Tumor Necrosis Factor- (cid:1) Induces Endothelial Dysfunction in Lepr db Mice Molecular Cardiology

Background —We hypothesized that the inflammatory cytokine tumor necrosis factor- (cid:1) (TNF) produces endothelial dysfunction in type 2 diabetes. Methods and Results —In m Lepr db control mice, sodium nitroprusside and acetylcholine induced dose-dependent vasodilation, and dilation to acetylcholine was blocked by the NO synthase inhibitor N G -monomethyl- L -arginine. In type 2 diabetic (Lepr db ) mice, acetylcholine- or flow-induced dilation was blunted compared with m Lepr db , but sodium nitroprusside produced comparable dilation. In Lepr db mice null for TNF (db TNF (cid:1) /db TNF (cid:1) ), dilation to acetylcholine or flow was greater than in diabetic Lepr db mice and comparable to that in controls. Plasma concentration of TNF was significantly increased in Lepr db versus m Lepr db mice. Real-time polymerase chain reaction and Western blotting showed that mRNA and protein expression of TNF and nuclear factor- (cid:2) B were higher in Lepr db mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor- (cid:2) B and TNF expression in the Lepr db mice. Immunostaining results show that TNF in mouse heart is localized predominantly in vascular smooth muscle cells rather than in endothelial cells and macrophages. Superoxide generation was elevated in vessels from Lepr db mice versus controls. Administration of the superoxide scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF restored endothelium-dependent dilation in Lepr db mice. set by the Laboratory Animal Care Committee at Texas A&M University. Heterozygote controls (m Lepr db ), homozygote type 2 diabetes (Lepr db ), and Lepr db null for TNF (db TNF (cid:1) /db TNF (cid:1) ) mice were purchased from the Jackson Laboratory (Bar Harbor, Me) and maintained on a normal rodent chow diet. Our studies used 12- to 14-week-old, 15- to 25-g m Lepr db and 25- to 50-g Lepr db and db TNF (cid:1) /db TNF (cid:1) mice of either sex. We used the same strain (C57BL/ 6J) of m Lepr db and db TNF (cid:1) /db TNF (cid:1) mice to match the backgrounds of Lepr db mice. The cross (db TNF (cid:1) /db TNF (cid:1) ) of Lepr db with TNF knockout mice is heterozygous for Lepr db and homozygous for TNF knockout mice (TNF (cid:1) / (cid:1) ). These db TNF (cid:1) /db TNF (cid:1) mice show the phenotypes of hyperglycemia and obesity, the diabetic phenotype that is consistent with the penetrance of the leptin receptor mutation. The obese mice from the second round of breeding of Lepr db and TNF (cid:1) / (cid:1) were used in experimentation. vasoactive responses in type 2 diabetes, the aforementioned vasodi- latory functions were examined in the presence of the O 2 (cid:1) scavenger TEMPOL (a membrane-permeable O 2 (cid:1) dismutase mimetic; 1 mmol/L, 60-minute incubation). The contributions of NAD(P)H oxidase, xanthine oxidase, and mitochondrial respiratory chain in generating O 2 (cid:1) were assessed by treating the vessels with an NAD(P)H oxidase inhibitor apocynin (10 (cid:5) mol/L), a xanthine oxidase inhibitor allopurinol (10 (cid:5) mol/L), or the mitochondrial respiratory chain inhibitor rotenone (1 (cid:5) mol/L) for a 60-minute incubation, separately. Ebselen (10 (cid:5) mol/L) and catalase (1000 U/mL) were also used (60-minute incubation) to determine whether ONOO (cid:1) and H 2 O 2 were involved in endothelial dysfunction. All drugs were administered extraluminally in these functional studies. NF- (cid:2) B protein expressions were with the use of TNF primary antibodies (Santa nitrotyrosine primary antibodies (Abcam), and NF- (cid:2) B primary antibodies (Abcam) in m Lepr db , Lepr db , and Lepr db mice treated with anti-TNF (0.625 mg/mL per kilogram per day IP for 3 days), the NAD(P)H oxidase inhibitor (80 (cid:5) g/d IP for 10 days; to antagonize RAGE signaling). Horseradish goat as (Abcam). Signals were visualized by enhanced chemilumines- cence (ECL, Amersham) and quantified by Quantity One (Bio-Rad Versadoc imaging system). Statistical comparisons of vasomotor responses under various treatments were performed with 2-way ANOVA, and intergroup differences were tested with Bonferroni inequality. the