Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium

Abstract

The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.

Keywords: Actin cytoskeleton; Acute lung injury; Adherens junction; Barrier function; Cell polarity; Endosome; Endothelium; Internalization; Rab5 GTPase; VE-cadherin.

Fig. 1

Effect of LPS on the activity and expression of Rab5a in HPMECs. a Expression of active and total Rab5a was measured by pulldown assay and Western blotting (WB) using polyclonal antibodies against Rab5a. Representative blots showing active Rab5a (upper panel), relative GAPDH (2nd panel), total Rab5a (3rd panel), and GAPDH expression (lower panel) are shown. HPMECs were cultured and treated with LPS (1 μg/ml) for various time intervals (1, 6, and 12 h). b Quantitative data of active and total Rab5a expression normalized to GAPDH expression. The data are presented as the mean ± SE (n = 3). For active Rab5a, ^# p < 0.05 versus the 0 h group, ^$ p < 0.05 versus the 1 h group; for total Rab5a, *p < 0.05 versus the 0 h group, ^{^} p < 0.05 versus the 1 h group. c HPMECs were cultured on coverslips and then treated with 1 μg/ml LPS for 6 h, fixed, and stained with an anti-Rab5a polyclonal antibody. The images represent three separate experiments. Green, Rab5a; blue, DNA stained with DAPI (nuclei). Scale bar, 25 μm

Fig. 2

Effect of LPS on the expression and localization of VE-cadherin and the actin cytoskeleton in HPMECs. a The expression levels of the VE-cadherin and F-actin in the membrane and cytosol fractions and whole cell lysate were detected by WB. Representative blots showing the expression of VE-cadherin in the cell membrane (upper panel), in the cytosol (2nd panel), and in the whole cells (3rd panel), and the expressions of F-actin (4th panel) and GAPDH (lower panel) are shown. HPMECs were treated with 1 μg/ml LPS for various time intervals. b Histogram showing the quantification of VE-cadherin in the membrane fraction (white bars) and in the cytosol fraction (grid bars) from three independent experiments. The data are presented as the mean ± SE *p < 0.05 versus the 0 h membrane group, ^{^} p < 0.05 versus the 1 h membrane group; ^# p < 0.05 versus the 0 h cytosolic group, ^$ p < 0.05 versus the 1 h cytosolic group. c, d Confocal microscopic analysis of VE-cadherin (c) and F-actin (d) in HPMECs after LPS treatment. HPMECs were cultured on Transwell inserts for 5–7 days. The HPMECs were grown to about 90 % confluence. They were stimulated with 1 μg/ml LPS for 6 h and imaged by confocal laser scanning microscopy (CLSM). Green indicates cells labeled with VE-cadherin; red, F-actin; blue, DAPI. Arrows indicate intercellular gaps and arrowheads indicate fibers. Scale bar 25 µm. The photographs represent three separate experiments

Fig. 3

HPMEC barrier dysfunction after LPS treatment. a HPMECs were cultured and stimulated with LPS at 1 μg/ml, and real-time cell growth curves were subsequently observed and taken from four independent experiments using the iCELLigence system. b A time histogram shows the normalized cell index (CI) with LPS treatment for 1, 6, and 12 h. The data are presented as the mean ± SE. n = 4, *p < 0.05 versus the 0 h group, ^{^} p < 0.05 versus the 1 h group. c HPMECs were grown to confluence on 0.4-μm Transwell inserts and stimulated with 1 μg/ml LPS for different time intervals (1, 6, and 12 h). FITC-dextran was added to the upper wells at a concentration of 1 mg/mL. After 0.5 h of incubation, 50 μl of medium from the bottom chamber was aspirated and measured in a fluorescence plate reader. The FITC-dextran permeability is expressed as the fold increase ±SE (n = 3). *p < 0.05 versus the 0 h group, ^{^} p < 0.05 versus the 1 h group

Fig. 4

Transfection of Rab5a siRNA and the WT Rab5a plasmid into HPMECs. a Efficiency of Rab5a siRNA and WT Rab5a plasmid transfection in HPMECs. HPMECs were cultured on coverslips and transfected with either Cy3-tagged Rab5a siRNA for 12 h using the X-tremeGENE siRNA Transfection Reagent or the GFP-tagged WT Rab5a plasmid for 48 h using the X-tremeGENE HP DNA Transfection Reagent. The localization of Rab5a siRNA and Rab5a WT in HPMECs was detected using CLSM. Similar results were obtained in three separate experiments. Blue, DNA staining with DAPI (nuclear); red, Cy3; green, GFP. Scale bar 25 μm. b Rab5a expression in HPMECs after transfection with Rab5a siRNA was assayed. HPMECs were transfected with NC siRNA (control siRNA), Rab5a siRNA, NC plasmid (empty plasmid), or the Rab5a–GFP WT plasmid. Representative bands showing Rab5a (upper panel) and GAPDH expression (lower panel) are shown. A histogram illustrates the quantitative analysis of the protein levels of endogenous Rab5a, normalized to GAPDH expression. The data are presented as the mean ± SE. n = 3. *p < 0.05 versus the control and NC siRNA groups

Fig. 5

Rab5a regulates the LPS-induced disruption of VE-cadherin and actin cytoskeleton remodeling in HPMECs. a The expression levels of VE-cadherin and F-actin in the cell membrane, cell cytosol fractions, and whole cells were determined by WB. Representative blots showing the expression of VE-cadherin in the membrane (upper panel), cytosol (2nd panel), and whole cells (3rd panel), F-actin (4th panel), and GAPDH (5th panel) are shown. HPMECs were transfected with or without Rab5a siRNA or the Rab5a WT plasmid and then treated with 1 μg/ml LPS for 6 h. b Quantitative data showing VE-cadherin protein levels in the cell membrane and cytosol, normalized to GAPDH expression. The data are presented as the mean ± SE n = 3. *p < 0.05 versus the membrane control group, ^{^} p < 0.05 versus the membrane LPS group; ^# p < 0.05 versus the cytosolic control group, ^$ p < 0.05 versus the cytosolic LPS group. c Localization of VE-cadherin in HPMECs following Rab5a siRNA or WT plasmid transfection. After pretransfection with NC siRNA, Rab5a siRNA, the NC plasmid, or the WT Rab5a plasmid, confluent HPMECs were stimulated with 1 μg/ml LPS for 6 h. VE-cadherin localization was detected by CLSM. Green, VE-cadherin; blue, DAPI. Arrows indicate intercellular gaps. Scale bar 25 µm. The photographs represent three independent experiments. d Quantification of the area of intercellular gaps as a percentage of the total area imaged. The data are presented as the mean ± SE based on three independent experiments. *p < 0.05 versus the NC siRNA group, ^{^} p < 0.05 versus the NC siRNA plus LPS treatment group. e Localization of the expression of F-actin in HPMECs following Rab5a siRNA or WT plasmid transfection. After pretransfection with NC siRNA, Rab5a siRNA, the NC plasmid, or the Rab5a WT plasmid, HPMECs were stimulated or not stimulated with 1 μg/ml LPS for 6 h. The actin cytoskeleton was stained with rhodamine-phalloidin and observed via CLSM. Red, F-actin; blue, DAPI. Arrows indicate stress fibers. Scale bar 25 µm. The photographs represent three independent experiments

Fig. 6

Knockdown of Rab5a inhibits LPS-induced VE-cadherin internalization in HPMECs. HPMECs were transfected with NC siRNA or Rab5a siRNA and stimulated with 1 μg/ml LPS for 6 h, and then incubated with anti-VE-cadherin antibodies against the extracellular domain of VE-cadherin (VE-cadherin-N) at 4 °C for 1 h. The VE-cadherin internalization was monitored by the uptake of external VE-cadherin antibodies at 37 °C and visualized in fixed cells using secondary fluorescent antibodies (red). The cells were also stained with Rab5a antibodies (green). Colocalization between VE-cadherin and Rab5a was displayed as yellow color in these overlays. Scale bar 7.5 µm. The photographs represent three independent experiments

Fig. 7

Rab5a regulates the LPS-induced disruption of human endothelial cell polarity. a Inhibitory effect of Rab5a siRNA on the LPS-induced disruption of HPMEC polarity. HPMECs cultured on Transwell inserts and transfected with NC siRNA, Rab5a siRNA, the NC plasmid, or the WT Rab5a plasmid were treated with 1 μg/ml of LPS for 6 h. The cells were then labeled with the apical protein marker Podxl. Red, Podxl; blue, DAPI. Arrows indicate Podxl localized at the apical surface of the cell. Arrowheads indicate Podxl distribution at the basal rather than apical surface of the cell. Z-axis, scale bar 5 µm. b Quantification of the apical/basal fluorescence intensity of the Podxl protein over time in a histogram. The apical or basal fluorescence intensity was calculated by integrating the area under the apical or basal peak defined via membrane labeling with Podxl using ImageJ software. The mean apical/basal fluorescence intensity was calculated in ten cells from three independent experiments. *p < 0.05 versus the NC siRNA group; ^p < 0.05 versus the NC siRNA plus LPS treatment group. c Inhibitory effect of Rab5a siRNA on the LPS-induced disruption of the F-actin cytoskeleton. Photograph of red fluorescence showing the distribution of F-actin in HPMECs (upper panel). Following pretransfection with NC siRNA, Rab5a siRNA, the NC plasmid, and the WT Rab5a plasmid, confluent HPMECs were stimulated with 1 μg/ml LPS for 6 h. The F-actin cytoskeleton was stained with rhodamine-phalloidin. Red, F-actin; blue, DAPI. Scale bar, 10 µm. Quantification of the F-actin fluorescence intensity is indicated in the corresponding histograms below the fluorescence images, which represent the intensity profiles of F-actin staining along the dotted lines in the photomicrographs, emphasizing differences in the cross-cellular F-actin expression

Fig. 8

Rab5a regulates LPS-induced HPMEC dysfunction. a HPMECs were transfected with NC siRNA or Rab5a siRNA for 48 h and then stimulated with 1 μg/ml LPS for 6 h. Real-time cell growth curves were generated using the iCELLigence system. The data are shown as the mean ± SE from three independent experiments. b Quantification of the normalized CI in the histogram. After transfection with NC siRNA and Rab5a siRNA, the cells were stimulated with LPS for 6 h. The data are presented as the mean ± SE, n = 3. *p < 0.05 versus the control group; ^p < 0.05 versus the NC siRNA plus LPS treatment group. c HPMECs were cultured on 0.4-μm Transwell inserts and transfected with NC siRNA or Rab5a siRNA for 48 h and then stimulated with 1 μg/ml LPS for 6 h. FITC-dextran was added to the upper wells at a concentration of 1 mg/ml. After 0.5 h of incubation, 50 μl of medium from the bottom chamber was aspirated and analyzed using a fluorescence plate reader. FITC-dextran permeability was expressed as fold increase ± SE (n = 3). *p < 0.05 versus the control group; ^p < 0.05 versus the NC siRNA plus LPS treatment group

Fig. 9

Rab5a expression is elevated by LPS treatment in mouse lungs in vivo. a C57BL/6 mice received i.p. injections with saline or 20 mg/kg LPS. OTC-embedded lung sections were prepared 24 h after LPS challenge. Rab5a expression in the endothelium was analyzed by CLSM. Green indicates labeling of Rab5a; red, CD31; blue, DAPI. Scale bar 7.5 µm. The figures represent three experiments. Note: a floating nucleus at the upper left of the image in the LPS group is a contaminted white blood cell's nucleus in the vascular lumen. b C57BL/6 mice were i.p. injected with saline or 20 mg/kg LPS. Lung tissues were collected after 24 h of treatment. Rab5a expression was analyzed via WB. Representative blots showing Rab5a (upper panel) and β-actin expression (lower panel) are shown. Lower panel shows quantitative data corresponding to Rab5a protein levels normalized to β-actin expression. The data are presented as the mean ± SE n = 3. *p < 0.05 versus the control group

Fig. 10

Silencing Rab5a with siRNA attenuates LPS-induced endothelial barrier dysfunction in vivo. a The distribution of Cy3-tagged Rab5a siRNA in lung tissue samples isolated from C57BL/6 mice. OTC-embedded lung sections were prepared 6 days after transfection and stained with DAPI. The Cy3-expressing cells in lung tissues were visualized under a confocal microscope. Red, Cy3; blue, DAPI. Scale bar 25 µm. b Rab5a expression in lung tissues after transfection with Rab5a siRNA. C57BL/6 mice were injected via the tail vein with NC siRNA or Rab5a siRNA. Lung tissue samples were collected 3 and 6 days later. Rab5a expression was analyzed via WB. Representative blots showing Rab5a (upper panel) and β-actin expression (lower panel) are included. Lower panel shows quantitative data of Rab5a protein levels, normalized to β-actin. The data are presented as the mean ± SE n = 3. *p < 0.05 versus the control group. c C57BL/6 mice were injected via the tail vein with NC siRNA or Rab5a siRNA and then challenged with 20 mg/kg LPS via i.p. injection. Evans blue tracer (40 mg/kg) was injected 24 h after the LPS challenge. The lungs of the mice were harvested 2 h after Evans blue dye injection, and pulmonary transvascular permeability was measured. The data are presented as the mean ± SE, n = 4. *p < 0.05 versus the control group; ^p < 0.05 versus the NC siRNA plus LPS treatment group. d C57BL/6 mice were injected via the tail vein with NC siRNA or Rab5a siRNA, and 20 mg/kg LPS was injected 5 days after transfection. The mice were killed 24 h after LPS injection. Lung tissue samples were fixed with 4 % paraformaldehyde, dissected, and subjected to H&E staining (original magnification ×200, ×400). e Mice in the control and Rab5a siRNA groups were injected with 40 mg/kg LPS i.p., and their survival was monitored. Differences in mortality were assessed using the log-rank test. The experiments were performed with ten mice in each of the two groups. *p < 0.05 versus the control group