Indika Edirisinghe

Activation of Nrf2 in Endothelial Cells Protects Arteries From Exhibiting a Proinflammatory State

Objective—Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries. Methods and Results—En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38–VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38–VCAM-1 signaling at the susceptible site in wild-type but not Nrf2−/− animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1. Conclusions—Nrf2 prevents ECs at the atheroprotected site from exhibiting a proinflammatory state via the suppression of p38–VCAM-1 signaling. Pharmacological activation of Nrf2 reduces EC activation at atherosusceptible sites and may provide a novel therapeutic strategy to prevent or reduce atherosclerosis.

Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice

A variety of mouse models have been used to study the pathogenesis of pulmonary emphysema/chronic obstructive pulmonary disease. The effect of cigarette smoke (CS) is believed to be strain dependent, because certain mouse strains are more susceptible or resistant to development of emphysema. However, the molecular basis of susceptibility of mouse strains to effects of CS is not known. We investigated the effect of CS on lungs of most of the commonly used mouse strains to study the molecular mechanism of susceptibility to effects of CS. C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice were exposed to CS for 3 consecutive days, and various parameters of inflammatory and oxidative responses were assessed in lungs of these mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to lung inflammatory and oxidant responses to CS exposure. The mouse strain that was more susceptible to effects of CS showed augmented lung inflammatory cell influx, activation of NF-kappaB and p38 MAPK, and increased levels of matrix metalloproteinase-9 and NF-kappaB-dependent proinflammatory cytokines compared with resistant mouse strains. Similarly, decreased levels of glutathione were associated with increased levels of lipid peroxidation products in susceptible mouse strains compared with resistant strains. Hence, we identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains could be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.

Deacetylases and NF-kappaB in redox regulation of cigarette smoke-induced lung inflammation: epigenetics in pathogenesis of COPD.

Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders including chronic obstructive pulmonary disease (COPD), due to its effect on pro-inflammatory gene transcription. Cigarette smoke-mediated oxidative stress activates NF-kappaB-dependent transcription of pro-inflammatory mediators either through activation of inhibitor kappaB-alpha kinase (IKK) and/or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-kappaB-co-activator complex formation results in targeted increase in chromatin modifications, such as histone acetylation leading to inflammatory gene transcription. NF-kappaB-dependent gene expression, at least in part, is regulated by changes in deacetylases such as histone deacetylases (HDACs) and sirtuins. Cigarette smoke and oxidants also alter the activity of HDACs and sirtuins by post-translational modifications by protein carbonylation and nitration, and in doing so further induce gene expression of pro-inflammatory mediators by chromatin modifications. In addition, cigarette smoke/oxidants can reduce glucocorticoid sensitivity by attenuating HDAC2 activity and expression, which may account for the glucocorticoid insensitivity in patients with COPD. Understanding the mechanisms of NF-kappaB regulation, and the balance between histone acetylation and deacetylation may lead to the development of novel therapies based on the pharmacological manipulation of IKK and deacetylases in lung inflammation and injury.

IKKα Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.

Berries: anti-inflammatory effects in humans.

A sustained pro-inflammatory state is a major contributing factor in chronic disease development, progression, and complication, including the most commonly known diseases: cardiovascular disease, Alzheimers, and type 2 diabetes. Fruits, such as berries, contain polyphenol compounds purported to have anti-inflammatory activity in humans. Among the most notable polyphenols in berries are anthocyanins, responsible for their distinctive colors of red, blue, and purple. Berries have been studied widely for their antioxidant properties; however, preclinical data suggest important effects on inflammatory pathways. Correspondingly, the effects of berries, including extracts and purified anthocyanins, have been the subject of a number of human trials. This review aims to evaluate the current state of the human science on berry (products) as a source of dietary polyphenols, particularly anthocyanins, to modulate inflammatory status. Identifying dietary strategies that manage the modern-day inflammatory burden has important implications for chronic disease risk reduction and informing dietary guidelines aimed at achieving and maintaining health.

