Arturo Rosso

OBJECTIVE: Inflammatory stimuli released into atherosclerotic plaque microenvironment regulate vessel formation by modulating gene expression and translation. microRNAs are a class of short noncoding RNAs, acting as posttranscriptional regulators of protein-coding genes involved in various biological processes, including vascular cell biology. Among them, microRNA-221/222 (miR-221/222) seem to negatively modulate vascular remodeling by targeting different target genes. Here, we investigated their potential contribution to inflammation-mediated neovessel formation. METHODS AND RESULTS: We used quantitative real-time RT-PCR amplification to analyze expression of 7 microRNAs previously linked to vascular biology, such as miR-17-5p, miR-21, miR-126, miR-210, miR-221, miR-222, and miR-296 and found high levels of expression for all of them in quiescent endothelial cells. However, miR-126, miR-221, miR-222, and miR-296 turned out to be down-modulated in endothelial cells exposed to inflammatory stimuli. Applying a gain-of-function approach, we demonstrated that, among them, only miR-222 was involved in inflammation-mediated vascular remodeling. In addition, we identified signal transducer and activator of transcription 5A (STAT5A) as a bona fide target of miR-222 and observed that miR-222 negatively correlated with STAT5A expression in human endothelial cells from advanced neovascularized atherosclerotic lesions. CONCLUSIONS: We identified STAT5A as a novel miR-222 target, and this finding opens up new perspectives for treatment of vascular diseases.

Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor β common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor β common subunit with JAK2 is ligand-dependent.Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN. Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor β common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor β common subunit with JAK2 is ligand-dependent. Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN.

Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1alpha, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1alpha, possibly in the form of homodimers, binds to the sis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1alpha heterodimers, bind to the prolactin-inducible element of the beta-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1alpha activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins.

BACKGROUND: Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated-ghrelin (UnAG) to reduce ischemia-induced tissue damage in a mouse model of peripheral artery disease. METHODS AND RESULTS: We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen-actived protein kinase signaling UnAG protected against reactive oxygen species-induced cell injuries by inducing the expression of superoxide dismutase-2 (SOD-2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR-221/222, previously linked to muscle regeneration processes, was up-regulated and negatively correlated with p57(Kip2) expression in UnAG-treated mice. UnAG, unlike AG, promoted cell-cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG-induced p38/mitogen-actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD-2-depleted SCs. By siRNA technology, we also demonstrated that SOD-2 is the antioxidant enzyme involved in the control of miR-221/222-driven posttranscriptional p57(Kip2) regulation. Loss-of-function experiments targeting miR-221/222 and local pre-miR-221/222 injection in vivo confirmed a role for miR-221/222 in driving skeletal muscle regeneration after ischemia. CONCLUSIONS: These results indicate that UnAG-induced skeletal muscle regeneration after ischemia depends on SOD-2-induced miR-221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species-mediated skeletal muscle damage.