Binase penetration into alveolar epithelial cells does not induce cell death

Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.     

Reduction of caveolin 1 gene expression in lung carcinoma cell lines

Caveolae are plasma membrane microdomains that have been implicated in organizing and concentrating certain signaling molecules. Caveolins, constitute the main structural proteins of caveolae. Caveolae are abundant in terminally differentiated cell types. However, caveolin-1 is down-regulated in transformed cells and may have a potential tumor suppressor activity. In the lung, caveolae are present in the endothelium, smooth muscle cells, fibroblasts as well as in type I pneumocytes. The presence of caveolae and caveolin expression in the bronchial epithelium, although probable, has not been investigated in human. We were interested to see if the bronchial epithelia express caveolins and if this expression was modified in cancer cells. We thus tested for caveolin-1 and -2 expression several bronchial epithelial primary cell lines as well as eight lung cancer cell lines and one larynx tumor cell line. Both caveolin-1 and -2 are expressed in all normal bronchial cell lines. With the exception of Calu-1 cell line, all cancer cell lines showed very low or no expression of caveolin-1 while caveolin-2 expression was similar to the one observed in normal bronchial epithelial cells.     

Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells

Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.     

Bacterial penetration of bladder epithelium through lipid rafts

Type 1 fimbriated Escherichia coli represents the most common human uropathogen, owing much of its virulence to invasion of the uroepithelium, which is highly impermeable due to the preponderance of uroplakins and highly ordered lipid components. We sought to elucidate the molecular basis for E. coli invasion of the bladder epithelium by employing human 5637 bladder epithelial cells, and we found the following: (i) intracellular E. coli associated with caveolae and lipid raft components; (ii) RNA(i) reduction of caveolin-1 expression inhibited bacterial invasion; (iii) a signaling molecule required for E. coli invasion was located in lipid rafts and physically associated with caveolin-1; (iv) bacterial invasion was inhibited by lipid raft disrupting/usurping agents. In the mouse bladder, the E. coli type 1 fimbrial receptor, uroplakin Ia, was located in lipid rafts, and lipid raft disruptors inhibited E. coli invasion. Cumulatively, E. coli uroepithelial invasion occurs through lipid rafts, which, paradoxically, contribute to bladder impermeability.     

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.     

Src-induced phosphorylation of caveolin-2 on tyrosine 19. Phospho-caveolin-2 (Tyr(P)19) is localized near focal adhesions,remains associated with lipid rafts/caveolae,but no longer forms a high molecular mass hetero-oligomer with caveolin-1

Caveolin-2 is the least well studied member of the caveolin gene family. It is believed that caveolin-2 is an "accessory protein" that functions in conjunction with caveolin-1. At the level of the ER, caveolin-2 interacts with caveolin-1 to form a high molecular mass hetero-oligomeric complex that is targeted to lipid rafts and drives the formation of caveolae. However, caveolin-2 is not required for caveolae formation, implying that it may fulfill some unknown regulatory role. Here, we present the first evidence that caveolin-2 is a phosphoprotein. We show that caveolin-2 undergoes Src-induced phosphorylation on tyrosine 19. To study this phosphorylation event in vivo, we generated a novel phospho-specific antibody probe that only recognizes phosphocaveolin-2 (Tyr(P)(19)). We then used NIH-3T3 cells stably overexpressing c-Src to examine the localization and biochemical properties of phosphocaveolin-2 (Tyr(P)(19)). Our results indicate that phosphocaveolin-2 (Tyr(P)(19)) is localized near focal adhesions, remains associated with lipid rafts/caveolae, but no longer forms a high molecular mass hetero-oligomer with caveolin-1. Instead, phosphocaveolin-2 (Tyr(P)(19)) behaves as a monomer/dimer in velocity gradients. Thus, we conclude that the tyrosine phosphorylation of caveolin-2 (Tyr(P)(19)) may function as a signal that is recognized by the cellular machinery to induce the dissociation of caveolin-2 from caveolin-1 oligomers. We also demonstrate that (i) insulin-stimulation of adipocytes and (ii) integrin ligation of endothelial cells can both induce the tyrosine phosphorylation of caveolin-2 (Tyr(P)(19)). During integrin ligation, phosphocaveolin-2 (Tyr(P)(19)) co-localizes with activated FAK at focal adhesions. Thus, phosphocaveolin-2 (Tyr(P)(19)) may function as a docking site for Src homology domain-2 (SH2) domain containing proteins during signal transduction. In support of this notion, we identify several SH2 domain containing proteins, namely c-Src, NCK, and Ras-GAP, that interact with caveolin-2 in a phosphorylation-dependent manner. Furthermore, our co-immunoprecipitation experiments show that caveolin-2 and Ras-GAP are constitutively associated in c-Src expressing NIH-3T3 cells, but not in untransfected NIH-3T3 cells.     

Inhibition of volume-regulated anion channels by dominant-negative caveolin-1

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity.     

Mutual regulation between metabotropic glutamate type 1alpha receptor and caveolin proteins: from traffick to constitutive activity

In this paper, a molecular and functional interaction between metabotropic glutamate receptor type 1alpha (mGlu1alpha receptor) and caveolin-1 or caveolin-2beta is described. An overlapping pattern of staining for mGlu1alpha receptor with caveolin-1 and caveolin-2 by confocal laser microscopy in transiently transfected HEK-293 cells is observed. The presence of mGlu1alpha receptor in caveolin-enriched membrane fractions was demonstrated by flotation gradient analysis in the absence of detergents and the interaction between mGlu1alpha receptor with caveolin-1 and with caveolin-2beta was demonstrated by coimmunoprecipitation experiments. In HEK-293 cells, caveolin-2beta accumulates surrounding lipid droplets when single expressed but coexpression with mGlu1alpha receptor changed dramatically the subcellular localization of caveolin-2beta, directing it from lipid droplets to the cell surface. At the membrane level, the interaction between caveolin-1 and mGlu1alpha receptor could abrogate the constitutive activity exhibited by mGlu1alpha receptor. Overall, these results show that mGlu1alpha receptor interacts with caveolins and that this interaction is physiologically relevant for receptor function. Interestingly, we provide evidence that caveolin-1 is not just acting as a scaffolding protein for the mGlu1alpha receptor but that also regulates mGlu1alpha receptor constitutive activity.     

GM-CSF provides autocrine protection for murine alveolar epithelial cells from oxidant-induced mitochondrial injury

Exposure of mice to hyperoxia induces alveolar epithelial cell (AEC) injury, acute lung injury and death. Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung protects against these effects, although the mechanisms are not yet clear. Hyperoxia induces cellular injury via effects on mitochondrial integrity, associated with induction of proapoptotic members of the Bcl-2 family. We hypothesized that GM-CSF protects AEC through effects on mitochondrial integrity. MLE-12 cells (a murine type II cell line) and primary murine type II AEC were subjected to oxidative stress by exposure to 80% oxygen and by exposure to H(2)O(2). Exposure to H(2)O(2) induced cytochrome c release and decreased mitochondrial reductase activity in MLE-12 cells. Incubation with GM-CSF significantly attenuated these effects. Protection induced by GM-CSF was associated with Akt activation. GM-CSF treatment also resulted in increased expression of the antiapoptotic Bcl-2 family member, Mcl-1. Primary murine AEC were significantly more tolerant of oxidative stress than MLE-12 cells. In contrast to MLE-12 cells, primary AEC expressed significant GM-CSF at baseline and demonstrated constitutive activation of Akt and increased baseline expression of Mcl-1. Treatment with exogenous GM-CSF further increased Akt activation and Mcl-1 expression in primary AEC. Conversely, suppression of AEC GM-CSF expression by use of GM-CSF-specific small interfering RNA resulted in decreased tolerance of oxidative stress, Furthermore, silencing of Mcl-1 prevented GM-CSF-induced protection. We conclude that GM-CSF protects alveolar epithelial cells against oxidative stress-induced mitochondrial injury via the Akt pathway and its downstream components, including Mcl-1. Epithelial cell-derived GM-CSF may contribute to intrinsic defense mechanisms limiting lung injury.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Deletion of the transmembrane transporter ABCG1 results in progressive pulmonary lipidosis

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.     

Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro

Lipopolysaccharide (LPS)-induced lung inflammation is known to increase pulmonary intercellular adhesion molecule-1 (ICAM-1) expression. In the present study, L2 cells, a cell line of alveolar epithelial cells, were stimulated with LPS, and ICAM-1 expression was studied. ICAM-1 protein on L2 cells peaked at 6 (38% increase; P < 0.01) and 10 (48% increase; P < 0.001) h after stimulation with Escherichia coli and Pseudomonas aeruginosa LPS, respectively. ICAM-1 mRNA expression was markedly increased, with a peak at 2-4 (E. coli) and 4-6 (P. aeruginosa) h. Adherence assays of neutrophils to LPS-stimulated L2 cells showed a threefold increase in adherence (P < 0.001). Pretreatment of the neutrophils with anti-lymphocyte function-associated antigen-1 and anti-Mac-1 antibodies reduced adherence by 54% (P < 0.001). Analysis of immunofluorescence staining for ICAM-1 showed an exclusive apical expression of ICAM-1. These results indicate that LPS upregulates functional active ICAM-1 on the apical part of the membrane in rat pneumocytes.     

Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins

Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa. We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P. aeruginosa invasion. Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P. aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001). UO124, a negative control for UO126, had no effect on bacterial internalization. Infection of cells with an internalization-defective flhA mutant of P. aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P. aeruginosa. An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P. aeruginosa invasion by approximately 40% (P=0.035). Together, these data suggest that P. aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.     

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells.     

Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell

Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.     

The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103–122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro¹¹⁰ residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.     

Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.     

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.