Background-Ghrelin is a novel growth hormone-releasing peptide that has been shown to improve cachexia in heart failure and cancer and to ameliorate the hemodynamic and metabolic disturbances in septic shock. Because cytokine-induced inflammation is critical in these pathological states and because the growth hormone secretagogue receptor has been identified in blood vessels, we examined whether ghrelin inhibits proinflammatory responses in human endothelial cells in vitro and after administration of endotoxin to rats in vivo. Methods and Results-Human umbilical vein endothelial cells (HUVECs) were treated with or without tumor necrosis factor-␣ (TNF-␣), and induction of proinflammatory cytokines and mononuclear cell adhesion were determined. Ghrelin (0.1 to 1000 ng/mL) inhibited both basal and TNF-␣-induced cytokine release and mononuclear cell binding. Intravenous administration of ghrelin also inhibited endotoxin-induced proinflammatory cytokine production in rats in vivo. Ghrelin inhibited H 2 O 2 -induced cytokine release in HUVECs, suggesting that the peptide blocks redox-mediated cellular signaling. Moreover, ghrelin inhibited basal and TNF-␣-induced activation of nuclear factor-B. Des-acyl ghrelin had no effect on TNF-␣-induced cytokine production in HUVECs, suggesting that the antiinflammatory effects of ghrelin require interaction with endothelial growth hormone secretagogue receptors. Conclusions-Ghrelin inhibits proinflammatory cytokine production, mononuclear cell binding, and nuclear factor-B activation in human endothelial cells in vitro and endotoxin-induced cytokine production in vivo. These novel antiinflammatory actions of ghrelin suggest that the peptide could play a modulatory role in atherosclerosis, especially in obese patients, in whom ghrelin levels are reduced.

Non-phagocytic NAD(P)H oxidases have been impliinduced stimulation of NAD(P)H oxidase activity in nonphagocytic cell types could represent a mechanism by which oxidant-mediated injury is amplified in vivo and identify these NAD(P)H oxidase enzymes as therapeutic targets for the amelioration of the biological effects of chronic oxidant stress. EXPERIMENTAL PROCEDURESCell Culture-SMC were prepared from 250 to 300-g male HarlanSprague Dawley rats as previously described (10, 11). Aortic adventitial fibroblasts were prepared from C57 BL/6 and from gp91 phox knock-out mice (Jackson Lab, Bar Harbor, ME) as described (12, 13). The cells were grown in minimum essential media supplemented with 10% fetal bovine serum and penicillin (100 IU/ml) and streptomycin (100 g/ml) in a humidified atmosphere containing 5% CO 2 at 37°C. Stocks were subcultured at subconfluence by trypsinization. All experiments were performed on cells between passages 8 and 20 grown to 70% to 95% confluence in 24-well plates, 35-or 60-mm dishes, or 4-well chamber slides.Superoxide Measurement by Enhanced Chemiluminescence-Lucigenin luminescence measurements were carried out using 5 M lucigenin, a concentration which is unlikely to induce redox cycling (14). Cells were grown to subconfluence in 35-mm dishes and growth arrested in serum-free medium for 24 h. After exposure to H 2 O 2 at the indicated concentrations and times, cells were washed twice with PBS, and 5 M lucigenin was added. Immediately afterward, NADPH (0.1 mM) was added, and after dark adaptation, luminescence was measured every 15 s for 5 min in a luminometer (Zylux Corp). Changes in luminescence were expressed as relative luminescent units per 10 5 cells per minute. No changes in luminescence were observed in the absence of NADPH, and addition of H 2 O 2 to lucigenin in PBS in the absence of cells did not increase chemiluminescence. Some experiments were also performed with coelenterazine (5 M), a structurally distinct luminescent probe which reportedly is not capable of redox cycling via autoxidation (15). Data were analyzed by ANOVA followed by Bonferroni t testing.Intracellular O 2 . Determination by Confocal Microscopy-SMC grown on chamber slides were treated as indicated under "Results" and in the figure legends, washed, and incubated with dihydroethidium (HE, 5

The role of reactive oxygen species, such as superoxide anions (O 2) and hydrogen peroxide (H 2 O 2), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H 2 O 2 , rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene,-galactosidase (AdLacZ). Infection with AdCat resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat, cellular catalase activity was increased by 50-to 100-fold, and intracellular H 2 O 2 concentration was reduced, as compared with control. Infection with AdCat reduced [ 3 H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.370.09 disintegrations per minute per cell [AdLacZ] versus 0.220.08 disintegrations per minute per cell [AdCat], P0.05). Five days after infection with 100 MOI of AdCat, cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 7808413 [AdCat], P0.05 versus 233 7003032 [noninfected] or 222 4105332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCat as compared with control. Infection with AdCat resulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H 2 O 2 importantly modulates survival and proliferation of vascular smooth muscle cells. (Circ Res. 1999;85:524-533.) Key Words: catalase apoptosis vascular smooth muscle cell cell proliferation hydrogen peroxide P roliferation of vascular smooth muscle cells is an important contributing factor in the pathophysiology of hyper-tension and atherosclerosis, as well as in coronary artery restenosis after angioplasty and stent placement. 1,2 However, the factors that induce proliferation of vascular smooth muscle cells, which normally exist in the arterial wall in a state of quiescence, are unknown. 3 Recently, it has been reported that reactive oxygen species, such as superoxide anions (O 2) and hydrogen peroxide (H 2 O 2), are capable of stimulating vascular smooth muscle cell proliferation. 4,5 These oxidants were shown to be rapidly produced by smooth muscle cells after exposure to platelet-derived growth factor or angiotensin II, factors that stimulate smooth muscle cell growth. 5,6 In addition, the production of reactive oxygen species in the blood vessel wall is enhanced in experimental models of hypercholesterolemia, hypertension, diabetes, and balloon injury to the coronary arteries. 7-10 Moreover, in angiotensin II-induced hypertension, excess free radical production was predominantly localized...

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, has been reported to have beneficial effects on cardiac function. The authors used the Langendorff model of ischemia/reperfusion (I/R) injury in isolated rat heart to determine whether ghrelin exerts direct cardioprotective effects. Also, the capacity of ghrelin to bind to sarcolemmal membrane fractions before and after ischemia and reperfusion was examined. Compared with vehicle administration, administration of ghrelin (100-10,000 pM) during the reperfusion period resulted in improvement in coronary flow, heart rate, left ventricular systolic pressure, and left ventricular end-diastolic pressure. Ghrelin also enhanced the rates of left ventricular contraction and relaxation after ischemia following reperfusion. Administration of ghrelin during reperfusion reduced myocardial release of lactate dehydrogenase and myoglobin, indicating protection against cardiomyocyte injury. In addition, ghrelin attenuated the depletion of myocardial ATP resulting from ischemia and reperfusion. A receptor-binding assay demonstrated that maximum binding capacity of ghrelin to sarcolemmal membranes was significantly increased after ischemia and was further increased after I/R. However, Scatchard analysis showed that the affinity of ghrelin for its receptor was not altered. The authors have concluded that administration of ghrelin during reperfusion protects against myocardial I/R injury. The cardioprotective effects are independent of growth hormone release and likely involve binding to cardiovascular receptors, a process that is upregulated during I/R.

Background-Low-level endotoxemia (ie, Ն50 pg/mL) in apparently healthy subjects was recently identified as a powerful, independent risk factor for atherosclerosis. Methods and Results-We treated human saphenous veins (HSVs) with low levels of endotoxin. Release of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA. Superoxide was determined by using the fluorescent probe dihydroethidium (HE), and monocyte binding was assessed with calcein-labeled U-937 cells. Three-to 4-fold increases in MCP-1 and IL-8 release were observed at endotoxin concentrations of 100 pg/mL; these increases were inhibited by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin. Studies in cultured endothelial cells suggest that the mechanism is related to inhibition of isoprenylation (ie, geranylgeranylation) rather than cholesterol formation. Endotoxin produced dose-dependent increases in HE fluorescence that were inhibited by the superoxide dismutase mimics Tiron and MnTBAP. Endotoxin potently induced U-937 cell binding to HSV; binding was inhibited by both Tiron and atorvastatin. Toll-like receptor-4 expression was detected in cultured HSV endothelial and smooth muscle cells and in intact HSV. Key Words: toll-like receptor-4 Ⅲ superoxide Ⅲ monocytes Ⅲ saphenous veins Ⅲ atorvastatin A therosclerosis of coronary arteries and coronary bypass grafts frequently occurs in patients without traditional cardiac risk factors. 1 Inflammatory markers have been correlated with increased risk of cardiovascular events, 2 suggesting that chronic inflammation might play a major role in the development of atherosclerosis and restenosis in many patients. 3,4 The factors responsible for vascular inflammation in atherosclerosis, however, are largely unknown. Conclusions-ClinicallyOne potentially important proatherosclerotic factor is circulating endotoxin, a unique glycolipid that comprises most of the outer leaflet of the outer wall of Gram-negative bacteria (GNB 1 ). 5 This complex molecule, found exclusively in GNB, consists of a highly variable carbohydrate portion and a unique lipid A region that is highly conserved across many GNB species. 5 Epidemiologic evidence suggests that endotoxemia at levels of Ն50 pg/mL constitutes a strong risk factor for the development of atherosclerosis, particularly among smokers. 6,7 Moreover, chronic infections conferred an increased risk of atherosclerosis even in low-risk subjects who lacked conventional vascular disease risk factors. 6,7 Low levels of endotoxemia in apparently healthy subjects might result from chronic or recurrent infection associated with the breaching of epithelial barrier function (eg, periodontitis, smoker's bronchitis, diverticulitis, etc). The circulating endotoxin activates inflammatory cells (ie, macrophages and neutrophils) by binding to CD14 through a process that is modulated by lipopolysaccharide-binding protein. 8 CD14 has no intracellular domain and interacts with Toll-like receptor-4 (TLR-4) ...

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (≤1 ng/ml) caused production of chemotactic cytokines in HCAEC. Endotoxin-induced cytokine production was maximal at LPS-binding protein:soluble CD14 ratios <1, typically observed in individuals with subclinical infection; higher LPS-binding protein:soluble CD14 ratios were inhibitory. Endotoxin potently activated HCASMC, with cytokine release >10-fold higher in magnitude at >10-fold lower threshold concentrations (10–30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.

Background-Abdominal aortic aneurysms (AAAs) in humans are associated with locally increased oxidative stress and activity of NADPH oxidase. We investigated the hypothesis that vitamin E, an antioxidant with documented efficacy in mice, can attenuate AAA formation during angiotensin II (Ang II) infusion in apolipoprotein E-deficient mice. Methods and Results-Six-month-old male apolipoprotein E-deficient mice were infused with Ang II at 1000 ng/kg per minute for 4 weeks via osmotic minipumps while consuming either a regular diet or a diet enriched with vitamin E (2 IU/g of diet). After 4 weeks, abdominal aortic weight and maximal diameter were determined, and aortic tissues were sectioned and examined using biochemical and histological techniques. Vitamin E attenuated formation of AAA, decreasing maximal aortic diameter by 24% and abdominal aortic weight by 34% (PϽ0.05, respectively). Importantly, animals treated with vitamin E showed a 44% reduction in the combined end point of fatalϩnonfatal aortic rupture (PϽ0.05). Vitamin E also decreased aortic 8-isoprostane content (a marker of oxidative stress) and reduced both aortic macrophage infiltration and osteopontin expression (PϽ0.05, respectively). Vitamin E treatment had no significant effect on the extent of aortic root atherosclerosis, activation of matrix metalloproteinases 2 or 9, serum lipid profile, or systolic blood pressure. Key Words: aneurysm Ⅲ vitamin E Ⅲ oxidative stress Ⅲ vascular inflammation Ⅲ NADPH oxidase Ⅲ osteopontin A bdominal aortic aneurysms (AAAs) occur in Ϸ3% of humans Ͼ65 years of age and are characterized by localized structural deterioration of the aortic wall, leading to progressive aortic dilation. The most dreaded complication of AAA is rupture, the likelihood of which is directly related to aneurysm diameter. 1 Although open surgical repair can improve outcomes in patients with large AAAs, the procedure is associated with significant operative risks and complications, particularly in the presence of comorbid conditions common in these patients. Percutaneous repair techniques (ie, stent grafting) have been developed, but not all patients are candidates, and these procedures are also associated with significant complications. 2 The development of effective medical therapy for AAA has been hampered by lack of understanding of the mechanisms responsible for aneurysm growth and rupture. Conclusions-VitaminStudies published over the past decade support the view that inflammation plays a key role in the pathogenesis of AAAs. [3][4][5][6][7] One of the known causes and consequences of inflammation is an increase in local levels of oxidative stress.Indeed, Miller et al have shown that human aneurysmal aorta displays clear evidence of increased oxidative damage and pro-oxidant enzyme expression/activity compared with adjacent nonaneurysmal tissue. 8 Whether oxidative stress is merely associated with AAAs, or whether it contributes to the pathogenesis of the disease, remains to be determined.In the present study, we examined the effects of ...

Human cytomegalovirus (CMV) is a possible co-factor in atherogenesis and vascular occlusion, but its ability to actively infect medium and large blood vessels is unclear. A vascular explant model was adapted to investigate CMV infection in human coronary artery, internal mammary artery (IMA), and saphenous vein (SV). Vascular explants were inoculated with CMV Towne or low-passage clinical isolate and examined in situ for CMV cytopathic effect and immediate-early and early antigens, as indicators of active infection. At 5 to 7 days after inoculation, we found that CMV Towne actively infected eight of eight different atherosclerotic blood vessel explants (coronary artery, n = 4; SV and IMA grafts, n = 4), whereas it only infected 2 of 14 nonatherosclerotic blood vessel explants (SV, n = 10; IMA, n = 4) (P = 0.001). The CMV clinical isolate actively infected none of six sets of nonatherosclerotic SV explants at 5 to 7 days after inoculation. The active CMV infections involved adventitial and, less frequently, intimal cells. A small subset of infected cells in atherosclerotic tissue expresses the endothelial cell marker CD31. Smooth muscle cells residing in both atherosclerotic and nonatherosclerotic blood vessels were free of active CMV infections even after all vascular tissue layers were exposed to the virus. In contrast, active CMV Towne infection was evident at 2 days after inoculation in smooth muscle cells and endothelial cells previously isolated from the SV tissues. We conclude that active CMV infection is enhanced in atherosclerotic blood vessels compared to atherosclerosis-free vascular equivalents, and this viral activity is restricted to subpopulations of intimal and adventitial cells.