Walter C. Hubbard

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.

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.

Preparation and assay of monohydroxy-eicosatetraenoic acids

Abstract 5-, 8-, 9-, 11-, 12-, and 15-hydroxy-eicosatetraenoic acids (HETEs) were prepared from arachidonic acid by reaction with H2O2 in the presence of Cu2+ ions. They were separated by high-performance liquid chromatography on silica gel (μPorasil), using a linear solvent gradient from hexane to chloroform: only the 8- and 9-isomers were not resolved. Multi-milligram quantities of highly purified HETEs could be easily generated by this method, which thus provides a useful tool to study the biological activity of these compounds. Octadeuterated analogs of HETEs prepared from octadeuterated arachidonic acid by this procedure were suitable for use as internal standards in stable isotope dilution assays, by combined gas chromatography and mass spectrometry, with selected ion monitoring. The detection limit of the HETEs was less than 1 ng.

Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production.

Eosinophil infiltration into the airways has been implicated in the pathophysiology of asthma. To improve our understanding of the function of eosinophils in asthma, we have compared the phenotype and function of eosinophils obtained simultaneously from blood and bronchoalveolar lavage (BAL) of allergic subjects 19 hours after segmental lung allergen challenge. Eosinophils were purified by discontinuous density gradient centrifugation, and their distribution at various layers was quantitated. Eosinophils at the 1.080 to 1.085 gm/ml interfaces from blood and BAL (purity > 70%) were analyzed by immunofluorescence and flow cytometry for several surface markers including adhesion-activation antigens. Eosinophils in BAL from antigen-challenged sites were markedly increased compared with control diluent-challenged BAL sites (0.3% +/- 1% vs 28.1% +/- 9.7%, n = 12, p < 0.002), and a greater percentage were hypodense (specific gravity < 1.080 gm/ml) than in peripheral blood (51.3 +/- 5.3 vs 19.0 +/- 4.4, n = 15, p < 0.01). In vitro, resting and activated BAL eosinophils biosynthesized less thromboxane B2 than blood eosinophils. Although both BAL and blood eosinophils expressed similar levels of Fc gamma RII (CD32), CD11a, and CD45, resting levels of Mo-1 (CD11b) were upregulated on BAL eosinophils (mean fluorescence intensity, 316% +/- 48% of blood eosinophils, n = 5, p < 0.05). Blood eosinophils stimulated in vitro with 1 mumol/L platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine achieved levels of CD11b expression similar to those of BAL eosinophils. In contrast, CD11b expression on BAL eosinophils could not be further increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin receptor subtypes in human and guinea‐pig pulmonary tissues

1 In this study the endothelin (ET) receptor subtypes mediating contractions produced by ET‐1 in human and guinea‐pig pulmonary tissues were investigated. In addition the receptor responsible for ET‐1‐induced prostanoid release in human bronchus was determined. 2 In human bronchus and human pulmonary artery ET‐1 (0.1 nm–0.3 μm) was a potent and effective contractile agent (pD2 = 7.58 ± 0.15, n = 6, and 8.48 ±0.11, n = 7, respectively). BQ‐123 (1–10 μm), a potent and selective ETA receptor antagonist, potently antagonized ET‐1‐induced contraction in human pulmonary artery (pKB = 6.8 with 1 μm BQ‐123, n = 7) but had no effect in human bronchus (n = 6). 3 Sarafotoxin S6c (0.1 nm–0.1 μm), the ETB‐selective agonist, did not contract human pulmonary artery (n = 5), but potently and effectively contracted human bronchus: pD2 = 8.41 ± 0.17, maximum response = 74.4 ± 3.1% of 10 μm carbachol; n = 5. BQ‐123 (1–10 μm) did not antagonize sarafotoxin S6c‐induced contraction in human bronchus (n = 5). 4 ET‐1 potently contracted guinea‐pig trachea, bronchus, pulmonary artery and aorta (pD2 = 8.15 ± 0.14, 7.72 ± 0.12, 8.52 ± 0.12, and 8.18 ± 0.12, respectively, n = 6–14). BQ‐123 (0.1–10 μm) antagonized ET‐1‐induced contractions in guinea‐pig pulmonary artery (pKB = 6.7 with 1 μm BQ‐123, n = 6), aorta (pKB = 7.1 with 1 μm BQ‐123, n = 6) and trachea (pKB = 6.2 with 1 μm BQ‐123, n = 6) but was without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nm–0.1 μm) did not contract guinea‐pig aorta (n = 4) or guinea‐pig pulmonary artery (n = 6) but potently and effectively contracted guinea‐pig bronchus: pD2 = 8.55 ± 0.1; maximum contraction = 63.6 ± 3.1% of 10 μm carbachol, n = 4. Sarafotoxin S6c (0.1 nm–0.1 μm) was a much less effective agonist in guinea‐pig trachea: maximum contraction = 13.9 ± 2.5% of 10 μm carbachol, n = 4; P < 0.0001, compared to bronchus. Contractions produced by sarafotoxin S6c in guinea‐pig bronchus or trachea were unaffected by BQ‐123 (10 μm, n = 4). 5 Significant differences were observed in the efficacy, relative to carbachol, but not the potency of sarafotoxin S6c in guinea‐pig airways, with a much greater maximum contractile response in bronchus (69.6 ± 2.4% of 10 μm carbachol, n = 6) or lower region of the trachea (48.5 ± 5.9% of 10 μm carbachol, n = 6) than in the middle region of the trachea (14.4 ± 4.0% of 10 μm carbachol, n = 6) or the upper region of the trachea (19.3 ± 2.7% of 10 μm carbachol, n = 6). There were minimal regional differences in either ET‐1‐induced contraction or the potency of BQ‐123 (3 μm) for inhibition of responses to ET‐1 in guinea‐pig airways. 6 Release of various prostanoids in human bronchus induced by ET‐1 (0.3 μm) was essentially abolished with 10 μm BQ‐123. 7 These data provide evidence that distinct ET receptors mediate ET‐1‐induced contraction in human pulmonary artery, guinea‐pig pulmonary artery and guinea‐pig aorta (ETA subtype) compared with human bronchus and guinea‐pig bronchus (non‐ETA, perhaps ETB subtype). Contractions to ET‐1 in guinea‐pig trachea appear to involve both ETA and non‐ETA (ETB?) receptor subtypes. Furthermore, regional differences appear to exist in the relative distribution of ET receptor subtypes in guinea‐pig airways. In human bronchus ET‐1‐induced prostanoid release, unlike the contractile response, appears to be mediated via ETA receptor activation.

Immunologically induced neuromodulation of guinea pig nodose ganglion neurons

The influence of specific antigen challenge on the excitability of C-cells in nodose ganglia isolated from actively sensitized guinea pigs was evaluated using intracellular recording techniques. Antigen (ovalbumin) caused a significant depolarization (approximately 8 mV) of the resting membrane potential. Antigen exposure had differing effects on the membrane input impedance; decreasing it in 15 neurons, increasing it in 6 neurons, and having no effect in 8 neurons. About 20% of guinea pig nodose C-cells reveal a long-lasting after-spike hyperpolarization (AHPslow). Antigen challenge reversibly blocked the AHPslow in 4 of 18 neurons studied in 18 ganglia. About 30% of the nodose ganglion neurons display a time- and voltage-dependent inward rectification at membrane potentials more negative than -75 mV. Exposing the ganglion to the sensitizing antigen consistently blocked this response in 8 of 8 neurons. Histological assessment of toluidine blue stained cells revealed that the nodose ganglion contained approximately 100 mast cells. Exposing the ganglion to ovalbumin stimulated mast cell degranulation, as measured by a decrease in number of stained cells, and evoked the release of histamine, PGD2, and immunoreactive peptidoleukotrienes from the tissue. The results support the hypothesis that endogenous inflammatory mediators released during the immediate hypersensitivity (allergic) reactions can modulate the excitability of primary C-fiber afferents. Mechanisms underlying antigen-induced neuromodulation of these neurons include depolarization of the resting membrane potential, changes in membrane resistance, blockade of a time- and voltage-dependent anomalous rectifier, and, in some cells, blockade of the AHPslow.

Prostaglandin levels in human colorectal mucosa: Effects of sulindac in patients with familial adenomatous polyposis

Recent evidence suggests that nonsteroidalantiinflammatory drugs (NSAIDs) may prevent colorectalcancer. The mechanism of action of NSAIDs inchemoprevention is unknown but may be linked to theireffect on mucosal prostaglandin levels. Levels of fivemajor prostaglandin metabolites were measured by gaschromatography-mass spectrometry in biopsy specimens offlat rectal mucosa from four patients with familial adenomatous polyposis (FAP) before and aftersulindac therapy and from five healthy individuals. Theprostaglandin present at highest concentration in rectalmucosa from FAP and control subjects was prostaglandin E2. The concentration of thromboxaneB2 alone was significantly elevated in FAPpatients compared to controls (P = 0.016). In FAPpatients treated with sulindac, all prostaglandinmetabolite levels were significantly reduced compared to pretreatmentlevels (P < 0.05) except prostaglandin D2(P = 0.07). Prostaglandins D2, E2,F2α, and 6-keto-F1αlevels also were significantly reduced in FAP patients on sulindac compared to healthy controls (P< 0.05). However, interpatient heterogeneity ofresponse to sulindac was evident with changes rangingfrom +19% to –89%, and the patient with thegreatest reductions after sulindac developed colorectal cancerafter 35 months of therapy. Sulindac treatment, at drugdoses shown to regress colorectal adenomas in FAPpatients, has heterogeneous effects on the level ofmajor prostaglandins in their rectal mucosa and maynot prevent colorectal cancer due to uncoupling ofprostaglandin levels and carcinogenesis.

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.

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer(12:0) or Cer(8:0); 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramides proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge

Background Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported.