Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch

Konstantin G. Birukov, Jeffrey R. Jacobson, Alejandro A. Flores, Shui Q. Ye, Anna A. Birukova, Alexander D. Verin, and Joe G. N. Garcia

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224

Submitted 8 October 2002 ; accepted in final form 9 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 µM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.

mechanical strain; thrombin; permeability; cytoskeleton; actin; myosin; phosphorylation


MECHANICAL VENTILATION at high tidal volumes is associated with compromised blood-gas barrier and increased lung permeability and may culminate in ventilator-induced lung injury syndrome or pulmonary edema (13). Clinical studies and in vivo models of mechanical ventilation, including animal studies and isolated perfused lungs exposed to mechanical ventilation at excessive tidal volumes, suggest an activation of inflammatory events such as macrophage accumulation and increased cytokine production (39, 47, 53). Studies of cellular mechanisms underlying ventilator-induced lung injury suggest involvement of multiple cell types (alveolar epithelium, neutrophils, alveolar macrophages, and lung endothelium) in this process (for review see Ref. 13) and demonstrate several potential pathways leading to blood-gas barrier dysfunction. These pathways include activation of IL-8 production by alveolar epithelium (58) and lung macrophages (41), leukocyte infiltration, and alveolar epithelial injury or death (52).

Direct measurements of interstitial/vascular distension in the mechanically ventilated lung are not currently available because of the complexity of local distension patterns in the lung parenchyma. This is especially true during mechanical ventilation of injured lung when inflammation-induced occlusion of a part of the alveolar tree results in overdistension of the functional alveoli. However, studies of alveolar epithelial cell cultures exposed to mechanical strain in vitro suggest that a 25% increase in cell surface area corresponding to 8-12% linear distension likely correlates with physiological levels of mechanical strain experienced by alveolar epithelium, whereas cyclic stretch (CS) resulting in a 37-50% increase in cell surface area corresponding to 17-22% linear distension is relevant to pathophysiological conditions produced by mechanical ventilation and causes progressive cell death (52). In vitro endothelial cell culture models exposed to CS at an average of 15-20% linear elongation demonstrate vascular cell activation characterized by VEGF expression (42) and fibroblast growth factor release (6) as well as production of inflammatory cytokine IL-8 and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1) (37). Fewer data are available on endothelial cell physiological cellular responses at different amplitudes; however, studies by Okada and colleagues (37) demonstrate that activation of IL-8 and MCAF/MCP-1 production is amplitude dependent and reached maximal values at 15% average elongation, which is related to activation of pulmonary endothelium by excessive mechanical ventilation. On the other hand, findings by Yang and coworkers (63) suggest differential regulation of matrix metalloproteinase-1 expression by small and high mechanical strains, suggesting the importance of specific stretch amplitudes for mechanosensitive regulation of vascular remodeling. Thus clinical observations and studies using animal and cell culture models suggest that mechanical strain beyond 15% linear elongation may be considered pathophysiological.

Similar to alveolar epithelium, lung vascular endothelial cells experience mechanical strain resulting from respiratory cycles and intraluminal blood pressure. However, modulation of the pulmonary endothelial barrier by mechanical forces, although suggestive, has not yet been evaluated. Regulation of the functionally complex, semiselective vascular endothelial barrier suggests an essential role for the endothelial cell cytoskeleton in maintaining equilibrium between barrier-disrupting contractile forces and barrier-protective cell-adhesive processes (14). Increased endothelial cell permeability in response to thrombin stimulation is associated with dramatic actomyosin cytoskeletal rearrangement, activation of endothelial cell contraction, gap formation (14, 21, 22, 57), and phosphorylation of regulatory myosin light chains (MLC) mediated by MLC kinase or via Rho kinase-dependent mechanisms (9, 25). In turn, endothelial cell barrier enhancement induced by barrier-protective factors, such as platelet-derived phospholipid sphingosine-1-phosphate (S1P) or hepatocyte growth factor (HGF), also requires actomyosin remodeling that is, however, different from that induced by thrombin and includes a small GTPase rac-dependent formation of a prominent cortical actin rim, disappearance of central stress fibers, peripheral accumulation of phosphorylated MLC, and enhancement of endothelial cell adherens junctions (18, 27), suggesting that spatially defined MLC phosphorylation and actomyosin activity may be a critical component of endothelial cell barrier regulation (18). Although a large list of barrier-disruptive (i.e., thrombin, histamine, tumor necrosis factor, interleukins) and barrier-protective (i.e., S1P, HGF) chemical agonists is now described in the literature (18, 27, 33, 40, 55), a role of mechanical factors persisting in pulmonary circulation, such as shear stress and CS in the regulation of vascular permeability, is not well known. Shear stress is imposed on the surface of the endothelial monolayer underlying pulmonary vasculature by blood flow. Studies from systemic circulation suggest a barrier-protective effect of physiological laminar flow on the vascular endothelium (11), and recent studies by our laboratory (4) and others (12, 43) suggest that laminar shear stress induces significant cytoskeletal changes in pulmonary endothelial cells that reflect barrier-protective effect of physiological flow on the pulmonary endothelial cell. Effects of CS on endothelial cell barrier are not yet studied; however, mechanical strain has been shown to have a dramatic effect on endothelial cell cytoskeletal organization (30, 34, 60), and actin cytoskeleton has been demonstrated to play a critical role in CS-induced cell reorientation (46).

