Abstract
Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability.
Keywords: vascular endothelial growth factor, hepatocyte growth factor
mechanical ventilation at high tidal volumes triggers several pathways, including increased cytokine production (44, 47, 65), leukocyte infiltration, cell membrane disruption, and endothelial barrier dysfunction (54, 60, 64), and leads to ventilator-induced lung injury (VILI) and pulmonary edema. Experimental models of mechanical ventilation suggest activation of inflammatory events and increased vascular leak (44, 46). Direct measurements of interstitial and/or vascular distension in the mechanically ventilated lung are not currently available because of the complexity of local distension patterns in the lung parenchyma, further complicated by uneven regional lung distension observed during inflammation and lung injury. Several groups, including ours, have established cell culture models related to in vivo VILI conditions and reproduced cellular responses to VILI, such as increased cytokine production and exacerbation of agonist-induced endothelial barrier dysfunction by high amplitude cyclic stretch (CS; Refs. 7, 20, 47, 65).
Increased levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in pulmonary circulation have been detected in animal models (15) and in patients with acute respiratory distress syndrome (ARDS) at different stages of acute lung injury (ALI; Refs. 42, 48, 53). VEGF regulates vascular permeability to water and proteins, and VEGF overexpression in the lungs as well as injection of purified VEGF increases endothelial permeability in vivo (26, 50). The barrier-disruptive effects of VEGF in the pulmonary endothelium have been associated with the activation of mitogen-activated (MAP) kinases and Rho-dependent signaling (33, 55). In turn, HGF exhibits protective effects against vascular leak in vivo (18), which may be associated with stimulation of MAP-kinases ERK1/2 and p38, phosphatidylinositol 3-kinase (PI3-kinase), and Rac-GTPase, leading to enhancement of the peripheral actin cytoskeleton and increased interactions between adherens junction proteins α/β-catenin and VE-cadherin (8, 35).
Among the variety of signaling pathways activated in the vascular endothelium by biomechanical forces, including MAP kinase cascades (ERK1/2, JNK, and p38), nonreceptor tyrosine kinases (p60Src and FAK), integrin-mediated signaling, and ion channels (4, 14, 17, 19, 23, 24, 27, 34, 37, 45), small GTPases Rac and Rho appear to play an essential role in endothelial recognition of “physiological” and “pathological” levels of CS by endothelial cells (ECs; Refs. 29–31). We have previously shown the differential effects of physiological and pathological magnitudes of CS on thrombin-induced EC barrier disruption (10, 51). Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation, which is critical for the EC recovery phase. These studies suggest a critical role of amplitude-dependent CS in Rac/Rho GTPase balance and mechanochemical regulation of the lung EC barrier.
In the current study, we evaluated the barrier-protective effects of HGF in pulmonary EC models of VEGF-induced endothelial barrier dysfunction. We tested the potential synergistic effects of HGF and physiological levels of CS and studied their protective effects on pulmonary endothelial barrier dysfunction induced by pathological mechanochemical stimulation.
MATERIALS AND METHODS
Cell culture and reagents.
Rac and Rho antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); di-phospho-myosin light chain (MLC), phospho-p21-activated kinase (PAK) 1, and PAK1 antibodies were obtained from Cell Signaling (Beverly, MA); and phospho-myosin-associated phosphatase type (MYPT) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). All reagents for immunofluorescence staining were purchased from Molecular Probes (Eugene, OR). Unless specified, biochemical reagents were obtained from Sigma (St. Louis, MO). Human pulmonary artery endothelial cell (HPAECs) were obtained from Lonza (Allendale, NJ), and bovine pulmonary artery endothelial cell (BPAECs) were obtained from the American Type Tissue Culture Collection (Rockville, MD; culture line-CCL 209) and cultured as described previously (5, 7, 11). Comparative experiments did not reveal significant differences between HPAECs and BPAECs in cellular responses to HGF or VEGF. All experimental data, including immunofluorescence studies obtained for HPAECs, were reproduced in BPAEC culture. Immunofluorescence experiments shown in this study were conducted using HPAECs; all other data depict experiments conducted using BPAECs.
Cell culture under CS.
All CS experiments were performed using the FX-4000T Flexcell Tension Plus system (Flexcell International, McKeesport, PA) equipped with a 25-mm BioFlex Loading station, as described previously (7, 51). Experiments were performed in the presence of culture medium containing 2% FBS to decrease basal activation of cellular signaling, including Rac and Rho activities. Briefly, ECs were seeded at standard densities (8 × 105 cells/well) onto collagen I-coated flexible bottom BioFlex plates. After 48 h of culture, each plate received fresh medium, was mounted onto the Flexecell system, and was exposed for 2 h to either low magnitude (5% elongation) or high magnitude (18% elongation) CS with a frequency of 15 cycles/min to recapitulate the mechanical stresses experienced by the alveolar endothelium during normal respiration and high tidal volume mechanical ventilation, respectively (7, 58, 59). At 2 h, cells were treated with the agonists of interest with continuous exposure to CS. Control BioFlex plates with static EC culture were placed in the same cell culture incubator. Two-hour CS preconditioning was chosen based on previously published studies (7, 10, 51), which showed that at this time point cell orientation is complete and most of the intracellular signaling activated during the initial stage of stretch-induced cellular remodeling returns to basal levels. At the end of the experiment, cell lysates were collected for Western blot analysis or CS-exposed endothelial monolayers were fixed with 3.7% formaldehyde and used for immunofluorescence staining as described previously (6, 11).
Quantitative analysis of paracellular gap formation and enhancement of peripheral F-actin rim.
Paracellular gap formation and enhancement of peripheral F-actin rim were quantitatively analyzed as described previously (7, 9, 12). At least 20 microscopic fields for each experimental condition were analyzed. 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. Similarly, to analyze peripheral F-actin rim cell areas within 3-μm range from cell-cell interface were manually marked out, and images were differentially segmented between areas of polymerized actin and background. Peripheral areas of F-actin immunoreactivity were captured based on image grayscale levels, and immunofluorescence signal intensity was measured and expressed in arbitrary units per microscopic field. The values were statistically processed using Sigma Plot 7.1 (SPSS Science) software.
Small interfering RNA-based knockdown of Rho and Rac in pulmonary ECs.
To reduce the content of endogenous Rho or Rac, cells were treated with gene-specific small interfering RNA (siRNA) duplexes, as described previously (6). In brief, predesigned Rac1-specific and Rho-specific siRNAs of standard purity were ordered from Ambion (Austin, TX) in purified, desalted, deprotected, annealed double-strand form. The following 21-bp duplexes of siRNA were used: for Rac1: sense, 5′-GGAGAUUGGUGCUGUAAAAtt-3′ and antisense, 5′-UUUUACAGCACCAAUCUCCtt-3′; and for RhoA: sense, 5′-GGUGGAUGGAAAGCAGGUAtt-3′ and antisense, 5′-UACCUGCUUUCCAUCCACCtc-3′. Nonspecific, nontargeting siRNA duplex #1 from Dharmacon (Lafayette, CO) was used as a control treatment. Cells were grown to 70% confluence, and the transfection of siRNA (final concentration of 100 nM) was performed using DharmaFECT1 transfection reagent (Dharmacon) according to the manufacturer's protocol. Cells were grown to 70% confluence, and the transfection of siRNA (final concentration of 50 nM) was performed using DharmaFECT1 transfection reagent (Dharmacon) according to manufacturer's protocol. After 48 h, cells were harvested and used for experiments.
Measurement of transendothelial electrical resistance.
Measurements of transendothelial electrical resistance (TER) across confluent HPAEC monolayers were taken using the electrical cell-substrate impedance sensing system (ECIS; Applied Biophysics, Troy, NY) as described previously (8, 11).
