Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin

Abstract

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Keywords: Rab GTPase; acute respiratory distress syndrome; endocytosis; endothelium; vascular endothelial-cadherin.

Figure 1.

Constitutive activation of Rab4 enhances endothelial barrier function and attenuates LPS- or Pseudomonas aeruginosa–induced permeability in vitro and in vivo. (A–D) Equivalent numbers of lung microvascular endothelial cells (LMVECs) were transiently transfected with green fluorescent protein (GFP; triangles) or Rab4^wt (B), Rab4^Q67L (C) or Rab4^S22N (D) cDNA (squares). After 48 hours, changes in endothelial monolayer resistance were measured, in the presence (open symbols) and absence (solid symbols) of LPS (1 μg/ml). Overexpression was assessed by immunoblot analysis of cell lysates using antibodies directed against GFP, with a loading control of actin (A). (E) Adult (8–10 wk) C57/BL6 male mice were injected with liposomes containing cDNA encoding GFP, Rab4^wt, Rab4^Q67L, or Rab4^S22N. At 44 hours after injection, mice were injected with P. aeruginosa PA103 (10⁷ CFU, intratracheally) for a further 4 hours, and lung weights were measured before and after a 72-hour drying period. Arrows indicate addition of LPS. Data are presented as mean (±SD); n = 4–5; *P < 0.05 versus vehicle, ^#P < 0.05 versus GFP with respective treatment.

Figure 2.

Rab4 localizes to cell surface vascular endothelial (VE)-cadherin, and Rab4 activation attenuates LPS-induced internalization of VE-cadherin from the cell surface. (A and B) Equivalent numbers of LMVECs were transiently transfected with GFP or Rab4^wt, Rab4^Q67L, or Rab4^S22N cDNA. At 48 hours, cells were treated with LPS (1 μg/ml) for 6 hours. (A) Cell surface expression of VE-cadherin was determined with whole-cell indirect ELISA using chemiluminescence. Nonspecific binding was assayed using IgG (rcu, relative chemiluminescence units). (B) Expression of VE-cadherin, Rab4 and early endosome antigen 1 (EEA1) was measured in whole cell lysates by immunoblot analysis. Blots were stripped and reprobed for vinculin as loading control, and densitometry analysis was performed. (C) LMVECs were grown to confluence on gelatin-coated glass coverslips and treated with vehicle (saline) or LPS (1 μg/ml, 6 h). Cells were then fixed, permeabilized, and immunofluorescently stained for Rab4 and VE-cadherin, followed by Texas red– and fluorescein isothiocyanate (FITC)-labeled secondary antibodies, respectively. Images were captured via microscopy at 100× magnification. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. Arrowheads indicate areas of colocalization, and arrows indicate areas of VE-cadherin internalization from cell surface. Representative images are presented (i). Colocalization of VE-cadherin with Rab4 was quantified using Image J colocalization software (National Institutes of Health, Bethesda, MD) (ii). Data are presented as mean (±SD); n = 3 (B and C) and 6 (A). *P < 0.05 versus vehicle, ^#P < 0.05 versus GFP, ^†P < 0.05 versus Rab4^wt.

Figure 3.

Inhibition of pro-lysosomal Rab GTPase Rab9, but not Rab7, enhances endothelial barrier function and attenuates LPS- or P. aeruginosa–induced permeability in vitro and in vivo. (A, B, and D) Equivalent numbers of LMVECs were transiently transfected with GFP (triangles), Rab7^T22N (B), or Rab9^S21N (D) cDNA (squares). After 48 hours, changes in endothelial monolayer resistance were measured using electrical cell impedance sensor (ECIS) in the presence (open symbols) and absence (solid symbols) of LPS (1 μg/ml). Overexpression was assessed by immunoblot analysis of cell lysates using antibodies directed against GFP, with a loading control of actin (A). (C and E) Adult (8–10 wk old) C57/BL6 male mice were injected with liposomes containing cDNA encoding GFP, Rab7^T22N, or Rab9^S21N. At 44 hours after injection, mice were injected with P. aeruginosa PA103 (10⁷ CFU, intratracheally) for a further 4 hours, and lung weights were measured before and after a 72-hour drying period. Arrows indicate addition of LPS. Data are presented as mean (±SD); n = 4–5; *P < 0.05 versus vehicle, ^#P < 0.05 versus GFP.

Figure 4.

Inhibition of prolysosomal Rab GTPase Rab9, but not Rab7, increases VE-cadherin expression at the cell surface. (A) Equivalent numbers of LMVECs were transiently transfected with GFP, Rab7^T22N, or Rab9^S21N cDNA. At 48 hours, cells were treated with LPS (1 μg/ml) for 6 hours. Cell surface expression of VE-cadherin was determined with whole-cell indirect ELISA using chemiluminescence. Nonspecific binding was assayed using IgG. (B) Expression of Rab7, Rab9, and lysosomal-associated membrane protein 1 (LAMP1) was measured in whole-cell lysates from LMVECs exposed to LPS (1 μg/ml) for 6 hours by immunoblot analysis. Blots were stripped and reprobed for vinculin as loading control, and densitometry analysis was performed. (C and D) LMVECs were grown to confluence on gelatin-coated glass coverslips and treated with vehicle (saline) or LPS (1 μg/ml, 6 h). Cells were then fixed, permeabilized, and immunofluorescently stained for Rab7 (C) or Rab9 (D) and VE-cadherin, followed by Texas red– and FITC-labeled secondary antibodies, respectively. Images were captured via microscopy at 100× magnification. Nuclei were stained with DAPI. Scale bars, 20 μm. Arrowheads indicate areas of colocalization, and arrows indicate areas of VE-cadherin internalization from cell surface. Representative images are presented (i). Colocalization of VE-cadherin with Rab7 or Rab9 was quantified using Image J cololcalization software (ii). Data are presented as mean (±SD); n = 3 (B, C, and D) and 6 (A). *P < 0.05 versus vehicle, ^#P < 0.05 versus GFP.

Figure 5.

Protection from LPS-induced endothelial permeability, mediated by Rab4 activity, is dependent on extracellular signal–regulated kinase (ERK) phosphorylation. (A and B) Equivalent numbers of LMVECs were transiently transfected with GFP, Rab4^wt, Rab4^Q67L, or Rab4^S22N cDNA (A) or GFP, Rab7^T22N, or Rab9^S21N cDNA (B). At 48 hours, cells were treated with LPS (1 μg/ml) for 6 hours. Phosphorylation of p38, ERK1/2, or p70 was assessed in whole-cell lysates by immunoblot analysis with an antibody specific to each phosphorylated protein. Blots were stripped and reprobed for total protein expression and vinculin as a loading control. Representative blots are shown. (C–F) Equivalent numbers of LMVECs were transiently transfected with GFP or Rab4^S22N cDNA. At 48 hours, cells were pretreated with chemical inhibitors, U0126 (DMSO vehicle, 10 μM) (C), SB203580 (DMSO vehicle, 10 nM) (D), or rapamycin (DMSO vehicle, 10 nM) (E) for 30 minutes followed by treatment with LPS (1 μg/ml) for 6 hours. Cell surface expression of VE-cadherin was determined with whole-cell indirect ELISA using chemiluminescence. Nonspecific binding was assayed using IgG. Changes in endothelial monolayer resistance were measured using ECIS (F [i]), and percentage drop in monolayer resistance was calculated (F [ii]). Data are presented as mean (±SD); n = 3 (A, B, and F) and 4 (C–E). *P < 0.05 versus GFP, ^#P < 0.05 versus vehicle.