In this work, we demonstrated an essential role of cytoskeletal rearrangement and MLC phosphorylation in CS-induced cytoskeletal rearrangement and investigated effects of chronic CS preconditioning at magnitudes relevant to physiological and pathophysiological conditions on the endothelial cell barrier properties under basal conditions and after agonist stimulation. Our data demonstrate that CS preconditioning of pulmonary endothelium at high amplitude cyclic stretch (18% elongation) enhances barrier-disruptive effects of thrombin, whereas endothelial cell exposure to a physiologically relevant magnitude of CS (5% elongation) revealed a barrier-protective effect. Finally, the results of gene profiling suggest phenotypic changes of human pulmonary artery endothelial cells (HPAEC) exposed to CS. The results of this study suggest an important mechanism for differential phenotypic regulation of pulmonary endothelium barrier properties by physiological and pathophysiological mechanical stimulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. Anti-diphospho-MLC-specific antibodies that recognize phosphorylated Ser19 and Thr18 of myosin regulatory light chains were utilized and have been described previously (40). Anti-phospho-specific p38, anti-phospho-specific extracellular signal-regulated kinase (ERK)1/2, and horseradish peroxidase-conjugated secondary antibodies were obtained from New England BioLabs (Beverly, MA). Anti-p38 antiserum was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-tyrosine (PY20) and anti-ERK1/2 antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). The following reagents were also commercially obtained: the MAP or ERK kinase (MEK) inhibitor U-O126 (Promega, Madison, WI); Rho kinase inhibitor Y-27632 (Tocris Cookson, Ballwin, MO); MLC kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-7) and p38 MAP kinase inhibitor SB-203580 (Calbiochem, La Jolla, CA); and Texas red phalloidin (Molecular Probes, Eugene, OR).

Cell culture. HPAEC were obtained from Clonetics, Bio-Whittaker (Frederick, MD). Cells were maintained in complete culture medium consisting of Clonetics EBM basic medium containing 10% bovine serum, supplemented with a set of nonessential amino acids, endothelial cell growth factors, and 100 U/ml of penicillin/streptomycin (Clonetics, BioWhittaker), and incubated at 37°C in a humidified 5% CO2 incubator. Cells were used for CS experiments at passages 6-8.

Cell culture under CS. All CS experiments were performed with the FX-4000T Flexercell Tension Plus system (Flexcell International, McKeesport, PA) equipped with a 25-mm BioFlex loading station designed to provide uniform radial and circumferential strain across a membrane surface along all radii. BioFlex loading station is composed of a single plate and six planar 25-mm cylinders per plate centered beneath each well of the BioFlex plate, and the top surface is just below the BioFlex membrane surface. Each BioFlex membrane is stretched over the post when under vacuum pressure, creating a single-plane uniformly stretched circle. The radial and circumferential strain was experimentally determined by the vendor (Flexcell International) by stamping the BioFlex membrane with a dot pattern followed by labeling the distance between each pair of dots and measuring their change relative to vacuum levels. Experimental measurements demonstrated that the part of the membrane stretching over the post (25-mm diameter) received uniform strain in the radial direction that was proportional to vacuum level. To ensure that only endothelial cells exposed to controlled levels of strain were used for further analysis, a 5-mm-thick peripheral portion of the cells was removed at the end of the CS experiment with a stainless steel instrument. All CS experiments were performed in the presence of complete culture medium containing 10% fetal bovine serum. HPAEC were seeded at standard densities (8 x 105 cells/well) onto collagen I-coated flexible-bottomed BioFlex plates. Both static HPAEC cultures and cells exposed to CS were seeded onto identical plates to ensure standard culture conditions. After 48 h of culture, medium was changed in each plate, and experimental plates with endothelial cell monolayers were mounted onto the Flexercell system and exposed to CS of desired magnitude (5 or 18% elongation) and duration (0-48 h). Control BioFlex plates with static endothelial cell culture were placed in the same cell culture incubator. When necessary, HPAEC were preincubated with protein kinase inhibitors (5 µM Y-27632, 10 µM U-0126, 20 µM SB-203580, 10 µM ML-7, or 50 µM forskolin) during 1 h before CS. At the end of the experiment, cells were lysed in SDS buffer for gel electrophoresis and Western blot analysis or fixed with 3.7% formaldehyde, and the actin cytoskeleton was visualized with immunofluorescent staining protocol with Texas red-conjugated phalloidin.

Western immunoblotting. Protein extracts were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (30 V for 18 h or 90 V for 2 h), and the membranes were incubated with specific antibodies of interest. Immunoreactive proteins were detected with the enhanced chemiluminescence detection system (ECL) according to the manufacturer's directions (New England BioLabs). The relative intensities of the protein in the bands were quantified by scanning densitometry.

Cell viability. In the last 30 min of CS exposure, ethidium homodimer-1 and calcein-AM (LIVE/DEAD kit; Molecular Probes) were added to the cells at a final concentration of 0.23 and 0.12 µM, respectively. Ethidium homodimer-1 (excitation ~495 nm and emission ~635 nm), which is excluded by the intact plasma membrane of live cells, enters cells with damaged membranes and undergoes a fluorescence enhancement on binding to nucleic acids. Calcein-AM (excitation ~495 nm and emission ~515 nm), a cell-permeant dye, is retained within live cells. After termination of CS experiments, control and CS-exposed wells were examined with an inverted epifluorescence microscope (x20 objective, TE-300; Nikon), and images of the cells were captured with a digital imaging system (Sony DKC-5000 LCD camera, Adobe Photoshop 5.1 software). Images were acquired from three random locations in the well, six wells for each experimental condition, at both emissions for visualizing ethidium homodimer-1 and calcein-AM. The differentially stained cells were counted, and the numbers of nonviable cells were expressed as a percentage of the total number of cells. Results across all wells in an experimental group were averaged and are expressed as means ± SD. Comparisons between specific experimental groups were evaluated with unpaired Student's t-test.

Immunoblot detection of ERK1/2 and p38 MAP kinases. Activation of ERK1/2 and p38 MAP kinases was assessed by determining the phosphorylation status of the proteins by immunoblotting with a phospho-specific antibody. Briefly, after being exposed to CS, cells were washed with ice-cold PBS twice and scraped in SDS-PAGE sample buffer containing 500 mM Tris, pH 6.8, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2% SDS, 20% 2-mer-captoethanol, and 10% glycerol, boiled for 5 min, and samples were resolved on 8% SDS-PAGE and transferred to Immobilon membrane (Millipore, Bedford, MA). The membrane was probed with appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody followed by immunodetection using ECL reagents (New England Bio-Labs) and autoradiography. To ensure equal loading, the membranes were stripped and reprobed with anti-ERK-1/2 or anti-p38 antibody. After protein detection on the Western blots by ECL, autoradiography films were subjected to scanning densitometry and analyzed with ImageQuant software.

MLC phosphorylation in intact endothelium. Phosphorylation profiles of regulatory MLC were analyzed by Western blotting using anti-diphospho-MLC antibody and an ECL detection kit (New England BioLabs). The relative intensities of the protein in the bands were quantified by scanning densitometry.

Immunofluorescent staining. After CS, cells were fixed in 3.7% formaldehyde solution in PBS for 10 min at 4°C, washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min at 4°C, and blocked with 2% BSA in PBS for 20 min. Actin filaments were stained with Texas red-conjugated phalloidin (Molecular Probes) diluted in the blocking buffer. After the immunostaining procedure, cell preparations were analyzed with a Nikon video-imaging system consisting of a phase-contrast inverted microscope connected to a digital camera and image processor. The images were recorded and processed with Adobe Photoshop 5.5 software. For quantitative analysis of thrombin-mediated gap formation, endothelial cells grown on BioFlex plates were left static or exposed to CS followed by 5-min thrombin stimulation. After CS was discontinued, culture medium from BioFlex plates was aspirated and endothelial cell monolayers were fixed with 3.7% formaldehyde solution in PBS and used for immunofluorescent staining with Texas red-conjugated phalloidin. Elastic membranes with cells were then excised from the BioFlex plates, mounted onto large coverslips, and inspected by fluorescent microscopy. Images from at least five microscopic fields were processed with Adobe Photoshop 5.5, gaps were outlined, and a number of gaps per microscopic field and their individual surface areas were measured with ImageQuant 5.2 software. Integral gap surface areas were calculated for each microscopic field and used for statistical analysis as a morphological parameter of thrombin-induced endothelial cell barrier dysfunction. Control analysis of endothelial cell monolayer integrity was also performed in static and CS-stimulated HPAEC cultures without thrombin stimulation, and results suggest that only a few gaps per 10 microscopic fields were typically found in static and stretched HPAEC without thrombin stimulation.

Measurements of transendothelial monolayer resistance. Transmonolayer electrical resistance of HPAEC grown on gold electrodes was measured with the electrical cell impedance sensor technique as previously described (19, 45) using the electrical cell-substrate impedance sensing (ECIS) system (Applied Biophysics, Troy, NY). After 48 h of CS preconditioning at 5 or 18% elongation, HPAEC from experimental or static plates were trypsinized, counted, and seeded at equal densities (2 x 105 cells/ml) into eight-well ECIS plates (400 µl/well). ECIS plates were placed into the cell culture incubator for 16 h to allow cell spreading and attachment. Electrical resistance increased immediately after the cells attached to and covered the electrodes, and the resistance achieved a steady state when the endothelial cells became confluent. The experiments were conducted after the electrical resistance achieved a steady state. Resistance data were normalized to the initial voltage and plotted as normalized resistance.

Expression profiling and data analysis. Endothelial cell cultures placed in the FlexCell unit were exposed to 5 and 18% elongation for 0, 6, 24, and 48 h. Expression profiling was carried out using the Affymetrix GeneChip System in the Gene Expression Profiling Core, Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine. Briefly, endothelial cell total RNA from each time point was extracted with TRIzol reagent (GIBCO BRL) and purified with RNeasy Mini kit (Qiagen, Santa Clarita, CA). Total RNA (7.5 µg) from each time point was converted into double-stranded cDNA by using SuperScript Choice system (GIBCO BRL) with an oligo(dT) primer containing T7 RNA polymerase promoter (Genset). The double-stranded cDNA was purified by phenol/chloroform extraction and then used for in vitro transcription using the BioArray HighYield RNA transcript labeling kit (Enzo). Biotin-labeled cRNA was purified by RNeasy Mini kit and fragmented. cRNA samples were hybridized to an Affymetrix HG-U95A chip for 16 h. The chip was washed and stained on the Affymetrix Fluidics Station 400 using instructions and reagents provided by Affymetrix. This involved removal of nonhybridized material and then incubation with phycoerythrin-streptavidin to detect bound cRNA. The signal intensity was amplified by a second staining with biotin-labeled anti-streptavidin antibody and followed phycoerythrin-streptavidin staining. Microplates were scanned and fluorescence intensities were quantified with the Agilent GeneArray scanner. Data analysis was carried out with the Affymetrix Total Desktop Mining Solution, including Microarray Suite, MicroDB, Data Mining Tool, and Genespring analysis software package.