Transwell permeability assays.
Permeability for FITC-labeled 2,000-kDa dextran was assessed in Transwell assays using in the vitro vascular permeability assay kit (Chemicon International, Billerica, MA), according to the manufacturer's instructions. Briefly, cells were grown to confluence onto collagen-coated inserts. Experiments were conducted in EC culture medium containing 2% FBS. EC monolayers were treated with HGF of VEGF for 1.5 h, followed by addition of FITC-dextran on top of the cells. In experiments with combined HGF of VEGF treatment, ECs were pretreated with HGF for 1.5 h and then treated with VEGF for another 1.5 h, followed by addition of FITC-dextran for 30 min. The extent of permeability was determined by measurements of fluorescence of the plate well solution.
GTPase activation assays.
The GTPase activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology (Billerica, MA), as we have described previously (6). In brief, after stimulation, cell lysates were collected, and GTP-bound Rac or Rho was captured using pull-down assays with immobilized PAK1-PBD or rhotekin-RBD, respectively, according to the manufacturer's protocol. The levels of activated small GTPases as well as total Rac or Rho content were evaluated by Western blot analysis.
Immunoblotting.
After stimulation, cells were lysed, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with specific antibodies as described previously (6). The relative intensities of immunoreactive protein bands were quantified by scanning densitometry using Image Quant software (Molecular Dynamics, Sunnyvale, CA). Levels of phosphorylated or activated proteins were normalized to the total target protein levels and expressed in relative densitometry units.
Statistical analysis.
Results are means ± SD of 3–7 independent experiments. Stimulated samples were compared with the controls using the unpaired Student's t-test. For multiple-group comparisons, one-way ANOVA, followed by the post hoc Fisher's test, was used. P < 0.05 was considered statistically significant.
RESULTS
Effects of VEGF on endothelial permeability and Rho pathway activation.
Measurements of permeability across pulmonary EC monolayers demonstrate that VEGF significantly decreased TER in human pulmonary artery ECs, reflecting an EC barrier compromise, with maximal response at 30 min of stimulation with 200 ng/ml VEGF (Fig. 1A). VEGF-induced EC permeability changes were associated with time-dependent activation of Rho-GTPase and phosphorylation of its downstream target MLC (Fig. 1, B and C). These data show that, although a rapid transient increase in transendothelial resistance was observed after 5–10 min of VEGF stimulation and may be associated with transient elevation of Rac activity (21), the most prominent and sustained effect of VEGF treatment was a decline in TER reflecting endothelial barrier dysfunction. Prevailing hyperpermeability response to VEGF may also be explained by activation of additional signaling mechanisms driving EC permeability such as activation of MLC kinase, tyrosine kinases, p38 stress MAP kinase, and ERK1/2 MAP kinases (3).
Fig. 1.
Effect of VEGF on pulmonary endothelial cell (EC) barrier function. A: ECs were plated on gold microelectrodes. At time indicated by arrow, ECs were treated with either vehicle or VEGF (50, 100, 200, or 500 ng/ml) and used for measurements of transendothelial electrical resistance (TER). B and C: cells were treated with VEGF (200 ng/ml) for indicated periods of time. B: Rho activity was measured using an in vitro activation assay described in materials and methods. C: levels of phosphorylated myosin light chain (MLC) in the total lysates were determined by Western blot analysis using di-phospho-MLC antibodies. Results represent 3–5 independent experiments.
Effects of HGF on endothelial permeability and Rac pathway activation.
HGF induced pronounced dose-dependent TER increases, reflecting an EC barrier-protective response, with maximal effect at 30 ng/ml (Fig. 2A). HGF-induced EC barrier enhancement achieved its maximal level at 15 min of stimulation and was associated with time-dependent activation of Rac-GTPase (Fig. 2B). HGF at higher concentrations (50 and 75 ng/ml) did not cause a further increase in TER (data not shown). Consistent with the Rac activation profile, HGF induced activation of downstream Rac target PAK1, as detected by increased phosphorylation at the PAK1 autophosphorylation site (13; Fig. 2C).
