Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macro-autophagy (mitophagy), processes that may be influenced by pro-apoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homolog-induced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)-Cer also increased in CS-exposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16-Cer was sufficient to trigger necroptosis. Inhibition of Cer-generating acid sphingomyelinase reduced both CS-induced PINK1 phosphorylation and necroptosis. When exposed to CS, Pink1-deficient ( Pink1-/-) mice, which are protected from airspace enlargement compared with wild-type littermates, had blunted C16-Cer elevations and less lung necroptosis. CS-exposed Pink1-/- mice also exhibited significantly increased levels of lignoceroyl (C24)-DHC, along with increased expression of Cer synthase 2 ( CerS2), the enzyme responsible for its production. This suggested that a combination of high C24-DHC and low C16-Cer levels might protect against CS-induced necroptosis. Indeed, CerS2-/- mice, which lack C24-DHC at the expense of increased C16-Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24-DHC, as protective against CS toxicity by enhancing autophagy, whereas C16-Cer accumulation contributes to mitochondrial damage and PINK1-mediated necroptosis, which may be amplified by the inhibition of C24-DHC-producing CerS2.-Mizumura, K., Justice, M. J., Schweitzer, K. S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W. C., Pewzner-Jung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Smoking exposure induces human lung endothelial cell adaptation to apoptotic stress.

Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.

Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease.

Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of ß-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders.

Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible.

X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible.

Dihydroceramide-based response to hypoxia.

To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.

Combined extraction of acyl carnitines and 26:0 lysophosphatidylcholine from dried blood spots: prospective newborn screening for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder that affects the nervous system, and the adrenal cortex. Newborn screening for X-ALD has been proposed to allow improved diagnosis along with prospective monitoring and treatment for this severe disorder. Newborn dried whole blood spot (DBS) 26:0 lysophosphatidyl choline was validated as a diagnostic marker for X-ALD and other peroxisomal disorders of peroxisomal ß-oxidation. In this study, we developed a new one step extraction procedure that simultaneously extracts acyl carnitines and the lysophosphatidyl cholines from DBS. Further analysis of these metabolites has been performed by two different high throughput LC-MS/MS methods. The 26:0 lysophosphatidyl choline levels in this study were consistent with previously published values and discriminate between healthy and abnormal profiles. There is a very minor modification to the original acyl carnitine extraction procedure and our data indicates that there is no significant effect on acyl carnitine levels in DBS. Our new method potentially can be complementary to the current newborn screening panel. It successfully combines the existing method for acyl carnitine analysis and 26:0 lysophosphatidyl choline that can be applied for prospective X-ALD newborn screening.

Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice.

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages.

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema.

RATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) method.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD) has until now been limited in implementation because of the lack of an accepted standard methodology. We have previously reported a technique using LC-MS/MS analysis that could provide the basis for screening of newborns for X-ALD. The target analyte diagnostic for X-ALD and other peroxisomal disorders of peroxisomal beta-oxidation is 1-hexacosanoyl-2-lyso-sn-3-glycero-phosphorylcholine (26:0-lyso-PC). We report here the validation of the analytical method using an authentic standard of the target compound. The method possesses sensitivity of <1.0fmole injected on column with a correlation coefficient (R(2)) of 0.9987. A tetradeuterated analog of 26:0-lyso-PC served as the internal standard. The sensitivity of this clinical method was confirmed using 17 newborn samples of individuals with peroxisomal disorders retrieved from state newborn screening programs. These samples were run masked with over 1000 newborn samples. All affected individuals were identified with one exception. One sample which was retrieved as an affected did not have the biochemical or genetic abnormality of X-ALD and thus is considered an error in sample identity. These studies clearly show that the method is highly sensitive and accurate in identifying individuals with a defect in peroxisomal beta-oxidation such as X-ALD.

Apoptotic sphingolipid signaling by ceramides in lung endothelial cells.

The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate-chain length (C(8:0)) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-alpha-generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C(8:0) ceramide- and TNF-alpha (plus cycloheximide)-induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C(16:0) ceramide in response to C(8:0) and TNF-alpha treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-alpha-stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.

Reduced retina microglial activation and improved optic nerve integrity with minocycline treatment in the DBA/2J mouse model of glaucoma.

