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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>k</it>-nearest neighbours learning (<it>k</it>NN). The method can be executed by expert- and data-driven input queries to guide the search for biologically meaningful biosignatures. The resulting <it>integrated kNN </it>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 <it>in vitro </it>experimental follow-up, which provides additional evidence of the potential validity of the top biosignature.</p> <p>Conclusion</p> <p>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.</p

Adenosine Stimulates the Migration of Human Endothelial Progenitor Cells. Role ofCXCR4 and MicroRNA-150

International audienceBACKGROUND: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

Quantitative PCR primers.

<p>Annealing temperatures are indicated.</p

Expression of Ado receptors in EPC.

<p>A. mRNA expression profiles of the 4 Ado receptors in EPC were obtained by quantitative PCR. Significant differences were observed (n = 3, P = 0.004), the A₃ subtype being predominant and the A₁ subtype not detected (ND). Expression values were normalized to β-actin. B–D. Ado regulates the expression of its own receptors in EPC. mRNA expression was assessed by quantitative PCR at different times after treatment with 10 µmol/L Ado (n = 3). Ado transiently increased A_2A and A_2B expression (P = 0.003 and P = 0.002, respectively) and decreased A₃ expression (P = 0.01). * P≤0.01 vs control.</p

Adenosine enhances the migration of EPC.

<p>EPC were treated by 10 µmol/L Ado for 6 hours before transfer to the top of a Boyden chamber. The bottom of the chamber was filled with culture medium containing either recombinant SDF-1α (A and B) or conditioned medium from cardiac fibroblasts (C). The amount of cells that migrated through the membrane was assessed by fluorescence after 16 hours. A. SDF-1α induced EPC migration and Ado enhanced this effect (P = 0.02, two-way ANOVA. n = 3). B. Blocking CXCR4 with a neutralizing antibody (10 µg/mL) for one hour before exposure to 50 ng/mL SDF-1α for 16 hours in a Boyden chamber inhibited the migration in both untreated and Ado-treated cells. * P<0.05 vs control. # P<0.01 vs Ado (n = 3). C. Ado enhances the migration of EPC induced by conditioned medium from cardiac fibroblasts. EPC were treated by 10 µmol/L Ado or vehicle for 6 hours before transfer to the top of a Boyden chamber. The bottom of the chamber was filled with cell-free conditioned medium from fibroblasts. In some samples, EPC were pre-incubated for 1 hour with anti-CXCR4 blocking antibodies before transfer to the chambers. The amount of EPC that migrated through the membrane was assessed by fluorescence after 16 hours. * P<0.01 vs control. # P<0.001 vs Ado (n = 7).</p

Adenosine enhances cell surface expression of CXCR4.

<p>EPC were treated with Ado and CXCR4 expression was assessed by flow cytometry. A. Representative histogram of EPC treated by 10 µmol/L Ado for 6 hours. Dark grey: isotype control. Light grey: CXCR4 staining of control cells. White: CXCR4 staining of Ado-treated cells. B. Kinetic of CXCR4 expression on EPC treated by 10 µmol/L Ado. * P<0.001 vs control (n = 10). C. Dose-dependent effect of Ado on CXCR4 expression on EPC harvested 6 hours after treatment. * P<0.05 vs control. ** P<0.01 vs control (n = 3).</p