Loss of SOX18/CLAUDIN5 disrupts the pulmonary endothelial barrier in ventilator-induced lung injury

Abstract

Mechanical strain contributes to ventilator-induced lung injury (VILI) through multi-factorial and complex mechanisms that remain unresolved. Prevailing evidence suggests that the loss of pulmonary endothelial tight junctions (TJs) plays a critical role. TJs are dynamically regulated by physiologic and hemodynamic forces to stabilize the endothelial barrier. The transcription factor sex-determining region Y-box (SOX)-18 is important in regulating blood vessel development and vascular permeability through its ability to regulate the transcription of Claudin-5, an endothelial TJ protein. Previously, we demonstrated that SOX18 expression is increased by shear stress in the pulmonary endothelium. Therefore, in this study, we investigated how mechanical strain mediated through cyclic stretch affects the SOX18/Claudin-5 regulatory axis. Our data demonstrate that SOX18 and Claudin-5 are downregulated in human lung microvascular endothelial cells (HLMVEC) exposed to cyclic stretch and the mouse lung exposed to high tidal mechanical ventilation. Overexpression of SOX18 reduced the loss of Claudin-5 expression in HLMVEC with cyclic stretch and preserved endothelial barrier function. Additionally, overexpression of Claudin-5 in HLMVEC ameliorated barrier dysfunction in HLMVEC exposed to cyclic stretch, although SOX18 expression was not enhanced. Finally, we found that the targeted overexpression of SOX18 in the pulmonary vasculature preserved Claudin-5 expression in the lungs of mice exposed to HTV. This, in turn reduced lung vascular leak, attenuated inflammatory lung injury, and preserved lung function. Together, these data suggest that enhancing SOX18 expression may prove a useful therapy to treat patients with ventilator-induced lung injury.

Keywords: ALI; SOX-18; claudin-5; endothelial barrier function; mechanical stress; tight junctions.

FIGURE 1

Exposing human lung microvascular endothelial cells to pathologic cyclic stretch reduces Sox18 expression. HLMVECs were analyzed by immunofluorescent staining using a VE-cadherin antibody after being subjected to 18% cyclic stretch for 8 h, replated on coverslips, and fixed at the indicated times. mRNA from HLMVECs were subjected to cDNA synthesis and SYBR green qRT-PCR using specific primers directed to SOX18 sequence and normalized to Beta-2-Microglobulin, a housekeeping gene. Protein extracts prepared from HLMVECs were immunoblotted using SOX18 antibody. VE-cadherin is reduced in the HLMVECs subjected to cyclic stretch (A). Transendothelial resistance recovery is significantly diminished in cells stretched at 18% for 8 h (B). qRT-PCR analysis demonstrated a significant decrease of SOX18 mRNA after cyclic stretch (C,E). Densitometric analysis showed a significant decrease in SOX18 protein levels after 8 h of cyclic stretch (F) but not 4 h (D). *p < 0.05 versus Static. N = 10 (A); N = 4 (B,D); N = 5 (F); N = 6 (C,E).

FIGURE 2

SOX18 overexpression prevents the loss of barrier function in human lung microvascular endothelial cells exposed to cyclic stretch. SOX18 overexpression was induced with an adenovirus (AdSOX18) containing SOX18 and GFP as a reporter (each with its own promoter). HLMVECs were treated with AdSOX18 for 48 h with an MOI of 40 unless otherwise indicated. HLMVEC lysates were analyzed by immunoblot using SOX18 antibody. HLMVECs were analyzed by immunofluorescent staining using VE-cadherin antibody after being subjected to 18% cyclic stretch for 8 h. Fluorescent microscopy shows a proportional increase in adenoviral expression as indicated by the increase in green light fluorescence from the GFP reporter (A). SOX18 overexpression was confirmed by immunoblotting (B). Densitometric analysis shows a significant increase in SOX18 expression after treatment of HLMVECs with AdSOX18 in both static and subjected to cyclic stretch cells relative to the non-adenovirus pretreated cells (C). The transendothelial resistance reduction in HLMVECs subjected to cyclic stretch is ameliorated in cells overexpressing SOX18 (D). Overexpression of SOX18 prevents VE-cadherin loss after cyclic stretch in HLMVECs (E). *p < 0.05 versus Static (C,E) or AdSOX18 (D), †p < 0.05 versus Stretch (C) or Static + AdSOX18 (E), ‡p < 0.05 versus Stretch. N = 14–20 (C); N = 4 (D); N = 21–31 (E).

