Alveolar type II epithelial cell FASN maintains lipid homeostasis in experimental COPD.

Alveolar epithelial type II (AEC2) cells strictly regulate lipid metabolism to maintain surfactant synthesis. Loss of AEC2 cell function and surfactant production are implicated in the pathogenesis of the smoking-related lung disease chronic obstructive pulmonary disease (COPD). Whether smoking alters lipid synthesis in AEC2 cells and whether altering lipid metabolism in AEC2 cells contributes to COPD development are unclear. In this study, high-throughput lipidomic analysis revealed increased lipid biosynthesis in AEC2 cells isolated from mice chronically exposed to cigarette smoke (CS). Mice with a targeted deletion of the de novo lipogenesis enzyme, fatty acid synthase (FASN), in AEC2 cells (FasniΔAEC2) exposed to CS exhibited higher bronchoalveolar lavage fluid (BALF) neutrophils, higher BALF protein, and more severe airspace enlargement. FasniΔAEC2 mice exposed to CS had lower levels of key surfactant phospholipids but higher levels of BALF ether phospholipids, sphingomyelins, and polyunsaturated fatty acid-containing phospholipids, as well as increased BALF surface tension. FasniΔAEC2 mice exposed to CS also had higher levels of protective ferroptosis markers in the lung. These data suggest that AEC2 cell FASN modulates the response of the lung to smoke by regulating the composition of the surfactant phospholipidome.

Association of urine mitochondrial DNA with clinical measures of COPD in the SPIROMICS cohort.

BACKGROUNDMitochondrial dysfunction, a proposed mechanism of chronic obstructive pulmonary disease (COPD) pathogenesis, is associated with the leakage of mitochondrial DNA (mtDNA), which may be detected extracellularly in various bodily fluids. Despite evidence for the increased prevalence of chronic kidney disease in COPD subjects and for mitochondrial dysfunction in the kidneys of murine COPD models, whether urine mtDNA (u-mtDNA) associates with measures of disease severity in COPD is unknown.METHODSCell-free u-mtDNA, defined as copy number of mitochondrially encoded NADH dehydrogenase-1 (MTND1) gene, was measured by quantitative PCR and normalized to urine creatinine in cell-free urine samples from participants in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Urine albumin/creatinine ratios (UACR) were measured in the same samples. Associations between u-mtDNA, UACR, and clinical disease parameters - including FEV1 % predicted, clinical measures of exercise tolerance, respiratory symptom burden, and chest CT measures of lung structure - were examined.RESULTSU-mtDNA and UACR levels were measured in never smokers (n = 64), smokers without airflow obstruction (n = 109), participants with mild/moderate COPD (n = 142), and participants with severe COPD (n = 168). U-mtDNA was associated with increased respiratory symptom burden, especially among smokers without COPD. Significant sex differences in u-mtDNA levels were observed, with females having higher u-mtDNA levels across all study subgroups. U-mtDNA associated with worse spirometry and CT emphysema in males only and with worse respiratory symptoms in females only. Similar associations were not found with UACR.CONCLUSIONU-mtDNA levels may help to identify distinct clinical phenotypes and underlying pathobiological differences in males versus females with COPD.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov ( NCT01969344).FUNDINGUS NIH, National Heart, Lung and Blood Institute, supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from AstraZeneca/MedImmune, Bayer, Bellerophon Therapeutics, Boehringer-Ingelheim Pharmaceuticals Inc., Chiesi Farmaceutici S.p.A., Forest Research Institute Inc., GlaxoSmithKline, Grifols Therapeutics Inc., Ikaria Inc., Novartis Pharmaceuticals Corporation, Nycomed GmbH, ProterixBio, Regeneron Pharmaceuticals Inc., Sanofi, Sunovion, Takeda Pharmaceutical Company, and Theravance Biopharma and Mylan.

Mitophagy-dependent macrophage reprogramming protects against kidney fibrosis.

Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators mitofusin-2 (MFN2) and Parkin downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1-/- or Prkn-/- BM-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-ß1-treated Pink1-/- BMDMs exhibited increased superoxide levels, along with reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2, MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-ß1-treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to our knowledge to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating the PINK1/MFN2/Parkin-mediated pathway.

Fatty acid synthase downregulation contributes to acute lung injury in murine diet-induced obesity.

The prevalence of obesity is rising worldwide and obese patients comprise a specific population in the intensive care unit. Acute respiratory distress syndrome (ARDS) incidence is increased in obese patients. Exposure of rodents to hyperoxia mimics many of the features of ARDS. In this report, we demonstrate that high fat diet induced obesity increases the severity of hyperoxic acute lung injury in mice in part by altering fatty acid synthase (FASN) levels in the lung. Obese mice exposed to hyperoxia had significantly reduced survival and increased lung damage. Transcriptomic analysis of lung homogenates identified Fasn as one of the most significantly altered mitochondrial associated genes in mice receiving 60% compared to 10% fat diet. FASN protein levels in the lung of high fat diet mice were lower by immunoblotting and immunohistochemistry. Depletion of FASN in type II alveolar epithelial cells resulted in altered mitochondrial bioenergetics and more severe lung injury with hyperoxic exposure, even upon the administration of a 60% fat diet. This is the first study to show that a high fat diet leads to altered FASN expression in the lung and that both a high fat diet and reduced FASN expression in alveolar epithelial cells promote lung injury.

Circulating cell death biomarker TRAIL is associated with increased organ dysfunction in sepsis.

BACKGROUND: In sepsis, there may be dysregulation in programed cell death pathways, typified by apoptosis and necroptosis. Programmed cell death pathways may contribute to variability in the immune response. TRAIL is a potent inducer of apoptosis. Receptor-interacting serine/threonine protein kinase-3 (RIPK3) is integral to the execution of necroptosis. We explored whether plasma TRAIL levels were associated with in-hospital mortality, organ dysfunction, and septic shock. We also explored the relationship between TRAIL and RIPK3. METHODS: We performed an observational study of critically ill adults admitted to intensive care units at 3 academic medical centers across 2 continents, using 1 as derivation and the other 2 as validation cohorts. Levels of TRAIL were measured in the plasma of 570 subjects by ELISA. RESULTS: In all cohorts, lower (<28.5 pg/ml) versus higher levels of TRAIL were associated with increased organ dysfunction (P ≤ 0.002) and septic shock (P ≤ 0.004). Lower TRAIL levels were associated with in-hospital mortality in 2 of 3 cohorts (Weill Cornell-Biobank of Critical Illness, P = 0.012; Brigham and Women's Hospital Registry of Critical Illness, P = 0.011; Asan Medical Center, P = 0.369). Lower TRAIL was also associated with increased RIPK3 (P ≤ 0.001). CONCLUSION: Lower levels of TRAIL were associated with septic shock and organ dysfunction in 3 independent ICU cohorts. TRAIL was inversely associated with RIPK3 in all cohorts. FUNDING: NIH (R01-HL055330 and KL2-TR002385).

A phase I trial of low-dose inhaled carbon monoxide in sepsis-induced ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a prevalent disease with significant mortality for which no effective pharmacologic therapy exists. Low-dose inhaled carbon monoxide (iCO) confers cytoprotection in preclinical models of sepsis and ARDS. METHODS: We conducted a phase I dose escalation trial to assess feasibility and safety of low-dose iCO administration in patients with sepsis-induced ARDS. Twelve participants were randomized to iCO or placebo air 2:1 in two cohorts. Four subjects each were administered iCO (100 ppm in cohort 1 or 200 ppm in cohort 2) or placebo for 90 minutes for up to 5 consecutive days. Primary outcomes included the incidence of carboxyhemoglobin (COHb) level ≥10%, prespecified administration-associated adverse events (AEs), and severe adverse events (SAEs). Secondary endpoints included the accuracy of the Coburn-Forster-Kane (CFK) equation to predict COHb levels, biomarker levels, and clinical outcomes. RESULTS: No participants exceeded a COHb level of 10%, and there were no administration-associated AEs or study-related SAEs. CO-treated participants had a significant increase in COHb (3.48% ± 0.7% [cohort 1]; 4.9% ± 0.28% [cohort 2]) compared with placebo-treated subjects (1.97% ± 0.39%). The CFK equation was highly accurate at predicting COHb levels, particularly in cohort 2 (R2 = 0.9205; P < 0.0001). Circulating mitochondrial DNA levels were reduced in iCO-treated participants compared with placebo-treated subjects. CONCLUSION: Precise administration of low-dose iCO is feasible, well-tolerated, and appears to be safe in patients with sepsis-induced ARDS. Excellent agreement between predicted and observed COHb should ensure that COHb levels remain in the target range during future efficacy trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02425579. FUNDING: NIH grants P01HL108801, KL2TR002385, K08HL130557, and K08GM102695.

Beclin-1 regulates cigarette smoke-induced kidney injury in a murine model of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), associated with cigarette smoke-induced (CS-induced) emphysema, contributes significantly to the global health care burden of disease. Although chronic kidney disease (CKD) may occur in patients with COPD, the relationship between COPD and CKD remains unclear. Using a murine model of experimental COPD, we show that chronic CS exposure resulted in marked kidney injury and fibrosis, as evidenced by histological and ultrastructural changes, altered macrophage subpopulations, and expression of tissue injury, fibrosis, and oxidative stress markers. CS induced mitochondrial dysfunction, and increased autophagic flux in kidney tissues and in kidney tubular epithelial (HK-2) cells, as determined by LC3B turnover assays. Mice heterozygous for Beclin-1 (Becn1+/-) were protected from the development of kidney tissue injury and renal fibrosis in response to CS exposure, and displayed impaired basal and inducible mitochondrial turnover by mitophagy. Interestingly, CS caused a reduction of Beclin-1 expression in mouse kidneys and kidney tubular epithelial cells, attributed to increased autophagy-dependent turnover of Beclin-1. These results suggest that Beclin-1 is required for CS-induced kidney injury and that reduced levels of Beclin-1 may confer renoprotection. These results identify the kidney as a target for CS-induced injury in COPD and the Beclin-1-dependent autophagy pathway as a potential therapeutic target in CKD.

Circulating RIPK3 levels are associated with mortality and organ failure during critical illness.

BACKGROUND: Necroptosis is a form of programmed necrotic cell death that is rapidly emerging as an important pathophysiological pathway in numerous disease states. Necroptosis is dependent on receptor-interacting protein kinase 3 (RIPK3), a protein shown to play an important role in experimental models of critical illness. However, there is limited clinical evidence regarding the role of extracellular RIPK3 in human critical illness. METHODS: Plasma RIPK3 levels were measured in 953 patients prospectively enrolled in 5 ongoing intensive care unit (ICU) cohorts in both the USA and Korea. RIPK3 concentrations among groups were compared using prospectively collected phenotypic and outcomes data. RESULTS: In all 5 cohorts, extracellular RIPK3 levels in the plasma were higher in patients who died in the hospital compared with those who survived to discharge. In a combined analysis, increasing RIPK3 levels were associated with elevated odds of in-hospital mortality (odds ratio [OR] 1.7 for each log10-unit increase in RIPK3 level, P < 0.0001). When adjusted for baseline severity of illness, the OR for in-hospital mortality remained statistically significant (OR 1.33, P = 0.007). Higher RIPK3 levels were also associated with more severe organ failure. CONCLUSIONS: Our findings suggest that elevated levels of RIPK3 in the plasma of patients admitted to the ICU are associated with in-hospital mortality and organ failure. FUNDING: Supported by NIH grants P01 HL108801, R01 HL079904, R01 HL055330, R01 HL060234, K99 HL125899, and KL2TR000458-10. Supported by Samsung Medical Center grant SMX1161431.

RIPK3 mediates pathogenesis of experimental ventilator-induced lung injury.

In patients requiring ventilator support, mechanical ventilation (MV) may induce acute lung injury (ventilator-induced lung injury [VILI]). VILI is associated with substantial morbidity and mortality in mechanically ventilated patients with and without acute respiratory distress syndrome. At the cellular level, VILI induces necrotic cell death. However, the contribution of necroptosis, a programmed form of necrotic cell death regulated by receptor-interacting protein-3 kinase (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL), to the development of VILI remains unexplored. Here, we show that plasma levels of RIPK3, but not MLKL, were higher in patients with MV (i.e., those prone to VILI) than in patients without MV (i.e., those less likely to have VILI) in two large intensive care unit cohorts. In mice, RIPK3 deficiency, but not MLKL deficiency, ameliorated VILI. In both humans and mice, VILI was associated with impaired fatty acid oxidation (FAO), but in mice this association was not observed under conditions of RIPK3 deficiency. These findings suggest that FAO-dependent RIPK3 mediates pathogenesis of acute lung injury.

RIPK3 promotes kidney fibrosis via AKT-dependent ATP citrate lyase.

Renal fibrosis is a common pathogenic response to injury in chronic kidney disease (CKD). The receptor-interacting protein kinase-3 (RIPK3), a regulator of necroptosis, has been implicated in disease pathogenesis. In mice subjected to unilateral ureteral obstruction-induced (UUO-induced) or adenine diet-induced (AD-induced) renal fibrosis, models of progressive kidney fibrosis, we demonstrate increased kidney expression of RIPK3. Mice genetically deficient in RIPK3 displayed decreased kidney fibrosis and improved kidney function relative to WT mice when challenged with UUO or AD. In contrast, mice genetically deficient in mixed-lineage kinase domain-like protein (MLKL), a downstream RIPK3 target, were not protected from UUO-induced kidney fibrosis. We demonstrate a pathway by which RIPK3 promotes fibrogenesis through the AKT-dependent activation of ATP citrate lyase (ACL). Genetic or chemical inhibition of RIPK3 suppressed the phosphorylation of AKT and ACL in response to TGF-ß1 in fibroblasts. Inhibition of AKT or ACL suppressed TGF-ß1-dependent extracellular matrix production and myofibroblast differentiation in fibroblasts. Pharmacological inhibition of ACL suppressed UUO-induced kidney fibrosis. RIPK3 expression was highly regulated in human CKD kidney. In conclusion, we identify a pathway by which RIPK3 promotes kidney fibrosis independently of MLKL-dependent necroptosis as a promising therapeutic target in CKD.

Carbon monoxide inhibits Fas activating antibody-induced apoptosis in endothelial cells.

BACKGROUND: The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (ie., Fas/CD95), forming a death-inducing signaling complex (DISC). The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury. Carbon monoxide, an enzymatic product of heme oxygenase-1, exerts antiapoptotic effects at low concentration in vitro and in vivo. METHODS: Using mouse lung endothelial cells (MLEC), we examined the antiapoptotic potential of carbon monoxide against apoptosis induced by the Fas/CD95-activating antibody (Jo2). Carbon monoxide was applied to cell cultures in vitro. The expression and/or activation of apoptosis-related proteins and signaling intermediates were determined using Western Immunoblot and co-immunoprecipitation assays. Cell death was monitored by lactate dehydrogenase (LDH) release assays. Statistical significance was determined by student T-test and a value of P < 0.05 was considered significant. RESULTS: Treatment of MLEC with Fas-activating antibody (Jo2) induced cell death associated with the formation of the DISC, and activation of caspases (-8, -9, and -3), as well as the pro-apoptotic Bcl-2 family protein Bax. Exposure of MLEC to carbon monoxide inhibited Jo2-induced cell death, which correlated with the inhibition of DISC formation, cleavage of caspases-8, -9, and -3, and Bax activation. Carbon monoxide inhibited the phosphorylation of the Fas-associated death domain-containing protein, as well as its association with the DISC. Furthermore, carbon monoxide induced the expression of the antiapoptotic protein FLIP and increased its association with the DISC.CO-dependent cytoprotection against Fas mediated apoptosis in MLEC depended in part on activation of ERK1/2-dependent signaling. CONCLUSIONS: Carbon monoxide has been proposed as a potential therapy for lung and other diseases based in part on its antiapoptotic effects in endothelial cells. In vitro, carbon monoxide may inhibit both Fas/caspase-8 and Bax-dependent apoptotic signaling pathways induced by Fas-activating antibody in endothelial cells. Strategies to block Fas-dependent apoptotic pathways may be useful in development of therapies for lung or vascular disorders.

Autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.

The molecular and cellular mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD) remain incompletely understood. We have investigated the potential role of macro-autophagy, a cellular homeostatic mechanism, in COPD and cigarette smoke-induced lung-cell injury. Autophagy is a dynamic process for the turnover of organelles and proteins, which regenerates metabolic precursors through the lysosomal-dependent catabolism of cellular macromolecules. It is typically associated with survival pathways, especially in nutrient deficiency states. The role of autophagy in human diseases is less clear, and has been associated with both protective and detrimental consequences, depending on the disease model. While autophagy is considered cytoprotective, this process is often found in association with cell death, and the relationships between autophagy and cell death remain ambiguous. We have found elevated autophagy in COPD lung specimens, as well as in response to cigarette smoke exposure in vitro and in vivo. In our studies, the activation of autophagic proteins was associated with epithelial cell apoptosis in response to cigarette smoke, with pathogenic implications in COPD. Further studies are needed to determine the functional significance of autophagy in COPD and other diseases of the lung.

Protection against ischemia/reperfusion injury in cardiac and renal transplantation with carbon monoxide, biliverdin and both.

Both carbon monoxide (CO) and biliverdin, products of heme degradation by heme oxygenase, have been shown to attenuate ischemia/reperfusion (I/R) injury. We hypothesized in this study that dual-treatment with CO and biliverdin would induce enhanced protective effects against cold I/R injury. Heterotopic heart and orthotopic kidney transplantation were performed in syngeneic Lewis rats after 24-h cold preservation in UW solution. While monotherapy with CO (20 ppm) or biliverdin (50 mg/kg, ip) did not alter the survival of heart grafts, dual-treatment increased survival to 80% from 0% in untreated recipients, with a significant decrease of myocardial injury and improved cardiac function. Similarly, dual-treatment significantly improved glomerular filtration rates of renal grafts and prolonged recipient survival compared to untreated controls. I/R injury-induced up-regulation of pro-inflammatory mediators (e.g. TNF-alpha, iNOS) and extravasation of inflammatory infiltrates were significantly less with dual-treatment than untreated controls. In addition, dual-treatment was effective in decreasing lipid peroxidation and improving graft blood flow through the distinctive action of biliverdin and CO, respectively. The study shows that the addition of byproducts of heme degradation with different mechanisms of action provides enhanced protection against transplant-associated cold I/R injury of heart and kidney grafts.