Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch
2006; Elsevier BV; Volume: 168; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.050431
ISSN1525-2191
AutoresAnna A. Birukova, Santipongse Chatchavalvanich, Alexander Rios, Kamon Kawkitinarong, Joe G. N. Garcia, Konstantin G. Birukov,
Tópico(s)Hemoglobin structure and function
ResumoVentilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier. Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier. Pathological lung overdistention is associated with mechanical ventilation at high-tidal volumes and compromises the blood-gas barrier, increases lung permeability, and may culminate in ventilator-induced lung injury and pulmonary edema.1Dos Santos CC Slutsky AS Invited review: mechanisms of ventilator-induced lung injury: a perspective.J Appl Physiol. 2000; 89: 1645-1655Crossref PubMed Scopus (405) Google Scholar, 2Uhlig S Ventilation-induced lung injury and mechanotransduction: stretching it too far?.Am J Physiol. 2002; 282: L892-L896Google Scholar Clinical studies suggest that vascular leak observed in ventilator-induced lung injury patients is associated with increased levels of edemagenic agents and inflammatory cytokines such as thrombin, histamine, tumor necrosis factor-α, interleukin-8, and interleukin-1.1Dos Santos CC Slutsky AS Invited review: mechanisms of ventilator-induced lung injury: a perspective.J Appl Physiol. 2000; 89: 1645-1655Crossref PubMed Scopus (405) Google Scholar, 3Narimanbekov IO Rozycki HJ Effect of IL-1 blockade on inflammatory manifestations of acute ventilator-induced lung injury in a rabbit model.Exp Lung Res. 1995; 21: 239-254Crossref PubMed Scopus (121) Google Scholar, 4Pugin J Dunn I Jolliet P Tassaux D Magnenat JL Nicod LP Chevrolet JC Activation of human macrophages by mechanical ventilation in vitro.Am J Physiol. 1998; 275: L1040-L1050PubMed Google Scholar, 5Vlahakis NE Schroeder MA Limper AH Hubmayr RD Stretch induces cytokine release by alveolar epithelial cells in vitro.Am J Physiol. 1999; 277: L167-L173PubMed Google Scholar However, the significance of the interactions between the edemagenic agents and pathological mechanical distension of the lung tissue in progression of ventilator-induced lung injury-associated vascular leak and pulmonary edema has been only recently recognized. 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Consistent with these findings, in vitro models of pulmonary cells exposed to pathophysiological regimen of mechanical stretch and edemagenic agonists may provide vital information about molecular mechanisms of mechanochemical regulation of lung endothelial or epithelial permeability. 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95: 892-901Crossref PubMed Scopus (135) Google Scholar Recent studies showed that Rac and Rho activities in vascular endothelial and smooth muscle cells can be regulated by mechanical forces.19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar, 20Shikata Y Rios A Kawkitinarong K DePaola N Garcia JG Birukov KG Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells.Exp Cell Res. 2005; 304: 40-49Crossref PubMed Scopus (143) Google Scholar, 21Tzima E del Pozo MA Shattil SJ Chien S Schwartz MA Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment.EMBO J. 2001; 20: 4639-4647Crossref PubMed Scopus (439) Google Scholar, 22Katsumi A Milanini J Kiosses WB del Pozo MA Kaunas R Chien S Hahn KM Schwartz MA Effects of cell tension on the small GTPase Rac.J Cell Biol. 2002; 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285: L785-L797Google Scholar In this study, we tested the hypothesis that CS related to physiological and pathological lung mechanical ventilation is critically involved in the regulation of agonist-induced disruption and recovery of pulmonary EC monolayer integrity. Using a cell culture model of human lung ECs exposed to CS at physiologically and pathologically relevant magnitudes in vitro, we investigated magnitude-dependent effects of CS on the recovery of EC monolayer integrity after thrombin challenge and linked these effects with activation of the small GTPases Rho and Rac. RhoA and Rac1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), a paxillin antibody was obtained from BD Transduction Laboratories (San Diego, CA), a cortactin antibody was purchased from Upstate Biotechnology (Lake Placid, NY), horseradish peroxidase-linked anti-mouse and anti-rabbit IgG antibodies were obtained from Cell Signaling Inc. (Beverly, MA). Rho and Rac activation kits were purchased from Upstate Biotechnology. Rac inhibitor NSC-23766 was purchased from Calbiochem (La Jolla, CA). All