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

Modification of cell wall polysaccharide guides cell division in <i>Streptococcus mutans</i>

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning. [Figure not available: see fulltext.

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30–50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions

Active trafficking of alpha 1 antitrypsin across the lung endothelium.

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency

Mechanism of α-1 antitrypsin endocytosis by lung endothelium

The integrity of lung alveoli is maintained by proper circulating levels of α-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT’s antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin’s protection against developing cigarette smoke-induced emphysema. Sohrab, S., Petrusca, D. N., Lockett, A. D., Schweitzer, K. S., Rush, N. I., Gu, Y., Kamocki, K., Garrison, J., Petrache, I. Mechanism of α-1 antitrypsin endocytosis by lung endothelium

Secretory pathways leading to A1AT transcytosis across cultured pulmonary endothelial monolayers.

<p>(A–D) Immunoblot showing effect of inhibition of classical secretory pathway with tunicamycin (A–C; at the indicated doses; 18 h) or brefeldin A (D; 1 µg/mL, 60 min) on intracellular A1AT (A, B, D) and secretion of A1AT (A, C) detected by Western blotting and quantified by densitometry, using vinculin as loading control. Secreted A1AT was measured in equal volumes of supernatants (which were concentrated 25-fold). (E) Pre-inhibition of the classical secretory pathway with tunicamycin (1 h) enhances A1AT (3 h) basolateral secretion, measured by Western blotting of concentrated supernatants, suggesting the utilization of a non-classical secretory pathway. Bands are from the same immunoblot. (F) Alternative secretion of A1AT (100 µg/mL, 2 h) by endothelial cells via microparticles release, as detected by Western blotting of A1AT in endothelial microparticles isolated from supernatants via ultracentrifugation (representative blot of n = 3). (G) Time course of basolateral EMP release from endothelial cells treated with A1AT (100 µg/mL). Bars represent mean+SEM; *p<.05, #p = .07 vs. control; n = 3.</p

A1AT trafficking across cultured pulmonary endothelial and epithelial bilayers.

<p>(A) Co-culture schematic showing pulmonary epithelial cells cultured on the bottom of the transwell membrane and pulmonary endothelial cells cultured on the top. Endothelial cells only were exposed to A1AT. (B) Phalloidin staining (top and epithelial cells) and brightfield microscopy (bottom) showing confluent monolayers of endothelial and epithelial cells seeded on transwell inserts. (C) Immunoblotting of A1AT in cell lysates showing intracellular presence of A1AT in endothelial and epithelial cells and immunoblot of secreted A1AT from concentrated bottom supernatant (representative blots of n = 3). Bands shown are from the same immunoblot. (D). Schematic showing A1AT treatment of the basolateral media or apical surface of NHBE cells differentiated at ALI. (E). Confocal microscopy of NHBE cells differentiated at ALI after 2 h incubation with fluorescently labeled A1AT (green, 20 µM) added to either the basolateral media (i) or apical surface (ii). Only the basolaterally applied A1AT was observed to enter the cells. Arrows indicate ciliated side of the epithelium (cilia stained in white, nuclei in blue). (F). Densitometric quantification of epithelial cell lysates and ASL collected at the indicated times after adding 20 µM of A1AT in either the basolateral (solid line, triangles) or the apical (solid line, circles) compartment from 3 different lung donors measured by Western blotting (mean+SEM; n = 3). Plot (dashed line, squares) showing the relative A1AT present in the ASL after the basolateral application experiment. All fold changes are relative to time 0 before the addition of A1AT. (G) Concentration of A1AT in ASL of ALI cultures after A1AT (20 µM) addition to the basolateral compartment. ASL was collected with 250 µl PBS washes. A1AT quantification was made by customized ELISA and corrected for wash dilution (n = 3 different lung donors). (H) Levels of intracellular and secreted A1AT measured by ELISA (n = 2) in NHBE cells treated with conditioned endothelial media (containing 43.4 nM endothelial-secreted A1AT, 2 h);. n = 1.</p