Claudin 4 knockout mice: normal physiological phenotype with increased susceptibility to lung injury.

Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly upregulated in wild-type mice, whereas Cldn4 KO mice showed greater degrees of lung injury. RNA sequencing, in conjunction with differential expression and upstream analysis after ventilator-induced lung injury, suggested Egr1, Tnf, and Il1b as potential mediators of increased lung injury in Cldn4 KO mice. These results demonstrate that claudin 4 has little effect on normal lung physiology but may function to protect against acute lung injury.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Troglitazone attenuates TGF-ß1-induced EMT in alveolar epithelial cells via a PPARγ-independent mechanism.

Peroxisome proliferator activated receptor γ (PPARγ) agonists are effective antifibrotic agents in a number of tissues. Effects of these agents on epithelial-mesenchymal transition (EMT) of primary alveolar epithelial cells (AEC) and potential mechanisms underlying effects on EMT have not been well delineated. We examined effects of troglitazone, a synthetic PPARγ agonist, on transforming growth factor (TGF)-ß1-induced EMT in primary rat AEC and an alveolar epithelial type II (AT2) cell line (RLE-6TN). TGF-ß1 (2.5 ng/mL) induced EMT in both cell types, as evidenced by acquisition of spindle-like morphology, increased expression of the mesenchymal marker α-smooth muscle actin (α-SMA) and downregulation of the tight junctional protein zonula occludens-1 (ZO-1). Concurrent treatment with troglitazone (or rosiglitazone), ameliorated effects of TGF-ß1. Furthermore, following stimulation with TGF-ß1 for 6 days, troglitazone reversed EMT-related morphological changes and restored both epithelial and mesenchymal markers to control levels. Treatment with GW9662 (an irreversible PPARγ antagonist), or overexpression of a PPARγ dominant negative construct, failed to inhibit these effects of troglitazone in AEC. Troglitazone not only attenuated TGF-ß1-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3ß, but also inhibited nuclear translocation of ß-catenin, phosphorylation of Smad2 and Smad3 and upregulation of the EMT-associated transcription factor SNAI1. These results demonstrate inhibitory actions of troglitazone on TGF-ß1-induced EMT in AEC via a PPARγ-independent mechanism likely through inhibition of ß-catenin-dependent signaling downstream of TGF-ß1, supporting a role for interactions between TGF-ß and Wnt/ß-catenin signaling pathways in EMT.

Ligand-independent transforming growth factor-ß type I receptor signalling mediates type I collagen-induced epithelial-mesenchymal transition.

Evidence suggests epithelial-mesenchymal transition (EMT) as one potential source of fibroblasts in idiopathic pulmonary fibrosis. To assess the contribution of alveolar epithelial cell (AEC) EMT to fibroblast accumulation in vivo following lung injury and the influence of extracellular matrix on AEC phenotype in vitro, Nkx2.1-Cre;mT/mG mice were generated in which AECs permanently express green fluorescent protein (GFP). On days 17-21 following intratracheal bleomycin administration, ~4% of GFP-positive epithelial-derived cells expressed vimentin or α-smooth muscle actin (α-SMA). Primary AECs from Nkx2.1-Cre;mT/mG mice cultured on laminin-5 or fibronectin maintained an epithelial phenotype. In contrast, on type I collagen, cells of epithelial origin displayed nuclear localization of Smad3, acquired spindle-shaped morphology, expressed α-SMA and phospho-Smad3, consistent with activation of the transforming growth factor-ß (TGFß) signalling pathway and EMT. α-SMA induction and Smad3 nuclear localization were blocked by the TGFß type I receptor (TßRI, otherwise known as Alk5) inhibitor SB431542, while AEC derived from Nkx2.1-Cre;Alk5(flox/KO) mice did not undergo EMT on collagen, consistent with a requirement for signalling via Alk5 in collagen-induced EMT. Inability of a pan-specific TGFß neutralizing antibody to inhibit effects of collagen together with absence of active TGFß in culture supernatants is consistent with TGFß ligand-independent activation of Smad signalling. These results support the notion that AECs can acquire a mesenchymal phenotype following injury in vivo and implicate type I collagen as a key regulator of EMT in AECs through signalling via Alk5, likely in a TGFß ligand-independent manner.

Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

Mechanisms of alveolar epithelial translocation of a defined population of nanoparticles.

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Characterization of mouse alveolar epithelial cell monolayers.

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance (R(T)) and equivalent short-circuit current (I(EQ)) of 1.8 kOmega.cm(2) and 5.3 microA/cm(2), respectively, after 8 days in culture. Amiloride (10 microM), ouabain (0.1 mM), and pimozide (10 microM) decreased MAECM I(EQ) to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of Na(+) in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 microeq.cm(-2).h(-1), respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations.

Polystyrene nanoparticle trafficking across alveolar epithelium.

We investigated trafficking of polystyrene nanoparticles (PNP; 20 and 100 nm; carboxylate, sulfate, or aldehyde-sulfate modified [negatively charged] and amidine-modified [positively charged]) across rat alveolar epithelial cell monolayers (RAECM). Apical-to-basolateral fluxes of nanoparticles were estimated as functions of apical PNP concentration ([PNP]) and temperature. Uptake of nanoparticles into RAECM was determined using confocal microscopy. Fluxes increased as charge density became less negative/more positive, with positively charged PNPs trafficking 20-40 times faster than highly negatively charged PNP of comparable size. Trafficking rates decreased with increasing PNP diameter. PNP fluxes tended to level off at high apical [PNP]. Fluxes at 4 degrees C were significantly lower than those at 37 degrees C. Confocal microscopy revealed nanoparticles localized to cell cytoplasm, whereas cell junctions and nuclei appeared free of PNP. These data indicate that (1) trafficking of PNP across RAECM is strongly influenced by charge density, size, and temperature, (2) PNP translocate primarily transcellularly, and (3) PNP translocation requires cellular energy.

Nanoparticle effects on rat alveolar epithelial cell monolayer barrier properties.

Inhaled nanoparticles have been reported to contribute to deleterious effects on human health. In this study, we investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged; 30 nm) and single-wall carbon nanotubes (SWCNT) on alveolar epithelial cell barrier properties. Transmonolayer resistance (R(t)) and equivalent short-circuit current (I(eq)) of primary rat alveolar epithelial monolayers were measured in the presence and absence of varying concentrations of apical nanoparticles. In some experiments, apical-to-basolateral fluxes of radiolabeled mannitol or inulin were determined with or without apical UAPS exposure and lactate dehydrogenase (LDH) release was analyzed after UAPS or SWCNT exposure. Results revealed that exposure to UAPS decreased R(t) and I(eq) significantly over 24 h, although neither mannitol nor inulin fluxes changed. Positively charged QD decreased R(t) significantly (with subsequent recovery), while negatively charged QD did not. R(t) decreased significantly after SWCNT exposure (with subsequent recovery). On the other hand, PNP exposure had no effects on R(t) or I(eq). No significant increases in LDH release were observed after UAPS or SWCNT exposure. These data indicate that disruption of alveolar epithelial barrier properties due to apical nanoparticle exposure likely involves alteration of cellular transport pathways and is dependent on specific nanoparticle composition, shape and/or surface charge.

Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells.

Oxidized l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 microg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2-* production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2-* by xanthine oxidase-lumazine and H2O2 by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species.

VEGF increases paracellular transport without altering the solvent-drag reflection coefficient.

Vascular endothelial growth factor (VEGF) increases microvascular permeability and has been implicated in the development of numerous pathologies including diabetic retinopathy (DR), hypoxia/ischemia, and tumor biology. The transport pathways by which water and solutes cross the endothelium in response to VEGF, however, are not completely understood. We measured, in real time, bovine retinal endothelial cell (BREC) hydraulic conductivity (Lp), 70 kDa dextran permeability (Pe), and the solvent-drag reflection coefficient (sigma) before and after addition of 50 ng/ml VEGF. The diffusional permeability coefficient for dextran (Pd) was measured before pressure gradient application. The sudden application of a 10-cm H2O hydrostatic pressure gradient induced water and solute fluxes that decayed to steady-state values after approximately 2 h. Subsequently, the addition of VEGF significantly increased Lp and Pe by 4.3-fold +/- 0.7-fold and 3.0-fold +/- 0.3-fold, respectively, after 110 min; however, the reflection coefficient remained approximately constant throughout the experiment (approximately 0.8). These observations suggest that water and dextran utilize common paracellular channels across BREC monolayers. Furthermore, the addition of VEGF increases the number or availability of channels but does not alter the selectivity of the monolayer to 70 kDa dextran.

A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells.

A sudden increase in the transmural pressure gradient across endothelial monolayers reduces hydraulic conductivity (L(p)), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70-kDa dextran, L(p), and the solvent-drag reflection coefficients (sigma) during the sealing process. The diffusional permeability coefficients for albumin (1.33 +/- 0.18 x 10(-6) cm/s) and dextran (0.60 +/- 0.16 x 10(-6) cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran (P(ealb) and P(edex)) were measured after the application of a 10 cmH(2)O pressure gradient; during the first 2 h of pressure application, P(ealb), P(edex), and L(p) were significantly reduced by 2.0 +/- 0.3-, 2.1 +/- 0.3-, and 3.7 +/- 0.3-fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens-1 (ZO-1) was significantly increased at cell-cell contacts after the application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered sigma despite significantly elevating both L(p) and effective permeability. Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin. Furthermore, the structures of the TJ determine transport rates but do not define the selectivity of the monolayer to solutes (sigma).

MEMS shear stress sensors for cardiovascular diagnostics.

Coronary artery disease is the leading cause of morbidity and mortality in the industrialized nations. Both biochemical and biomechanical stimuli modulate the pathogenesis of coronary artery diseases. Shear stress acting on the lumen of blood vessels intimately modulates the biological activities of vascular endothelial cells (ECs). We hereby develop microelectro mechanical system (MEMS)-based sensors at the dimension comparable to a single EC to monitor realtime shear stress in fluidic channel. Our goal is to fabricate sensors for ex vivo or in vivo shear stress measurement at Reynolds number commonly encountered in human circulation. The MEMS sensors were designed based on the previously described heat transfer principles. The polysilicon was doped with phosphorous to render the sensing element a high resistivity at 2.5 KOmega. The development of backside wire bonding enabled the application for the vascular geometry. The small dimension (80x2 mum) and the gain amplitude at 71 KHz offered an entry point to measure shear stress with high spatial and temporal resolution.

Detection of oxidized low density lipoproteins by indium oxide nanowires-based field effect transistor.

Oxidized low density lipoprotein (oxLDL) particles are known to initiate the development of coronary artery disease. Field effect transistors (FET) made of indium oxide (In2O3) nanowires have been demonstrated as sensitive chemical sensors for gaseous molecules such as NO2 and NH3. We hereby, apply In2O3 nanowires FETs to differentiate oxLDL from native LDL (nLDL), cytochrome-c and bovine serum albumin in terms of the transistor characteristic curves: I(D)-V(GS) (current versus drain-source voltage) and I(D)-V(GS) (current versus gate-source voltage). Measurements under static and flow conditions revealed that exposure of nanowires to these redox proteins selectively changed the FET conductivity and the gating effect of the transistors. To enhance the selectivity for oxLDL detection, surface chemistry modification with antioxLDL antibody was performed.

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin.

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.