Adenosine stimulates the migration of human endothelial progenitor cells. Role of CXCR4 and microRNA-150.

BACKGROUND: Administration of endothelial progenitor cells (EPC) represents a promising option to regenerate the heart after myocardial infarction, but is limited because of low recruitment and engraftment in the myocardium. Mobilization and migration of EPC are mainly controlled by stromal cell-derived factor 1α (SDF-1α) and its receptor CXCR4. We hypothesized that adenosine, a cardioprotective molecule, may improve the recruitment of EPC to the heart. METHODS: EPC were obtained from peripheral blood mononuclear cells of healthy volunteers. Expression of chemokines and their receptors was evaluated using microarrays, quantitative PCR, and flow cytometry. A Boyden chamber assay was used to assess chemotaxis. Recruitment of EPC to the infarcted heart was evaluated in rats after permanent occlusion of the left anterior descending coronary artery. RESULTS: Microarray analysis revealed that adenosine modulates the expression of several members of the chemokine family in EPC. Among these, CXCR4 was up-regulated by adenosine, and this result was confirmed by quantitative PCR (3-fold increase, P<0.001). CXCR4 expression at the cell surface was also increased. This effect involved the A(2B) receptor. Pretreatment of EPC with adenosine amplified their migration towards recombinant SDF-1α or conditioned medium from cardiac fibroblasts. Both effects were abolished by CXCR4 blocking antibodies. Adenosine also increased CXCR4 under ischemic conditions, and decreased miR-150 expression. Binding of miR-150 to the 3' untranslated region of CXCR4 was verified by luciferase assay. Addition of pre-miR-150 blunted the effect of adenosine on CXCR4. Administration of adenosine to rats after induction of myocardial infarction stimulated EPC recruitment to the heart and enhanced angiogenesis. CONCLUSION: Adenosine increases the migration of EPC. The mechanism involves A(2B) receptor activation, decreased expression of miR-150 and increased expression of CXCR4. These results suggest that adenosine may be used to enhance the capacity of EPC to revascularize the ischemic heart.

MicroRNA-16 affects key functions of human endothelial progenitor cells.

The capacity of EPCs to repair injured tissues is limited. The role of miRNAs in EPCs is largely unknown. We tested whether miRNAs may be useful to enhance the regenerative capacity of EPCs. Early EPCs were isolated from human PBMCs, and late EPCs were amplified from enriched human peripheral CD34(+) cells. Expression profiles of miRNAs and mRNAs were obtained by microarrays. Among the miRNAs differentially expressed between early and late EPCs, five members of the miR-16 family (miR-15a/-15b/-16/-103/-107) were overexpressed in early EPCs. Web-accessible databases predicted 375 gene targets for these five miRNAs. Among these, two regulators of cell cycle progression (CCND1 and CCNE1) and one associated gene (CDK6) were less expressed in early EPCs. Administration of anti-miR-16 in early EPCs enhanced the expression of these three genes, and administration of pre-miR-16 in late EPCs decreased their expression. In early EPCs, antagonism of miR-16 allowed for cell-cycle re-entry, stimulated differentiation, enhanced IL-8 secretion, and promoted the formation of capillary-like structures by HUVECs. In conclusion, miR-16 regulates key biological pathways in EPCs. This may have important implications to enhance the capacity of EPCs to repair injured tissues.

Monocyte chemotactic protein 3 is a homing factor for circulating angiogenic cells.

AIMS: Circulating angiogenic cells (CAC) participate in cardiac repair. CAC recruitment to the ischaemic heart is mainly induced by the chemokine (C-X-C motif) receptor 4 (CXCR4)/stromal-cell derived factor-1α axis. However, CAC mobilization is only partly prevented by CXCR4 blockade, indicating that other mechanisms are involved. Since the expression of monocyte chemotactic protein 3 (MCP3) is increased in ischaemic hearts, we hypothesized that it may participate in CAC mobilization. METHODS AND RESULTS: CAC were obtained from peripheral blood mononuclear cells of healthy volunteers. In vitro migration of CAC was concentration-dependently increased by recombinant MCP3 (one-fold increase, P= 0.001), and this effect was inhibited by antibodies neutralizing the chemokine (C-C motif) receptor 1 (CCR1). CCR1 expression at the surface of CAC was confirmed by flow cytometry. Conditioned medium from heparan sulfate-activated macrophages, which contained MCP3, induced the migration of CAC (one-fold increase, P= 0.01). This increase was partly inhibited by CCR1 antibodies. The migration of CAC was also stimulated by macrophage inflammatory protein 3ß. This effect was blocked by CCR7 antibodies and was of lower magnitude than that of MCP3. MCP3 induced the formation of blood vessels in Matrigel plugs implanted in mice (1.5-fold increase, P< 0.001). This effect was abrogated by anti-CCR1 antibodies. CONCLUSION: This study shows that MCP3 stimulates the migration of CAC and angiogenesis, suggesting that MCP3 may be useful to improve cardiac repair.