reagents used for immunofluorescent staining were purchased from Molecular Probes (Eugene, OR). Unless specified, all other reagents, including sphingosine 1-phosphate and human thrombin, were obtained from Sigma Chemical Co. (St. Louis, MO). Human pulmonary artery endothelial cells (HPAECs) were obtained from Clonetics, BioWhittaker Inc. (Frederick, MD). Cells were maintained in complete culture medium consisting of Clonetics EBM basic medium containing 10% fetal bovine serum and supplemented with a set of nonessential amino acids, endothelial cell (EC) growth factors, and 100 U/ml penicillin/streptomycin (Clonetics, BioWhittaker Inc.) and incubated at 37°C in humidified 5% CO2 incubator. Cells were used for CS experiments at passages 6 to 8. All CS experiments were performed using FX-4000T Flexercell Tension Plus system (Flexcell International, McKeesport, PA) equipped with a 25-mm BioFlex loading station, as previously described.19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar, 20Shikata Y Rios A Kawkitinarong K DePaola N Garcia JG Birukov KG Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells.Exp Cell Res. 2005; 304: 40-49Crossref PubMed Scopus (143) Google Scholar Experiments were performed in the presence of culture medium containing 2% fetal bovine serum. Briefly, HPAECs were seeded at standard densities (8 × 105 cells/well) onto collagen I-coated flexible bottom BioFlex plates. Both static HPAEC cultures and cells exposed to CS were seeded onto identical plates to ensure standard culture conditions. After 48 hours of culture, the medium was changed in each plate, and experimental plates with EC monolayers were mounted onto the Flexercell system and exposed to CS of desired magnitude (5% or 18% elongation) and duration (0 to 48 hours). Control BioFlex plates with static EC culture were placed in the same cell culture incubator. When necessary, static controls and CS-exposed HPAECs were treated with thrombin and incubated for 5, 15, 30, or 50 minutes of continuous exposure to both stimuli. At the end of the experiment, cell lysates were collected for Rac and Rho activation assays or for gel electrophoresis and Western blot analysis; alternatively, CS-exposed endothelial monolayers were fixed with 3.7% formaldehyde and used for immunohistochemistry. Predesigned Rac-specific small interfering RNA (siRNA) of standard purity was purchased from Ambion, Inc. (Austin, TX) in purified, desalted, deprotected, and annealed double-strand form. The following 21-bp duplexes of siRNA were used: sense 5′-GGAGAUUGGUGCUGUAAAAtt-3′ and anti-sense 5′-UUUUACAGCACCAAUCUCCtt-3′. Nonspecific, nonsilencing FI-luciferase GL2 duplex fluorescently labeled on the sense strand with 5′-fluorescein (Dharmacon Research, Lafayette, CO) was used as a control treatment. HPAECs were grown to 70% confluence, and the transfection of siRNA (final concentration, 100 nmol/L) was performed using GeneSilencer transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's protocol. Forty-eight hours later cells were used for the experiments. After exposure to CS, ECs were fixed in 3.7% formaldehyde solution in phosphate-buffered saline (PBS) for 10 minutes at 4°C, washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 30 minutes at room temperature, and blocked with 2% bovine serum albumin in PBS for 30 minutes. Incubation with antibody of interest was performed in blocking solution (2% bovine serum albumin in PBS) for 1 hour at room temperature followed by staining with Alexa 488-conjugated secondary antibodies. Actin filaments were stained with Texas Red-conjugated phalloidin diluted in the blocking solution. After immunostaining, the slides were analyzed using a Nikon video imaging system (Nikon Instech Co., Japan) consisting of a inverted microscope Nikon Eclipse TE300 connected to SPOT RT monochrome digital camera and image processor (Diagnostic Instruments, Sterling Heights, MI). The images were acquired using SPOT 3.5 acquisition software (Diagnostic Instruments) and processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). Quantitative analysis of paracellular gap formation was performed as previously described.19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar, 24Birukova AA Birukov KG Smurova K Adyshev DM Kaibuchi K Alieva I Garcia JG Verin AD Novel role of microtubules in thrombin-induced endothelial barrier dysfunction.FASEB J. 2004; 18: 1879-1890Crossref PubMed Scopus (173) Google Scholar, 25Birukova AA Smurova K Birukov KG Usatyuk P Liu F Kaibuchi K Ricks-Cord A Natarajan V Alieva I Garcia JG Verin AD Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms.J Cell Physiol. 2004; 201: 55-70Crossref PubMed Scopus (154) Google Scholar The 16-bit images were analyzed using MetaVue 4.6 software (Universal Imaging, Downington, PA). Paracellular gaps were manually marked out, and images were differentially segmented between gaps and cells based on image grayscale levels. The gap formation was expressed as a ratio of the gap area to the area of the whole image. The values were statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago, IL) software. For each experimental condition at least 10 microscopic fields in each independent experiment were analyzed from the different areas of the plate (both central and peripheral). The representative images of EC monolayers closer to the plate periphery were taken to illustrate amplitude-dependent effects of CS on thrombin-induced gap formation, cortactin translocation, and focal adhesion remodeling. Immunoblot detection of proteins of interest was performed as described previously.19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar, 26Birukov KG Leitinger N Bochkov VN Garcia JG Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins.Microvasc Res. 2004; 67: 18-28Crossref PubMed Scopus (56) Google Scholar Briefly, protein extracts were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (100 V for 1 hour), and probed with Rho or Rac antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence according to the manufacturer's protocol (Amersham, Little Chalfont, UK). The relative intensities of the protein in the bands were quantified by scanning densitometry. Rho and Rac activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology, as we have previously described.14Birukova AA Smurova K Birukov KG Kaibuchi K Garcia JGN Verin AD Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction.Microvasc Res. 2004; 67: 64-77Crossref PubMed Scopus (230) Google Scholar, 18Birukov KG Bochkov VN Birukova AA Kawkitinarong K Rios A Leitner A Verin AD Bokoch GM Leitinger N Garcia JG Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac.Circ Res. 2004; 95: 892-901Crossref PubMed Scopus (135) Google Scholar Briefly, ECs pre-exposed to CS at selected magnitude and duration were treated with vehicle or stimulated with thrombin for desired periods of time. Then, cell lysates were collected, and GTP-bound Rac or Rho were captured using pulldown assays with immobilized PBD domain and rhotekin, respectively, according to the manufacturer's protocols. The levels of activated small GTPases as well as total Rac and Rho content were evaluated by Western blot analysis and quantified by scanning densitometry of the autoradiography films. The levels of activated proteins Rac or Rho were normalized to total Rac or Rho level for densitometry evaluations. Results are expressed as means ± SD of three to five independent experiments. Stimulated samples were compared with controls by unpaired Student's t-test. For multiple-group comparisons, one-way analysis of variance followed by the post hoc Fisher's test were used. P < 0.05 was considered statistically significant. We have previously shown magnitude-dependent effects of CS on the acute phase of thrombin-induced EC barrier disruption.19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar In this study, we examined the effects of 5% CS and 18% CS on restoration of EC monolayer integrity after thrombin challenge. Human pulmonary ECs were exposed to 5% or 18% CS for 2 hours followed by continuous thrombin (50 nmol/L) stimulation for 5 minutes (peak of barrier disruption) or 50 minutes (recovery phase). Cells exposed to 2 hours of 5% CS or 18% CS without agonist stimulation revealed similar patterns of cytoskeletal arrangement characterized by circumferential F-actin rim and few central stress fibers oriented in a perpendicular direction to the main distension vector (Figure 1A, top). Consistent with our previous studies,19Birukov KG Jacobson JR Flores AA Ye SQ Birukova AA Verin AD Garcia JG Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.Am J Physiol. 