Fig. 2.
Effect of hepatocyte growth factor (HGF) on pulmonary EC barrier function. A: ECs were grown on gold microelectrodes followed by treatment with either vehicle or HGF (10, 20, or 30 ng/ml, indicated by arrow), and TER was measured over the time. B and C: cells were treated with HGF (30 ng/ml) for indicated periods of time. B: Rac activity was measured using a Rac-GTP pull-down assay. C: levels of p21-activated kinase PAK autophosphorylation in total lysates were determined by Western blot analysis with phospho-PAK1 antibodies. Results represent 3–7 independent experiments. RDU, relative densitometry units.
Effects of HGF on VEGF-induced endothelial barrier compromise.
We (8) recently demonstrated that HGF induces attenuation of the thrombin-induced Rho pathway of EC barrier dysfunction via Rac-dependent mechanisms. In the current study, we evaluated the barrier-protective effects of HGF on VEGF-induced barrier compromise. EC pretreatment with HGF caused a pronounced barrier-protective response indicated by the TER increase (Fig. 3A) and significantly attenuated VEGF-induced TER decline. The protective effect of HGF against VEGF-induced EC hyperpermeability was further examined using the solute flux assay. EC monolayers grown on semipermeable membranes were pretreated with HGF alone or with HGF followed by VEGF stimulation, and permeability for FITC-labeled dextran was assessed using Transwell assays. Similar to the TER results, VEGF alone caused dramatic permeability increase, whereas HGF significantly reduced basal EC monolayer permeability and markedly attenuated VEGF-induced permeability for FITC-labeled dextran (Fig. 3B).
Fig. 3.
Effects of HGF on VEGF-induced EC permeability. A: at time indicated by first arrow, confluent pulmonary ECs were preincubated with HGF (30 ng/ml) followed by VEGF (200 ng/ml) challenge, indicated by second arrow, and TER changes were monitored over time. B: pulmonary EC monolayers grown in Transwell plates were incubated with VEGF (200 ng/ml) with or without HGF (30 ng/ml) pretreatment, and the amount of dextran that crossed the EC monolayer was determined by measurements of FITC-labeled dextran fluorescence values in bottom chambers. Data are means ± SD of 3 independent experiments (*P < 0.05).
Importantly, the protective effects of HGF against VEGF-induced EC barrier dysfunction were accompanied by HGF-induced inhibition of Rho activation upon VEGF challenge (Fig. 4A). HGF inhibited Rho kinase-mediated phosphorylation of the myosin-binding subunit of MYPT1 and VEGF-induced MLC phosphorylation (Fig. 4, B and C), the two markers of the activated Rho pathway. The levels of autophosphorylated PAK1 in EC treated with a combination of HGF and VEGF were elevated compared with those in untreated cells or to EC treated with VEGF alone (Fig. 4D), suggesting increased levels of Rac activity in the ECs upon combined VEGF and HGF treatment. The HGF protective effects against VEGF-induced barrier dysfunction were also associated with HGF-mediated reduction of stress fibers and levels of stress fiber-associated diphospho-MLC. These observations were further conformed by quantitative analysis of phosphorylated MLC colocalized with F-actin fibers in control and treated ECs [5.96 ± 2.91 arbitrary units of fluorescence intensity (UFI) in cells pretreated with HGF before VEGF stimulation vs. 31.94 ± 13.98 UFI in EC treated with VEGF alone, P < 0.05; vehicle control = 4.95 ± 1.08 UFI]. Presented data reflect attenuation of VEGF-induced actomyosin contractility in pulmonary ECs, which is central to gap formation and agonist-induced paracellular permeability (Fig. 4E).
Fig. 4.
