Mechanosensitive Rap1 activation promotes barrier function of lung vascular endothelium under cyclic stretch

Abstract

Mechanical ventilation remains an imperative treatment for the patients with acute respiratory distress syndrome, but can also exacerbate lung injury. We have previously described a key role of RhoA GTPase in high cyclic stretch (CS)-induced endothelial cell (EC) barrier dysfunction. However, cellular mechanotransduction complexes remain to be characterized. This study tested a hypothesis that recovery of a vascular EC barrier after pathologic mechanical stress may be accelerated by cell exposure to physiologic CS levels and involves Rap1-dependent rearrangement of endothelial cell junctions. Using biochemical, molecular, and imaging approaches we found that EC pre- or postconditioning at physiologically relevant low-magnitude CS promotes resealing of cell junctions disrupted by pathologic, high-magnitude CS. Cytoskeletal remodeling induced by low CS was dependent on small GTPase Rap1. Protective effects of EC preconditioning at low CS were abolished by pharmacological or molecular inhibition of Rap1 activity. In vivo, using mice exposed to mechanical ventilation, we found that the protective effect of low tidal volume ventilation against lung injury caused by lipopolysaccharides and ventilation at high tidal volume was suppressed in Rap1 knockout mice. Taken together, our results demonstrate a prominent role of Rap1-mediated signaling mechanisms activated by low CS in acceleration of lung vascular EC barrier restoration.

FIGURE 1:

Effects of different regimens of CS on human pulmonary EC cytoskeletal remodeling and activation of RhoA pathway. (A) Effects of 4 and 18% CS on actin cytoskeleton and adherens junctions. After 6-h CS stimulation, pulmonary EC monolayers were fixed and used for immunofluorescence staining for F-actin using Texas Red phalloidin (red, top panels) and VE-cadherin antibody (green, bottom panels). Bar = 10 µm. Bar graphs represent quantitative analysis of F-actin and VE-cadherin signal intensity. Data are expressed as mean ± SD of three independent experiments, five fields per condition; *, p < 0.05. (B) Time course of MYPT and MLC phosphorylation in pulmonary ECs exposed to 4 and 18% CS. After CS stimulation for indicated periods of time, cells were lysed, and phosphorylation of corresponding proteins was detected by Western blot with p-MYPT1 (Thr⁸⁵⁰) and pp-MLC antibodies. Equal protein loading was confirmed by membrane reprobing with α-tubulin and pan-MYPT antibodies. Bar graphs depict quantitative densitometry analysis of Western blot data; n = 4; *, p < 0.05 vs. corresponding time points from 4% CS group. (C) Time course of MLC phosphorylation in pulmonary ECs exposed to 4% CS, 18% CS, or 4–18% CS switch. Cells were exposed to 6-h 4% CS, 6-h 18% CS, or 3-h 4% CS followed by 3-h 18% CS. Thrombin stimulation was performed in the last 5–15 min of CS exposure. MLC phopshorylation was detected by Western blot with pp-MLC antibodies. Equal protein loading was confirmed by membrane reprobing with α-tubulin antibody. Bar graphs represent analysis of Western blot data; n = 5; *, p < 0.05 vs. 4% or 4–18% CS.

FIGURE 2:

Effects of different patterns of applied CS on thrombin-induced cytoskeletal remodeling and MLC phosphorylation in human pulmonary ECs. Pulmonary ECs were treated by 18% CS or 4% CS for 6 h; alternatively, 3-h 4% CS was introduced either before or after 3-h 18% CS. Thrombin (0.05 U/ml) was added in the last 10 min of CS stimulation. (A) Visualization of F-actin using immunofluorescence staining with Texas Red phalloidin. Paracellular gaps are marked by arrows. Bar = 10 µm. Bar graphs represent quantitative analysis of gap formation. Data are expressed as mean ± SD; n = 3; *, p < 0.05. (B) Visualization of cell junctions using immunofluorescence staining with VE-cadherin antibody. Bar = 10 µm. (C) Thrombin-induced MLC phosphorylation in pulmonary ECs exposed to different patterns of CS was evaluated by Western blot with pp-MLC antibodies. Equal protein loading was confirmed by membrane reprobing with β-actin antibody. Bar graphs represent analysis of Western blot data; n = 4; *, p < 0.05.

