApoE KO mouse). In human aortic aneurysms, the expression of miR-143 and -145 was significantly decreased compared with control aortas. In addition, overexpression of miR-143 and -145 decreased neointimal formation in a rat model of acute vascular injury. An in-depth analysis of the miR-143/145 KO mouse model showed that this miR cluster is expressed mostly in the SMC compartment, both during development and postnatally, in vessels and SMC-containing organs. Loss of miR-143 and miR-145 expression induces structural modifications of the aorta, because of an incomplete differentiation of VSMCs. In conclusion, our results show that the miR-143/145 gene cluster has a critical role during SMC differentiation and strongly suggest its involvement in the reversion of the VSMC differentiation phenotype that occurs during vascular disease.

Reciprocal Regulation of MicroRNA-1 and Insulin-Like Growth Factor-1 Signal Transduction Cascade in Cardiac and Skeletal Muscle in Physiological and Pathological Conditions

Background— MicroRNAs (miRNAs/miRs) are small conserved RNA molecules of 22 nucleotides that negatively modulate gene expression primarily through base paring to the 3′ untranslated region of target messenger RNAs. The muscle-specific miR-1 has been implicated in cardiac hypertrophy, heart development, cardiac stem cell differentiation, and arrhythmias through targeting of regulatory proteins. In this study, we investigated the molecular mechanisms through which miR-1 intervenes in regulation of muscle cell growth and differentiation. Methods and Results— On the basis of bioinformatics tools, biochemical assays, and in vivo models, we demonstrate that (1) insulin-like growth factor-1 (IGF-1) and IGF-1 receptor are targets of miR-1; (2) miR-1 and IGF-1 protein levels are correlated inversely in models of cardiac hypertrophy and failure as well as in the C2C12 skeletal muscle cell model of differentiation; (3) the activation state of the IGF-1 signal transduction cascade reciprocally regulates miR-1 expression through the Foxo3a transcription factor; and (4) miR-1 expression correlates inversely with cardiac mass and thickness in myocardial biopsies of acromegalic patients, in which IGF-1 is overproduced after aberrant synthesis of growth hormone. Conclusions— Our results reveal a critical role of miR-1 in mediating the effects of the IGF-1 pathway and demonstrate a feedback loop between miR-1 expression and the IGF-1 signal transduction cascade.

MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

PDGF enhances podosome formation and cell migration by regulating expression of the microRNAs miR-143 and -145, which target PDGF-R, PKC-ε, and fascin.

A Cell-Based High-Content Screening Assay Reveals Activators and Inhibitors of Cancer Cell Invasion

Cyclin-dependent kinase 5 is identified as an anticancer target because its activity promotes invasiveness of cancer cells. Finding Targets for Inhibiting Cancer Cell Metastasis Cancer cells escape from the primary tumor site in part because of invadopodia, actin-rich, foot-like cellular processes that degrade the surrounding extracellular matrix (ECM). Invasiveness of cancer cells is often correlated to the presence of invadopodia. Quintavalle et al. developed a high-throughput method of screening the effects of compounds on the formation of invadopodia and ability to degrade the ECM. They found that the chemotherapeutic agent paclitaxel increased the formation of invadopodia and ability to degrade the ECM, suggesting that its use might promote invasiveness, especially if used before the main therapy or in patients with tumors resistant to the cytotoxicity of this drug. In contrast, invasive behavior was inhibited by a group of compounds that inhibited the cyclin-dependent kinase Cdk5, which was found to phosphorylate the actin regulatory protein caldesmon, thereby triggering its degradation and leading to the formation of invadopodia. Thus, these results identify potential undesirable cellular effects of a chemotherapeutic agent and a possible therapeutic target for restricting invasiveness in cancer cells. Acquisition of invasive cell behavior underlies tumor progression and metastasis. To further define the molecular mechanisms underlying invasive behavior, we developed a high-throughput screening strategy to quantitate invadopodia, which are actin-rich membrane protrusions of cancer cells that contribute to tissue invasion and matrix remodeling. We tested the LOPAC 1280 collection of pharmacologically active agents in a high-content, image-based assay and identified compounds that inhibited invadopodium formation without overt toxicity, as well as compounds that increased invadopodia number. The chemotherapeutic agent paclitaxel increased both the number of invadopodia and the invasive behavior of various human cancer cell lines, effects that have potential clinical implications for its use before surgical removal of a primary tumor (neoadjuvant therapy) or in patients with chemoresistant tumors. Several compounds that inhibited invasion have been characterized as cyclin-dependent kinase (Cdk) inhibitors, and loss-of-function experiments determined that Cdk5 was the relevant target. We further determined that Cdk5 promoted both invadopodium formation and cancer cell invasion by phosphorylating and thus decreasing the abundance of the actin regulatory protein caldesmon.

