Vitamin E inhibits abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice.

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% (P<0.05, respectively). Importantly, animals treated with vitamin E showed a 44% reduction in the combined end point of fatal+nonfatal aortic rupture (P<0.05). Vitamin E also decreased aortic 8-isoprostane content (a marker of oxidative stress) and reduced both aortic macrophage infiltration and osteopontin expression (P<0.05, 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. CONCLUSIONS: Vitamin E ameliorates AAAs and reduces the combined end point of fatal+nonfatal aortic rupture in this animal model. These findings are consistent with the concept that oxidative stress plays a pivotal role in Ang II-driven AAA formation in hyperlipidemic mice.

Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells.

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 (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.

Ghrelin inhibits proinflammatory responses and nuclear factor-kappaB activation in human endothelial cells.

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-alpha (TNF-alpha), and induction of proinflammatory cytokines and mononuclear cell adhesion were determined. Ghrelin (0.1 to 1000 ng/mL) inhibited both basal and TNF-alpha-induced cytokine release and mononuclear cell binding. Intravenous administration of ghrelin also inhibited endotoxin-induced proinflammatory cytokine production in rats in vivo. Ghrelin inhibited H2O2-induced cytokine release in HUVECs, suggesting that the peptide blocks redox-mediated cellular signaling. Moreover, ghrelin inhibited basal and TNF-alpha-induced activation of nuclear factor-kappaB. Des-acyl ghrelin had no effect on TNF-alpha-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-kappaB 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.

Neonatal vulnerability to ischemia and reperfusion: Cardioplegic arrest causes greater myocardial apoptosis in neonatal lambs than in mature lambs.

OBJECTIVES: Apoptosis is a mechanism for deletion of injured or obsolete cells that is distinct from necrosis and mediated by mitochondrial release of cytochrome c caspase activation. Because myocardial apoptosis is a part of normal fetal and postnatal maturation, we hypothesize that neonatal myocardium is more vulnerable to undergo myocardial apoptosis than mature myocardium after cardioplegic arrest. METHODS: Newborn and mature lambs (n = 5 in each group) underwent cardiopulmonary bypass, antegrade crystalloid hyperkalemic cardioplegic arrest for 60 minutes, and a 6-hour recovery period. Myocardium was examined by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL), Western blotting, in vitro kinase assays, and fluorometric assays of the activity of caspases 3, 8, and 9. Myocardium from nonoperated control subjects (n = 5 in each age group) was also obtained. RESULTS: More TUNEL-positive nuclei were present in the newborn postcardioplegic myocardium (P =.04). Caspase 3, 8, and 9 activities were 1.6-fold, 1.5-fold, and 1.4-fold greater in the newborn postcardioplegic myocardium (P =.04, P =.01, and P =.01, respectively). The Bax/Bcl-2 ratio was higher in the newborn postcardioplegic myocardium (P =.04). Apoptosis signal-regulating kinase 1 activity and cleaved caspase 3 levels were higher in the newborn postcardioplegic myocardium (P =.02 and P =.009). Mitochondrial release of cytochrome c was greater in the newborn postcardioplegic myocardium (P =.009). CONCLUSIONS: The increased Bax/Bcl-2 ratio in the newborn myocardium suggests a proapoptotic state that is manifested by greater TUNEL staining, cytochrome c release, and cleavage of caspase 3. Increased apoptosis signal-regulating kinase 1 activity suggests greater oxidative stress, immature mechanisms to ameliorate oxidative stress, or both in the neonatal myocardium. Mitochondrial release of cytochrome c suggests that apoptosis-related mitochondrial dysfunction might contribute to early postoperative myocardial dysfunction in the neonate.

Protective effects of ghrelin on ischemia/reperfusion injury in the isolated rat heart.

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.

Enhanced cytomegalovirus infection in atherosclerotic human blood vessels.

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.

Low-level endotoxin induces potent inflammatory activation of human blood vessels: inhibition by statins.

BACKGROUND: Low-level endotoxemia (ie, >or=50 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. CONCLUSIONS: Clinically relevant levels of endotoxin, as reported in ambulatory populations, have profound inflammatory effects on intact HSV. Inhibition of endotoxin-induced vascular inflammation might contribute to the beneficial effects of statins in treating atherosclerosis.

Activation of NAD(P)H oxidase by lipid hydroperoxides: mechanism of oxidant-mediated smooth muscle cytotoxicity.

Oxidized lipids, such as 13-hydroperoxyoctadecadienoic acid (13-HPODE), have been implicated in the pathogenesis of atherosclerosis. 13-HPODE, a constituent of oxidized low-density lipoproteins, can induce cytotoxicity of vascular smooth muscle cells (SMC), which may facilitate plaque destabilization and/or rupture. 13-HPODE-induced cytotoxicity has been linked to oxidative stress, although the mechanisms by which this occurs are unknown. In the present study, we show that 13-HPODE and 9-HPODE (10-30 microM) increased superoxide (O2*-) production and induced cytotoxicity in SMC. The 13-HPODE-induced increase in O2*- was blocked by transfecting the cells with antisense oligonucleotides against p22phox, suggesting that the O2*- was produced by NAD(P)H oxidase. Similar concentrations of the corresponding HPODE reduction products, 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE, neither increased O2*- production nor induced cytotoxicity, while 4-hydroxy nonenal (4-HNE), an unsaturated aldehyde lipid peroxidation product, induced cytotoxicity without increasing O2*- production. Treatment with superoxide dismutase or Tiron to scavenge O2*-, or transfection with p22phox antisense oligonucleotides to inhibit O2*- production, attenuated 13-HPODE-induced cytotoxicity, but not that induced by 4-HNE. These findings suggest that activation of NAD(P)H oxidase, and production of O2*-, play an important role in lipid hydroperoxide-induced smooth muscle cytotoxicity.