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Background-Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. Methods and Results-To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2-deficient mice to a model of chronic normobaric hypoxia. COX-2-null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2-deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2-deficient mice had significant upregulation of the endothelin-1 receptor (ET A ) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET A receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2-deficient PASMCs with iloprost, a prostaglandin I 2 analog, and prostaglandin E 2 abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. Conclusions-Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET A receptor expression and increased PASMC hypertrophy. COX-2-deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2-derived prostaglandin I 2 or prostaglandin E 2 .

Background Isoflurane may be protective in pre-clinical models of lung injury but its use in patients with lung injury remains controversial and the mechanism of its protective effects remains unclear. We hypothesized that this protection is mediated at the level of alveolar tight junctions and investigated the possibility in a two-hit model of lung injury that mirrors human acute respiratory distress syndrome. Methods Wild-type mice were treated with isoflurane one hour after exposure to nebulized endotoxin (n=8) or saline control (n=9) then allowed to recover for 24 hrs prior to mechanical ventilation (MV, tidal volume 15 mL/kg, 2 hrs) producing ventilator-induced lung injury. Mouse lung epithelial cells were similarly treated with isoflurane one hour after exposure to lipopolysaccharide. Cells were cyclically stretched the following day to mirror the MV protocol used in vivo. Results Mice treated with isoflurane following exposure to inhaled endotoxin and prior to MV exhibited significantly less physiologic lung dysfunction. These effects appeared to be mediated by decreased vascular leak, but not altered inflammatory indices. Mouse lung epithelial cells treated with lipopolysaccharide and cyclic stretch and lungs harvested from mice following treatment with lipopolysaccharide and MV had decreased levels of a key tight junction protein (i.e. zona occludens 1) that was rescued by isoflurane treatment. Conclusions Isoflurane rescued lung injury induced by a two-hit model of endotoxin exposure followed by MV by maintaining the integrity of the alveolar-capillary barrier possibly by modulating the expression of a key tight junction protein.

High-mobility group box 1 (HMGB1) is a nuclear protein that has been found to be a critical mediator of lethality in endotoxemia and sepsis. During the systemic inflammatory response, circulating levels of HMGB1 are increased, but in a delayed fashion compared with early inflammatory mediators. To counteract the inflammatory response of endotoxemia, a secondary anti-inflammatory response ensues in an attempt to prevent inflammation-induced tissue injury. One such cytoprotective gene that is induced during endotoxemia is heme oxygenase (HO)-1. HO-1, and its products of heme metabolism, possess anti-inflammatory and antioxidant properties to counter the damaging effects of endotoxemia. In the present study, we wanted to determine whether tissue and circulating levels of HMGB1 are increased further in the absence of HO-1 during endotoxemia, and whether this increase may contribute to the pathobiology of endotoxemia. Lung inflammation, HMGB1 protein levels, and expression of HMGB1 in inflammatory cells were increased in HO-1 2/2 mice compared with HO-1 1/1 mice. After the administration of LPS, tissue levels of HMGB1 were not increased further in HO-1 2/2 mice; however, circulating levels of HMGB1 were higher when compared with HO-1 1/1 mice. HO-1 2/2 mice treated with a carbon monoxide-releasing molecule or biliverdin showed a reduction in plasma HMGB1, which was associated with a marked improvement in survival. HO-1 2/2 mice given HMGB1-neutralizing antibody showed improvement in survival compared with control antibody. These data suggest that exaggerated circulating levels of HMGB1 contribute to endotoxin-induced mortality in the absence of HO-1.

NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2−/−) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2−/− mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.

BackgroundThe architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.ObjectivesTo determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.Methodology/Principal FindingsAdministration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.Conclusions/SignificanceWe describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

SYNOPSIS The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with gram-negative bacteria that elaborate endotoxin. The high mobility group A1 (HMGA1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule, minor groove binder (MGB) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. Therefore we hypothesized that netropsin would improve survival from murine endotoxemia by attenuating NOS2 induction through interference with HMGA1-DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxemia in wild type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1-DNA binding site was critical for this effect. EMSAs demonstrated that netropsin interferes with HMGA1-NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, nuclear Overhauser effect spectroscopy (NOESY) data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1 binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modeling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes important for the development of critical illness.

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis.