Exposure to Ambient Air Fine Particulate Matter Prevents VEGF-Induced Mobilization of Endothelial Progenitor Cells from the Bone Marrow

Background: Exposure to ambient fine particulate matter air pollution (PM2.5; < 2.5 µm in aerodynamic diameter) induces endothelial dysfunction and increases the risk for cardiovascular disease. Endothelial progenitor cells (EPCs) contribute to postnatal endothelial repair and regeneration. In humans and mice, EPC levels are decreased upon exposure to elevated levels of PM2.5

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages

<div><p>Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.</p></div

miR-223 mimic reduces TLR4 expression in macrophages stimulated with LPS.

<p>(A) WT BMM have about 21,000-fold greater expression of miR-223 by TaqMan probe-based qPCR compared to miR223KO BMMs. (B) miR223KO BMMs electroporated with miR-223 mimic at 500nM have approximately 290,000-fold higher expression than miR223KO BMMs electroporated in the presence of 500nM of mimic control oligo. (C) qPCR of total cDNA (RNA used in B) shows that the miR-223 mimic (“mimic”) significantly decreases mTLR4 and mFBXW7 (pan-detecting primer) in the presence of LPS, compared to BMM not receiving miR-223 mimic (“Ctrl,” control oligo) relative to beta-2 microglobulin (B2M).</p

Cohort 1—Patient Characteristics—MicroRNA Arrays.

<p>Cohort 1—Patient Characteristics—MicroRNA Arrays.</p

Heatmap of miRNA expression for those with a significant difference between groups.

<p>The heatmap is organized by group (lean or obese) on x-axis and increasing significance between groups from top to bottom on the y-axis. Red indicates an increase and green/black a decreased relative expression per sample (n = 6 per group).</p

Pathway analysis of miR-223 gene targets using IPA^® software by Ingenuity Systems (Qiagen).

<p>KEGG Canonical pathways are labeled (CP). All targets are depicted by the official gene symbol and function of protein product can be found in legend. Genes symbols are: ADIPOQ, Adiponectin; PIK3C2A, phosphoinositide-3-kinase (PI3K), class 2, alpha polypeptide; IRS1, insulin receptor substrate 1; INSR, insulin receptor; PDPK1, 3-phosphoinositide dependent protein kinase-1; RPS6KB1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; PRKACB, protein kinase A, cAMP-dependent, catalytic, beta.</p

Pearson correlation coefficients—miRNA expression and patient parameters.

<p>Pearson correlation coefficients—miRNA expression and patient parameters.</p

miR-223 null macrophages have enhanced inflammatory signaling and increased FBXW7 levels.

<p>(A) Effect of LPS (100ng/ml) on mmu-miR-223 levels in the mouse macrophage cell line Raw264.7 and in primary mouse macrophages from C57BL/6J mice as measured by qPCR, relative to snoRNA234. (B) LPS treatment dramatically decreases FBXW7 mRNA levels in primary mouse macrophages at 24 hours (normalized to B2M) by qPCR. (C) Immunoblot data examining response of primary macrophages from WT and miR-223 knockout mice (KO) to LPS and combined IFNγ/LPS; 50μg of whole cell lysate was loaded per lane and run on a denaturing SDS PAGE gel. Densitometry and statistics are presented in (D). (E) hFBXW7 3’-UTR luciferase reporter assay experiment. Data is expressed as luciferase-hFBXW7 3’-UTR activity over β-galactosidase activity as measured by luminescence. “+miR-223” indicates co-transfection of miR-223 mimic. “WT UTR” indicates a vector containing un-mutated hFBXW7 3’-UTR and “Mutated UTR” indicates a vector containing hFBXW7 3’-UTR that has been mutated at all four miR-223 seed sequences. All values are expressed as ± SEM; * indicates p<0.05 via student t-test.</p

Murine obesity demonstrated increased miR-223 expression and repressed FBXW7 levels.

<p>(A) mmu-miR-223 levels were significantly increased in the whole adipose and liver tissues, but not in skeletal muscle. (B) FBXW7α and -β gene expression by qPCR in WT and <i>ob/ob</i> mice whole adipose tissue, white column—alpha isoform; black column—beta isoform. (C) FBXW7α and -β gene expression by qPCR in WT and <i>ob/ob</i> mice whole liver, white column—alpha isoform; black column—beta isoform.</p