TGFβ Triggers miR-143/145 Transfer From Smooth Muscle Cells to Endothelial Cells, Thereby Modulating Vessel Stabilization

RATIONALE The miR-143/145 cluster is highly expressed in smooth muscle cells (SMCs), where it regulates phenotypic switch and vascular homeostasis. Whether it plays a role in neighboring endothelial cells (ECs) is still unknown. OBJECTIVE To determine whether SMCs control EC functions through passage of miR-143 and miR-145. METHODS AND RESULTS We used cocultures of SMCs and ECs under different conditions, as well as intact vessels to assess the transfer of miR-143 and miR-145 from one cell type to another. Imaging of cocultured cells transduced with fluorescent miRNAs suggested that miRNA transfer involves membrane protrusions known as tunneling nanotubes. Furthermore, we show that miRNA passage is modulated by the transforming growth factor (TGF) β pathway because both a specific transforming growth factor-β (TGFβ) inhibitor (SB431542) and an shRNA against TGFβRII suppressed the passage of miR-143/145 from SMCs to ECs. Moreover, miR-143 and miR-145 modulated angiogenesis by reducing the proliferation index of ECs and their capacity to form vessel-like structures when cultured on matrigel. We also identified hexokinase II (HKII) and integrin β 8 (ITGβ8)-2 genes essential for the angiogenic potential of ECs-as targets of miR-143 and miR-145, respectively. The inhibition of these genes modulated EC phenotype, similarly to miR-143 and miR-145 overexpression in ECs. These findings were confirmed by ex vivo and in vivo approaches, in which it was shown that TGFβ and vessel stress, respectively, triggered miR-143/145 transfer from SMCs to ECs. CONCLUSIONS Our results demonstrate that miR-143 and miR-145 act as communication molecules between SMCs and ECs to modulate the angiogenic and vessel stabilization properties of ECs.

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction CascadeNovelty and Significance

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate &bgr;-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of &bgr;-adrenergic receptors leads to impaired cardiac function, and &bgr;-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. Objective: To determine whether miR-133 affects &bgr;-adrenergic receptor signaling during progression to heart failure. Methods and Results: Based on bioinformatic analysis, &bgr;1-adrenergic receptor (&bgr;1AR) and other components of the &bgr;1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective &bgr;1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic &bgr;1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. Conclusions: miR-133 controls multiple components of the &bgr;1AR transduction cascade and is cardioprotective during heart failure.

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction Cascade

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate &bgr;-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of &bgr;-adrenergic receptors leads to impaired cardiac function, and &bgr;-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. Objective: To determine whether miR-133 affects &bgr;-adrenergic receptor signaling during progression to heart failure. Methods and Results: Based on bioinformatic analysis, &bgr;1-adrenergic receptor (&bgr;1AR) and other components of the &bgr;1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective &bgr;1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic &bgr;1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. Conclusions: miR-133 controls multiple components of the &bgr;1AR transduction cascade and is cardioprotective during heart failure.

Heart failure: Targeting transcriptional and post-transcriptional control mechanisms of hypertrophy for treatment

Heart failure (HF) is a syndrome caused by diminished heart function that arises from pathologies like hypertension, infarction, and diabetes. Neurohormonal, cardiorenal and cardiocirculatory models have been developed to explain HF but they have not provided sufficient understanding for the elaboration of therapies to conquer the syndrome. In fact, even though progress has been made in improving survival, HF remains a frequent cause of hospitalization and death. Since in most forms of HF, development of the disorder is associated with an alteration of cardiomyocyte structure, perceived as an increase in heart mass due to cell hypertrophy, effort is being directed to address hypertrophy as a therapeutic target. Here, we outline recent understanding of two gene-silencing regulatory mechanisms underlying cardiomyocyte hypertrophy, i.e., transcriptional control by HDACs, and post-transcriptional control by microRNAs.

MicroRNA 143-145 deficiency impairs vascular function.

MicroRNAs are required for vascular smooth muscle growth, differentiation and function. MiR143-145 modulates cytoskeletal dynamics and acquisition of the contractile phenotype by smooth muscle cells. Lack of this miRNA cluster results in decreased blood pressure and reduced vasocontraction. As all these observations point to a key role for miR143-145 in the vasculature, we investigated whether miR143-14S deficiency is associated with impaired vascular tone. Vasocontraction was assessed in isolated aortic rings from miR143-145 KO and wild type animals incubated with increasing concentrations of phenylephrine (10−9M to 10−5M) or KCl 0.3M. In both cases, aortic vessel contraction was dramatically reduced in miR143-145 KO animals compared to controls. Next, aortic rings were pre-contracted with phenylephrine (EC60: 10−7M) and concentration responses for acetylcholine were obtained. A significantly reduced vasodilation was observed in miR143-145 KO animals compared to controls and similar results were obtained when an exogenous donor of nitric oxide (sodium nitroprusside) was used. Endothelial nitric oxide synthase or guanylate cyclase mRNA expression were not different between the animal groups thus suggesting to investigate the effect of other vasodilators. Isoprenaline mediated vasodilation was significantly reduced in miR143-145 KO animals compared to controls in the absence or in the presence of the guanylate cyclase inhibitor ODQ (10−4M), suggesting that also beta adrenergic vasodilation is impaired following miR143-145 deficiency. Finally, the effect of a stable mimetic prostacyclin, namely iloprost, was investigated and again a reduced vasodilation was observed in miR143-145 KO animals. MiR143-145 deficiency is associated not only with altered vasocontraction but also with impaired vasodilation, which probably reflects the impaired VSMC differentiation phenotype reported in miR143-145 KO animals.

T cell costimulation blockade blunts pressure overload-induced heart failure

Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF.