Prostacyclin post-treatment improves LPS-induced acute lung injury and endothelial barrier recovery via Rap1

Protective effects of prostacyclin (PC) or its stable analog beraprost against agonist-induced lung vascular inflammation have been associated with elevation of intracellular cAMP and Rac GTPase signaling which inhibited the RhoA GTPase-dependent pathway of endothelial barrier dysfunction. This study investigated a distinct mechanism of PC-stimulated lung vascular endothelial (EC) barrier recovery and resolution of LPS-induced inflammation mediated by small GTPase Rap1. Efficient barrier recovery was observed in LPS-challenged pulmonary EC after prostacyclin administration even after 15 h of initial inflammatory insult and was accompanied by the significant attenuation of p38 MAP kinase and NFκB signaling and decreased production of IL-8 and soluble ICAM1. These effects were reproduced in cells post-treated with 8CPT, a small molecule activator of Rap1-specific nucleotide exchange factor Epac. By contrast, pharmacologic Epac inhibitor, Rap1 knockdown, or knockdown of cell junction-associated Rap1 effector afadin attenuated EC recovery caused by PC or 8CPT post-treatment. The key role of Rap1 in lung barrier restoration was further confirmed in the murine model of LPS-induced acute lung injury. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, and Evans blue extravasation and live imaging of vascular leak over 6 days using a fluorescent tracer. The data showed significant acceleration of lung recovery by PC and 8CPT post-treatment, which was abrogated in Rap1a(-/-) mice. These results suggest that post-treatment with PC triggers the Epac/Rap1/afadin-dependent mechanism of endothelial barrier restoration and downregulation of p38MAPK and NFκB inflammatory cascades, altogether leading to accelerated lung recovery

Microtubule Dynamics Control HGF-Induced Lung Endothelial Barrier Enhancement

Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.</p

Microtubule dynamics control HGF-induced lung endothelial barrier enhancement.

Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics

Expression of phosphorylation-deficient stathmin attenuates HGF-induced stimulation of peripheral MT network formation.

<p>Cells grown on coverslips were transfected with empty vector (Em. Vec.) or STMN-S63A and stimulated with HGF (50 ng/ml, 10 min). A: MT network was visualized by immunofluorescence staining of methanol-fixed cells with an antibody against β-tubulin. Transfected cells were detected by staining with His-tag antibody. Insets show magnified images with details of MT structure in non-transfected and STMN-S63A transfected cells. Bar  =  10 µm. Results are representative of three independent experiments. B: Bar graphs depict results of quantitative analysis of peripheral microtubules; n = 3; 10 cells from each experiment; *P<0.05.</p

Involvement of Rac pathway in HGF-induced MT-associated signaling.

<p><b>A–D</b>: HPAEC were subjected to pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) or knockdown of Rac1 or PAK1 as described in Methods and stimulated with HGF (50 ng/ml) for the indicated periods of time. <b>A</b>: Rac activation was determined by Rac-GTP pulldown assay. The content of activated Rac was normalized to the total Rac content in EC lysates. <b>B</b>: Time-dependent stimulation of stathmin phosphorylation and increase in tubulin acetylation was detected by western blot. <b>C</b>: Effect of preincubation with c-Met inhibitor on HGF-induced stathmin phosphorylation was evaluated by western blot with phospho-S⁶³-stathmin antibody. <b>D and E</b>: HGF-induced stathmin phosphorylation and tubulin acetylation in cells with Rac1 (<b>D</b>) and PAK1 (<b>E</b>) knockdown were evaluated by western blot. siRNA-induced target protein depletion was confirmed by membrane probing with Rac1 or PAK1 antibody. Equal protein loading in all assays was confirmed by membrane probing with β-actin antibody. <b>F</b>: Bar graphs depict the quantitative densitometry analysis of western blot data from four independent experiments; *P <0.05, RDU: relative density units.</p

Role of truncated oxidized phospholipids in acute endothelial barrier dysfunction caused by particulate matter

