Large impact of low concentration oxidized LDL on angiogenic potential of human endothelial cells: a microarray study

Abstract

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.

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

(A) – basal expression of scavenger receptors in HCAECs evaluated by qPCR. (B) – ox-LDL induced expression of scavenger receptors in HCAECs by qPCR. (C) - 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.

Figure 2. Effects of ox-LDL on genes implicated in angiogenesis.

(A) - 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) - validation of microarray data on select genes (white bars) by qPCR (black bars); (C) – tube formation on matrigel by HCAECs in presence of 5 µg/ml ox-LDL (16 hrs); (D) - Graph summarizing data on matrigel angiogenesis.

Figure 3. Effects of LOX-1 abrogation on choroid angiogenesis.

Eyes of 8 week old wild type (C57BL) and LOX-1 KO (C57BL background) mice were subjected to laser photocoagulation (see Materials and Methods). 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.

Figure 4. Bi-directional effects of ox-LDL on genes implicated in apoptosis.

(A) - Differentially expressed genes involved in regulation of apoptosis (white bars) and validation of microarray data by qPCR (black bars). (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. (C) – 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.

Figure 5. Ox-LDL signaling is partially mediated by miR-21.

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

Figure 6. Schematics of pro-angiogenic signaling of ox-LDL based on microarray data partially validated by qPCR.

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.