Inducible Nitric Oxide Synthase Contributes to Ventilator-induced Lung Injury

Abstract

Rationale: Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation. Objectives: This study investigated changes in lung NOS expression and activity in a mouse model of ventilatorinduced lung injury. Methods: C57BL/6J (wild-type [WT]) and iNOSdeficient (iNOS Ϫ/Ϫ ) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay. Results: iNOS mRNA and protein expression was absent in iNOS Ϫ/Ϫ mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS Ϫ/Ϫ mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS Ϫ/Ϫ mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS Ϫ/Ϫ mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS Ϫ/Ϫ mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine. Conclusion: Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury.Keywords: inducible nitric oxide synthase; lung permeability; mechanical ventilation Nitric oxide (NO) is involved in many physiologic and pathologic conditions, such as blood vessel relaxation, neurotransmission, and host defense. NO also plays a critical role in tissue injury in the context of various inflammatory conditions (1). NO is produced by three isoforms (neuronal, endothelial, and inducible) of NO synthase (NOS). Endothelial NOS (eNOS), which is calcium/calmodulin (Ca 2ϩ /CaM)-dependent and activated by agonists (e.g., acetylcholine), produces a low level of NO output, whereas inducible NOS (iNOS) is independent of Ca 2ϩ /CaM, is transcriptionally regulated by proinflammatory products (e.g., LPS) and cytokines (interleukin 1␤ [IL-1␤], tumor necrosis factor ␣ [TNF-␣], IFN-␥), and results in sustained and elevated (Received in original form November 19, 2004; accepted in final form May 20, 2005) Supported by awards from the National Heart, Lung, and Blood Institute (NIH R01 HL049441 and P50 HL 73994).* These authors contributed equally to the work presented in this article. release of NO (2). Therefore, overproduction of NO, in particular in the setting of superoxide production (3), leads to oxidative stress and tissue injury in conditions such as endotoxin-induced acute lung injury (ALI) (4).ALI is characterized by a severe inflammatory process, pr...

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

“…Neither PDE2A nor iNOS were evaluated in these studies. The inability of HV T ventilation alone to increase BAL inflammation or protein is consistent with previous work showing that isolated VILI decreases endothelial barrier function with only small changes in BAL leukocytes and protein (33).…”

“…Although we previously showed that 4 h of 20 ml/kg HV T ventilation increased protein extravasation and lung water in this preparation by a sGCdependent mechanism, HV T -induced increased lung PDE2A expression was examined only at 40 and 80 min in bufferperfused mouse lungs (39). Thus it is possible that an increase in PDE2A from HV T alone was not sustained for 4 h. A similar situation may exist for iNOS, which was previously shown to increase at 2 h in this intact mouse VILI model (33).…”

“…7B). Experiments utilizing NO synthase inhibitors or iNOS knockout mice in IT LPS or VILI models underscore the critical importance of iNOS in these injuries (24,31,33,42). For example, in IT LPS, NO production from iNOS was upstream of MIP-2 and TNF-␣ (31, 42) and contributed significantly to alveolar neutrophil influx (31,42), and alveolar-capillary barrier dysfunction (24,31,42).…”

“…LPS, through Toll-like receptor-4 signaling, was shown to cause increased NO from phosphorylated endothelial (eNOS) (11) and inducible nitric oxide synthase (iNOS) (24,31) in alveolar macrophages, neutrophils, epithelium, and endothelium. NO from both eNOS and iNOS was found to play a major pathogenic role in ALI from LPS (24,31,42) and in ventilator-induced lung injury (VILI) (33,39). These injurious effects of endogenous NO have been attributed to a reaction with superoxide to generate peroxynitrite (31,33).…”

“…NO from both eNOS and iNOS was found to play a major pathogenic role in ALI from LPS (24,31,42) and in ventilator-induced lung injury (VILI) (33,39). These injurious effects of endogenous NO have been attributed to a reaction with superoxide to generate peroxynitrite (31,33). In both LPS-induced ALI (47) and VILI (39), however, NO also activates soluble guanylyl cyclase (sGC), increasing production of cGMP in multiple cell types including endothelium (39), epithelium (9,17), and macrophages (3).…”