Dysregulated miR-361-5p/VEGF Axis in the Plasma and Endothelial Progenitor Cells of Patients with Coronary Artery Disease

<div><p>Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3′-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs <i>in vitro</i>, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.</p></div

Mesenchymal Stem Cells from Human Umbilical Cord Express Preferentially Secreted Factors Related to Neuroprotection, Neurogenesis, and Angiogenesis

<div><p>Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases such as infarcted myocardia and strokes because of their ability to promote endogenous angiogenesis and neurogenesis <i>via</i> a variety of secreted factors. MSCs found in the Wharton’s jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. We isolated MSCs from Wharton’s jelly and bone marrow (WJ-MSCs and BM-MSCs, respectively) and compared their secretomes. It was found that WJ-MSCs expressed more genes, especially secreted factors, involved in angiogenesis and neurogenesis. Functional validation showed that WJ-MSCs induced better neural differentiation and neural cell migration <i>via</i> a paracrine mechanism. Moreover, WJ-MSCs afforded better neuroprotection efficacy because they preferentially enhanced neuronal growth and reduced cell apoptotic death of primary cortical cells in an oxygen-glucose deprivation (OGD) culture model that mimics the acute ischemic stroke situation in humans. In terms of angiogenesis, WJ-MSCs induced better microvasculature formation and cell migration on co-cultured endothelial cells. Our results suggest that WJ-MSC, because of a unique secretome, is a better MSC source to promote <i>in vivo</i> neurorestoration and endothelium repair. This study provides a basis for the development of cell-based therapy and carrying out of follow-up mechanistic studies related to MSC biology.</p> </div

Suppression levels of miR-361-5p, not miR-484, restored VEGF expression and EPC functions.

<p>(<b>A</b>) Overexpression of miR-361-5p in PB-EPCs down-regulated VEGF levels. *: <i>p</i><0.05, by <i>Student's</i> T test. (<b>B</b>) Overexpression of miR-484 in PB-EPCs neither down-regulated VEGF levels (<i>middle panel</i>) nor inhibited cell motility (<i>right panel</i>). **: <i>p</i><0.01 by <i>Student's</i> T test. (<b>C</b>) miR-361-5p transfectants showed reduced microtubule formation (left), cellular migration (<i>middle</i>), and cell proliferation (<i>right</i>) abilities. Representative pictures are shown in <i>Suppl. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098070#pone-0098070-g001" target="_blank">Fig. 1</a> online. *: <i>p</i><0.05, **: <i>p</i><0.01 by <i>Student's</i> T test. (<b>D–F</b>) Restoring miR-361-5p level in CAD-EPCs repairs EPC functions. (<i>D</i>): RT-qPCR shows that miR-361-5p oligonucleotide antagomir transfection represses CAD-EPC miR-361-5p to a level similar to that in normal PB-EPC controls. EPC vasculogenesis (<i>E</i>) and migration (<i>F</i>) abilities were also measured. *: <i>p</i><0.05 by one-way ANOVA test followed by Tukey's post-hoc test.</p

Reduced VEGF levels and angiogenic activities in EPCs from CAD patients.

<p>(<b>A</b>) Morphology of healthy and diseased EPCs. (<b>B</b>) Expression of indicated molecules in EPCs by flow cytometric analyses. (<b>C</b>) Different angiogenic abilities between healthy and diseased EPCs. EPCs from peripheral blood of healthy individuals (PB-EPCs) migrate faster and form better microvasculature structures <i>in vitro</i> than those from patients with CAD (CAD-EPCs). EPCs from different sources were subjected to Transwell cell migration assays or onto MatriGel for tube formation assays. Migrated cells were stained (representative pictures are shown, left lower panel) and counted (right panel, n = 3). Tube lengths of formed microvascular structure were also measured (middle panel, n = 3). *: <i>p</i><0.05 by <i>Student's</i> T test. (<b>D</b>) Cell proliferation assays show PB-EPCs grow faster in vitro. Cultured EPCs were subjected into MTT assays for monitoring cell proliferation rate. *: <i>p</i><0.05 by <i>Student's</i> T test. (<b>E</b>) Hierarchical VEGF mRNA expression levels in CAD-EPC and healthy PB-EPC showed by RT-qPCR. Mean gene expression levels of EPC genes were compared to the average CT values of GAPDH and beta-actin controls. Results are expressed as mean±standard deviation. ***: <i>p</i><0.001 by Mann-Whitney <i>U</i> test. (<b>F</b>) Reduced serum VEGF protein levels in CAD patients determined by ELISA assays. ***: <i>p</i><0.001 by Mann-Whitney <i>U</i> test. (<b>G</b>) Treating PB-EPCs with the Avastin anti-VEGF mAb suppresses <i>in vitro</i> microtubular formation, cell migration, and cell proliferation activities. *: <i>p</i><0.05, **: <i>p</i><0.01 by <i>Student's</i> T test.</p