PURPOSE: In the context of the retinal ganglion cell (RGC) axon degeneration in the optic nerve that occurs in glaucoma, microglia become activated, then phagocytic, and redistribute in the optic nerve head. The authors investigated the potential contribution of retinal microglia activation to glaucoma progression in the DBA/2J chronic mouse glaucoma model. METHODS: The authors treated 6-week-old DBA/2J mice for 25 weeks with minocycline, a tetracycline derivative known to reduce microglia activation and to improve neuronal survival in other models of neurodegenerative disease. They quantified RGC numbers and characterized microglia activation, gliosis, and both axonal integrity and retrograde tracer transport by RGCs in mice systemically treated with minocycline or vehicle only. RESULTS: Minocycline reduced microglial activation and improved RGC axonal transport and integrity, yet it had no effect on the characteristic age-related ocular changes that lead to chronically elevated pressure and did not alter Müller or astrocyte gliosis. Specifically, minocycline increased the fraction of microglia with resting ramified morphology and reduced levels of Iba1 mRNA and protein, a microglia-specific calcium ligand linked to activation. The reduction in microglial activation was coupled to significant improvement in RGC axonal transport, as measured by neuronal retrograde tracing from the superior colliculus. Finally, minocycline treatment significantly decoupled RGC axon loss from increased intraocular pressure. CONCLUSIONS: These observations suggest that in glaucoma, retina and optic nerve head microglia activation may be a factor in the early decline in function of the optic nerve and its subsequent degeneration.

The Diversity of Chemoprotective Glucosinolates in Moringaceae (Moringa spp.).

Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera, the drumstick tree, produces unique GS but little is known about GS variation within M. oleifera, and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M. longituba. We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low (M. longituba), and by far the highest was M. arborea, a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.

Intermittent hypoxia induces hyperlipidemia in lean mice.

Obstructive sleep apnea, a syndrome leading to recurrent intermittent hypoxia (IH), has been associated previously with hypercholesterolemia, independent of underlying obesity. We examined the effects of experimentally induced IH on serum lipid levels and pathways of lipid metabolism in the absence and presence of obesity. Lean C57BL/6J mice and leptin-deficient obese C57BL/6J-Lep(ob) mice were exposed to IH for five days to determine changes in serum lipid profile, liver lipid content, and expression of key hepatic genes of lipid metabolism. In lean mice, exposure to IH increased fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, phospholipids (PLs), and triglycerides (TGs), as well as liver TG content. These changes were not observed in obese mice, which had hyperlipidemia and fatty liver at baseline. In lean mice, IH increased sterol regulatory element binding protein 1 (SREBP-1) levels in the liver, increased mRNA and protein levels of stearoyl-coenzyme A desaturase 1 (SCD-1), an important gene of TG and PL biosynthesis controlled by SREBP-1, and increased monounsaturated fatty acid content in serum, which indicated augmented SCD-1 activity. In addition, in lean mice, IH decreased protein levels of scavenger receptor B1, regulating uptake of cholesterol esters and HDL by the liver. We conclude that exposure to IH for five days increases serum cholesterol and PL levels, upregulates pathways of TG and PL biosynthesis, and inhibits pathways of cholesterol uptake in the liver in the lean state but does not exacerbate the pre-existing hyperlipidemia and metabolic disturbances in leptin-deficient obesity.

Newborn Screening for X-Linked Adrenoleukodystrophy.

Early diagnosis of males with X-linked adrenoleukodystrophy (X-ALD) is essential for preventing loss of life due to adrenal insufficiency and for timely therapy of the childhood cerebral form of X-ALD with hematopoietic cell transplantation. This article describes X-ALD, the current therapies, the history of the development of the newborn screening test, the approval by the Secretary of Health and Human Services for the addition of X-ALD newborn screening to the recommended uniform panel of disorders screened as newborns (RUSP) and the successful implementation of X-ALD newborn screening in the state of New York beginning on 30 December 2013. Follow-up guidelines that have been established in New York are outlined. Based on the success of newborn screening in New York, and early results in Connecticut, where X-ALD newborn screening started in December 2015, and in California, where X-ALD newborn screening began in September 2016, we are confident and hopeful that X-ALD newborn screening will expand to include all US states and to countries that have established neonatal screening programs. The Minster of Health in the Netherlands has approved the addition of X-ALD to the newborn screening program with a start date expected in 2017. The states, such as Massachusetts, Illinois, Minnesota, New Jersey, Florida and Washington, that have legislative approval will commence screening as soon as budgetary resources, testing and follow-up procedures are in place.

Structural and functional characterization of endothelial microparticles released by cigarette smoke.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

Ceramide synthases expression and role of ceramide synthase-2 in the lung: insight from human lung cells and mouse models.

Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.

Lung endothelial monocyte-activating protein 2 is a mediator of cigarette smoke-induced emphysema in mice.

Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke-induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.