Gene Expression Analyses of Mouse Aortic Endothelium in Response to Atherogenic Stimuli

Abstract

Objective Endothelial cells are central to the initiation of atherosclerosis, yet there has been limited success in studying their gene expression in the mouse aorta. To address this, we developed a method for determining the global transcriptional changes that occur in the mouse endothelium in response to atherogenic conditions and applied it to investigate inflammatory stimuli. Approach and Results We characterized a method for the isolation of endothelial cell RNA with high purity directly from mouse aortas and adapted this method to allow for the treatment of aortas ex vivo before RNA collection. Expression array analysis was performed on endothelial cell RNA isolated from control and hyperlipidemic prelesion mouse aortas, and 797 differentially expressed genes were identified. We also examined the effect of additional atherogenic conditions on endothelial gene expression, including ex vivo treatment with inflammatory stimuli, acute hyperlipidemia, and age. Of the 14 most highly differentially expressed genes in endothelium from prelesion aortas, 8 were also perturbed significantly by ≥1 atherogenic conditions: 2610019E17Rik, Abca1, H2-Ab1, H2-D1, Pf4, Ppbp, Pvrl2, and Tnnt2. Conclusions We demonstrated that RNA can be isolated from mouse aortic endothelial cells after in vivo and ex vivo treatments of the murine vessel wall. We applied these methods to identify a group of genes, many of which have not been described previously as having a direct role in atherosclerosis, that were highly regulated by atherogenic stimuli and may play a role in early atherogenesis.

Deficiency of ATP-Binding Cassette Transporters A1 and G1 in Endothelial Cells Accelerates Atherosclerosis in Mice

“…Abca1 is upregulated in aortic arch ECs following a pro-atherogenic stimulus, 31 suggesting a particular anti-atherogenic role for the Abca1 transporter in this region of the aorta. Interestingly, a pro-atherogenic stimulus involving either LPS or pro-atherogenic diet feeding increased NF-κB activation particularly at sites of the aorta exposed to disturbed blood flow, suggesting a link between EC cholesterol accumulation and NF-κB activation in these regions.…”

“…Moreover, endothelial Abca1 as well as Abcg1 expression is upregulated by laminar blood flow in the thoracic aorta in mice, 30 and endothelial Abca1 expression is upregulated in the aortic arch by treatment with a pro-atherogenic stimulus. 31 …”

Applications and Limitations of Mouse Models for Understanding Human Atherosclerosis

“…Variables between studies include the model, the method ( en face , frozen sections, cholesterol content), the staining procedure (lipid, immune), the diet, the length of the study, the age of the mice, the vivarium conditions, and the number of mice studied. Because of the small size of mouse lesions, quantitative analyses of cell composition and gene expression are difficult (Erbilgin et al, 2013; Zhao et al, 2007). On the other hand, most human studies used clinical definitions for atherosclerosis.…”

Atherosclerosis

“…81 In a gene expression analysis of mouse aortic endothelium in response to hypercholesterolemia, inflammation, or aging, 14 genes were significantly different between these different atherogenic stimulations and normal condition. 82 Angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, is abundant in endothelial cells. Findings using whole body genetic deficiency of Angptl2, bone marrow transplantation, and endothelial specific overexpression of Angptl2, demonstrated that endothelial cell-derived Angptl2 contributed to atherosclerotic lesion formation, attributed to endothelial dysfunction.…”