smRNA-seq reveals candidate anti-VEGF miRNAs in both EPCs and the plasma of CAD patients.

<p>(<b>A</b>) A Venn diagram showing overlapping miRNAs between smRNA-seq data and bioinformatics prediction results. (<b>B</b>) A table highlighting candidate VEGF-targeting miRNAs highly expressed in PB-EPCs. #: miRNAs known to target VEGF directly; arrows: miRNAs picked for further RT-qPCR validation. (<b>C–D</b>) RT-qPCR showing expression levels of 8 miRNAs in EPCs (<i>C</i>) and the plasma (<i>D</i>) of normal controls (PB) and CAD patients. *: <i>p</i><0.05; **: <i>p</i><0.01: ***: <i>p</i><0.001 by Mann-Whitney <i>U</i> test.</p

The miR-361-5p/VEGF pair contributes to CAD-EPC activities.

<p>(<b>A</b>) Structure of the VEGF transcript and the predicted binding site between the VEGF 3′UTR (untranslated region) and miR-361-5p. (<b>B</b>) A luciferase reporter plasmid containing either the miR-361-5p binding site (WT) or a mutant (Mut) binding site were co-transfected with miR-361-5p. The luciferase activity was assessed 2 days post-transfection (<i>right panel</i>). The expression levels of miR-361-5p were detected by RT-qPCR (<i>left panel</i>). *: <i>p</i><0.05 by one-way ANOVA test followed by Tukey's post-hoc test. (<b>C–D</b>) Neutralization of VEGF in miR-361-5p-reduced (by antagomirs) CAD-EPCs suppresses cellular activities. miR-361-5p and VEGF levels in treated CAD-EPCs were determined by RT-qPCR (<i>C</i>). Transwell migration (<i>D, left panel</i>) and tube formation (<i>D, right panel</i>) assays were conducted on CAD-EPCs treated with the indicated antagomirs and/or the Avastin anti-VEGF mAb. Representative pictures are in <i>Suppl. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098070#pone-0098070-g002" target="_blank">Figs. 2A-B</a> online, and the quantitative results of the images are shown. *: <i>p</i><0.05 by one-way ANOVA test followed by Tukey's post-hoc test.</p

Preferential neuroprotection effects of WJ-MSCs.

<p>(<b>A</b>) Schematic representation of the transmembranous stem cell co-culture system using an oxygen-glucose deprivation (OGD) model. (<b>B</b>) Immunofluorescence staining of the rat primary cortical cells subjected to OGD alone (left), to co-culture with BM-MSCs (middle), or to co-culture with WJ-MSCs (right) at 72 hours post-OGD. Neuronal marker MAP2 is shown in red, the astroglial marker GFAP in green, and DAPI nuclear staining in blue. Scale bar: 20 µm. (<b>C</b>) Quantification of cell death and apoptosis rate using PI and TUNEL staining, respectively, at 72 hours post-OGD. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (<b>D</b>) Quantification of total neurite length (left) and neurite branch point numbers (right). (<b>E</b>) Percentage of neuron number (left) and astrocyte number (right) after 72 hours co-cultured with BM-MSCs or WJ-MSCs post-OGD.</p

Higher neural induction ability of WJ-MSCs.

