Profibrotic Transcriptome Signature Macrophage Epoxygenase Determines a

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2 ) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4 2 / 2 macrophages have relatively increased peroxisome proliferator-activated receptor- g levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibro-nectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4 2 / 2 rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography–tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic RNA sequencing (RNA-seq). We show that rodent macrophages predominantly express the human CYP2J2 ortholog ( Cyp2j4 in rats; Cyp2j6 in mice). By using zinc-finger nuclease (ZFN) technology in the rat, we generated an inbred rat strain deficient for functional Cyp2j4 . We show that Cyp2j4 2 / 2 bone marrow–derived macrophages (BMDMs) have reduced EET production (11,12- and 14,15-EETs) and adopt a profibrotic phenotype with transcriptional activation of extracellular among which fibronectin is the most Epoxygenase-derived EETs regulate fibronectin in and human macrophages. Cyp2j4 2 / 2 show enhanced , , and and following left kidney. In quantitative mass [LC-MS/ MS]) in control and kidneys increase in renal I collagen and fibronectin in / 2 compared controls in a model glomerular inflammation.

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