Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations

The dietary polyphenolic compound resveratrol, by activating the protein deacetylase enzyme silent information regulator 2/sirtuin 1 (SIRT1), prolongs life span in evolutionarily distant organisms and may mimic the cytoprotective effects of dietary restriction. The present study was designed to elucidate the effects of resveratrol on cigarette smoke-induced vascular oxidative stress and inflammation, which is a clinically highly relevant model of accelerated vascular aging. Cigarette smoke exposure of rats impaired the acetylcholine-induced relaxation of carotid arteries, which could be prevented by resveratrol treatment. Smoking and in vitro treatment with cigarette smoke extract (CSE) increased reactive oxygen species production in rat arteries and cultured coronary arterial endothelial cells (CAECs), respectively, which was attenuated by resveratrol treatment. The smoking-induced upregulation of inflammatory markers (ICAM-1, inducible nitric oxide synthase, IL-6, and TNF-alpha) in rat arteries was also abrogated by resveratrol treatment. Resveratrol also inhibited CSE-induced NF-kappaB activation and inflammatory gene expression in CAECs. In CAECs, the aforementioned protective effects of resveratrol were abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicked the effects of resveratrol. Resveratrol treatment of rats protected aortic endothelial cells against cigarette smoking-induced apoptotic cell death. Resveratrol also exerted antiapoptotic effects in CSE-treated CAECs, which could be abrogated by knockdown of SIRT1. Resveratrol treatment also attenuated CSE-induced DNA damage in CAECs (comet assay). Thus resveratrol and SIRT1 exert antioxidant, anti-inflammatory, and antiapoptotic effects, which protect the endothelial cells against the adverse effects of cigarette smoking-induced oxidative stress. The vasoprotective effects of resveratrol will likely contribute to its antiaging action in mammals and may be especially beneficial in pathophysiological conditions associated with accelerated vascular aging.     

Selective I kappa B kinase expression in airway epithelium generates neutrophilic lung inflammation

To determine whether NF-kappaB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IkappaB kinase 1 (cIKK1) or IkappaB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-kappaB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing beta-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-kappaB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-kappaB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-kappaB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-kappaB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-kappaB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-kappaB activation by these kinases. These data show that selective expression of IkappaB kinases in airway epithelium results in NF-kappaB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-kappaB in lung epithelium may be beneficial in treating inflammatory lung diseases.     

Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig.

The pathogenesis of pulmonary hypertension in patients with chronic obstructive pulmonary disease is not understood. We have previously shown increased levels of mediators that control vasoconstriction (endothelin-1), vascular cell proliferation (endothelin-1 and vascular endothelial growth factor), and vasodilation (endothelial nitric oxide synthase) in the intrapulmonary arteries of animals exposed to cigarette smoke. To determine whether these mediators could be implicated in the structural remodeling of the arterial vasculature and increased pulmonary arterial pressure caused by chronic cigarette smoke exposure, guinea pigs were exposed to daily cigarette smoke for 6 mo. Pulmonary arterial pressures were measured. Intrapulmonary artery structure was analyzed by morphometry, artery mediator protein expression by immunohistochemistry, and artery mediator gene expression by laser capture microdissection and real-time RT-PCR. We found that the smoke-exposed animals developed increases in pulmonary arterial pressure and increased muscularization of the small pulmonary arteries. Gene expression and protein levels of all three mediators were increased, and pulmonary arterial pressure correlated both with the levels of mediator production and with the degree of arterial muscularization. We conclude that chronic smoke exposure produces increased vasoactive mediator expression in the small intrapulmonary arteries and that these mediators are associated with vascular remodeling as well as increased pulmonary arterial pressure. These findings support the idea that hypertension in chronic obstructive pulmonary disease is a result of direct cigarette smoke-mediated effects on the vasculature and suggest that interference with endothelin and VEGF production and activity or augmentation of nitric oxide levels may be beneficial.     

Functions and regulation of NF-kappaB RelA during pneumococcal pneumonia

Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation. To determine the roles of the NF-kappaB subunit RelA in facilitating these events, we infected RelA-deficient mice (generated on a TNFR1-deficient background) with Streptococcus pneumoniae. RelA deficiency decreased cytokine expression, alveolar neutrophil emigration, and lung bacterial killing. S. pneumoniae killing was also diminished in the lungs of mice expressing a dominant-negative form of IkappaBalpha in airway epithelial cells, implicating this cell type as an important locus of NF-kappaB activation during pneumonia. To study mechanisms of epithelial RelA activation, we stimulated a murine alveolar epithelial cell line (MLE-15) with bronchoalveolar lavage fluid (BALF) harvested from mice infected with S. pneumoniae. Pneumonic BALF, but not S. pneumoniae, induced degradation of IkappaBalpha and IkappaBbeta and rapid nuclear accumulation of RelA. Moreover, BALF-induced RelA activity was completely abolished following combined but not individual neutralization of TNF and IL-1 signaling, suggesting either cytokine is sufficient and necessary for alveolar epithelial RelA activation during pneumonia. Our results demonstrate that RelA is essential for the host defense response to pneumococcus in the lungs and that RelA in airway epithelial cells is primarily activated by TNF and IL-1.     

