Macrophage epoxygenase determines a profibrotic transcriptome signature.

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(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ 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. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) 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 macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.

Implication of sestrin3 in epilepsy and its comorbidities.

Epilepsy is a serious neurological disorder affecting about 1% of the population worldwide. Epilepsy may arise as a result of acquired brain injury, or as a consequence of genetic predisposition. To date, genome-wide association studies and exome sequencing approaches have provided limited insights into the mechanisms of acquired brain injury. We have previously reported a pro-epileptic gene network, which is conserved across species, encoding inflammatory processes and positively regulated by sestrin3 (SESN3). In this study, we investigated the phenotype of SESN3 knock-out rats in terms of susceptibility to seizures and observed a significant delay in status epilepticus onset in SESN3 knock-out compared to control rats. This finding confirms previous in vitro and in vivo evidence indicating that SESN3 may favour occurrence and/or severity of seizures. We also analysed the phenotype of SESN3 knock-out rats for common comorbidities of epilepsy, i.e., anxiety, depression and cognitive impairment. SESN3 knock-out rats proved less anxious compared to control rats in a selection of behavioural tests. Taken together, the present results suggest that SESN3 may regulate mechanisms involved in the pathogenesis of epilepsy and its comorbidities.

Camk2n1 Is a Negative Regulator of Blood Pressure, Left Ventricular Mass, Insulin Sensitivity, and Promotes Adiposity.

Metabolic syndrome is a cause of coronary artery disease and type 2 diabetes mellitus. Camk2n1 resides in genomic loci for blood pressure, left ventricle mass, and type 2 diabetes mellitus, and in the spontaneously hypertensive rat model of metabolic syndrome, Camk2n1 expression is cis-regulated in left ventricle and fat and positively correlates with adiposity. Therefore, we knocked out Camk2n1 in spontaneously hypertensive rat to investigate its role in metabolic syndrome. Compared with spontaneously hypertensive rat, Camk2n1-/- rats had reduced cardiorenal CaMKII (Ca2+/calmodulin-dependent kinase II) activity, lower blood pressure, enhanced nitric oxide bioavailability, and reduced left ventricle mass associated with altered hypertrophic networks. Camk2n1 deficiency reduced insulin resistance, visceral fat, and adipogenic capacity through the altered cell cycle and complement pathways, independent of CaMKII. In human visceral fat, CAMK2N1 expression correlated with adiposity and genomic variants that increase CAMK2N1 expression associated with increased risk of coronary artery disease and type 2 diabetes mellitus. Camk2n1 regulates multiple networks that control metabolic syndrome traits and merits further investigation as a therapeutic target in humans.

Complement Factor B Is a Determinant of Both Metabolic and Cardiovascular Features of Metabolic Syndrome.

CFB (complement factor B) is elevated in adipose tissue and serum from patients with type 2 diabetes mellitus and cardiovascular disease, but the causal relationship to disease pathogenesis is unclear. Cfb is also elevated in adipose tissue and serum of the spontaneously hypertensive rat, a well-characterized model of metabolic syndrome. To establish the role of CFB in metabolic syndrome, we knocked out the Cfb gene in the spontaneously hypertensive rat. Cfb-/- rats showed improved glucose tolerance and insulin sensitivity, redistribution of visceral to subcutaneous fat, increased adipocyte mitochondrial respiration, and marked changes in gene expression. Cfb-/- rats also had lower blood pressure, increased ejection fraction and fractional shortening, and reduced left ventricular mass. These changes in metabolism and gene expression, in adipose tissue and left ventricle, suggest new adipose tissue-intrinsic and blood pressure-independent mechanisms for insulin resistance and cardiac hypertrophy in the spontaneously hypertensive rat. In silico analysis of the human CFB locus revealed 2 cis-regulated expression quantitative trait loci for CFB expression significantly associated with visceral fat, circulating triglycerides and hypertension in genome-wide association studies. Together, these data demonstrate a key role for CFB in the development of spontaneously hypertensive rat metabolic syndrome phenotypes and of related traits in humans and indicate the potential for CFB as a novel target for treatment of cardiometabolic disease.

New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein.

The Wistar Kyoto (WKY) rat and the spontaneously hypertensive (SHR) rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN) and metabolic syndrome, respectively. Novel transgenic (Tg) strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP) under the rat elongation factor 1 alpha (EF1a) promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminaryin vitroandin vivoimaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.