FIGURE 3:

Effects of pulmonary EC exposure to different CS patterns on Rap1 activation and association between VE-cadherin and p120-catenin. (A) Time course of Rap1 activation in pulmonary ECs exposed to 18% CS or 4% CS. Pull down of GTP-bound Rap1 reflecting Rap1 activation levels was performed as described in Materials and Methods. Rap1 content in total cell lysates was used as a normalization control. Bar graphs represent analysis of Western blot data; n = 4; *, p < 0.05 vs. 4% CS. (B) Western blot analysis of p120-catenin and VE-cadherin content in the membrane/cytoskeletal fractions of EC exposed to 6-h 18% CS, 3-h 4% CS switched to 3-h 18% CS, or 6-h 4% CS. The content of p120-catenin and VE-cadherin in the total cell lysates was used as a normalization control. Bar graphs represent analysis of Western blot data; n = 5; *, p < 0.05. (C) HPAECs were exposed to 6-h 18% CS or 3-h 4% CS switched to 3-h 18% CS with or without pretreatment with GGTI-298 (15 µm, 30 min) before CS stimulation, and surface protein biotinylation assay was performed for VE-cadherin. VE-cadherin content in total cell lysate is presented as input. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05. (D) HPAECs treated with nonspecific or Rap1-specific siRNA 48 h before CS stimulation were exposed to 6-h 18% CS or 3-h 4% CS switched to 3-h 18% CS. Surface protein biotinylation assay was performed for VE-cadherin. Inset demonstrates efficiency of siRNA-induced Rap1 protein depletion. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05. (E) Coimmunoprecipitation of VE-cadherin and p120-catenin from cell lysates of ECs exposed to 6-h 18%CS, 3-h 4% CS switched to 3-h 18% CS, or 6-h 4% CS (left panel) or ECs pretreated with GGTI-298 before CS stimulation (right panel). Antibody to p120-catenin was used for immunoprecipitation. Membrane reprobing for p120-catenin was used as a normalization control. Inset shows Western blot analysis of siRNA-induced Rap1 depletion. Bar graph depicts quantitative analysis of immunoprecipitated protein complexes; n = 4; *, p < 0.05. (F) Rap1 knockdown attenuates VE-cadherin immunofluorescence at cell junctions of ECs exposed to 4% CS and promotes further disruption of VE-cadherin positive cell contacts in ECs exposed to 18%CS. Bar = 5 µm. Insets depict higher magnification images of cell junction areas. (G) Rap1 knockdown increases accumulation of FITC-avidin tracer on the substrate underlying EC monolayers. Bar = 10 µm. Bar graphs depict quantitative analysis of FITC signal expressed as mean ± SD of four independent experiments, five fields per condition; *, p < 0.05.

FIGURE 4:

Effects of pharmacologic and molecular inhibition of Rap1 on down-regulation of Rho signaling caused by thrombin and high-magnitude CS in ECs preconditioned at 4% CS. Pulmonary ECs were exposed to 4% CS or 18% CS for 6 h, or to 3-h 4% CS switched to 3-h 18% CS. Thrombin (0.05 U/ml) was added for the last 10 min of CS experiment. Rap1 inhibitor (GGT-298, 15 µM) was added for 30 min before the 18% CS or before switching to 18% CS. (A) Visualization and quantitative analysis of FITC-avidin binding to the bottoms of the plates with CS-stimulated EC monolayers. Rap1 inhibitor attenuated 4% CS-induced protection of EC barrier integrity as detected by XPerT permeability assay. Bar = 20 µm. (B) Inhibition of Rap1 by pretreatment with GGTI-298 abolished down-regulation of RhoA activity caused by EC exposure to 4% CS. (C) Rap1 inhibitor attenuated suppression of RhoA-dependent phosphorylation of MYPT and MLC caused by 4% CS preconditioning. (D) Effect of siRNA-induced Rap1 knockdown on 4% CS-induced suppression of MYPT and MLC phosphorylation caused by thrombin. Right panel shows efficiency of siRNA-induced Rap1 protein depletion. Membrane reprobing for α-tubulin was used as a normalization control. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05 vs. nonspecific RNA.

FIGURE 5:

Effects of pharmacologic Rap1 activation on EC permeability in pulmonary ECs exposed to 18% CS and thrombin. (A) Pulmonary ECs were exposed to 18% CS for 6 h with or without 8CPT pretreatment (75 µM, 30 min before 18% CS) followed by thrombin stimulation (10 min). EC monolayers were then fixed and used for immunofluorescence staining with VE-cadherin antibody. Bar = 5 µm. Bar graph represents quantitative analysis of peripheral VE-cadherin signal intensity expressed as mean ± SD; n = 4 independent experiments; *, p < 0.05. (B) 18% CS-induced increase of pMYPT and pMLC levels was inhibited by 8CPT pretreatment. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05 vs. 18% CS. (C) 18% CS-induced increase in EC permeability was inhibited by 8CPT pretreatment as detected by XPerT permeability assay; n = 4 independent experiments; *, p < 0.05. Bar = 10 µm. (D) Ectopic expression of afadin attenuated thrombin-induced MLC phosphorylation in ECs exposed to 4 and 18% CS. The ECs were transfected with empty vector or afadin plasmid (WT) 48 h before the CS stimulation. Right panel depicts protein expression of recombinant GFP-tagged afadin in pulmonary ECs. Membrane reprobing for α-tubulin was used as a normalization control. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05.

Figure 6:

Low-magnitude CS switch attenuates inflammatory signaling activated by LPS and high-magnitude CS. Pulmonary ECs were primed by LPS for 15 min before exposure to 6-h 18% CS or 3-h 4% CS followed by 3-h 18% CS. (A) 18% CS-stimulated phosphorylation of p-NFκB and expression of ICAM1, VCAM1, and E-selectin. These effects were reduced in ECs preconditioned at 4% CS before 18% CS. (B) Intracellular localization of NFκB was detected by immunofluorescence imaging. ECs were exposed to 4% CS or to 4% CS before or after 18% CS. Bar = 10 µm. (C) Western blot analysis of ICAM1 expression. Inclusion of 4% CS before or after 18% CS reduced ICAM1 expression in pulmonary ECs activated by LPS and 18% CS. Membrane reprobing for α-tubulin was used as a normalization control. Bar graphs represent analysis of Western blot data; *, p < 0.05 vs. 18% CS.

FIGURE 7:

Testing protective role of Rap1 in clinically relevant cell models of ALI: inflammatory stimulation combined with exposure to CS. Pulmonary ECs were primed by LPS for 15 min followed by exposure to 4% CS, 18% CS, or 4–18% CS switch. ICAM1 expression monitored by Western blot analysis of cell lysates was used as a marker of EC inflammatory activation. (A) 4% CS attenuated ICAM1 expression caused by LPS priming and initial exposure to 18% CS, as determined by Western blot. Preincubation with Rap1 inhibitor abolished inhibitory effects of 4% CS on ICAM1 expression. (B) Knockdown of Rap1 using gene-specific siRNA attenuated the inhibitory effect of 4% CS preconditioning on expression of ICAM stimulated by LPS and 18% CS, as determined by Western blot. (C) EC primed with LPS for 15 min were treated with vehicle or Rap1 activator 8CPT (75 µM, 30 min) followed by exposure to 18% CS. Phospho-NFκB and ICAM1 levels were analyzed by Western blot as parameters of EC inflammation. For all experiments, membrane reprobing for α-tubulin was used as a normalization control. Bar graphs represent analysis of Western blot data; n = 3; *, p < 0.05 vs. 18% CS. (D) Immunofluorescence analysis of effects of 18% CS and switch from 4% CS to 18% CS on actin cytoskeleton, adherens junctions, and EC barrier disruption in LPS-primed ECs. F-actin remodeling was evaluated by immunofluorescence staining with Texas Red phalloidin. Formation of paracellular gaps (marked by arrows) was observed in LPS-primed ECs exposed to 18% CS (6 h). Disruption of EC monolayers was attenuated in LPS-primed ECs preconditioned at 4% CS (3 h) before 18% CS switch (3 h). Bar graph depicts quantitative analysis of gap formation; n = 3; *, p < 0.05. Knockdown of Rap1 using gene-specific siRNA attenuated the protective effect of 4% CS preconditioning on monolayer integrity. Bar = 10 µm. (E) Immunofluorescence analysis of VE-cadherin at the cell junctions in pulmonary ECs primed with LPS. Left two panels: 18% CS; right two panels: switch from 4% CS to 18% CS. Bar = 10 µm. Bar graphs show quantitative analysis of VE-cadherin signal intensity. Data are expressed as mean ± SD of three independent experiments, five fields per condition; *, p < 0.05.

FIGURE 8:

Effect of switch from HTV to LTV on parameters of LPS-induced lung injury. C57B6 WT mice were subjected to 4-h HTV, 4-h LTV, or a switch of 1-h HTV to 3-h LTV in the presence or absence of concurrent administration of TRAP6 or in the presence or absence of 18-h LPS treatment by intratracheal infusion before ventilation procedures. (A) Live imaging analysis of lung vascular leak after intravenous injection of TRAP6 and different ventilation schemes. TRAP6-induced accumulation of fluorescent Angiosense 680 EX imaging agent in the lungs was detected by a Xenogen IVIS 200 Spectrum imaging system after termination of mechanical ventilation as described in Materials and Methods and presented in arbitrary colors. (B) Live imaging analysis of lung vascular leak after intratracheal injection of LPS and different ventilation schemes. (C, D) Mice exposure to LTV (4 h), HTV (4 h), or HTV (1 h)-LTV (3 h) switch was performed 18 h after LPS administration (intratracheal). At the end of experiment, BAL collection was performed, and measurements of total cell count (C) and protein concentration (D) were performed in BAL samples. (E) The BAL levels of the mouse KC and MIP2 were evaluated using ELISA. Data are expressed as mean ± SD of four independent experiments; *, p < 0.05 vs. LPS alone.

FIGURE 9:

Role or Rap1 in the protective effect of HTV-LTV switch on LPS-induced lung injury. LPS administration (intratracheal) of Rap1−/− mice and matched wild-type controls was performed 18 h before exposure to different regimens of mechanical ventilation. The experiment was terminated after 4 h of mechanical ventilation. (A) BAL cell count measurements. (B) BAL protein content measurements. Data are expressed as mean ± SD of four independent experiments; *, p < 0.05. (C) Evans blue dye (30 ml/kg, i/v) was injected 30 min before termination of the experiment. Photographs depict lung vascular permeability assessed by Evans blue accumulation in the lung tissue.