Statistical analysis. ANOVAs with a Student-Newman-Keuls test were used to compare the means of two or more different treatment groups. Results are expressed as means ± SE. Differences between two groups were considered statistically significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cytoskeletal response of HPAEC to CS. Endothelial cells grown on flexible-bottomed plates revealed rapid reorientation of actin microfilaments on exposure to CS with rearrangement in the direction perpendicular to the main distension vector (Fig. 1). Actin remodeling was observed to occur in two phases with early changes (10 min after onset of CS exposure) characterized by an increased number of stress fibers oriented perpendicular to the main distension vector. At later time points (1 h), when endothelial cell reorientation became evident, a partial dissolution of centrally localized stress fibers and concomitant enhancement of cortical actin rim was observed. This cytoskeletal arrangement specific for an endothelial cell monolayer cultured under CS was further maintained in endothelial cells exposed to CS for up to 96 h.



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Fig. 1. Cyclic stretch (CS)-induced cytoskeletal remodeling in human pulmonary artery endothelial cells (HPAEC). HPAEC grown on collagen I-coated BioFlex plates were subjected to CS at 18% elongation at 30 cycles/min for the indicated periods of time. F-actin staining was performed with Texas red-conjugated phalloidin. Increased stress fiber formation and alignment of actin filaments in a direction perpendicular to the distension vector were noticeable at 10 min of stretch exposure. Partial disappearance of central stress fibers and enhancement of cortical actin rim is observed at later time points. Bar = 10 µm. Results are representative of 4 independent experiments.

 

Actin cytoskeletal rearrangement involves actin polymerization and assembly of functional actomyosin complex, which includes phosphorylation of regulatory MLC. Analysis of endothelial cells subjected to CS revealed that CS induced rapid and transient increases in MLC phosphorylation that were significant at 10 min, followed by a gradual decline to basal levels by 2 h (Fig. 2). The time course of transient MLC phosphorylation correlated with the dynamics of actin cytoskeleton rearrangement shown in Fig. 1. Short-term CS also induced transient activation of ERK1/2 and p38 MAP kinases (Fig. 2), consistent with previous reports (10, 23, 28). To investigate the potential involvement of p38 and ERK1/2 activation and MLC phosphorylation in CS-mediated cytoskeletal reorientation, endothelial cells were pretreated with MEK and p38 MAP kinase inhibitors U-O126 and SB-203580, respectively. Figure 3 demonstrates that MAP kinase inhibitors did not affect CS-induced cytoskeletal rearrangement, whereas reduction in MLC phosphorylation via pretreatment with the Rho kinase inhibitor Y-27632 (Fig. 3, bottom left) or forskolin (Fig. 3, bottom right) abolished CS-induced F-actin reorientation. These results suggest a critical role for MLC phosphorylation in CS-mediated actin cytoskeletal remodeling.



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Fig. 2. Transient mitogen-activated protein (MAP) kinase activation and myosin light chain (MLC) phosphorylation in HPAEC exposed to CS. Activation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases in response to CS at 18% elongation and CS-induced MLC diphosphorylation was detected by Western blot as described in MATERIALS AND METHODS. Increase in phosphorylation state indicates time-dependent activation with a peak at 30 min for ERK1/2 and 10 min for p38. Peak of MLC diphosphorylation was observed by 30 min of CS. Results are representative of 3 independent experiments.

 


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Fig. 3. Effects of protein kinase inhibitors on CS-induced F-actin rearrangement. HPAEC were preincubated 1 h before the CS experiment as outlined in MATERIALS AND METHODS. Pretreatment with Y-27632 (5 µM, 30 min) and forskolin (50 µM, 30 min) attenuated CS-mediated F-actin rearrangement. Bar = 10 µm. Results are representative of 4 independent experiments. SB, SB-203580.

 

Magnitude-dependent effects of acute and long-term CS on thrombin-induced endothelial cell MLC phosphorylation and gap formation. Examination of the effects of high- and low-magnitude CS on thrombin-induced MLC phosphorylation revealed that 15 min of exposure of HPAEC to 5% CS resulted in a 2.1-fold increase in MLC phosphorylation compared with static endothelial cells challenged with the same dose of thrombin (Fig. 4, A and B). Furthermore, HPAEC exposed to 18% CS during 15 min revealed an even greater (3.2-fold) increase in MLC phosphorylation on thrombin stimulation compared with static culture. Interestingly, increased MLC phosphorylation in response to thrombin was also observed in HPAEC exposed to chronic (48 h) CS (Fig. 4C). Results of quantitative densitometry of Western blots probed with anti-diphospho-MLC antibody suggest a 2.1-fold and 3.1-fold increase in MLC phosphorylation in endothelial cells exposed to CS at 5 and 18% elongation on thrombin challenge, respectively, when compared with static culture. Both basal and thrombin-induced MLC phosphorylation in CS-preconditioned and static endothelial cell cultures was partially attenuated by the MLC kinase inhibitor ML-7 and was completely abolished by the Rho kinase inhibitor Y-27632 (Fig. 4D), suggesting involvement of MLC kinase- and Rho-dependent mechanisms in CS-induced MLC phosphorylation.



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Fig. 4. Stretch-dependent enhancement of thrombin-induced MLC phosphorylation. HPAEC exposed to 15-min CS at 5 or 18% elongation were stimulated with 50 nM thrombin for an additional 5 min. MLC phosphorylation was assessed by Western blot of the cell lysates with diphospho-MLC antisera (A) and quantified by scanning densitometry (B) as described in MATERIALS AND METHODS. C: increased MLC phosphorylation in HPAEC exposed to 48-h CS at 5 or 18% elongation followed by 50 nM thrombin stimulation. D: effects of HPAEC pretreatment with 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-7; 10 µM, 30 min) or Y-27632 (5 µM, 30 min) on MLC phosphorylation in HPAEC exposed to 15 min of 18% CS and stimulated with thrombin (50 nM, 5 min). In each experiment, equal loadings were confirmed by reprobing the membranes with pan-MLC antibody (A). Each point represents the means ± SE of 3 experiments. Results are significant at *P < 0.05. VEH, vehicle; Arb. arbitrary; ppMLC, diphospho-MLC; OD, optical density; pan-MLC, total MLC.

 

In the next series of experiments, we examined morphological changes of endothelial cell monolayers exposed to 48 h of CS at 5 or 18% elongation followed by 5-min stimulation with thrombin. Immunofluorescent staining of endothelial cell monolayers revealed distinct cytoskeletal arrangement of unstimulated CS-preconditioned endothelial cells (Fig. 5, top right) with prominent peripheral actin rim and a reduced number of central stress fibers compared with more random microfilament alignment in static endothelial cells (Fig. 5, top left). Endothelial cell monolayers exposed to 48 h of CS at 18% elongation exhibited an increased number of gaps with larger gap areas after thrombin challenge (Fig. 5, middle right) compared with thrombin-stimulated static culture (Fig. 5, bottom left). Endothelial cell monolayers preconditioned at 5% elongation revealed fewer gaps and a more preserved monolayer structure compared with 18% CS-preconditioned endothelial cell culture (Fig. 5, bottom right). Quantitative analysis of CS-induced gap formation in static and CS-preconditioned HPAEC cultures revealed that the integral gap surface area in endothelial cells preconditioned at 5% CS and exposed to thrombin for 5 min (2,435 ± 320 µm2 per microscopic field) was higher than in static endothelial cells exposed to thrombin (937 ± 15.7 µm2 per microscopic field), but maximal gap formation was observed after thrombin stimulation of CS exposed to 18% CS (4,811 ± 440 µm2 per microscopic field). Importantly, neither short-term (10-60 min) nor long-term CS preconditioning (48 h) compromised HPAEC monolayer integrity in the absence of thrombin (Fig. 1 and Fig. 5, top right).



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Fig. 5. CS preconditioning potentiates thrombin-induced gap formation. After 48 h of CS preconditioning at 18 or 5% elongation, cells were stimulated with 50 nM thrombin for 5 min, and actin cytoskeleton remodeling and gap formation were compared with those in static thrombin-stimulated HPAEC culture. F-actin was detected with staining with Texas redconjugated phalloidin. Arrows depict gaps in endothelial cell monolayer observed in static and CS-exposed endothelial cell cultures as result of thrombin stimulation. Results are representative of 4 independent experiments. Bar = 10 µm.

 

Thus our results demonstrate a key role for MLC phosphorylation in CS-induced cytoskeletal reorientation (Fig. 3) and suggest synergistic effects of thrombin and CS on MLC phosphorylation (Fig. 4) that are linked to increased gap formation in endothelial cells stimulated with thrombin and CS (Fig. 5). Next, we directly assessed effects of inhibition of MLC phosphorylation on CS- and thrombin-induced gap formation. HPAEC exposed to 48 h of CS at 18% elongation were preincubated with the Rho kinase inhibitor Y-27632 (5 µM, 30 min) before thrombin challenge. Figure 6 demonstrates that gap formation induced by thrombin and further potentiated by CS exposure was significantly attenuated by endothelial cell pretreatment with Y-27632. Quantitative analysis of gap formation (Fig. 6B) depicts a dramatic decrease in integral gap surface area in static endothelial cells and cells exposed to CS when pretreated with Y-27632 before thrombin challenge. Figure 6C shows that the barrier-protective effect of Y-27632 correlates with inhibition of MLC phosphorylation in both static and CS-preconditioned endothelial cells. Thus our results demonstrate a critical role for MLC phosphorylation in regulation of actomyosin cytoskeleton and endothelial cell barrier integrity by mechanical and chemical stimuli and suggest a Rho/Rho kinase- and MLC-dependent mechanism for pulmonary endothelial cell barrier regulation by CS in a magnitude-dependent manner.



