Giulio Pompilio

MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand ephrin-A3

MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.

Circulating microRNAs are new and sensitive biomarkers of myocardial infarction

Aims Circulating microRNAs (miRNAs) may represent a novel class of biomarkers; therefore, we examined whether acute myocardial infarction (MI) modulates miRNAs plasma levels in humans and mice. Methods and results Healthy donors (n = 17) and patients (n = 33) with acute ST-segment elevation MI (STEMI) were evaluated. In one cohort (n = 25), the first plasma sample was obtained 517 ± 309 min after the onset of MI symptoms and after coronary reperfusion with percutaneous coronary intervention (PCI); miR-1, -133a, -133b, and -499-5p were ∼15- to 140-fold control, whereas miR-122 and -375 were ∼87–90% lower than control; 5 days later, miR-1, -133a, -133b, -499-5p, and -375 were back to baseline, whereas miR-122 remained lower than control through Day 30. In additional patients (n = 8; four treated with thrombolysis and four with PCI), miRNAs and troponin I (TnI) were quantified simultaneously starting 156 ± 72 min after the onset of symptoms and at different times thereafter. Peak miR-1, -133a, and -133b expression and TnI level occurred at a similar time, whereas miR-499-5p exhibited a slower time course. In mice, miRNAs plasma levels and TnI were measured 15 min after coronary ligation and at different times thereafter. The behaviour of miR-1, -133a, -133b, and -499-5p was similar to STEMI patients; further, reciprocal changes in the expression levels of these miRNAs were found in cardiac tissue 3–6 h after coronary ligation. In contrast, miR-122 and -375 exhibited minor changes and no significant modulation. In mice with acute hind-limb ischaemia, there was no increase in the plasma level of the above miRNAs. Conclusion Acute MI up-regulated miR-1, -133a, -133b, and -499-5p plasma levels, both in humans and mice, whereas miR-122 and -375 were lower than control only in STEMI patients. These miRNAs represent novel biomarkers of cardiac damage.

Exogenous High-Mobility Group Box 1 Protein Induces Myocardial Regeneration After Infarction via Enhanced Cardiac C-Kit+ Cell Proliferation and Differentiation

High-mobility group box 1 protein (HMGB1) is a chromatin protein that is released by inflammatory and necrotic cells. Extracellular HMGB1 signals tissue damage, stimulates the secretion of proinflammatory cytokines and chemokines, and modulates stem cell function. The present study examined exogenous HMGB1 effect on mouse left-ventricular function and myocyte regeneration after infarction. Myocardial infarction was induced in C57BL/6 mice by permanent coronary artery ligation. After 4 hours animals were reoperated and 200 ng of purified HMGB1 was administered in the peri-infarcted left ventricle. This intervention resulted in the formation of new myocytes within the infarcted portion of the wall. The regenerative process involved the proliferation and differentiation of endogenous cardiac c-kit+ progenitor cells. Circulating c-kit+ cells did not significantly contribute to HMGB1-mediated cardiac regeneration. Echocardiographic and hemodynamic parameters at 1, 2, and 4 weeks demonstrated a significant recovery of cardiac performance in HMGB1-treated mice. These effects were not observed in infarcted hearts treated either with the unrelated protein glutathione S-transferase or a truncated form of HMGB1. Thus, HMGB1 appears to be a potent inducer of myocardial regeneration following myocardial infarction.

Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia

This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of fibroblast growth factor 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total 16 mg: 4 × 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of fibroblast growth factor 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total 16 mg: 4 x 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.

Identification of Myocardial and Vascular Precursor Cells in Human and Mouse Epicardium

During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit+ and CD34+ cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit+ and CD34+ cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit+ cells 3 days after coronary ligation; at this time point, epicardial c-kit+ cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit+, together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

Myoendothelial Differentiation of Human Umbilical Cord Blood–Derived Stem Cells in Ischemic Limb Tissues

Abstract— Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34− cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.

Diagnostic potential of circulating miR-499-5p in elderly patients with acute non ST-elevation myocardial infarction

BACKGROUND Geriatric patients with acute non-ST elevation myocardial infarction (NSTEMI) can frequently present atypical symptoms and non-diagnostic electrocardiogram. The detection of modest cardiac troponin T (cTnT) elevation is challenging for physicians needing to routinely triage these patients. Unfortunately, non-coronary diseases, such as acute heart failure (CHF), may cause cTnT elevation. Circulating microRNAs (miRs) have emerged as biomarkers of MI. However, their diagnostic potential needs to be determined in elderly NSTEMI patients. METHODS 92 NSTEMI patients (82.6 ± 6.9 years old; complicated by CHF in 74% of cases) and 81 patients with acute CHF without AMI (81.3 ± 6.8 years old) were enrolled at presentation. A third group comprised 99 age-matched healthy control subjects (CTR). Plasma levels of miR-1, -21, -133a, -208a, -423-5p and -499-5p were analyzed. RESULTS MiR-1, -21 -133a and -423-5p showed a 3- to 10-fold increase and miR-499-5p exhibited >80-fold increase in acute NSTEMI patient vs. CTR. MiR-499-5p and -21 showed a significantly increased expression in NSTEMI vs. CHF. Interestingly, mir-499-5p was comparable to cTnT in discriminating NSTEMI vs. CTR and CHF patients. Its diagnostic accuracy was higher than conventional and hs-cTnT in differentiating NSTEMI (n=31) vs. acute CHF (n=32) patients with modest cTnT elevation at presentation (miR-499-5p AUC=0.86 vs. cTnT AUC=0.68 and vs. hs-cTnT AUC=0.70). CONCLUSIONS Circulating miR-499-5p is a sensitive biomarker of acute NSTEMI in the elderly, exhibiting a diagnostic accuracy superior to that of cTnT in patients with modest elevation at presentation.

Cyclophilin A: a key player for human disease.

Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis-trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies.

HMGB1 Attenuates Cardiac Remodelling in the Failing Heart via Enhanced Cardiac Regeneration and miR-206-Mediated Inhibition of TIMP-3

Aims HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts. Methods and Results Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit+ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20–25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206. Conclusions HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.

Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin

AIMS Bone marrow mesenchymal stromal cell (BMStC) transplantation into the infarcted heart improves left ventricular function and cardiac remodelling. However, it has been suggested that tissue-specific cells may be better for cardiac repair than cells from other sources. The objective of the present work has been the comparison of in vitro and in vivo properties of adult human cardiac stromal cells (CStC) to those of syngeneic BMStC. METHODS AND RESULTS Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC. CONCLUSION Our findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.