<p>(<b>A</b>) An interaction network of secreted factors. Factors that pertained to angiogenesis, vasculogenesis, neurite outgrowth, and neuron migration are connected. Genes in red are abundant in WJ-MSCs and genes in green are abundant in BM-MSCs. (<b>B</b>) Differential chemotaxis effects of WJ-MSCs and BM-MSCs. MSCs cultured in MesenCult® medium were seeded in the lower part of Tranwell plates, while N2a cells were placed in the upper chambers (illustrated in left panel). Migrated N2a cells to the other side of the membrane were stained with Hoechst 33342 and counted. Data are mean ± SD (right panel; *<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>–<b>D</b>) Induction of N2a neural differentiation by MSCs. N2a cells were cultured with MSC conditioned medium, medium only (negative control) or retinoic acid (RA; positive control) for 4 days before the cellular lysates were subjected to Western blotting analysis (C) or the cells were fixed for immunofluorescence staining (D). Neural markers TUBB3 and NEFL were analyzed. Cell nuclei were stained with DAPI. Scale bars: 50 μm.</p

Transplantation of miR-361-5p^low CAD-EPCs improves blood perfusion in the ischemic hindlimb.

<p>(<b>A</b>) Schematic represeatation of experimental design. (<b>B</b>) miR-361-5p levels in tranfected CAD-EPCs determined by RT-qPCR. *: <i>p</i><0.05 by <i>Student's</i> T test. (<b>C</b>) Representative images of hindlimb blood flow measured by laser Doppler before operation (Pre-Op), immediately after hindlimb ischemia surgery (Post-Op), and 2 weeks after intramuscular injection of culture medium (EGM2), peripheral blood EPC-transfected with scramble oligonucleotides (Scr), or CAD-EPC-transfected with miR-361-5p oligonucleotide antagomirs (Anti-miR). (<b>D</b>) Quantitative analysis of blood flow expressed as perfusion ratio of the ischemic to the contralateral (non-operated) hindlimb. *: <i>p</i><0.05; **: <i>p</i><0.01: ***: <i>p</i><0.001 compared with control; n = 6. (<b>E</b>) Immunofluorescence staining on nude mice tissues 7 days after injection with PKH-26-labeled CAD-EPCs. Capillaries in the ischemic muscles were visualized by anti-CD31 immunostaining (green), and injected human EPCs were monitored by PKH-26 fluorescence (red). Mice receiving miR-361-5p-repressed EPCs had more CD31+/PKH-26+ double-positive cells (white arrowheads) in ischemic muscle than another 2 control mice groups (Scr and medium). DAPI: nuclear staining of live cells (blue). (<b>F</b>) Quantitative analysis of capillary densities and CD31+/PKH-26+ double-positive cells in ischemic muscle of mice hindlimb ischemia surgery. HPF: high power field; N.D.: not detectable; *: <i>p</i><0.05 by one-way ANOVA test followed by Tukey's post-hoc test. (<b>G</b>) A proposed model of EPC angiogenesis activities are regulated by the miR-361-5p-VEGF pathway.</p

FIR treatment improves angiogenic activities of ECFCs.

<p><b>(A)</b> Quantitative data from the Transwell cell migration assays (left) and tube formation assays (right) using HG-dfECFCs with or without FIR treatment. n = 3 independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test. <b>(B)</b> Quantitative data from the Transwell cell migration assays (left) and tube assays (right) using dmECFCs treated with FIR. n = 3 independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test. <b>(C)</b> The expression levels of miR-134 in dfECFCs from four individuals with or without FIR treatment. * <i>p</i> < 0.05 by one-way ANOVA followed by Tukey’s post-hoc test. <b>(D)</b> miR-134 expression in vector control and miR-134 overexpressed dfECFC with or without FIR treatment. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, by one-way ANOVA followed by Tukey’s <i>post-hoc</i> test. <b>(E)</b> Representative images (lower) and quantitative data (upper) from the Transwell cell migration assays and microvascular formation assays using vector control or miR-134 overexpressed dfECFCs with or without FIR treatment. n = 3 independent experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 by one-way ANOVA followed Tukey’s post-hoc test. Scale bar: 50 μm.</p