FIGURE 3

Exposing human lung microvascular endothelial cells to pathologic cyclic stretch reduces Claudin-5 expression. HLMVECs were exposed to 18% cyclic stretch. HLMVEC lysates were analyzed by immunoblot using a specific Claudin-5 antibody. qRT-PCR analysis showed that 4 h of cyclic stretch did not significantly reduce Claudin-5 expression levels (A), which matches the densitometric analysis of Claudin-5 protein levels (B). By contrast, we found a significant decrease of CLDN5 mRNA after 8 hrs of cyclic stretch at 18% (C), reflecting the densitometric analysis showing that Claudin-5 protein levels are significantly decreased (D). Densitometric analysis showed that overexpression of SOX18 prevents the cyclic stretch-dependent Claudin-5 decrease in HLMVECs (E). *p < 0.05 versus Static, †p < 0.05 versus Stretch. N = 4 (B); N = 6 (A,C,D); N = 14–20 (E).

FIGURE 4

Claudin five overexpression prevents the loss of barrier function in human lung microvascular endothelial cells exposed to cyclic stretch. A CLDN5/GFP coding adenovirus was used to overexpress Claudin-5 isoform 1. HLMVECs were treated with AdCLDN5 for 48 h with an MOI of 40 unless otherwise indicated. HLMVEC lysates were subjected to immunoblot analysis using Claudin-5 antibody. HLMVECs were analyzed by immunofluorescent staining using a VE-cadherin antibody. Treatment of HLMVECs with AdCLDN5 induced a proportional increase in claudin-5 as observed by increased GFP reporter fluorescence (A). Immunoblotting shows a similar proportional increase in Claudin-5 protein levels (B). Total Claudin-5 expression (iso1+iso2) is significantly increased in HLMVECs pretreated with AdCLDN5 and subjected or not to cyclic stretch relative to the non-adenovirus pretreated cells (C). Overexpression of Claudin-5 prevents VE-cadherin loss after cyclic stretch in HLMVECs (D). The transendothelial resistance reduction in HLMVECs subjected to cyclic stretch is ameliorated in cells overexpressing Claudin-5 (E). *p < 0.05 versus Static (C,D) or AdCLDN5 (E), †p < 0.05 versus Stretch (C) or Static + AdCLDN5 (D), ‡p < 0.05 versus Stretch. N = 4–6 (C); N = 30–42 (D); N = 3–4 (E).

FIGURE 5

Sox18 and Cldn5 expression are reduced in the mouse lungs subjected to high tidal mechanical ventilation. The mice were exposed or not to ventilation with high tidal volume (HTV; 30 ml/kg, 75 bpm, 0.5 FiO₂) for 8 h. Protein extracts prepared from lung tissue homogenates were subjected to immunoblot analysis using Sox18 and Claudin-5 antibodies. mRNA isolated from mice lung tissues were subjected to cDNA synthesis and SYBR green qRT-PCR using specific primers directed to Sox18 sequence and normalized to β-2-Microglobulin, a housekeeping gene. qRT-PCR analysis demonstrated a significant decrease of Sox18 expression (A), matching the reduced Sox18 protein levels as determined by densitometric analysis (B). Similarly, Cldn5 mRNA after cyclic stretch is diminished (C), correlating with a decrease in Cldn5 protein levels as measured by immunoblot and densitometric analysis (D). *p < 0.05 versus Control. N = 4–6 (A); N = 3–5 (B); N = 8–12 (C); N = 3–5 (D).

FIGURE 6

The over-expression of Sox18 preserves lung mechanics in mice exposed to high tidal mechanical ventilation. Mice were injected or not with pCMV6-SOX18 (SOX18) or control pDST-luciferase plasmids using in vivo-jetPEI® via the tail vein. After 64 h, the mice were exposed or not to ventilation with high tidal volumes (HTV; 30 ml/kg, 75 bpm, 0.5 FiO2) for 8 h. HLMVECs were treated with AdSOX18 for 48 h to overexpress SOX18 and subjected to cyclic stretch for 8h. Immunoblot analysis demonstrated a significant increase in Sox18 protein levels in the lungs of mice given the pCMV6-SOX18 plasmid (A). The analysis of dynamic pressure-volume relationships in the mouse lung indicated that HTV ventilation caused a loss of Optimal Respiratory System Compliance (B) and Respiratory System Elastance (C) which is prevented in SOX18 over-expressing mice. SOX18 over-expression attenuated the increase in BALF protein concentration in mice exposed to HTV (D). Total cell count in the bronchoalveolar lavage fluid (BALF) was elevated after HTV exposure, and this was significantly decreased by SOX18 over-expression (E). IL-1β secretion in the BALF was increased with HTV treatment, which was ameliorated by SOX18 overexpression (F). Immunoblotting analysis of HLMVECs showed that overexpression of SOX18 reduced the cyclic stretch-induced increase in pS536-NF-kB (G). *p < 0.05 versus Control, †p < 0.05 versus HTV alone. N = 4 (A); N = 3–6 (B,C); N = 6–14 (D); N = 6–12 (E); N = 43–7 (F); N = 7 (G).