eNAMPT Is a Novel Damage-associated Molecular Pattern Protein That Contributes to the Severity of Radiation-induced Lung Fibrosis.

The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and Nampt+/- heterozygous C57BL6 mice and nonhuman primates (NHPs, Macaca mulatta) were exposed to a single WTLI dose (9.8 or 10.7 Gy for NHPs, 20 Gy for mice). WT mice received IgG1 (control) or an eNAMPT-neutralizing polyclonal or monoclonal antibody (mAb) intraperitoneally 4 hours after WTLI and weekly thereafter. At 8-12 weeks after WTLI, NAMPT expression was assessed by immunohistochemistry, biochemistry, and plasma biomarker studies. RILF severity was determined by BAL protein/cells, hematoxylin and eosin, and trichrome blue staining and soluble collagen assays. RNA sequencing and bioinformatic analyses identified differentially expressed lung tissue genes/pathways. NAMPT lung tissue expression was increased in both WTLI-exposed WT mice and NHPs. Nampt+/- mice and eNAMPT polyclonal antibody/mAb-treated mice exhibited significantly attenuated WTLI-mediated lung fibrosis with reduced: 1) NAMPT and trichrome blue staining; 2) dysregulated lung tissue expression of smooth muscle actin, p-SMAD2/p-SMAD1/5/9, TGF-ß, TSP1 (thrombospondin-1), NOX4, IL-1ß, and NRF2; 3) plasma eNAMPT and IL-1ß concentrations; and 4) soluble collagen. Multiple WTLI-induced dysregulated differentially expressed lung tissue genes/pathways with known tissue fibrosis involvement were each rectified in mice receiving eNAMPT mAbs.The eNAMPT/TLR4 inflammatory network is essentially involved in radiation pathobiology, with eNAMPT neutralization an effective therapeutic strategy to reduce RILF severity.

Circulating eNAMPT as a biomarker in the critically ill: acute pancreatitis, sepsis, trauma, and acute respiratory distress syndrome.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) exhibits dual functionality - as an intracellular enzyme regulating nicotinamide adenine dinucleotide metabolism and as an extracellular secreted protein (eNAMPT) to function as a cytokine regulator of innate immunity via binding to Toll-Like receptor 4 and NF-κB activation. In limited preclinical and clinical studies, eNAMPT was implicated in the pathobiology of acute respiratory distress syndrome (ARDS) suggesting that eNAMPT could potentially serve as a diagnostic and prognostic biomarker. We investigated the feasibility of circulating eNAMPT levels to serve as a biomarker in an expanded cohort of patients with ARDS and ARDS-predisposing conditions that included acute pancreatitis, sepsis, and trauma with comparisons to controls. METHODS: A total of 671 patients and 179 healthy controls were included in two independent cohorts. Plasma and serum eNAMPT levels were quantified using one of two complementary Enzyme-linked Immunosorbent Assays. After log base 2 variance stabilizing transformation of plasma/serum eNAMPT measurements, differences between healthy controls and each disease cohort were compared using linear regression or a generalized estimating equation (GEE) model where applicable. Complementary analyses included sensitivity, specificity, positive predictive values, negative predictive values, and the area under the receiver operating curve. RESULTS: Compared to controls, circulating eNAMPT levels were significantly elevated in subjects with acute pancreatitis, sepsis, trauma, and ARDS (all p < 0.01). In the acute pancreatitis cohort, circulating eNAMPT levels positively correlated with disease severity (p < 0.01). CONCLUSIONS: Circulating eNAMPT levels are novel biomarker in the critically ill with acute pancreatitis, sepsis, trauma, and/or ARDS with the potential to reflect disease severity.

Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Direct Extracellular NAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling. Transcriptional Regulation by SOX and HIF-2α.

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-ß1 [transforming growth factor-ß1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between -628 bp and -328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.

Nicotinamide phosphoribosyltransferase purification using SUMO expression system.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via Toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-ß-d-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.

Development of a biomarker mortality risk model in acute respiratory distress syndrome.

BACKGROUND: There is a compelling unmet medical need for biomarker-based models to risk-stratify patients with acute respiratory distress syndrome. Effective stratification would optimize participant selection for clinical trial enrollment by focusing on those most likely to benefit from new interventions. Our objective was to develop a prognostic, biomarker-based model for predicting mortality in adult patients with acute respiratory distress syndrome. METHODS: This is a secondary analysis using a cohort of 252 mechanically ventilated subjects with the diagnosis of acute respiratory distress syndrome. Survival to day 7 with both day 0 (first day of presentation) and day 7 sample availability was required. Blood was collected for biomarker measurements at first presentation to the intensive care unit and on the seventh day. Biomarkers included cytokine-chemokines, dual-functioning cytozymes, and vascular injury markers. Logistic regression, latent class analysis, and classification and regression tree analysis were used to identify the plasma biomarkers most predictive of 28-day ARDS mortality. RESULTS: From eight biologically relevant biomarker candidates, six demonstrated an enhanced capacity to predict mortality at day 0. Latent-class analysis identified two biomarker-based phenotypes. Phenotype A exhibited significantly higher plasma levels of angiopoietin-2, macrophage migration inhibitory factor, interleukin-8, interleukin-1 receptor antagonist, interleukin-6, and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) compared to phenotype B. Mortality at 28 days was significantly higher for phenotype A compared to phenotype B (32% vs 19%, p = 0.04). CONCLUSIONS: An adult biomarker-based risk model reliably identifies ARDS subjects at risk of death within 28 days of hospitalization.

Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α-mediated Apoptosis in Human Lung Endothelial Cells.

Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α-induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α-induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF-α-induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α-induced human lung EC apoptosis, suggesting that NAMPT-dependent NAD+ generation is unlikely to be involved in regulation of TNF-α-induced EC apoptosis. We next confirmed that TNF-α-induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF-α-induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.

Transglutaminase 6: a protein associated with central nervous system development and motor function.

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second ß-barrel domain in man and a variant with truncated ß-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca(2+) and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca(2+) and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons.

Linkage of NAMPT promoter variants to eNAMPT secretion, plasma eNAMPT levels, and ARDS severity.

BACKGROUND AND OBJECTIVES: eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a novel DAMP and TLR4 ligand, is a druggable ARDS therapeutic target with NAMPT promoter SNPs associated with ARDS severity. This study assesses the previously unknown influence of NAMPT promoter SNPs on NAMPT transcription, eNAMPT secretion, and ARDS severity. METHODS AND DESIGN: Human lung endothelial cells (ECs) transfected with NAMPT promoter luciferase reporters harboring SNPs G-1535A, A-1001 C, and C-948A, were exposed to LPS or LPS/18% cyclic stretch (CS) and NAMPT promoter activity, NAMPT protein expression, and secretion assessed. NAMPT genotypes and eNAMPT plasma measurements (Days 0/7) were assessed in two ARDS cohorts (DISCOVERY n = 428; ALVEOLI n = 103). RESULTS: Comparisons of minor allelic frequency (MAF) in both ARDS cohorts with the 1000 Human Genome Project revealed the G-1535A and C-948A SNPs to be significantly associated with ARDS in Blacks compared with controls and trended toward significance in non-Hispanic Whites. LPS-challenged and LPS/18% CS-challenged EC harboring the -1535G wild-type allele exhibited significantly increased NAMPT promoter activity (compared with -1535A) with the -1535G/-948A diplotype exhibiting significantly increased NAMPT promoter activity, NAMPT protein expression, and eNAMPT secretion compared with the -1535A/-948 C diplotype. Highly significant increases in Day 0 eNAMPT plasma values were observed in both DISCOVERY and ALVEOLI ARDS cohorts (compared with healthy controls). Among subjects surviving to Day 7, Day 7 eNAMPT values were significantly increased in Day 28 non-survivors versus survivors. The protective -1535A SNP allele drove -1535A/-1001A and -1535A/-948 C diplotypes that confer significantly reduced ARDS risk (compared with -1535G, -1535G/-1001 C, -1535G/-948A), particularly in Black ARDS subjects. NAMPT SNP comparisons within the two ARDS cohorts did not identify significant association with either APACHE III scores or plasma eNAMPT levels. CONCLUSION: NAMPT SNPs influence promoter activity, eNAMPT protein expression/secretion, plasma eNAMPT levels, and ARDS severity. NAMPT genotypes are a potential tool for stratification in eNAMPT-focused ARDS clinical trials.

Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

Background: Progressive pulmonary fibrosis is a serious complication in subjects with sarcoidosis. The absence of reliable, non-invasive biomarkers that detect early progression exacerbates the difficulty in predicting sarcoidosis severity. To potentially address this unmet need, we evaluated a panel of markers for an association with sarcoidosis progression (HBEGF, NAMPT, IL1-RA, IL-6, IL-8, ANG-2). This panel encompasses proteins related to inflammation, vascular injury, cell proliferation, and fibroblast mitogenesis processes. Methods: Plasma biomarker levels and biomarker protein expression in lung and lymph nodes tissues (immunohistochemical studies) from sarcoidosis subjects with limited disease and progressive (complicated) sarcoidosis were performed. Gene expression of the protein-coding genes included in this panel was analyzed using RNAseq in sarcoidosis granulomatous tissues from lung and lymph nodes. Results: Except for IL-8, plasma levels of each biomarker-eNAMPT, IL-1RA, IL-6, ANG-2, and HBEGF-were significantly elevated in sarcoidosis subjects compared to controls. In addition, plasma levels of HBEGF were elevated in complicated sarcoidosis, while eNAMPT and ANG-2 were observed to serve as markers of lung fibrosis in a subgroup of complicated sarcoidosis. Genomic studies corroborated HBEGF and NAMPT among the top dysregulated genes and identified cytokine-related and fibrotic pathways in lung granulomatous tissues from sarcoidosis. Conclusion: These findings suggest HBEGF, eNAMPT, and ANG-2 may serve as potential novel indicators of the clinical severity of sarcoidosis disease.