Shp2 plays an important role in acute cigarette smoke-mediated lung inflammation

Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.     

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.     

Cigarette smoke-induced proinflammatory alterations in the endothelial phenotype: role of NAD(P)H oxidase activation

Although the cardiovascular morbidity and mortality induced by cigarette smoking exceed those attributable to lung cancer, the molecular basis of smoking-induced vascular injury remains unclear. To test the link between cigarette smoke, oxidative stress, and vascular inflammation, rats were exposed to the smoke of five cigarettes per day (for 1 wk). Also, isolated arteries were exposed to cigarette smoke extract (CSE; 0 to 40 microg/ml, for 6 h) in organoid culture. We found that smoking impaired acetylcholine-induced relaxations of carotid arteries, which could be improved by the NAD(P)H oxidase inhibitor apocynin. Lucigenin chemiluminescence measurements showed that both smoking and in vitro CSE exposure significantly increased vascular O(2)(*-) production. Dihydroethidine staining showed that increased O(2)(*-) generation was present both in endothelial and smooth muscle cells. CSE also increased vascular H(2)O(2) production (dichlorofluorescein fluorescence). Vascular mRNA expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and that of inducible nitric oxide synthase was significantly increased by both smoking and CSE exposure, which could be prevented by inhibition of NAD(P)H oxidase (diphenyleneiodonium and apocynin) or scavenging of H(2)O(2). In cultured endothelial cells, CSE elicited NF-kappaB activation and increased monocyte adhesiveness, which were prevented by apocynin and catalase. Thus we propose that water-soluble components of cigarette smoke (which are likely to be present in the bloodstream in vivo in smokers) activate the vascular NAD(P)H oxidase. NAD(P)H oxidase-derived H(2)O(2) activates NF-kappaB, leading to proinflammatory alterations in vascular phenotype, which likely promotes development of atherosclerosis, especially if other risk factors are also present.     

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.     

TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways

Astrocytes participate in CNS innate immune responses as evident by their ability to produce a wide array of inflammatory mediators upon exposure to diverse stimuli. Although we have established that astrocytes use TLR2 to signal inflammatory mediator production in response to Staphylococcus aureus, a common etiological agent of CNS infections, the signal transduction pathways triggered by this pathogen and how TLR2 expression is regulated remain undefined. Three disparate inhibitors that block distinct steps in the NF-kappaB pathway, namely SC-514, BAY 11-7082, and caffeic acid phenethyl ester, attenuated NO, TNF-alpha, and CXCL2 release from S. aureus-activated astrocytes. Among these proinflammatory mediators, autocrine/paracrine TNF-alpha was pivotal for augmenting TLR2 expression, since receptor levels were not elevated in astrocytes isolated from TNF-alpha knockout mice upon bacterial exposure. Since TLR2 is critical for signaling astrocytic cytokine production in response to S. aureus, we evaluated the effect of TNF-alpha loss on proinflammatory mediator release. Interestingly, among the molecules assayed, only NO production was significantly attenuated in TNF-alpha knockout astrocytes compared with wild-type cells. Similar results were obtained following LPS treatment, suggesting that TNF-alpha is an important regulator of astrocytic TLR2 expression and NO release in response to diverse microbial stimuli. In addition, NF-kappaB inhibitors attenuated TNF-alpha-induced TLR2 expression in astrocytes. Overall, this study suggests that two important anti-bacterial effector molecules, TLR2 and NO, are regulated, in part, by NF-kappaB-dependent autocrine/paracrine effects of TNF-alpha in astrocytes.     

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro‐inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF‐κB nuclear accumulation, SIRT1 deacetylase activity, IL‐8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF‐κB binding to the IL‐8 gene promoter thus increasing IL‐8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF‐κB. FoxO3 siRNA treatment increased IL‐8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF‐κB activity and inducing pro‐inflammatory responses.     

Identification of NAP1, a regulatory subunit of IkappaB kinase-related kinases that potentiates NF-kappaB signaling

The IkappaB kinase (IKK)-related kinase NAK (also known as TBK or T2K) contributes to the activation of NF-kappaB-dependent gene expression. Here we identify NAP1 (for NAK-associated protein 1), a protein that interacts with NAK and its relative IKK epsilon (also known as IKKi). NAP1 activates NAK and facilitates its oligomerization. Interestingly, the NAK-NAP1 complex itself effectively phosphorylated serine 536 of the p65/RelA subunit of NF-kappaB, and this activity was stimulated by tumor necrosis factor alpha (TNF-alpha). Overexpression of NAP1 specifically enhanced cytokine induction of an NF-kappaB-dependent, but not an AP-1-dependent, reporter. Depletion of NAP1 reduced NF-kappaB-dependent reporter gene expression and sensitized cells to TNF-alpha-induced apoptosis. These results define NAP1 as an activator of IKK-related kinases and suggest that the NAK-NAP1 complex may protect cells from TNF-alpha-induced apoptosis by promoting NF-kappaB activation.