Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta

Abstract

In the arterial circulation, regions of disturbed flow (DF), which are characterized by flow separation and transient vortices, are susceptible to atherogenesis, whereas regions of undisturbed laminar flow (UF) appear protected. Coordinated regulation of gene expression by endothelial cells (EC) may result in differing regional phenotypes that either favor or inhibit atherogenesis. Linearly amplified RNA from freshly isolated EC of DF (inner aortic arch) and UF (descending thoracic aorta) regions of normal adult pigs was used to profile differential gene expression reflecting the steady state in vivo. By using human cDNA arrays, approximately 2,000 putatively differentially expressed genes were identified through false-discovery-rate statistical methods. A sampling of these genes was validated by quantitative real-time PCR and/or immunostaining en face. Biological pathway analysis revealed that in DF there was up-regulation of several broad-acting inflammatory cytokines and receptors, in addition to elements of the NF-kappaB system, which is consistent with a proinflammatory phenotype. However, the NF-kappaB complex was predominantly cytoplasmic (inactive) in both regions, and no significant differences were observed in the expression of key adhesion molecules for inflammatory cells associated with early atherogenesis. Furthermore, there was no histological evidence of inflammation. Protective profiles were observed in DF regions, notably an enhanced antioxidative gene expression. This study provides a public database of regional EC gene expression in a normal animal, implicates hemodynamics as a contributory mechanism to athero-susceptibility, and reveals the coexistence of pro- and antiatherosclerotic transcript profiles in susceptible regions. The introduction of additional risk factors may shift this balance to favor lesion development.

Fig. 1.

(A and B) Illustration of the endothelial cell isolation procedure. About 10,000 cells were scraped from precisely defined regions (≈1 cm²) of the aortic arch (DF) and the descending thoracic aorta (UF). (C and D) A representative field of EC isolated by mechanical scraping and stained with anti-platelet-endothelial cell adhesion molecule 1 (PECAM-1) and anti-α-actin antibodies. EC purity (green) was routinely >99% with only occasional contamination by isolated SMC (red; arrow).

Fig. 2.

Comparison of QRT-PCR results with microarray predictions for selected genes: red, up-regulated in DF; green, down-regulated in DF; gray, unchanged; white, undetermined (high interanimal variability).

Fig. 3.

En face immunostaining in DF and UF regions. (A and B) Subcellular localization of NF-κB complex (p65 antibody) was visualized by 2D projection of confocal imaging through the EC layer. The staining was predominantly cytoplasmic with nuclear exclusion in both aortic regions. Also clearly illustrated are the EC morphologies characteristic of DF (A) and UF (B). (C and D) Epifluorescence microscopy for the prominent differentially expressed antioxidative enzyme GPX3 revealed strong expression in DF (C) in contrast to low expression in UF (D), consistent with microarray predictions.