Abstract-Inflammation is a condition that underscores many cardiovascular pathologies including endothelial dysfunction, but no link is yet established between the vascular pathology of the metabolic syndrome with a particular inflammatory cytokine. We hypothesized that impairments in coronary endothelial function in the obese condition the prediabetic metabolic syndrome is caused by TNF-␣ overexpression. To test this, we measured endothelium-dependent (acetylcholine) and -independent vasodilation (sodium nitroprusside) of isolated, pressurized coronary small arteries from lean control and Zucker obese fatty (ZOF, a model of prediabetic metabolic syndrome) rats. In ZOF rats, dilation to ACh was blunted compared with lean rats, but sodium nitroprusside-induced dilation was comparable. Superoxide (O 2 · Ϫ ) generation was elevated in vessels from ZOF rats compared with lean rats, and administration of the O 2 · Ϫ scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF-␣ restored endothelium-dependent dilation in the ZOF rats. Real-time PCR and Western blotting revealed that mRNA and protein of TNF-␣ were higher in ZOF rats than that in lean rats, whereas eNOS protein levels were reduced in the ZOF versus lean rats. Immunostaining showed that TNF-␣ in ZOF rat heart is localized in endothelial cells and vascular smooth muscle cells. Expression of NAD(P)H subunits p22 and p40-phox were elevated in ZOF compared with lean animals. Administration of TNF-␣ more than 3 days also induced expression of these NAD(P)H subunits and abrogated endothelium-dependent dilation. In conclusion, the results demonstrate the endothelial dysfunction occurring in the metabolic syndrome is the result of effects of the inflammatory cytokine TNF-␣ and subsequent production of

Background-We hypothesized that the inflammatory cytokine tumor necrosis factor-␣ (TNF) produces endothelial dysfunction in type 2 diabetes. Methods and Results-In m Leprdb 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 TNFϪ /db TNFϪ ), 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-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-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. NAD(P)H oxidase activity, protein expression of nitrotyrosine, and hydrogen peroxide production were increased in Lepr db mice (compared with controls), but these variables were restored to control levels by anti-TNF. Conclusion-Advanced glycation end products/receptor of advanced glycation end products and nuclear factor-B signaling play pivotal roles in TNF expression through an increase in circulating and/or local vascular TNF production in the Lepr db mouse with type 2 diabetes. Increases in TNF expression induce activation of NAD(P)H oxidase and production of reactive oxidative species, leading to endothelial dysfunction in type 2 diabetes. (Circulation. 2007;115: 245-254.)

Background-Poly(ADP-ribose) polymerase (PARP) was suggested to play a role in endothelial dysfunction that is associated with a number of cardiovascular diseases. We hypothesized that PARP may play an important role in atherogenesis and that its inhibition may attenuate atherosclerotic plaque development in an experimental model of atherosclerosis. Methods and Results-Using a mouse (apolipoprotein E [ApoE]Ϫ/Ϫ ) model of high-fat diet-induced atherosclerosis, we demonstrate an association between cell death and oxidative stress-associated DNA damage and PARP activation within atherosclerotic plaques. PARP inhibition by thieno[2,3-c]isoquinolin-5-one reduced plaque number and size and altered structural composition of plaques in these animals without affecting sera lipid contents. These results were corroborated genetically with the use of ApoE Ϫ/Ϫ mice that are heterozygous for PARP-1. PARP inhibition promoted an increase in collagen content, potentially through an increase in tissue inhibitor of metalloproteinase-2, and transmigration of smooth muscle cells to intima of atherosclerotic plaques as well as a decrease in monocyte chemotactic protein-1 production, all of which are markers of plaque stability. In PARP-1 Ϫ/Ϫ macrophages, monocyte chemotactic protein-1 expression was severely inhibited because of a defective nuclear factor-B nuclear translocation in response to lipopolysaccharide. Furthermore, PARP-1 gene deletion not only conferred protection to foam cells against H 2 O 2 -induced death but also switched the mode of death from necrosis to apoptosis. Conclusions-Our results suggest that PARP inhibition interferes with plaque development and may promote plaque stability, possibly through a reduction in inflammatory factors and cellular changes related to plaque dynamics. PARP inhibition may prove beneficial for the treatment of atherosclerosis.

Background-We tested whether tumor necrosis factor (TNF)-␣ increases arginase expression in endothelial cells as one of the primary mechanisms by which this inflammatory cytokine compromises endothelial function during ischemiareperfusion (I/R) injury. Methods and Results-Mouse hearts were subjected to 30 minutes of global ischemia followed by 90 minutes of reperfusion and their vasoactivity before and after I/R was examined in wild-type (WT), tumor necrosis factor knockout (TNF Ϫ/Ϫ ), and TNF 1.6 (TNF ϩϩ/ϩϩ ) mice. In WT mice, dilation to the endothelium-dependent vasodilator ACh was blunted in I/R compared with sham control. L-arginine or arginase inhibitor NOHA restored NO-mediated coronary arteriolar dilation in WT I/R mice. O 2 Ϫ production was reduced by eNOS inhibitor, L-NAME, or NOHA in WT I/R mice. In TNF Ϫ/Ϫ mice, I/R did not alter Ach-induced vasodilation and O 2 Ϫ production compared with sham mice. The increase in arginase expression that occurs during I/R in WT mice was absent in TNF Ϫ/Ϫ mice. Arginase expression was confined largely to the endothelium and independent of inflammatory cell invasion. Arginase activity was markedly lower in TNF Ϫ/Ϫ , but higher in WT I/R than that in WT sham mice. Conclusions-Our data demonstrate TNF-␣ upregulates expression of arginase in endothelial cells, which leads to O 2

Background We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function. Methodology/Principal findings we conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1 μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500 μg/mL, and 4-phenyl butyric acid (PBA), 5 mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expression enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox−/− mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox−/− mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction. Conclusion/Significance We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.

