The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1-phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR(1), we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR(1)) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar-capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR(1), intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR(1) regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR(1) inverse agonist, SB-649146, or S1PR(1)(+/-) mice exhibited reduced S1P/SEW-2871-mediated barrier protection after challenge with LPS. In contrast, S1PR(2)(-/-) knockout mice as well as mice with reduced S1PR(3) expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR(1) receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR(1) agonist concentration, and S1PR(1) expression in target tissues.

Rationale: We previously demonstrated pre-B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility. Objectives: To explore mechanistic participation of PBEF in ALI and ventilator-induced lung injury (VILI). Methods: Two models of VILI were utilized to explore the role of PBEF using either recombinant PBEF or PBEF 1/2 mice. Measurements and Main Results: Initial in vitro studies demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor with in vivo studies demonstrating marked increases in bronchoalveolar lavage (BAL) leukocytes (PMNs) after intratracheal injection in C57BL/6J mice. These changes were accompanied by increased BAL levels of PMN chemoattractants (KC and MIP-2) and modest increases in lung vascular and alveolar permeability. We next explored the potential synergism between rhPBEF challenge (intratracheal) and a model of limited VILI (4 h, 30 ml/kg tidal volume) and observed dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-a, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI-and VILI-associated gene modules (nuclear factor-kB, leukocyte extravasation, apoptosis, Toll receptor pathways). Heterozygous PBEF 1/2 mice were significantly protected (reduced BAL protein, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 ml/kg tidal volume) and exhibited significantly reduced expression of VILIassociated gene expression modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI. Conclusions: These studies implicate PBEF as a key inflammatory mediator intimately involved in both the development and severity of ventilator-induced ALI.

Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll–like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (z50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (z70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (z40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.

We explored the mechanistic involvement of the growth arrest and DNA damage-inducible gene GADD45a in lipopolysaccharide (LPS)- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated that GADD45a(-/-) mice are modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume VILI, with increases in microvascular permeability and bronchoalveolar lavage levels of inflammatory cytokines. Expression profiling of lung tissues from VILI-challenged GADD45a(-/-) mice revealed strong dysregulation in the B-cell receptor signaling pathway compared with wild-type mice and suggested the involvement of PI3 kinase/Akt signaling components. Western blot analyses of lung homogenates confirmed approximately 50% reduction in Akt protein levels in GADD45a(-/-) mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction, which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling.

Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1(-/-)) or with reduced expression of S1P receptors (S1PR1(+/-), S1PR2(-/-), and S1PR3(-/-)) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2×/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2×/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.

We have generated genetically engineered mice that are uniquely susceptible to lipopolysaccharide (LPS)-induced and mechanical ventilation-induced lung injury in a sex-specific and age-specific manner. These mice express a nonmuscle isoform of the myosin light chain kinase gene (nmMLCK2) targeted to the endothelium. Homozygous mice have significantly reduced fecundity and litter survival until weaning, and they are initially growth delayed but eventually exceed the size of wildtype littermates. Mice at all ages show increased protein transport across the lung barrier; however, the phenotype is most discernible in 8-12-week-old male mice. When subjected to a clinically relevant LPS-induced lung injury model, 8-12-week-old young females and 30-36-week-old males seem to be the most significantly injured group. In contrast, 30-36-week-old males remain the most significantly injured group when mechanically ventilated at high tidal volumes, which is a clinically relevant model of mechanical stress lung injury. These data reveal that nmMLCK2 overexpression in the endothelium exacerbates lung injury in vivo in a sexually dimorphic and age-dependent manner.Actin microfilaments generate force by virtue of their ability to contract away from their site of attachment to the plasma membrane, and they do so via traction of the myosin light chain head groups attached to actin filaments by cross-bridges. The cross-bridge pulling action of myosin involves a conformational change in the molecule initiated by energetic phosphorylation of Ser19 on the myosin light chains. 1 The key enzyme involved in this phosphorylation event is myosin light chain kinase (MLCK), which exists in different tissues, and it may be characterized broadly as skeletal/cardiac, smooth muscle, and nonmuscle myosin light chain kinase (nmMLCK) isoforms. Although biochemical evidence for the existence of a Ca 2+ /calmodulin-dependent nonmuscle isoform in cultured endothelial cell (EC) was suggested earlier, [ 2 ] and [ 3 ] the controversy as to whether this enzymatic activity is identical to the 110-kDa protein found abundantly in smooth muscle preparations was resolved when we provided conclusive proof of a larger MLCK protein isoform (210 kDa) by cloning the gene (MYLK) from a human EC-derived cDNA library. 4 The gene was subsequently mapped to chromosome 3q21. 5

Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif 2/2 mice to 20 Gy single-fraction thoracic radiation to investigate the agerelated role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8-to 16-month-old wild-type mice. In addition, radiated 8-to 16-month-old mif 2/2 mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H 2 O 2 ) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif 2/2 mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.