Patrizia Dentelli

microRNA-222 Controls Neovascularization by Regulating Signal Transducer and Activator of Transcription 5A Expression

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. Conclusion—We identified STAT5A as a novel miR-222 target, and this finding opens up new perspectives for treatment of vascular diseases.

p53 Mediates the Accelerated Onset of Senescence of Endothelial Progenitor Cells in Diabetes

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.

Thrombopoietin Stimulates Endothelial Cell Motility and Neoangiogenesis by a Platelet-Activating Factor–Dependent Mechanism

In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.

UNACYLATED GHRELIN RESCUES ENDOTHELIAL PROGENITOR CELL FUNCTION IN INDIVIDUALS WITH TYPE 2 DIABETES

OBJECTIVE Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects. RESEARCH DESIGN AND METHODS Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47phox and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model. RESULTS Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG. CONCLUSIONS These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.

Signal transducers and activators of transcription 3 signaling pathway. An Essential Mediator of Inflammatory Bowel Disease and Other Forms of Intestinal Inflammation

&NA; Crohns disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are characterized by mucosal immune cell activation that is driven by a cytokine imbalance. Several cytokines involved in IBD act through the activation of the signal transducers and activators of transcription (STAT) family. We investigated the activation of STAT3 in the mucosa of CD and UC patients, and evaluated whether this event is specific for IBD patients. Using immunofluorescence and immunoblotting, total and phosphorylated STAT3 levels were assessed in biopsy specimens, isolated lamina propria mononuclear cells, and peripheral blood mononuclear cells from patients with CD, UC, other forms of intestinal inflammation, and control subjects. Immunoblotting revealed phosphorylated STAT3 in mucosal biopsy specimens from patients with CD, UC, celiac disease, and acute self‐limited colitis, but not in the normal mucosa of control subjects. In IBD patients, STAT3 activation was confined to actively inflamed areas. Accordingly, activated STAT3 was detected in isolated lamina propria mononuclear cells from inflamed IBD tissues, but not in peripheral blood mononuclear cells from control subjects or IBD patients. Immunofluorescence demonstrated that the sources of activated STAT3 were macrophages and T lymphocytes, but not neutrophils. STAT3 activation also was detected in T cells infiltrating the duodenal mucosa of celiac disease patients. We conclude that STAT3 signaling occurs in both CD and UC, where it is strictly confined to areas of active inflammation and is limited to infiltrating macrophages and T cells. The occurrence of STAT3 signaling in other acute and chronic intestinal inflammatory conditions suggests that, rather than a specific feature of IBD, it represents a fundamental signaling pathway that is shared by multiple forms of gut inflammation.

MIR221/MIR222-driven post-transcriptional regulation of P27KIP1 and P57KIP2 is crucial for high-glucose- and AGE-mediated vascular cell damage

Aims/hypothesisMicroRNAs (miRNAs) are a novel group of small non-coding RNAs that regulate gene expression at the post-transcriptional level and act on their target mRNAs in a tissue- and cell-type-specific manner. Herein, the relevance of MIR221/MIR222 in high-glucose- and AGE-mediated vascular damage was investigated.MethodsFunctional studies were performed using human mature endothelial cells and endothelial progenitor cells subjected to high glucose or AGE. Quantitative real-time amplification was performed to analyse MIR221/MIR222 expression in these experimental conditions. Luciferase assay was used to identify MIR221/MIR222 targets. Functional studies were performed in vitro and in vivo in mice using gain- and loss-of-function approaches.ResultsUsing an in vivo mouse model we demonstrated that exposure to AGE and high glucose impaired vessel formation. Moreover, in vitro functional studies revealed that both high glucose and AGE inhibit cell-cycle progression by modulating the expression of P27KIP1 (also known as CDKN1B) and P57KIP2 (also known as CDKN1C), which encode cyclin-dependent kinase inhibitor 1B (p27, Kip1) (P27KIP1) and cyclin-dependent kinase inhibitor 1C (p57, Kip2) (P57KIP2), respectively. Crucial to AGE- and high-glucose-mediated cell-cycle arrest was the downregulation of MIR221/MIR222 expression. Luciferase assay showed that MIR221 and MIR222 specifically bind to the P27KIP1 and P57KIP2 mRNA 3′-untranslated regions, implicating P27KIP1 and P57KIP2 as MIR221/MIR222 targets. These results were confirmed by gain-of-function experiments in vitro, and by injecting mice with endothelial cells overexpressing MIR221 and MIR222.Conclusions/interpretationWe provide evidence that high-glucose- and AGE-induced inhibition of vascular cell proliferation is controlled by MIR221/MIR222-driven post-transcriptional regulation of P27KIP1 and P57KIP2. These data add further insight to the possible contribution of miRNAs in vascular damage mediated by a high-glucose environment.

Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD-2–Mediated miR-221/222 Expression

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 p57Kip2 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 p57Kip2 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.

Interleukin-3 Stimulates Migration and Proliferation of Vascular Smooth Muscle Cells A Potential Role in Atherogenesis

Background —Cytokines released by activated T lymphocytes are key regulators of chronic inflammatory response, including atherosclerosis. The aim of this study was to investigate the presence of interleukin-3 (IL-3) in lymphocytes infiltrating the atherosclerotic plaque and the effect of this cytokine on primary vascular smooth muscle cells (SMCs). Methods and Results —Twenty atherosclerotic carotid arterial specimens and 5 early atherosclerotic lesions from the internal carotid were manually minced to fragments, and T lymphocytes infiltrating the atherosclerotic plaque were isolated on solid-phase anti-CD3 polystyrene plates. Southern blot analysis demonstrated that in all samples, lymphocytes expressed IL-3 and IL-2 receptor &agr;-chain transcripts, indicating that in this context, the activated T lymphocytes may release IL-3. We further analyzed the expression of the IL-3 receptor and the biological effects exerted by the ligand on vascular SMCs. &bgr;-IL-3–transducing subunit was detected both on cultured SMCs and on endothelial cells and SMCs within atheroma. The analysis of the IL-3–induced biological effects demonstrated that it was able to trigger both mitogenic and motogenic signals. Moreover, we demonstrated that the addition of PD98059, a known inhibitor of the MAP–extracellular signaling-regulated/MAP kinase pathway, completely inhibited IL-3–mediated MAP kinase activation and IL-3–induced migration and proliferation. Finally, IL-3 was found to stimulate vascular endothelial growth factor (VEGF) gene transcription. Conclusions —IL-3, expressed by activated T lymphocytes infiltrating early and advanced atherosclerotic plaques, may sustain the atherosclerotic process either directly, by activating SMC migration and proliferation, or indirectly, via VEGF production.

Unacylated Ghrelin Induces Oxidative Stress Resistance in a Glucose Intolerance and Peripheral Artery Disease Mouse Model by Restoring Endothelial Cell miR-126 Expression

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia–subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation.

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.