Large Impact of Low Concentration Oxidized LDL on Angiogenic Potential of Human Endothelial Cells: A Microarray Study

<div><p>Oxidized LDL (ox-LDL) is a key factor in atherogenesis. It is taken up by endothelial cells primarily by ox-LDL receptor-1 (LOX-1). To elucidate transcriptional responses, we performed microarray analysis on human coronary artery endothelial cells (HCAECs) exposed to small physiologic concentration of ox-LDL- 5 µg/ml for 2 and 12 hours. At 12 hours, cultures treated with ox-LDL exhibited broad shifts in transcriptional activity involving almost 1500 genes (>1.5 fold difference, p<0.05). Resulting transcriptome was enriched for genes associated with cell adhesion (p<0.002), angiogenesis (p<0.0002) and migration (p<0.006). Quantitative PCR analysis revealed that LOX-1 expression in HCAECs is at least an order of magnitude greater than the expression of other major ox-LDL specific receptors CD36 and MSR1. In keeping with the data on LOX-1 expression, pre-treatment of HCAECs with LOX-1 neutralizing antibody resulted in across-the-board inhibition of cellular response to ox-LDL. Ox-LDL upregulated a number of pro-angiogenic genes including multiple receptors, ligands and transcription factors and altered the expression of a number of genes implicated in both stimulation and inhibition of apoptosis. From a functional standpoint, physiologic concentrations of ox-LDL stimulated tube formation and inhibited susceptibility to apoptosis in HCAECs. In addition, ox-LDL exposure resulted in upregulation of miR-1974, miR-1978 and miR-21 accompanied with significant over-presentation of their target genes in the downregulated portion of ox-LDL transcriptome. Our observations indicate that ox-LDL at physiologic concentrations induces broad transcriptional responses which are mediated by LOX-1, and are, in part, shaped by ox-LDL-dependent miRNAs. We also suggest that angiogenic effects of ox-LDL are partially based on upregulation of several receptors that render cells hypersensitive to angiogenic stimuli.</p> </div

Ox-LDL transcriptome and its dependence on LOX-1 in endothelial cells.

<p>(<b>A</b>) – basal expression of scavenger receptors in HCAECs evaluated by qPCR. (<b>B</b>) – ox-LDL induced expression of scavenger receptors in HCAECs by qPCR. (<b>C</b>) - Differentially expressed genes (1.5-fold, p<0.05) in HCAECs exposed to 5 µg/ml ox-LDL for 12 hrs. Blue lines represent genes up- or down-regulated more than 2-fold. Red triangles represent expression of these genes in cultures pre-treated with LOX-1 neutralizing antibody (TS92, 10 mg/ml) before ox-LDL exposure.</p

Schematics of pro-angiogenic signaling of ox-LDL based on microarray data partially validated by qPCR.

<p>Color coding: Shades of green – upregulation. Rred-inhibition; Grey –no change; Pro-angiogenic action of ox-LDL appears to be most consistently associated with VEGF and Notch pathways. Downstream target genes of VEGF also show corresponding shifts in expression patterns.</p

Ox-LDL signaling is partially mediated by miR-21.

<p>(<b>A</b>) - qPCR analysis of miR-21 expression in response to ox-LDL (left) and (<b>B</b>) -microarray expression of corresponding validated target genes; (<b>C</b>) - qPCR analysis of miR-221 expression in response to ox-LDL and (<b>D</b>) - microarray expression of corresponding validated target genes. (<b>E</b>) – the percentage of target genes for miR-1974, miR-1978 and miR-21 in downregulated and upregulated portions of ox-LDL induced transcriptome.</p

Bi-directional effects of ox-LDL on genes implicated in apoptosis.

<p>(<b>A</b>) - Differentially expressed genes involved in regulation of apoptosis (white bars) and validation of microarray data by qPCR (black bars). (<b>B</b>) –Viability of HCAECs measured by MTT assay in response to bleomycin (4 hours) in control cultures and cells pre-treated with 5 µg/ml ox-LDL for 12 hrs. (<b>C</b>) – TUNEL staining in HCAECs exposed to bleomycin (10 mU/ml for 4 hours) in control cultures and cells pre-treated with either 5 µg/ml ox-LDL or 5 µg/ml ox-LDL in presence of LOX-1 neutralizing antibody.</p

Effects of ox-LDL on genes implicated in angiogenesis.

<p>(<b>A</b>) - Differentially expressed genes involved in angiogenesis (Blue – exposure to ox-LDL alone; Red- pre-treatment with LOX-1 antibody followed by exposure to ox-LDL; (<b>B</b>) <b>-</b> validation of microarray data on select genes (white bars) by qPCR (black bars); (<b>C</b>) – tube formation on matrigel by HCAECs in presence of 5 µg/ml ox-LDL (16 hrs); (<b>D</b>) - Graph summarizing data on matrigel angiogenesis.</p

Differentially expressed microRNAs.

<p>Differentially expressed microRNAs.</p

Effects of LOX-1 abrogation on choroid angiogenesis.

<p>Eyes of 8 week old wild type (C57BL) and LOX-1 KO (C57BL background) mice were subjected to laser photocoagulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047421#s4" target="_blank">Materials and Methods</a>). Choroid angiogenesis was visualized in RPE-choroid-sclera flat mounts from animals perfused with 1 ml of PBS containing 50 mg/ml FITC-dextran after 7 days. Mounts were examined under a ZEISS LSM 510 laser confocal microscope and images of laser spots were captured. The images represent 3D reconstruction of the choroid neovascularization complex.</p

Phenotypic consequences of <i>OLR1 overexpression or inhibition</i>.

<p><b>A.</b> Wound healing assay. Upper panel - representative images of wound healing assay performed using HCC1143 cells transfected with either empty plasmid or <i>OLR1</i> cDNA vector; Lower panel – graph depicting the distance between edges of the wound after 36 hours of incubation. (*) – p<0.01; <b>B.</b> Adhesion assay. Upper panel- representative images of adherent non-transfected HCC1143 cells loaded with CellTracker Red CMTX (Invitrogen, Carlbad, CA) and applied to non-activated or activated (50 µg/ml oxLDL, 4 hrs) confluent HUVECs transfected with OLR1 Silencer or scrambled siRNA. Lower panel - graph depicting the number of adherent cells averaged from multiple fields of view in triplicate cultures. (*) – p<0.05 compared to non-activated control (“scrRNA”); (†) - p<0.05 compared to scrambled RNA; <b>C.</b> Colorimetric transendothelial migration assay. Upper panel – verification of the confluence of HUVECs on the membranes by staining cells with CellTracker Red CMTX. Lower panel – absorbance values of stain extracted from the cells migrated through TNFα-activated endothelial monolayer in presence of <i>OLR1</i> neutralizing antibody or human IgG (Control).</p

<i>Olr1</i> deletion results in broad inhibition of NF-κB target genes.

<p>A diagram depicting a set of overlapping genes between transformation and <i>Olr1</i> KO transcriptomes. From the set of 238 genes upregulated during transformation, 26 genes were found to be inhibited in <i>Olr1</i> KO mice. Vast majority of these genes carried NF-κB sites in their proximal promoter sequences. In total, 86 NF-κB target genes were found to be inhibited in <i>Olr1</i> KO mice with enrichment for regulation of apoptosis (p = 0.0002), proliferation (p = 0.00003), wound healing (p = 0.0002), defense response (p = 0.0011), immune response (p = 0.0003) and cell migration (p = 0.0009).</p