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Fig. 6. Inhibition of MLC phosphorylation attenuates CS-mediated enhancement of thrombin-induced gap formation. HPAEC exposed to 48-h CS at 18% elongation were incubated with vehicle or Y-27632 (5 µM, 30 min) and stimulated with 50 nM thrombin for an additional 5 min. F-actin staining was performed with Texas red phalloidin (A), and thrombin-induced gap formation in CS-preconditioned and static endothelial cell cultures was quantitated (B) as described in MATERIALS AND METHODS. Arrows in A depict gaps in endothelial cell monolayer as a result of thrombin stimulation. Each point in C represents the means ± SE of 3 experiments with 5 microscopic fields per each experiment. MLC phosphorylation in HPAEC samples (C) was detected by Western blot with anti-diphospho-MLC antibody, and equal protein loadings were verified by membrane reprobing with pan-MLC antibody as described in MATERIALS AND METHODS. Stat, static.

 

Pulmonary endothelial cell viability under 5 and 18% stretch. Exposure of primary alveolar epithelial cell culture to increased levels of CS resulted in significant cell death (up to 50%) (52), suggesting high sensitivity of lung epithelium to excessive stretch relevant to 80% and higher total lung capacity in vivo (51). We next examined the effect of CS (5 or 18% elongation for 2 and 6 h) on endothelial cell viability. Figure 7 demonstrates that exposure to 18% CS induces a modest, but statistically significant, increase in the number of dead cells by 2 h, whereas 5% CS does not affect cell viability at this time point, compared with static culture. After 6 h of CS, both 5 and 18% CS-conditioned endothelial cell cultures also showed modest, but statistically significant, increases in cell death rates (0.93 ± 0.17% and 2.91 ± 0.25%, respectively) compared with 0.48 ± 0.08% for static culture. Control stimulation of endothelial cells with tumor necrosis factor (TNF)-{alpha}, a known activator of endothelial cell apoptosis, induced moderate cell death rates (3.02 ± 0.21%) in HPAEC cultures, consistent with previously described low apoptotic potential of TNF-{alpha} toward human endothelial cells (32). These data suggest that 18% elongation, relevant to pathophysiological levels of CS, induced modest levels of cell death that were comparable with effects of TNF-{alpha}. The numbers of dead cells observed after 6 and 24 h of CS exposure were not significantly different (data not shown), suggesting cell adaptation involving cytoskeletal remodeling and cell reorientation or phenotypic selection of endothelial cell population resistant to excessive levels of mechanical strain.



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Fig. 7. Effect of low- and high-magnitude CS on HPAEC viability. Cells were subjected to CS at 5 or 18% elongation for 2 or 6 h, and cell viability was assessed as described in MATERIALS AND METHODS. Stimulation of static HPAEC culture with tumor necrosis factor (TNF)-{alpha} (6 h, 40 ng/ml) was used as a positive control for cell apoptosis. CS at 18% elongation resulted in greater rates of cell death compared with 5% CS or static conditions. Each point represents the means ± SE of 5 measurements. n/d, no difference.

 

Effects of CS preconditioning on transmonolayer HPAEC electrical resistance. Magnitude-dependent effects of CS on endothelial cell MLC phosphorylation and barrier integrity reported here were observed in both long-term and acute CS experiments. Several reports, including ours (3, 5), demonstrate phenotypic effects of long-term mechanical stimulation on specific gene activation and protein expression (8, 16). In an attempt to investigate potential phenotypic effects of long-term CS stimulation on human pulmonary endothelial cell barrier regulation, we exposed HPAEC to 48-h CS at 5 or 18% elongation, and after replating cells onto gold electrodes, measurements of transendothelial electrical resistance (TER) were performed, as described in MATERIALS AND METHODS. Measurements of basal transmonolayer resistance of static and CS-preconditioned HPAEC cultures did not reveal statistically significant differences. Figure 8A depicts representative TER records demonstrating enhancement of thrombin-induced decline in electrical resistance in HPAEC preconditioned with CS at 18% elongation compared with static culture. On the contrary, HPAEC preconditioned at 5% elongation (Fig. 8B) revealed a smaller decline and faster recovery after thrombin challenge, suggesting barrier-protective effect of HPAEC preconditioning at a physiologically relevant regimen of mechanical strain.



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Fig. 8. A and B: effect of CS preconditioning on thrombin-induced changes in HPAEC transmonolayer electrical resistance. Electrical resistance across the endothelial monolayer was measured using the electrical cell-substrate impedance sensing system as described in MATERIALS AND METHODS. CS preconditioning at high magnitude (18% elongation) potentiates barrier-disruptive effect of thrombin, whereas preconditioning at low magnitude (5% elongation) exhibits barrier-protective effect. Results are representative of 6 independent experiments.

 

Studies by our laboratory suggest that platelet-derived phospholipid, S1P, exhibits vascular endothelial barrier-protective properties toward endothelial monolayer (18). We next investigated whether the barrier-protective effect of S1P can be modulated by CS. Figure 9 demonstrates that a significant increase in TER induced by S1P was not affected by CS preconditioning at 5 or 18% elongation; however, addition of thrombin after S1P stimulation resulted in the decline of TER, which was dependent on the magnitude of CS preconditioning, consistent with results of HPAEC stimulation with thrombin alone (Fig. 8).



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Fig. 9. Effect of CS preconditioning on sphingosine-1-phosphate (S1P)-mediated barrier-protective effect. CS preconditioning did not affect S1P-mediated transendothelial electrical resistance (TER) increase, whereas thrombin challenge induced a more pronounced TER decline in HPAEC cultured under 18% CS and faster recovery of HPAEC cultured under 5% CS. Results are representative of 6 independent experiments.

 

Expression profiling. The results described above demonstrate sustained stretch-induced changes of endothelial cell barrier properties, suggesting potential phenotypic effects of mechanical stimulation of pulmonary endothelial cells at different CS magnitudes. Utilization of DNA microarray analysis allowed the identification of a number of CS-regulated genes. Table 1 demonstrates sustained upregulation of gene transcripts encoding a number of important signaling proteins potentially involved in endothelial cell signaling, cytoskeletal and barrier regulation that was observed after 6 h of CS exposure, remaining elevated up to 48 h. Analysis of HPAEC exposed to 18 and 5% CS suggests that activation of selected genes was also dependent on CS amplitude with maximal upregulation of Rho, proteinase-activated receptor 2 (PAR-2), and ZIP kinase expression at 18% CS. Thus our results suggest time- and amplitude-dependent upregulation of target genes by CS. Among upregulated genes, small GTPase rho may be an important regulatory protein that mediates increased MLC phosphorylation and gap formation via activation of the downstream effector Rho kinase (15, 59). To confirm CS effects on rho protein expression, endothelial cells exposed to 48 h of CS preconditioning at 18% elongation were analyzed by Western blot. Figure 10 depicts increased rho protein expression in endothelial cells exposed to long-term CS. The bottom panel demonstrates equal MLC and Rac levels in static and CS-preconditioned HPAEC samples.


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Table 1. List of stretch-regulated genes detected in HPAEC exposed to CS at 5 or 18% elongation

 


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Fig. 10. Effect of CS preconditioning on Rho, Rac, and MLC expression. HPAEC were exposed to 48-h CS at 18% elongation; Rho, Rac, and MLC protein bands were detected by Western blot (top) as described in MATERIALS AND METHODS, and protein expression was assessed by scanning densitometry (bottom). Each point represents the means ± SE of 5 measurements. Results are significant at *P < 0.05.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The barrier properties of the intact pulmonary endothelium is a highly regulated process that is dependent on endothelial cell response to a variety of chemical and mechanical stimuli. Mechanisms of agonist-mediated endothelial cell barrier dysfunction involve multiple signaling cascades such as MAP kinase pathway, phospholipase C-mediated activation of phosphoinositide turnover, and Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinases and tyrosine kinases (20, 22, 24, 26, 33, 45, 55, 64), that result in dramatic changes of cell contacts, cytoskeleton, and endothelial cell barrier (14, 29). Barrier-disruptive effects of edemagenic agonists such as thrombin are tightly linked to activation of actomyosin complex and initiation of cell contraction. A key mechanism of thrombin-induced barrier dysfunction is associated with activation of MLC kinase- and Rho-dependent MLC phosphorylation, actin stress fiber formation, actomyosin contraction, and gap formation (14, 17, 56). In contrast, potent barrier-protective effects of platelet-derived phospholipid S1P also involve specific cytoskeletal rearrangements such as significant enhancement of cortical actin ring and peripheral accumulation of diphospho-MLC (18), suggesting an essential role for actin cytoskeletal remodeling in bidirectional regulation of endothelial cell barrier properties. Although an increasing number of reports suggest a primary role of mechanical factors in the pathogenesis of ventilator-induced lung injury, precise mechanisms of stretch-mediated endothelial barrier dysfunction are not well understood. Mechanical stretch induces transient activation of intracellular signaling (1) and endothelial cell reorientation driven by rapid actin cytoskeletal remodeling (30, 34, 46, 60). In addition, CS has a profound effect on expression of the components of extracellular matrix, growth factors, and cytokines (6, 37, 42, 50, 58), which may have a significant impact on pulmonary endothelial cell physiological responses to chemical stimuli. Thus although molecular and cytoskeletal mechanisms of thrombin-induced barrier dysfunction and barrier-protective effects of S1P in static endothelial cell cultures have been recently investigated, an interaction of mechanical factors and agonist stimulation may be an important issue in understanding the agonist action on the intact vascular endothelium in vivo.

Our study as well as previously published reports suggest a rapid cytoskeletal reorganization and F-actin-dependent endothelial cell reorientation in response to CS. Interestingly, in contrast to shear stress-induced cytoskeletal reorientation that was abolished by p38 MAP kinase inhibition (2), CS-induced reorientation was independent of ERK1/2 and p38 MAP kinases but was completely abolished by Rho kinase inhibitor Y-27632 or forskolin, an agent known to reduce MLC kinase activity and MLC phosphorylation (44), suggesting a critical role for myosin phosphorylation in control of endothelial cell cytoskeleton by CS. The results of this study demonstrate a synergistic effect of pathologically relevant levels of CS on thrombin-induced gap formation and permeability changes and show that inhibition of MLC phosphorylation by Y-27632 abolished endothelial cell barrier disruption induced by mechanochemical stimulation. Together, these results suggest a key role for MLC phosphorylation and actomyosin contraction in mechanochemical regulation of pulmonary endothelial cell barrier.

Another mechanical factor, shear stress, which is imposed onto endothelial cell monolayer by blood flow, has a distinct effect on endothelial cell morphology and signaling. Recent studies of pulmonary endothelial cells exposed to laminar shear stress demonstrated that physiologically relevant levels of laminar shear stress induced activation of cortical actin cytoskeleton with spatially defined MLC phosphorylation, protein tyrosine phosphorylation, and peripheral translocation of the actin-binding protein cortactin, involved in Arp2/3-mediated regulation of cortical actin polymerization (54), and these events were directly associated with previously reported enhancement of transmonolayer electrical resistance (11, 43) and decreased permeability for macromolecules (61). Exposure of HPAEC to CS did not induce cortactin translocation to cell periphery observed in flow-stimulated endothelium (Birukov, unpublished data). Thus results of this study and previously published data illustrate a complex pattern of mechanochemical regulation of endothelial cell barrier and suggest potentially different mechanisms of endothelial cell cytoskeletal and barrier regulation by CS and shear stress.