Effect of HGF on VEGF-induced EC barrier dysfunction. Endothelial monolayers were pretreated with HGF (30 ng/ml, 15 min) and stimulated with VEGF (200 ng/ml at 30, 45, or 60 min). A: Rho activity after 30 min of VEGF challenge was measured using the Rho-GTP pull-down assay described in materials and methods. Bottom: total Rho content in EC lysates. B–D: phosphorylation of myosin-associated phosphatase type (MYPT; B), MLC (C), or PAK1 (D) in lung endothelial EC pretreated with HGF followed by VEGF challenge was detected by Western blot with phospho-specific antibodies. MLC and PAK1 phosphorylation was determined after 30 min of VEGF treatment. E: effect of HGF (30 ng/ml, 15 min) on VEGF-induced (200 ng/ml, 30 min) cytoskeletal remodeling and MLC phosphorylation. Double immunofluorescence staining was performed using di-phospho-MLC antibodies and Texas red phalloidin to detect F-actin. VEGF-induced stress fibers and HGF-mediated peripheral actin accumulation are marked by arrows. Results represent 3–5 independent experiments.
Effects of pathological CS on VEGF responses in lung endothelium.
In the following experiments, we tested the hypothesis that pathological CS promotes VEGF-induced lung endothelial barrier dysfunction. Consistent with the results of TER measurements, immunofluorescence staining of the actin cytoskeleton in VEGF-stimulated EC monolayers showed increased stress fiber and paracellular gap formation, indicating the involvement of contractile mechanisms in VEGF-induced EC permeability (Fig. 5A). Remarkably, preconditioning of ECs with pathological CS (18% CS, 2 h) promoted VEGF-induced paracellular gap formation (Fig. 5, A and B). VEGF-induced cytoskeletal remodeling was associated with increased phospohrylation of MLC, which was further increased in ECs subjected to combined stimulation with pathological CS and VEGF (Fig. 5C). Paracellular gap formation induced by 18% CS and VEGF was completely abolished by the Rho kinase inhibitor Y-27632 (Fig. 5A, right). Complementary experiments were performed using siRNA-based knockdown of Rho. After transfection, lung ECs were subjected to 18% CS for 2 h followed by VEGF challenge. Compared with control cells transfected with nonspecific RNA, Rho knockdown attenuated cell orientation under CS and inhibited VEGF-induced EC barrier disruption, as detected by decreased formation of stress fibers and paracellular gaps (Fig. 5D). These data suggest the critical role of the Rho-dependent pathway in VEGF- and pathological CS-mediated EC contraction and increased permeability, which lead to EC barrier failure.
Fig. 5.
Effect of high magnitude CS on VEGF-induced EC barrier compromise. Pulmonary ECs grown to confluence on Flexcell plates were exposed to pathological (18%) CS for 2 h and then stimulated with VEGF (200 ng/ml, 30 min). A: immunofluorescence staining using Texas red phalloidin was performed to detect F-actin. Paracellular gaps are marked by arrows. Right: VEGF-induced gap formation was attenuated by the Rho kinase inhibitor Y27632 (2.5 μM, 30 min). B: quantitative analysis of CS and VEGF-induced paracellular gap formation. Data are means ± SD of 7 independent experiments (*P < 0.05). C: phosphorylated MLC was detected by immunoblotting with di-phospho-MLC specific antibodies. Equal protein loading was confirmed by reprobing of membranes with antibodies to nonphosphorylated protein. D: ECs were transfected with Rho-specific or nonspecific (ns) small interfering (si) RNA for 48 h before CS experiments. Cytoskeletal remodeling in control and VEGF-treated monolayers was analyzed by inmmunifluorescence staining of F-actin. Paracellular gaps are marked by arrows.
Effects of physiological CS on HGF-induced cytoskeletal remodeling and Rac signaling.
In the following studies, we examined the combined effects of HGF and 5% CS on EC cytoskeletal remodeling. Consistent with the previous observations (8, 35), stimulation of pulmonary endothelial monolayers with HGF induced actin rearrangement, accompanied by a reduction in the number of central F-actin fibers and an accumulation of actin at the areas of the cell periphery (Fig. 6A, top). EC preconditioning at physiological CS (5% CS, 2 h) further promoted enhancement of the cortical F-actin rim (Fig. 6, A and B) induced by HGF. EC preconditioning at 5% CS also increased HGF-induced Rac signaling, as indicated by increased levels of PAK1 autophosphorylation (Fig. 6C). These data suggest the synergistic effects of physiological CS and HGF on the EC barrier-protective response.
Fig. 6.
Effect of low magnitude CS on HGF-induced EC barrier protection. A: confluent ECs were exposed to physiological (5%) CS for 2 h followed by HGF (30 ng/ml, 15 min) stimulation and immunofluorescence staining for F-actin. Areas of peripheral F-actin accumulation are marked by arrows. B: quantitative analysis of CS- and HGF-induced cortical actin rim formation. Data are means ± SD of 4 independent experiments (*P < 0.05). C: Western blot analysis of HGF-induced PAK1 autophosphorylation in static cultures and ECs exposed to low magnitude CS (5%, 2 h). Equal protein loading was confirmed by reprobing of membranes with antibodies to nonphosphorylated protein. Results represent 3–5 independent experiments.
Effects of physiological CS and HGF on VEGF-induced endothelial barrier disruption.
We have previously shown that physiological CS reduced barrier disruption and promoted recovery of pulmonary EC monolayer integrity after thrombin challenge (10). In the current study, we tested the potential protective effects of HGF and physiological CS levels using in vitro models of acute pulmonary endothelial barrier failure (Fig. 7). In static EC cultures, HGF pretreatment significantly attenuated EC contraction and barrier failure in response to VEGF (Fig. 7A). Remarkably, EC preconditioning at 5% CS (2 h) before HGF and VEGF treatment promoted EC monolayer barrier properties as indicated by further reduction of VEGF-induced gap formation and enhancement of the cortical actin rim, compared with static EC cultures (Fig. 7, A and B and insets). To test the effects of CS magnitude on the VEGF-induced EC barrier dysfunction, we exposed ECs to 5 or 18% CS before VEGF treatment. Analysis of cytoskeletal remodeling detected by F-actin staining shows that barrier disruption caused by VEGF in EC monolayers preconditioned at 5% CS was markedly reduced compared with the barrier disruption in VEGF-treated EC preconditioned at 18% CS (Fig. 7B, bottom). These observations were confirmed by quantitative analysis of paracellular gap formation (Fig. 7, C and D). The barrier-protective effect of physiological CS was also accompanied by a reduction of VEGF-induced MLC phosphorylation, compared with static EC cultures (Fig. 7E). Taken together, these data demonstrate the protective effects of physiological CS against VEGF-induced EC barrier dysfunction and suggest synergistic interactions between HGF and physiological stretch in the enhancement of the EC barrier properties.
Fig. 7.
Effects of physiological CS preconditioning on VEGF-mediated EC barrier dysfunction. A: EC monolayers grown to confluence on Flexcell plates were stimulated with VEGF (200 ng/ml, 30 min) with or without HGF pretreatment (30 ng/ml, 15 min) under static conditions. F-actin was visualized by immunofluorescence staining with Texas red phalloidin. B: EC monolayers grown on Flexcell plates were preconditioned at physiological (5% CS) or pathological (18% CS) levels of CS for 2 h and stimulated with VEGF (200 ng/ml, 30 min) with or without HGF pretreatment (30 ng/ml, 15 min). F-actin was visualized by immunofluorescence staining with Texas red phalloidin. Paracellular gaps are marked by arrows. A and B: VEGF-induced paracellular gaps are partially inhibited by HGF pretreatment of static ECs and completely inhibited by HGF in 5% CS-preconditioned ECs. Insets: high magnification of selected areas. C and D: quantitative analysis of paracellular gap formation induced by VEGF challenge of pulmonary ECs exposed to 5% CS with or without HGF pretreatment (C) or to 18% CS and 5% CS (D). Data are means ± SD of 4 independent experiments (*P < 0.05). E: phosphorylated MLC was detected by Western blot with specific antibodies. Equal protein loading was confirmed by reprobing of membranes with antibodies to nonphosphorylated MLC.
To further verify a role of Rac signaling in the protective effects of physiological CS and HGF against VEGF-induced EC barrier disruption, we utilized siRNA-mediated depletion of endogenous Rac. Rac knockdown abolished the protective effects of 5% CS (Fig. 8A) or combination of HGF and 5% CS (Fig. 8B) against VEGF-induced stress fiber and gap formation, compared with control ECs treated with nonspecific RNA. These data confirm the important role of the Rac pathway in the EC barrier protective responses induced by HGF and physiological CS.
Fig. 8.
Rac knockdown attenuates protective effects of physiological CS and HFG against VEGF-induced EC barrier disruption. A and B: human pulmonary ECs were transfected with Rac-specific or nonspecific siRNA. After 48 h of transfection, cells were preconditioned at physiological (5% CS, 2 h) followed by VEGF (200 ng/ml, 30 min) challenge alone (A) or with HGF pretreatment (30 ng/ml, 15 min) before VEGF stimulation (B). Cytoskeletal remodeling in control and VEGF-treated EC monolayers was analyzed by inmmunifluorescence staining for F-actin. Paracellular gaps are marked by arrows. Results represent 3 independent experiments.
DISCUSSION
The pathological mechanical forces experienced by lung tissues during mechanical ventilation at high tidal volume dramatically alter pulmonary endothelial responses to bioactive molecules and may further propagate VILI and pulmonary edema (49, 57, 63). These clinical observations and experimental studies led to the development of the two-hit model of ALI proposed by the National Heart, Lung, and Blood Institute working group (41).
Several studies, including our recent reports, have shown the direct effects of physiological and pathological mechanical stretch on Rac and Rho GTPase activity (7, 10, 29–31) and lung endothelial and epithelial barrier regulation (7, 10, 40, 51, 59, 66, 71). However, the amount of mechanistic information about the interactions between pathological or physiological levels of mechanical stretch and ALI-related growth factors in the regulation of pulmonary endothelial barrier is very limited. The main finding of this study is the synergistic effect of physiological CS and HGF in the inhibition of VEGF-induced endothelial hyper-permeability. Our results suggest that the mechanism of this protective effect is via Rac-dependent downregulation of the Rho pathway of barrier dysfunction.
The results of this study also show antagonistic relations between VEGF and HGF in the control of pulmonary EC permeability. VEGF is primarily produced by type II alveolar epithelial cells and is a survival factor for lung microvascular ECs (67). In healthy human subjects, VEGF is highly compartmentalized to the lung, with alveolar VEGF protein levels 500 times higher than those in plasma (25). Under conditions of stress or injury, such as in ALI or VILI, because of the anatomic proximity between alveolar epithelial and microvascular ECs, VEGF may literally spill onto the pulmonary ECs, increasing permeability and leading to interstitial and pulmonary edema (25, 43). VEGF increases in the lung have been shown in various lung pathologies including hydrostatic edema, ARDS, and LPS-induced lung injury (28, 69). In addition, high tidal volume ventilation may stimulate VEGF expression, and VEGF release by other organs (kidney and liver) contributes to VILI-related multiorgan failure (22). This study shows that, in addition to stimulation of VEGF production, CS of pulmonary ECs related to lung mechanical ventilation may also modulate, in a magnitude-dependent manner, VEGF-induced signaling and changes in EC barrier function.
Clinical studies (48, 53, 62) also show dramatic (up to 25-fold) elevation of HGF levels in plasma and BAL fluid in patients with ALI/ARDS. HGF stimulates alveolar epithelial proliferation and is likely involved in the alveolar epithelial repair process in the lung (70). In addition, HGF promotes cell survival and enhancement of lung endothelial barrier properties critical for restoration of lung vascular barrier function (8, 35, 39). Our data show that attenuation of VEGF-induced pulmonary EC permeability by HGF was observed at lower HGF concentrations compared with the VEGF concentrations that caused endothelial leak. These findings suggest that even lower levels of HGF induction during acute lung may be sufficient to counteract the lung vascular permeability induced by higher local VEGF concentrations.
Accumulating evidence suggests considerable heterogeneity between micro- and macrovascular ECs as well as between ECs from different vascular beds in functional responses to agonists, which may be driven by EC phenotype-specific cell signaling pathways (1, 36, 68). Previous studies (3) show that VEGF increased permeability in both microvascular and macrovascular pulmonary ECs. Although the dose dependence of permeability responses by pulmonary arterial and lung microvascular ECs to VEGF were different, they were mediated by same signaling pathways (3). Microvascular and microvascular ECs also exhibited similar patterns of rapid Rho activation in response to VEGF (55, 61), followed by later Rac activation essential for VEGF-induced cell migration (21). Barrier-protective responses of HGF have been observed in both human lung microvascular (52) and macrovascular (8, 35; this study) ECs. On the other hand, HGF increased permeability in human umbilical vein EC cultures (38), and intravitreal injections of HGF increased retinal vascular permeability in rat models (16). These findings reflect phenotypic differences and heterogeneity of permeability responses by ECs from different vascular beds. However, existing data strongly suggest similar permeability responses by microvascular and macrovascular pulmonary ECs to HGF and VEGF.
Our previous studies (8, 35) have described a role of Rac signaling in the potent barrier protective effects of HGF on pulmonary endothelium in static culture, leading to the enhancement of the peripheral actin cytoskeleton, focal adhesion redistribution, and increased areas of intercellular adherens junctions. The results of this study show the essential role of Rac-Rho crosstalk in the protective effects of HGF against VEGF-induced permeability. Furthermore, it was shown that physiological CS alone or in combination with HGF decreased VEGF-induced gap formation and activation of the Rho pathway.
The clinically relevant model of pulmonary ECs exposed to pathological or physiological CS levels and a combination of VEGF and HGF that was used in this study further supports an autoregulation mechanism of EC barrier control in ALI/ARDS via changes in the levels of VEGF and HGF production. This study also strongly suggests a Rac/Rho crosstalk as a switch of mechanochemical EC barrier regulation under pathological and physiological conditions. The precise mechanisms of mechanochemical coupling of signal pathways leading to the Rac and Rho activation are a subject of further investigations.
The synergistic effects of HGF and physiological CS on attenuation of VEGF-induced EC permeability described in this study are clinically relevant and provide mechanistic support at the cellular level for potential novel protective strategies for ARDS treatment using low tidal volume ventilation and HGF administration. Therapeutic strategies using HGF to ameliorate cardiovascular disease have been already suggested (2, 32, 39, 56).
In summary, this study demonstrated for the first time the synergy between the barrier-protective effects of HGF and physiological CS leading to cytoskeletal remodeling and barrier enhancement mediated by Rac-dependent mechanisms. Our results also demonstrate the protective effects of HGF and physiological CS preconditioning against the barrier-disruptive effects of VEGF and pathological CS, which involve downregulation of the Rho and stimulation of the Rac pathways, leading to increased EC barrier restoration. Thus our studies describe a novel mechanism of pulmonary EC barrier regulation by physiological and VILI-related mechanical and chemical stimuli.
GRANTS
This work was supported by National Heart, Lung, and Blood Institutes Grants HL-076259, HL-075349, and HL-58064; the American Lung Association Career Investigator Grant to K. G. Birukov; a National Scientist Developing Grant from the American Heart Association; and the American Lung Association Biomedical Research Grant to A. A. Birukova.
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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