Search

Objective- The objective of this study was to determine the basis of resistance to atherosclerosis of inbred mouse strain BALB/cJ. Approach and Results- BALB/cJ mice carry a naturally occurring null mutation of the gene encoding the transcription factor Zhx2, and genetic analyses suggested that this may confer resistance to atherosclerosis. On a hyperlipidemic low-density lipoprotein receptor null background, BALB/cJ mice carrying the mutant allele for Zhx2 exhibited up to a 10-fold reduction in lesion size as compared with an isogenic strain carrying the wild-type allele. Several lines of evidence, including bone marrow transplantation studies, indicate that this effect of Zhx2 is mediated, in part, by monocytes/macrophages although nonbone marrow-derived pathways are clearly involved as well. Both in culture and in atherosclerotic lesions, macrophages from Zhx2 null mice exhibited substantially increased apoptosis. Zhx2 null macrophages were also enriched for M2 markers. Effects of Zhx2 on proliferation and other bone marrow-derived cells, such as lymphocytes, were at most modest. Expression microarray analyses identified >1000 differentially expressed transcripts between Zhx2 wild-type and null macrophages. To identify the global targets of Zhx2, we performed ChIP-seq (chromatin immunoprecipitation sequencing) studies with the macrophage cell line RAW264.7. The ChIP-seq peaks overlapped significantly with gene expression and together suggested roles for transcriptional repression and apoptosis. Conclusions- A mutation of Zhx2 carried in BALB/cJ mice is responsible in large part for its relative resistance to atherosclerosis. Our results indicate that Zhx2 promotes macrophage survival and proinflammatory functions in atherosclerotic lesions, thereby contributing to lesion growth.

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.