Assessment of HPAEC viability under CS at different magnitudes revealed very low rates of cell death. However, endothelial cell monolayers exposed to 18% CS revealed low, but still significantly higher, rates of cell death (2.91 ± 0.25% vs. 0.93 ± 0.17% dead cells after 6 h of 5% CS). Control stimulation of static HPAEC with apoptotic cytokine TNF-{alpha} revealed a very moderate induction of cell death at 6 h of the experiment that was also comparable with HPAEC exposed to 18% CS. These results are consistent with studies of alveolar epithelial cell lines A549 and H441 that demonstrated insignificant rates of cell injury after 20% CS exposure (7, 58). However, studies of freshly isolated lung epithelial cultures that revealed 40-50% cell death on high-magnitude CS (38, 52) suggest potential cell specificity in responses to mechanical stimuli. Thus our results demonstrate marginal effects of CS on endothelial cell viability and suggest that the apoptotic processes are unlikely to be involved in CS-mediated barrier regulation in human pulmonary endothelial cells.

Phenotypic responses of vascular cells exposed to CS in vitro include increased expression of contractile and cytoskeletal proteins by smooth muscle cells (MLC kinase, smooth muscle myosin heavy chains, desmin, and h-caldesmon) (5, 48, 49) as well as upregulated expression of thrombin receptor PAR-1 (35). Increased expression of contractile proteins has been shown to contribute to increased contractility of CS-preconditioned vascular smooth muscle cells (48, 49). A number of bioactive proteins regulated by CS has now been identified in endothelial cells and macrophages and include IL-8, transforming growth factor-{beta}, VEGF, and MCP-1 (41, 62, 65). In the present study, microarray DNA analysis was performed to assess time and amplitude dependence of CS effect on global gene expression. This analysis revealed a novel group of genes regulated by CS that can contribute to CS-mediated endothelial cell barrier regulation. Small GTPase rho is involved in stress fiber formation and activation of actomyosin contraction via its downstream effector, Rho kinase. PAR-2 is a protease-activated receptor known to mediate endothelial cell contractile response to macrophage-released proteases (31), and ZIP kinase is known to phosphorylate MLC and activate smooth muscle contraction (36). Further studies are underway to analyze in more detail stretch-mediated control HPAEC phenotype, identify key proteins involved in stretch-mediated HPAEC phenotype, and precisely characterize the specific role of these proteins in CS-dependent modulation of endothelial cell barrier function.

Despite the broadly described effects of mechanical strain on specific protein expression, the effects of CS on endothelial cell physiological properties, such as agonist-mediated regulation of endothelial cell permeability, are not well known. Our results demonstrate pronounced and amplitude-dependent effects of CS preconditioning on agonist-induced permeability changes in HPAEC that were observed even 16 h after replating. These results are consistent with the proposed phenotypic effect of long-term CS on pulmonary endothelial cells, as cDNA microarray analysis of CS preconditioned HPAEC revealed significant effects of CS on specific gene expression (Table 1) that was further validated by Western blot (Fig. 10). We speculate that increased expression of contractile and regulatory proteins, including Rho and ZIP kinase in pulmonary endothelial cells preconditioned at 18% CS, may contribute to enhanced barrier-disruptive effect of thrombin on endothelial cell monolayer observed in our experiments (Figs. 8 and 9). Previous studies of smooth muscle cells exposed to CS demonstrated an increased cytoskeletal protein expression that can be detected after a few days of CS exposure (5, 48). Termination of mechanical stimulation resulted in reversal of cell phenotype but may require 24-72 h depending on the half-life of a particular marker protein (5). Thus our results demonstrate for the first time a sustained magnitude-dependent modulation of physiological responses of human pulmonary endothelial cells by long-term (48 h) CS preconditioning supporting phenotypic regulation of pulmonary endothelial cells by CS in a magnitude-dependent manner.

In summary, in this study, we described acute and long-term effects of low- and high-magnitude CS on pulmonary endothelial cell signaling, cytoskeletal remodeling, monolayer integrity, and barrier function. Here we report that acute (10-60 min) and long-term (48 h) CS potentiates thrombin-induced MLC phosphorylation and gap formation in a magnitude-dependent manner. Our results suggest a critical role for MLC kinase- and Rho kinase-dependent MLC phosphorylation in CS-induced cytoskeletal remodeling and mechanochemical endothelial cell barrier regulation. Furthermore, we demonstrate for the first time phenotypic effects of high- and low-magnitude CS preconditioning on human pulmonary endothelial cell barrier regulation. Thus amplitude-dependent modulation of agonist-mediated endothelial cell barrier disruption by CS may be an important mechanism associated with mechanical ventilation-induced pulmonary vascular leak.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-50533, HL-58064, and HL-69340.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. G. Birukov, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Univ. School of Medicine, 5501 Hopkins Bayview Circle, JHAAC, 5A.42, Baltimore, MD 21224 (E-mail: kbirukov{at}jhmi.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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