Background-Epidermal growth factor receptor (EGFR) transactivation is a mediator of angiotensin II (Ang II) signaling in cultured vascular smooth muscle cells isolated from large arteries. The present study used mouse mesenteric resistance arteries (MRAs) to investigate the role of EGFR transactivation under pressure-induced myogenic tone (MT). Methods and Results-Isolated MRAs were mounted in an arteriograph and stimulated by 25 to 125 mm Hg or with Ang II and KCl. Stepwise increases in pressure resulted in MT development associated with increased EGFR phosphorylation and release of heparin-binding EGF (HB-EGF), a membrane-bound growth factor that is shed on cleavage by metalloproteinases. EGF (50 ng/mL) potentiated MT (59Ϯ1% to 51Ϯ0.6% of passive diameter at 75 mm Hg). Pretreatment with the EGFR inhibitors AG1478 (5 mol/L) or PD153035 (1 mol/L) significantly decreased MT. However, EGFR inhibitors had no effect on Ang II-and KCl-induced contraction. MT was potentiated by HB-EGF, 50 ng/mL, which is bound to the cell membrane and released on cleavage by metalloproteinases. Neutralizing HB-EGF antibodies or heparin treatment to sequester HB-EGF resulted in significant inhibition of pressure-induced MT. MT increased matrix metalloproteinase (MMP) 2 and MMP-9 gelatinase activity assessed by zymography, and specific MMP 2/9 inhibitors significantly decreased MT. Conclusions-These novel findings suggest that the mechanism of pressure-induced MT involves metalloproteinases 2/9 activation with subsequent HB-EGF release and EGFR transactivation.

OBJECTIVE-We previously showed epidermal growth factor receptor (EGFR) transactivation to be key mechanism in the regulation of resistance artery myogenic tone. Type 2 diabetes is associated with microvascular complications. We hypothesized that elevated EGFR phosphorylation contributes to resistance artery dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS AND RESULTS-Diabetic db/db and nondiabetic (control) mice were treated with EGFR inhibitor (AG1478; 10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) for 2 weeks. Isolated coronary artery and mesenteric resistance artery (MRA) were mounted in an arteriograph. Pressure-induced myogenic tone was increased in MRA and coronary artery from diabetic mice and normalized by AG1478. Phenylephrine-induced contraction and nitric oxide donor-induced relaxation were similar in all groups. Endothelium-dependent relaxation in response to shear stress and acetylcholine of MRA and coronary artery from diabetic mice was altered and associated with reduced endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Treated diabetic mice with AG1478 improved coronary artery and MRA endothelial function and restored eNOS expression. Immunostaining and Western blot analysis showed increased endothelial and smooth muscle cell EGFR phosphorylation of MRA and coronary artery from diabetic mouse, which was reduced by AG1478. Primary cultured endothelial cells from resistance arteries treated with high glucose for 48 h showed an increase of EGFR phosphorylation associated with eNOS expression and phosphorylation decrease in response to calcium ionophore. Pretreatment of endothelial cells with AG1478 prevented the effect of high glucose.CONCLUSIONS-This study provides evidence of the role of elevated EGFR phosphorylation in coronary artery and MRA dysfunction in diabetic db/db mice. Therefore, EGFR should be a potential target for overcoming diabetic small artery complications.

Coronary artery disease in patients with hypertension is increasing worldwide and leads to severe cardiovascular complications. The cellular and molecular mechanisms that underlie this pathologic condition are not well understood. Experimental and clinical research indicates that immune cells and inflammation play a central role in the pathogenesis of cardiovascular diseases. Recently, it has been reported that CD4(+)CD25(+) regulatory T cells (Tregs) regulate heart fibrosis in hypertension. In this study, we determined the role of Tregs in coronary arteriolar endothelial dysfunction in angiotensin II-dependent hypertensive mice. Mice infused with angiotensin II had significantly increased blood pressure, as determined using telemetry, and apoptotic Treg numbers, as measured using flow cytometry. The mice displayed inflammation, assessed by macrophage activation/infiltration into coronary arterioles and the heart, and increased local tumor necrosis factor-α release, which participates in reduced coronary arteriolar endothelial-dependent relaxation in response to acetylcholine using an arteriograph. Hypertensive mice injected with Tregs isolated from control mice had significantly reduced macrophage activation and infiltration, reduced tumor necrosis factor-α release, and improved coronary arteriolar endothelium-dependent relaxation. Our novel data indicate that Tregs are important in the development of coronary arteriolar endothelial dysfunction in hypertension. These results suggest a new direction in the investigation of vascular disease in hypertension and could lead to a therapeutic strategy that involves immune system modulation using Tregs.