Neuronatin: A New Inflammation Gene Expressed on the Aortic Endothelium of Diabetic Mice

Nino Mzhavia Shuiqing Yu Shota Ikeda Tehua T. Chu Ira Goldberg Hayes M. Dansky OBJECTIVE-Identification of arterial genes and pathways altered in obesity and diabetes. RESEARCH DESIGN AND METHODS-Aortic gene expression profiles of obese and diabetic db/db, high-fat diet-fed C57BL/6J, and control mice were obtained using mouse Affymetrix arrays. Neuronatin (Nnat) was selected for further analysis. To determine the function of Nnat, a recombinant adenovirus (Ad-Nnat) was used to overexpress the Nnat gene in primary endothelial cells and in the mouse aorta in vivo. RESULTS-Nnat, a gene of unknown vascular function, was upregulated in the aortas of db/db and high-fat diet-fed mice. Nnat gene expression was increased in db/db mouse aorta endothelial cells. Nnat protein was localized to aortic endothelium and was selectively increased in the endothelium of db/db mice. Infection of primary human aortic endothelial cells (HAECs) with Ad-Nnat increased expression of a panel of nuclear factor-B (NF-B)-regulated genes, including inflammatory cytokines, chemokines, and cell adhesion molecules. Infection of mouse carotid arteries in vivo with the Ad-Nnat increased expression of vascular cell adhesion molecule 1 protein. Nnat activation of NF-B and inflammatory gene expression in HAECs was mediated through pathways distinct from tumor necrosis factor-. Nnat expression stimulated p38, Jun NH 2-terminal kinase, extracellular signal-related kinase, and AKT kinase phosphorylation. Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF- B-induced gene expression. - CONCLUSIONSNnat expression is increased in endothelial cells of obese and diabetic mouse blood vessels. The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases. Diabetes 57:27742783, 2008 Eation between diabetes and coronary heart dispidemiological data has shown a strong associease (1,2). Although insulin-mediated improved glucose control reduced cardiovascular events in subjects with type 1 diabetes (3), less definitive information is available relating diabetes control and atherosclerosis prevention in type 2 diabetes. Recent findings suggest that hyperglycemia is associated with increased arterial wall inflammation (4) and increased expression of vascular inflammatory molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-Selectin (SELE) (5,6); nuclear factor-B (NF-B) activation (7,8); and inflammatory cytokine production. Fundamental understanding of the effects of diabetes on arterial genes and pathways may contribute to the discovery of new strategies for the treatment of diabetic vascular diseases beyond blood glucose control. Animal models of diabetes have been used to study the effects of hyperglycemia and insulin resistance at different stages of disease progression (9,10). We (11,12) and others (13,14) have demonstrated that mouse models of type 2 diabetes such as leptin receptor mutant db/db and dietinduced obesity mice have impaired vascular function. Kim et al. (15) demonstrated that high-fat diet feeding increases expression of markers of vascular inflammation in mouse thoracic aortas. Also, apolipoprotein E knockout db/db mice have increased VCAM-1 expression in aorta (16) and greater aortic sinus atherosclerosis (16,17). However, not all investigators have observed changes in expression of adhesion molecules in db/db mouse aortas (18). A number of studies have focused on the effects of diabetes on vascular cells. Endothelial cells isolated from db/db aortas have increased inflammatory cytokine and chemokine expression and more monocyte adhesion (19 21). The reasons for the altered biology of these cells is thought by many to be due to hyperglycemia; increased glucose concentrations induce interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) secretion and adhesion molecule expression in endothelial cells. IL-6/IL6R complex can induce an inflammatory phenotype in endothelial cells, promoting SELE, ICAM-1, and VCAM-1 expression and monocyte adhesion (21,22). In the current study, we performed gene expression profiling of aortas from two mouse models of type 2 diabetes to identify new genes and pathways that contribute to diabetic vascular diseases. We found that neuronatin (Nnat), a gene with unknown vascular function, was upregulated in the aortas of both db/db and high-fat dietfed mice. Immunohistochemical studies localized Nnat to the vascular endothelium. To gain insight into the function of this molecule, the effects of adenovirusinduced Nnat expression in human aortic endothelial cells (HAECs) and mouse arteries were studied. RESEARCH DESIGN AND METHODS All procedures were approved by the institutional animal care and use committee. Male C57BL/6J (strain 000664), db/db (Leprdb/db strain 000642), and heterozygous littermate (db/) mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were maintained in a temperaturecontrolled barrier facility with a 12-h light/dark cycle and were given free access to food and water. db/db mice were killed at 16 20 weeks of age. To induce obesity and diabetes, C57BL/6J mice were fed a high-fat TD03584 diet (Harlan Tekland, Indianapolis, IN) for 16 20 weeks starting at the age of 10 weeks. Control mice were fed regular chow. The high-fat diet contained 35% fat and 37% carbohydrate. Mouse aorta endothelial cell isolation. Endothelial cells were isolated by sorting with magnetic beads using antiplatelet-endothelial cell adhesion molecule-1 (CD31) biotin-conjugated antibody (Millipore). In brief, mice were anesthetized and perfused with 1,000 units/ml heparin in PBS. Aortas were isolated, stripped of perivascular fat, and incubated in RPMI containing 2 mg/ml collagenase 2 and 2 units/ml dispase for 30 min at 37C. Cells were collected by centrifugation at 400g at 4C and washed twice with MACS (magnetic cell sorting) buffer (Hanks balanced salt solution [Ca/Mg free], 2 mmol/l EDTA, and 0.5% BSA). The cells suspension was run through 70- and 40-m cell strainers. Cells were incubated with CD31 antibody for 15 min, washed and incubated with streptavidin SA-Microbeads (Miltenyi Biotec) for another 15 min, washed again, and applied to an equilibrated column. After several washes, the column was removed from the magnetic holder, and cells were eluted with MACS buffer. Endothelial cells were collected by centrifugation at 400g and processed for RNA isolation. Expression of adenovirus-encoded Nnat in endothelial cells. HAECs were obtained from Cambrex Life Science. Cells were grown in endothelial cell growth medium EGM-2 to confluency and were infected with Ad-Nnat, Ad green fluorescent protein (Ad-GFP), or Ad-Empty at multiplicity of infection (MOI) 10 unless indicated otherwise. Gene expression was assessed by real-time PCR, and proteins were analyzed by Western (...truncated)