Particulate matter (PM) air pollution is a global environmental health problem contributing to more severe lung inflammation and injury. However, the molecular and cellular mechanisms of PM-induced exacerbation of lung barrier dysfunction and injury are not well understood. In the current study, we tested a hypothesis that PM exacerbates vascular barrier dysfunction via ROS-induced generation of truncated oxidized phospholipids (Tr-OxPLs). Treatment of human pulmonary endothelial cells with PM caused endothelial cell barrier disruption in a dose-dependent fashion. Biochemical analysis showed destabilization of cell junctions by PM via tyrosine phosphorylation and internalization of VE-cadherin. These events were accompanied by PM-induced generation of Tr-OxPLs, detected by mass spectrometry analysis. Furthermore, purified Tr-OxPLs: POVPC, PGPC and lyso-PC alone, caused a rapid increase in endothelial permeability and augmented pulmonary endothelial barrier dysfunction induced by submaximal doses of PM. In support of a role of TR-OxPLs-dependent mechanism in mediation of PM effects, ectopic expression of intracellular type 2 platelet-activating factor acetylhydrolase (PAFAH2), which specifically hydrolyzes Tr-OxPLs, significantly attenuated PM-induced endothelial hyperpermeability. In summary, this study uncovered a novel mechanism of PM-induced sustained dysfunction of pulmonary endothelial cell barrier which is driven by PM-induced generation of truncated products of phospholipid oxidation causing destabilization of cell junctions

Expression of phosphorylation-deficient stathmin attenuates HGF-induced EC barrier enhancement.

<p><b>A</b>: Endothelial monolayers transfected with phosphorylation-deficient stathmin (STMN-S63A) or empty vector (Em. Vec.) were stimulated with HGF (50 ng/ml). <b>A</b>: TER measurements were performed over 1.5 hrs. Bar graphs depict results of quantitative analysis of permeability data; n = 5; *P<0.05. <b>B</b>: Cortactin phosphorylation at Y⁴²¹ and tubulin acetylation at indicated time points of HGF treatment was monitored by Western blot. Probing for β-tubulin was used as a normalization control. Results are representative of three independent experiments. Bar graphs depict the quantitative densitometry analysis of western blot data; n = 4; *P <0.05, RDU: relative density units.</p

HGF stimulates peripheral MT growth.

<p>HPAEC grown on coverslips were stimulated with HGF (50 ng/ml, 10 min) with or without pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) followed by <b>A</b>: Immunofluorescence staining with an antibody against β-tubulin; <b>B</b>: Immunostaining with anti-EB1 antibody. Insets show high magnification images of cell periphery areas with microtubules or EB1-positive microtubule tips. Bar  =  5 µm. Results are representative of five independent experiments. Bar graphs depict results of quantitative analysis of peripheral microtubules (<b>A, right panel</b>) and peripheral EB1 (<b>B, right panel</b>) in methanol-fixed HPAEC; *P<0.05; n = 4; 6 images from each experiment. <b>C</b>: Live cell imaging of HPAEC expressing GFP-EB1 stimulated with HGF with or without pretreatment with c-Met inhibitor. Projection analysis of 20 consecutive images before and after HGF treatment shows changes in GFP-EB1 track length. Bar  =  2 µm. Quantification of GFP-EB1 track length is presented on right panels. Each pair of dots represents the median track length in a cell before and after thrombin treatment. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.</p

Role of Rac1 in HGF-induced stimulation of peripheral MT network formation.

<p><b>A</b>: Cells grown on coverslips were transfected with non-specific RNA or Rac1-specific siRNA and stimulated with HGF (50 ng/ml, 10 min) followed by immunofluorescence staining with an antibody against β-tubulin. Bar  =  5 µm. Magnified images (insets) show details of MT structure. Results are representative of four independent experiments. <b>B</b>: Fraction of peripheral MT was quantified as described in Methods; *P<0.05; n = 4; 6 images from each experiment. <b>C</b>: Projection analysis of 20 consecutive images in control (<b>top panel</b>) and Rac1 knockdown (<b>bottom panel</b>) live cells before and after HGF treatment shows changes in GFP-EB1 track length. Bar  =  2 µm. Quantification of GFP-EB1 track length is presented on right panels. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.</p