Huetran Duong

Protection against LPS-Induced Pulmonary Edema through the Attenuation of Protein Tyrosine Phosphatase–1B Oxidation

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins β-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and β-catenin, as well as enhanced β-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.

p18, a novel adaptor protein, regulates pulmonary endothelial barrier function via enhanced endocytic recycling of VE-cadherin

Vascular permeability is a hallmark of several disease states including acute lung injury (ALI). Endocytosis of VE‐cadherin, away from the interendothelial junction (IEJ), causes acute endothelial barrier permeability. A novel protein, p18, anchors to the endosome membrane and plays a role in late endosomal signaling via MAPK and mammalian target of rapamycin. However, the fate of the VE‐cadherin‐positive endosome has yet to be elucidated. We sought to elucidate a role for p18 in VE‐cadherin trafficking and thus endothelial barrier function, in settings of ALI. Endothelial cell (EC) resistance, whole‐cell ELISA, and filtration coefficient were studied in mice or lung ECs overexpressing wild‐type or nonendosomal‐binding mutant p18, using green fluorescent protein as a control. We demonstrate a protective role for the endocytic protein p18 in endothelial barrier function in settings of ALI in vitro and in vivo, through enhanced recycling of VE‐cadherin‐positive early endosomes to the IEJ. In settings of LPS‐induced ALI, we show that Src tethered to the endosome tyrosine phosphorylates p18 concomitantly with VE‐cadherin internalization and pulmonary edema formation. We conclude that p18 regulates pulmonary endothelial barrier function in vitro and in vivo, by enhancing recycling of VE‐cadherin‐positive endosomes to the IEJ.—Chichger, H., Duong, H., Braza, J., Harrington, E. O., p18, a novel adaptor protein, regulates pulmonary endothelial barrier function via enhanced endocytic recycling of VE‐cadherin. FASEB J. 29, 868–881 (2015). www.fasebj.org

SH2 Domain-Containing Protein Tyrosine Phosphatase 2 and Focal Adhesion Kinase Protein Interactions Regulate Pulmonary Endothelium Barrier Function

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients suffering from ALI.

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild‐type (PKCδ+/+) and null (PKCδ−/−) mice housed under normoxia (21% O2) or hyperoxia (∼95% O2). We noted a significantly greater number of TUNEL‐positive cells in lungs of hyperoxic PKCδ+/+ mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ+/+ mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ−/− hyperoxic mice, compared to lungs of PKCδ+/+ hyperoxic mice. In vitro, both hyperoxia and H2O2 promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H2O2 treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H2O2‐induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase‐mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H2O2‐induced LMVEC p38 activation. Conversely, overexpression of wild‐type PKCδ or the catalytically active PKCδ cleavage product greatly increased H2O2‐induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant‐induced apoptosis is due to increased PKCδ → p38 signaling, and we describe a PKCδ‐centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress. J. Cell. Physiol. 227: 1899–1910, 2012.

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Neovascularization in the pulmonary endothelium is regulated by the endosome: Rab4-mediated trafficking and p18-dependent signaling

Neovascularization, the formation of new blood vessels, requires multiple processes including vascular leak, migration, and adhesion. Endosomal proteins, such as Rabs, regulate trafficking of key signaling proteins involved in neovascularization. The novel endosome protein, p18, enhances vascular endothelial (VE)-cadherin recycling from early endosome to cell junction to improve pulmonary endothelial barrier function. Since endothelial barrier integrity is vital in neovascularization, we sought to elucidate the role for endosome proteins p18 and Rab4, Rab7, and Rab9 in the process of vessel formation within the pulmonary vasculature. Overexpression of wild-type p18 (p18(wt)), but not the nonendosomal-binding mutant (p18(N39)), significantly increased lung microvascular endothelial cell migration, adhesion, and both in vitro and in vivo tube formation. Chemical inhibition of mTOR or p38 attenuated the proneovascularization role of p18(wt). Similar to the effect of p18(wt), overexpression of prorecycling wild-type (Rab4(WT)) and endosome-anchored (Rab4(Q67L)) Rab4 enhanced neovascularization processes, whereas molecular inhibition of Rab4, by using the nonendosomal-binding mutant (Rab4(S22N)) attenuated VEGF-induced neovascularization. Unlike p18, Rab4-induced neovascularization was independent of mTOR or p38 inhibition but was dependent on p18 expression. This study shows for the first time that neovascularization within the pulmonary vasculature is dependent on the prorecycling endocytic proteins Rab4 and p18.