Regular ArticleANP attenuates inflammatory signaling and Rho pathway of lung endothelial permeability induced by LPS and TNFα☆
Introduction
The lung endothelium forms a semi-selective barrier between circulating blood and interstitial fluid, which is dynamically regulated by a counterbalance of barrier-protective and barrier disruptive mediators present in the circulation. Prevalence of barrier disruptive stimuli results in increased permeability and movement of fluid, macromolecules and leukocytes into the interstitium. Because of its enormous surface area, the lung vasculature is particularly sensitive to barrier dysregulation with increases in vascular permeability leading to alveolar flooding (pulmonary edema) during inflammatory processes such as sepsis and acute respiratory distress syndrome (ARDS). Mechanisms which govern increased vascular permeability are under intense investigation, and various models of agonist-induced endothelial cell (EC) permeability have been developed (Dudek and Garcia, 2001, Lum and Malik, 1996, Mehta and Malik, 2006). However, little is known about intracellular processes, which determine EC barrier preservation in acute lung injury (ALI), and effective barrier-protective substances for ALI treatment remain to be identified.
Natriuretic peptides (atrial, brain, and C-type) belong to a family of mediators that regulate a variety of physiological functions including vascular tone, plasma volume, and renal function. The investigations of the action of atrial natriuretic peptide (ANP) in the cardiovascular system have concentrated mainly on the cGMP-dependent diuretic, natriuretic and vasodilating ANP effects (see Baxter, 2004 for review). However, ANP exhibits much broader range of biological activities including effects on endothelial function and inflammation, which suggest its role in the regulation of the lung function in the settings of acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury (Eison et al., 1988, Mitaka et al., 1992). In vivo and in vitro models of lung injury show that ANP can protect endothelial barrier function apart from its vasodilatory and natriuretic action (Furst et al., 2005, Imamura et al., 1988, Irwin et al., 2005, Mitaka et al., 1992).
Lipopolysaccharide (LPS) is an important structural component of the outer membrane of Gram-negative bacteria which can induce systemic inflammation and sepsis in mammals. Genetic mapping and animal models have identified Toll-like receptor 4 (TLR4) as an essential receptor for LPS signaling (Lu et al., 2008). TLR play an important role in activating signal transduction pathways leading to the induction of the inflammatory response. After binding to a ligand, TLR sequentially recruits the adaptor molecules MyD88, IL-1R-associated kinase (IRAK), and TNF receptor-associated factor 6 (TRAF6). In turn, these adaptor molecules activate MAP kinases JNK, p38, ERK1/2 and IκB, a cytoplasmic inhibitor of NFκB (Lu et al., 2008). The MAPK cascade activation contributes to the production of proinflammatory cytokines. Activation of IκB kinase complex results in phosphorylation of its components IκBα and IκBβ, ubiquitination and degradation of IκB, and release of the active NFκB complex which then translocates to the nucleus and binds to the promoters of various genes, activating transcription (see Lu et al., 2008 for review). Therefore, phosphorylation of the MAPKs and IκB degradation is a necessary step in the signaling cascade leading to transcription of inflammatory target genes. It is important to note that in addition to regulation of gene expression, MAPK signaling is also involved in remodeling of EC cytoskeleton and permeability changes by edemagenic and inflammatory mediators (Bogatcheva et al., 2003).
In this work we studied effects of ANP on the pulmonary EC permeability and cytoskeletal remodeling induced by proinflammatory agonists LPS and TNFα and linked them with activation of stress MAPK and NFκB cascades.
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Cell culture and reagents
Human pulmonary artery endothelial cells (HPAEC) and cell culture basal medium with growth supplements were obtained from Lonza Inc (Allendale, NJ). Cells were cultured according to the manufacturer's protocol, and used at passages 5–9. ANP was purchased from AnaSpec Inc (San Jose, CA). TNFα was obtained from R&D Systems (Minneapolis, MN). Di-phospho-MLC, phospho-p38, phospho-Erk1/2, phospho-IκBα, and IκBα antibodies were obtained from Cell Signaling (Beverly, MA); phospho-MYPT1 antibodies were
Effects of ANP on pulmonary EC hyperpermeability induced by inflammatory mediators
Effects of ANP on endothelial barrier dysfunction induced by inflammatory mediators LPS and TNFα were evaluated by measurements of permeability across the human pulmonary EC monolayers grown on gold microelectrodes. Our previous report showed that both, LPS and TNFα significantly decreased transendothelial electrical resistance (TER) in human pulmonary EC, reflecting endothelial barrier compromise, with maximal response at 6 h of agonist stimulation (Birukova et al., 2007). In the current
Discussion
This study shows that ANP exhibits potent protective effects on the pulmonary vascular EC exposed to inflammatory mediators LPS and TNFα. Observed protective effects on the EC barrier may explain in part dramatic increases in survival rates in the ANP-treated mice with induced septic shock (Ladetzki-Baehs et al., 2007). These protective effects of ANP may be mediated by several mechanisms discussed below.
Attenuation of Rho pathway as vascular barrier-protective mechanism triggered by ANP has
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Supported by HL89257 from the National Heart, Lung, and Blood Institute, the American Heart Association Midwest Affiliate Grant-in-Aid, and the American Lung Association Biomedical Research Grant.