Adenosine reduces cell surface expression of toll-like receptor 4 and inflammation in response to lipopolysaccharide and matrix products.

Recent evidence suggests that Toll-like receptor 4 (TLR4) is not only involved in innate immunity but is also an important mediator of adverse left ventricular remodeling and heart failure following acute myocardial infarction (MI). TLR4 is activated by lipopolysaccharide (LPS) but also by products of matrix degradation such as hyaluronic acid and heparan sulfate. Although cardioprotective properties of adenosine (Ado) have been extensively studied, its potential to interfere with TLR4 activation is unknown. We observed that TLR4 pathway is activated in white blood cells from MI patients. TLR4 mRNA expression correlated with troponin T levels (R (2) = 0.75; P = 0.01) but not with levels of white blood cells and C-reactive protein. Ado downregulated TLR4 expression at the surface of human macrophages (-50%, P < 0.05). Tumor necrosis factor-α production induced by the TLR4 ligands LPS, hyaluronic acid, and heparan sulfate was potently inhibited by Ado (-75% for LPS, P < 0.005). This effect was reproduced by the A2A Ado receptor agonist CGS21680 and the non-selective agonist NECA and was inhibited by the A2A antagonist SCH58261 and the A2A/A2B antagonist ZM241,385. In contrast, Ado induced a 3-fold increase of TLR4 mRNA expression (P = 0.008), revealing the existence of a feedback mechanism to compensate for the loss of TLR4 expression at the cell surface. In conclusion, the TLR4 pathway is activated after MI and correlates with infarct severity but not with the extent of inflammation. Reduction of TLR4 expression by Ado may therefore represent an important strategy to limit remodeling post-MI.

Proof-of-principle investigation of an algorithmic model of adenosine-mediated angiogenesis.

BACKGROUND: We investigated an algorithmic approach to modelling angiogenesis controlled by vascular endothelial growth factor (VEGF), the anti-angiogenic soluble VEGF receptor 1 (sVEGFR-1) and adenosine (Ado). We explored its feasibility to test angiogenesis-relevant hypotheses. We illustrated its potential to investigate the role of Ado as an angiogenesis modulator by enhancing VEGF activity and antagonizing sVEGFR-1. RESULTS: We implemented an algorithmic model of angiogenesis consisting of the dynamic interaction of endothelial cells, VEGF, sVEGFR-1 and Ado entities. The model is based on a logic rule-based methodology in which the local behaviour of the cells and molecules is encoded using if-then rules. The model shows how Ado may enhance angiogenesis through activating and inhibiting effects on VEGF and sVEGFR-1 respectively. Despite the relative simplicity of the model, it recapitulated basic features observed in in vitro models. However, observed disagreements between our models and in vitro data suggest possible knowledge gaps and may guide future experimental directions. CONCLUSIONS: The proposed model can support the exploration of hypotheses about the role of different molecular entities and experimental conditions in angiogenesis. Future expansions can also be applied to assist research planning in this and other biomedical domains.

Predictive integration of gene functional similarity and co-expression defines treatment response of endothelial progenitor cells.

BACKGROUND: Endothelial progenitor cells (EPCs) have been implicated in different processes crucial to vasculature repair, which may offer the basis for new therapeutic strategies in cardiovascular disease. Despite advances facilitated by functional genomics, there is a lack of systems-level understanding of treatment response mechanisms of EPCs. In this research we aimed to characterize the EPCs response to adenosine (Ado), a cardioprotective factor, based on the systems-level integration of gene expression data and prior functional knowledge. Specifically, we set out to identify novel biosignatures of Ado-treatment response in EPCs. RESULTS: The predictive integration of gene expression data and standardized functional similarity information enabled us to identify new treatment response biosignatures. Gene expression data originated from Ado-treated and -untreated EPCs samples, and functional similarity was estimated with Gene Ontology (GO)-based similarity information. These information sources enabled us to implement and evaluate an integrated prediction approach based on the concept of k-nearest neighbours learning (kNN). The method can be executed by expert- and data-driven input queries to guide the search for biologically meaningful biosignatures. The resulting integrated kNN system identified new candidate EPC biosignatures that can offer high classification performance (areas under the operating characteristic curve>0.8). We also showed that the proposed models can outperform those discovered by standard gene expression analysis. Furthermore, we report an initial independent in vitro experimental follow-up, which provides additional evidence of the potential validity of the top biosignature. CONCLUSION: Response to Ado treatment in EPCs can be accurately characterized with a new method based on the combination of gene co-expression data and GO-based similarity information. It also exploits the incorporation of human expert-driven queries as a strategy to guide the automated search for candidate biosignatures. The proposed biosignature improves the systems-level characterization of EPCs. The new integrative predictive modeling approach can also be applied to other phenotype characterization or biomarker discovery problems.

Adenosine modifies the balance between membrane and soluble forms of Flt-1.

VEGFR-1 (or Flt-1) exists under a sFlt-1 or a mFlt-1 form. sFlt-1 is antiangiogenic, and mFlt-1 is proangiogenic. The cardioprotective nucleoside Ado is proangiogenic, but its effects on Flt-1 are unknown and were tested in this study. In primary human macrophages from healthy volunteers, Ado inhibited sFlt-1 expression induced by LPS (-43%, P=0.006), HS, and IL-1ß but not hypoxia. This effect was also observed in macrophages from patients with acute MI (-33%, P<0.001). It was reproduced by the A(2A) Ado receptor agonist CGS21680 and abrogated by the A(2A) antagonist SCH58261. Conversely, Ado increased mFlt-1 expression, thus switching sFlt-1 from the soluble toward the membrane form. This switch was also present in macrophages from acute MI patients (P<0.001). Assessment of HIF-1α nuclear translocation and activation together with siRNA experiments suggested that the effect of Ado on Flt-1 involves HIF-1α. In conclusion, Ado down-regulates sFlt-1 and up-regulates mFlt-1 production, an effect that indicates that Ado may be used to stimulate angiogenesis in the heart.

Adenosine up-regulates vascular endothelial growth factor in human macrophages.

It is known from animal models that the cardioprotective nucleoside adenosine stimulates angiogenesis mainly through up-regulation of vascular endothelial growth factor (VEGF). Since macrophages infiltrate the heart after infarction and because adenosine receptors behave differently across species, we evaluated the effect of adenosine on VEGF in human macrophages. Adenosine dose-dependently up-regulated VEGF expression and secretion by macrophages from healthy volunteers. VEGF production was also increased by blockade of extracellular adenosine uptake with dipyridamole. This effect was exacerbated by the toll-like receptor-4 ligands heparan sulfate, hyaluronic acid and lipopolysaccharide, and was associated with an increase of hypoxia inducible factor-1alpha expression, the main transcriptional inducer of VEGF in hypoxic conditions. The agonist of the adenosine A2A receptor CGS21680 reproduced the increase of VEGF and the antagonist SCH58261 blunted it. In conclusion, these results provide evidence that activation of adenosine A2A receptor stimulates VEGF production in human macrophages. This study suggests that adenosine is a unique pro-angiogenic molecule that may be used to stimulate cardiac repair.

Activation of the adenosine-A3 receptor stimulates matrix metalloproteinase-9 secretion by macrophages.

AIMS: Matrix metalloproteinase-9 (MMP-9) plays an important role in ventricular remodelling after acute myocardial infarction (MI). The cardioprotectant adenosine (Ado) may be involved in ventricular remodelling. We have shown that Ado inhibits the secretion of MMP-9 by human neutrophils. This study investigated the effect of Ado on MMP-9 production by human macrophages. METHODS AND RESULTS: Cells used in this study were monocytes of healthy volunteers, a human monocyte cell line, and leukocytes from patients following MI. Monocytes were differentiated into macrophages and treated with Ado. Ado enhanced MMP-9 secretion by human macrophages in a time- and dose-dependent manner. Increasing the level of endogenous Ado by inhibition of Ado deaminase or Ado transferase also increased MMP-9 secretion. Ado enhanced MMP-9 production when macrophages were activated by hypoxia or Toll-like receptor-4 ligands such as lipopolysaccharide, hyaluronan, and heparan sulfate. The effect of Ado was replicated by the A3 agonist IB-MECA and inhibited by silencing the A3 receptor. Ado improved monocyte capacity to migrate through a matrix of gelatin B, and this effect was blocked by inhibition of MMP-9 activity. The chemotactic capacity of macrophages was reduced by Ado through a loss of expression of the monocyte chemotactic protein-1 receptor. Finally, MMP-9 expression was higher in blood cells from patients with acute MI compared with healthy volunteers. CONCLUSION: Adenosine activates MMP-9 secretion by macrophages through its A3 receptor. The effect is in contrast to that observed in neutrophils, where Ado inhibits MMP-9 secretion by the A2a receptor. These observations may have important implications for therapeutic strategies targeting Ado receptors in the setting of MI.