2003; 285: L785-L797Google Scholar ECs exposed to pathologically relevant CS levels (18% elongation) showed a dramatic increase in the level of paracellular gap formation after 5 minutes of thrombin treatment (shown by arrows), as compared to ECs exposed to physiological CS levels (5% elongation) or ECs in static culture (Figure 1A, middle). Analysis of EC monolayer recovery (after 50 minutes of thrombin challenge) showed that static controls and ECs exposed to 18% CS exhibited more stress fiber bundles and larger paracellular gaps, whereas ECs exposed to 5% CS exhibited less central stress fibers with more pronounced peripheral F-actin staining and demonstrated nearly complete disappearance of paracellular gaps (Figure 1A, bottom). Quantitative analysis of thrombin-induced (50 nmol/L, 5 minutes) gap formation (Figure 1B) confirmed these observations and showed that total gap areas in static controls and or EC monolayers exposed to 5% CS represented 60 to 65% of total gap area observed in EC monolayers exposed to 18% CS. More importantly, physiological CS level accelerated paracellular gap resolution after thrombin stimulation (50 nmol/L, 50 minutes). As demonstrated in Figure 1B, EC monolayers preconditioned at 5% CS showed a 4.8-fold reduction in the gap area after 50 minutes of thrombin stimulation, as compared to a 2.5-fold reduction in static EC cultures and a 2.6-fold reduction in ECs exposed to 18% CS. Remodeling of EC focal adhesions plays an important role in the regulation of the endothelial barrier by chemical agonists27Shikata Y Birukov KG Birukova AA Verin AD Garcia JG Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion remodeling: role of Src and GIT.FASEB J. 2003; 17: 2240-2249Crossref PubMed Scopus (115) Google Scholar and has been described in ECs exposed to CS.20Shikata Y Rios A Kawkitinarong K DePaola N Garcia JG Birukov KG Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells.Exp Cell Res. 2005; 304: 40-49Crossref PubMed Scopus (143) Google Scholar, 28Geng WD Boskovic G Fultz ME Li C Niles RM Ohno S Wright GL Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells.J Cell Physiol. 2001; 189: 216-228Crossref PubMed Scopus (37) Google Scholar, 29Naruse K Yamada T Sai XR Hamaguchi M Sokabe M Pp125FAK is required for stretch dependent morphological response of endothelial cells.Oncogene. 1998; 17: 455-463Crossref PubMed Scopus (91) Google Scholar In this study, we examined focal adhesion rearrangement in ECs exposed to physiological and pathological CS levels and stimulated with thrombin. Focal adhesion-associated protein paxillin was used to monitor CS- and/or agonist-induced focal adhesion remodeling. Thrombin stimulation (50 nmol/L, 5 minutes) caused an increase in the number and size of adhesion plaques in static and CS-preconditioned ECs (Figure 2A, middle). However, during the recovery phase after thrombin stimulation (50 minutes), cells exposed to 5% CS revealed accumulations of paxillin-containing focal adhesions at the cell periphery, whereas ECs preconditioned at 18% CS displayed random distribution of focal adhesions of larger size and numbers, as compared to static cultures and ECs exposed to 5% CS (Figure 2, A and B). Results of this study show that 5% CS promoted EC monolayer recovery after thrombin stimulation, which was characterized by disappearance of paracellular gaps and restoration of peripheral actin cytoskeleton, whereas high-magnitude CS delayed these processes (Figure 1). We and others have previously shown that enhancement of EC barrier induced by laminar shear stress or chemical agonists is associated with peripheral accumulation of a regulator of actin polymerization, cortactin.18Birukov KG Bochkov VN Birukova AA Kawkitinarong K Rios A Leitner A Verin AD Bokoch GM Leitinger N Garcia JG Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac.Circ Res. 2004; 95: 892-901Crossref PubMed Scopus (135) Google Scholar, 20Shikata Y Rios A Kawkitinarong K DePaola N Garcia JG Birukov KG Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells.Exp Cell Res. 2005; 304: 40-49Crossref PubMed Scopus (143) Google Scholar, 30Panetti TS Differential effects of sphingosine 1-phosphate and lysophosphatidic acid on endothelial cells.Biochim Biophys Acta. 2002; 1582: 190-196Crossref PubMed Scopus (94) Google Scholar, 31Dudek SM Jacobson JR Chiang ET Birukov KG Wang P Zhan X Garcia JG Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase.J Biol Chem. 2004; 279: 24692-24700Crossref PubMed Scopus (245) Google Scholar, 32Endo A Nagashima K Kurose H Mochizuki S Matsuda M Mochizuki N Sphingosine 1-phosphate induces membrane ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and CrkII.J Biol Chem. 2002; 277: 23747-23754Crossref PubMed Scopus (77) Google Scholar, 33Birukov KG Birukova AA Dudek SM Verin AD Crow MT Zhan X DePaola N Garcia JG Shear stress-mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells.Am J Respir Cell Mol Biol. 2002; 26: 453-464Crossref PubMed Scopus (186) Google Scholar ECs exposed to 5% CS or 18% CS exhibited diffuse cortactin distribution before and after 5 minutes of thrombin (50 nmol/L) stimulation (data not shown). During
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