Rab5 GTPase-mediated VE-cadherin Endocytosis Regulates Lung Microvascular Endothelial Barrier Function

Background:The vascular endothelium is formed by a continuous endothelial cell monolayer, constituting a semi-selective barrier between blood and the interstitium that controls the exchange of macromolecules and fluid. Endothelial permeability is essential for the pathogenesis of acute and chronic inflammation underlying many diseases, such as acute lung injury and acute respiratory distress syndrome(ALI/ARDS), atherosclerosis, and cancer and so on. Endothelial barrier function is regulated by adherens and tight junctions. The assembly and organization of adherens junctions precede the formation of tight junctions, which have important functions, including the establishment and maintenance of cell–cell adhesion, actin cytoskeleton remodeling, and intracellular signaling. Vascular endothelial(VE)-cadherin is an endothelial-specific transmembrane component of adherens junction complexes that plays key roles in the maintenance of vascular integrity, including rearrangement of the cytoskeleton, establishment of cell polarity, and tight junction remodeling.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, remains unknown. Here, we demonstrated that lipopolysaccharide(LPS) simulation markedly enhanced the expression of Rab5 in human pulmonary microvascular endothelial cells(HPMECs), which is accompanied with VE-cadherin internalization, and actin cytoskeleton remodeling. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. Small interfering RNA(si RNA)-mediated suppression of Rab5 a expression attenuated the disruption of LPS-induced internalization of VE-cadherin, and the cell polarity in vitro. Moreover, Rab5 a siRNA suppressed the disruption of LPS-induced endothelial hyperpermeability and protected vascular endothelial barrier function from LPS injury both in vitro and in vivo. These results suggest that Rab5 a is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization and actin cytoskeleton remodeling. These findings provide evidence implicating that Rab5 a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.Objective:1. To explore the influence of LPS on the expression of Rab5 and Rab5 activity of HPMECs.2. To investigate the mechanism and effect of Rab5 on the regulation of VE-cadherin internalization and cell polarity of HPMECs.3. To study on the mechanism of Rab5 regulating microvascular endothelial barrier function in vivo and in vitro.Methods:1. HPMECs were cultured and stimulated with LPS, and the expression of Rab5 a and the level of Rab5 a GTPase activation were measured by western blot analysis.2. Cellular localization and expression of VE-cadherin, F-actin were detected by confocal and western blot.3. HPMECs were cultured on E-plate L8. The endothelial cell barrier function was measured using iCELLigence system.4. HPMECs were transfected with NC siRNA, Rab5 a si RNA, NC plasmid, and Rab5 a wild type plasmid, then localization and expression of VE-cadherin and F-actin were detected by confocal and western blot, the apical-basal localization of Podxl was detected by confocal.5. HPMECs were cultured on transwell filteres. After different stimulation, the monolayer permeability of HPMECs was determined using tracer flux assay.6. C57BL/6 mice were transfected with Cy3-tagged Rab5 a si RNA for 6 days, then lung, liver, spleen, and kidney tissues were removed and frozen in liquid nitrogen used for fluorescence microscopy to detect the distribution of Cy3-tagged Rab5 a si RNA.7. Sepsis models. C57BL/6 mice or transfected with Rab5 a siRNA mice were challenged with 20 mg/kg LPS i.p. on day 5. Then mice were sacrificed after LPS challenged 24 hours. Lung tissues were immediately removed and frozen in liquid nitrogen or paraffin-embedded, then used for fluorescence microscopy and histology.8. C57BL/6 mice or transfected with Rab5 a si RNA mice were challenged with 20 mg/kg LPS i.p. on day 5. 40 mg/kg Evans blue albumin(EBA) was injected into the tail vein 2 h before the termination of 24 h of LPS treatment to assess vascular leakage.9. Suvival study. The sepsis model of C57BL/6 mice or transfected with Rab5 a si RNA mice was induced by 40mg/kg LPS i.p. Subsequently, mice were monitored 5 d, 4 times daily.Results:1. The expression levels of Rab5 a and activity of Rab5 a GTP were increased after LPS stimulation in a time-dependent manner. LPS caused actin cytoskeleton polymerization and induced the formation of numerous stress fibers in the cell cortex.2. LPS decreased the expression of VE-cadherin in cell membrane and increased its expression in cytosol, induced the translocation of VE-cadherin. By colocalization with endosome marker EEA1 and Rab5, LPS induced the internalization of VE-cadherin. LPS also induced the rearrangement of actin cytoskeleton in HPMECs, and disrupted endothelial cell polarity and endothelial barrier function.3. Rab5 a regulated the internalization of VE-cadherin during LPS injury. After transfection with Rab5 a siRNA, the internalization of VE-cadherin was significantly suppressed following LPS treatment. Rab5 a si RNA inhibited LPS-induced VE-cadherin decrease in the membrane, and prevented disassembly of VE-cadherin after LPS stimulation. However, overexpression of Rab5 a by Rab5 a WT plasmid transfection had no obvious effect on the internalization of VE-cadherin induced by LPS.4. Rab5 a regulated the arrangement of F-actin and maintained the normal apical-basal expression of Podxl, and the normal apical-basal cell polarity.5. Real time cell electric impedance sensing by i CELLigence system and permeability assay showed that LPS induced endothelial barrier dysfunction and hyperpermeability was inhibited by knockdown of Rab5 a.6. Rab5 a expression was elevated by LPS stimulation in the lung tissue in vivo. Colocalization with the vascular endothelial marker CD31 demonstrated that Rab5 a expression was up-regulated in lung endothelium of sepsis models.7. Using Rab5 a siRNA knockdown the expression of Rab5 a in vivo, Rab5 a knockdown significantly inhibited LPS-induced lung injury and prevented lung vacular leakage by histology detection.Conclusion:1. LPS elevates the expression and activity of Rab5 a in the lung endothelium in vivo and in vitro.2. Rab5 a regulates VE-cadherin internalization and F-actin organization in HPMECs. And Rab5 a also regulates the apical-basal cell polarity and endothelial barrier function in HPMECs.3. Inhibition of Rab5 a expression through Rab5 a siRNA transfection prevents LPS-induced lung injury and lung endothelial permeability in vivo.