Hector Quijada

Pathologic Mechanical Stress and Endotoxin Exposure Increases Lung Endothelial Microparticle Shedding

Acute lung injury (ALI) results from infectious challenges and from pathologic lung distention produced by excessive tidal volume delivered during mechanical ventilation (ventilator-induced lung injury [VILI]) and is characterized by extensive alveolar and vascular dysfunction. Identification of novel ALI therapies is hampered by the lack of effective ALI/VILI biomarkers. We explored endothelial cell (EC)-derived microparticles (EMPs) (0.1-1 μm) as potentially important markers and potential mediators of lung vascular injury in preclinical models of ALI and VILI. We characterized EMPs (annexin V and CD31 immunoreactivity) produced from human lung ECs exposed to physiologic or pathologic mechanical stress (5 or 18% cyclic stretch [CS]) or to endotoxin (LPS). EC exposure to 18% CS or to LPS resulted in increased EMP shedding compared with static cells (∼ 4-fold and ∼ 2.5-fold increases, respectively). Proteomic analysis revealed unique 18% CS-derived (n = 10) and LPS-derived EMP proteins (n = 43). VILI-challenged mice (40 ml/kg, 4 h) exhibited increased plasma and bronchoalveolar lavage CD62E (E-selectin)-positive MPs compared with control mice. Finally, mice receiving intratracheal instillation of 18% CS-derived EMPs displayed significant lung inflammation and injury. These findings indicate that ALI/VILI-producing stimuli induce significant shedding of distinct EMP populations that may serve as potential ALI biomarkers and contribute to the severity of lung injury.

Nicotinamide phosphoribosyltransferase inhibitor is a novel therapeutic candidate in murine models of inflammatory lung injury.

We previously identified the intracellular nicotinamide phosphoribosyltransferase (iNAMPT, aka pre-B-cell colony enhancing factor) as a candidate gene promoting acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating nicotinamide phosphoribosyltransferase potently inducing NF-κB signaling in lung endothelium. iNAMPT also synthesizes intracellular nicotinamide adenine dinucleotide (iNAD) in response to extracellular oxidative stress, contributing to the inhibition of apoptosis via ill-defined mechanisms. We now further define the role of iNAMPT activity in the pathogenesis of ARDS/VILI using the selective iNAMPT inhibitor FK-866. C57/B6 mice were exposed to VILI (40 ml/kg, 4 h) or LPS (1.5 mg/kg, 18 h) after osmotic pump delivery of FK-866 (100 mg/kg/d, intraperitoneally). Assessment of total bronchoalveolar lavage (BAL) protein, polymorphonuclear neutrophil (PMN) levels, cytokine levels (TNF-α, IL-6, IL-1α), lung iNAD levels, and injury scores revealed that FK-866-mediated iNAMPT inhibition successfully reduced lung tissue iNAD levels, BAL injury indices, inflammatory cell infiltration, and lung injury scores in LPS- and VILI-exposed mice. FK-866 further increased lung PMN apoptosis, as reflected by caspase-3 activation in BAL PMNs. These findings support iNAMPT inhibition via FK-866 as a novel therapeutic agent for ARDS via enhanced apoptosis in inflammatory PMNs.

Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκ B Signaling and Inflammatory Lung Injury

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.

Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner

Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage, and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2+/+ but not in Nrf2−/− mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment.

The NAMPT promoter is regulated by mechanical stress, signal transducer and activator of transcription 5, and acute respiratory distress syndrome-associated genetic variants

Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.

Health Disparities in ARDS

Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critically ill patients occurring in the context of sepsis and other systemic inflammatory disorders. Health disparities correlated with socioeconomic status and health care access disproportionately affect racial and ethnic minorities and lead to differences in prevalence and outcomes of ARDS. In addition, the role of genetic and environmental factors in critical illness outcomes has been examined. Multiple studies have highlighted differences in allelic variant frequencies between European descendant and African descent individuals. These allelic variants play an important role in inflammatory and infectious pathways in conditions that predispose to ARDS. In this chapter, we address race and ethnicity in the context of the complex genetic and environmental interactions that influence disparities in outcomes in ARDS in African American and Hispanic/Latino populations, groups most affected by health care disparities. We postulate that multiple factors contribute to poor outcomes in ethnic and racial minorities with ARDS and that precision medicine can be a realistic approach to overcome those disparities.