Protective activity of processed tomato products on postprandial oxidation and inflammation: A clinical trial in healthy weight men and women

SCOPE This study was designed to evaluate the ability of tomato rich in lycopene to modify postprandial oxidative stress, inflammation, and endothelial function in healthy weight individuals. METHODS AND RESULTS Twelve women and 13 men (mean age = 27 ± 8 years; mean body mass index= 22 ± 2) consumed high-fat meals known to induce postprandial oxidative stress on two separate occasions containing either processed tomato product or non-tomato alternative. Blood samples were collected at 0, 30, 60, 90, 120 min, then hourly until 360 min. Flow-mediated dilation (FMD) was performed at 0 and 210 min. Endpoints included changes in glucose, insulin, lipids, oxidized low-density lipoprotein (OxLDL), inflammatory cytokines, and FMD. Both meals induced increases in plasma glucose, insulin, and lipid concentrations (p < 0.05). A trend for higher triglycerides at >240 min was observed after the tomato meal (p = 0.006). Tomato significantly attenuated high-fat meal-induced LDL oxidation (p < 0.05) and rise in interleukin-6 (p < 0.0001), a proinflammatory cytokine and inflammation marker. CONCLUSION The data indicate that consuming tomato products with a meal attenuates postprandial lipemia-induced oxidative stress and associated inflammatory response. The relevance of OxLDL and inflammation to vascular injury suggests a potentially important protective role of tomato in reducing cardiovascular disease risk. ClinicalTrials.gov Registration number - NCT00966550.

Effect of grape seed extract on blood pressure in subjects with the metabolic syndrome

This study was undertaken to determine whether grape seed extracts (GSE) that contain powerful vasodilator phenolic compounds lower blood pressure in subjects with the metabolic syndrome. The subjects were randomized into 3 groups-(a) placebo, (b) 150 mg GSE per day, and (c) 300 mg GSE per day-and treated for 4 weeks. Serum lipids and blood glucose were measured at the beginning of the study and at the end. Blood pressure was recorded using an ambulatory monitoring device at the start of the treatment period and at the end. Both the systolic and diastolic blood pressures were lowered after treatment with GSE as compared with placebo. There were no significant changes in serum lipids or blood glucose values. These findings suggest that GSE could be used as a nutraceutical in a lifestyle modification program for patients with the metabolic syndrome.

VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction

Vascular endothelial growth factor (VEGF) induces phosphorylation of VEGF receptor‐2 (VEGFR‐2) and activates the downstream signaling pathway resulting in endothelial cell migration, proliferation, and survival. Cigarette smoking is associated with abnormal vascular and endothelial function, leading to airspace enlargement. Herein, we investigated the mechanism of cigarette smoke (CS) ‐induced endothelial dysfunction by studying the VEGF‐VEGFR‐2 signaling in mouse lung and human endothelial cells. CS exposure caused oxidative stress, as shown by increased levels of 4‐hydroxy‐2‐nonenal‐adducts in mouse lung and reactive oxygen species generation in human lung microvascular endothelial cells (HMVEC‐Ls). Inhibition of VEGFR‐2 by a specific kinase inhibitor (NVP‐AAD777) enhanced the CS‐induced oxidative stress, causing augmented inflammatory cell influx and proinflammatory mediators release in mouse lung. The levels of endothelial nitric oxide synthase (eNOS) and phosphorylated (p) ‐eNOS in the lungs of mice exposed to CS and/or treated with VEGFR‐2 inhibitor were decreased. CS down‐regulated VEGFR‐2 expression, eNOS levels, and VEGF‐induced VEGFR‐2 phosphorylation in HMVEC‐Ls, resulting in impaired VEGF‐induced endothelial cell migration and angiogenesis. Overall, these data show that inhibition of VEGFR‐2 augmented CS‐induced oxidative stress and inflammatory responses leading to endothelial dysfunction. This explains the mechanism of endothelial dysfunction in smokers and has implications in understanding the pathogenesis of pulmonary and cardiovascular diseases.—Edirisinghe, I., Yang, S.‐E., Yao, H., Rajendrasozhan, S., Caito, S., Adenuga, D., Wong, C., Rahman, A., Phipps, R. P., Jin, Z.‐G., Rahman, I. VEGFR‐2 inhibition augments cigarette smoke‐induced oxidative stress and inflammatory responses leading to endothelial dysfunction. FASEB J. 22, 2297–2310 (2008)

Mechanism of the endothelium-dependent relaxation evoked by a grape seed extract

GSEs (grape seed extracts) which contain polyphenolic compounds cause an endothelium-dependent relaxation of blood vessels. The aim of the present study was to examine the mechanisms involved in this response. A well-characterized GSE was applied to rabbit aortic rings suspended in organ baths containing Krebs-Henseleit buffer maintained at 37 degrees C. In aortic rings pre-contacted with noradrenaline (norepinephrine), the extract produced a dose-dependent relaxation. The maximum relaxations elicited by the extract (71.9+/-1.0%) were similar to those elicited by acetylcholine (64.2+/-1.5%) (n=12 for each). As expected, the relaxations were abolished by removal of the endothelium and by prior incubation with L-NAME (N(G)-nitro-L-arginine methyl ester), confirming the essential role of eNOS (endothelial NO synthase) in the response. The responses to the GSE were also abolished by incubation with wortmannin and LY294002, which are inhibitors of PI3K (phosphoinositide 3-kinase). These compounds had no effect on the responses to acetylcholine. Using immunoblotting, we also demonstrated that the GSE induced the phosphorylation of both Akt and eNOS in HUVECs (human umbilical vein endothelial cells). Finally, the extract was modified by methylation of the hydroxy groups in the polyphenolic groups and was applied to the aortic rings. The modified extract failed to cause a relaxation. Taken together, these findings suggest that the endothelium-dependent relaxation induced by the GSE was mediated by activation of the PI3K/Akt signalling pathway through a redox-sensitive mechanism, resulting in phosphorylation of eNOS.

Effect of fatty acids on endothelium-dependent relaxation in the rabbit aorta

The metabolic syndrome, Type II (non-insulin-dependent) diabetes and obesity are associated with endothelial dysfunction and increased plasma concentrations of NEFAs (non-esterified fatty acids; free fatty acids). The present study was undertaken to define the inhibitory effects of saturated NEFAs on EDR (endothelium-dependent relaxation). Experiments were performed in rings of rabbit aorta to establish (i) dose-response relationships, (ii) the effect of chain length, (iii) the effect of the presence of double bonds, (iv) reversibility and time course of inhibition, and (v) the effect on nitric oxide production. Aortic rings were incubated (1 h) with NEFA-albumin complexes derived from lauric (C(12:0)), myristic (C(14:0)), palmitic (C(16:0)), stearic (C(18:0)) and linolenic (C(18:3)) acids. EDR induced by acetylcholine (0.1-10 mumol/l) was measured after pre-contraction with noradrenaline. Inhibition of EDR was dose-dependent (0.5-2 mmol/l NEFA), and the greatest inhibition (51%) was observed with stearic acid (2 mmol/l). Lauric acid had the smallest inhibitory effect. The inhibitory effects were always reversible and were evident after 15 min of incubation. Linolenic acid caused a significantly lower inhibition of EDR than stearic acid. SOD (superoxide dismutase) restored the inhibitory effect caused by NEFAs, suggesting the involvement of ROS (reactive oxygen species) in removing nitric oxide. The nitric oxide concentration measured after exposure of the rings to acetylcholine was lower after incubation with NEFAs than with Krebs buffer alone. This finding is consistent with removal of nitric oxide by ROS. This claim was supported by the demonstration of increased concentrations of nitrated tyrosine in the rings incubated with NEFAs.