Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

You-Hong Cheng,Brian F Volkman,Matthias Majetschak,Jonathan M Eby,Heather M Laporte,Raghavan Raju

doi:10.1371/journal.pone.0187949

You-Hong Cheng, Brian F Volkman + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0187949

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Journal: PLOS ONE	Publication Date: Nov 10, 2017
Citations: 15	License type: CC BY 4.0

Affiliation: Loyola University Chicago, Medical College of Wisconsin

Abstract

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3–68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05–0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03–3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3–68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3–68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3–68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

Highlights

Acute respiratory distress syndrome (ARDS) remains a major contributor to morbidity and mortality in critically ill patients [1,2,3,4]
To confirm that CXCR4 and atypical chemokine receptor 3 (ACKR3) are expressed in human primary pulmonary artery endothelial cell (hPPAEC) and to assess whether both receptors form heteromeric complexes, we performed Proximity ligation assays (PLA) to detect individual receptors and receptor-receptor interactions at single molecule resolution
We tested the effects of a panel of CXCR4 and ACKR3 ligands on hPPAEC monolayer permeability in transwell-permeability assays with Fluorescein isothiocyanate (FITC)-dextran (Fig 1B)

Summary

Introduction

Acute respiratory distress syndrome (ARDS) remains a major contributor to morbidity and mortality in critically ill patients [1,2,3,4]. Thrombin plays an important role in the pathogenesis of ARDS; in addition to functions of thrombin in the clotting cascade, thrombin fulfills diverse roles in inflammation and is well known to impair endothelial barrier function through activation of the G protein-coupled receptors (GPCR) protease-activated receptors (PARs) [7,8,9]. A large body of evidence suggests that PAR-1 is the major mediator of thrombin signaling in vascular endothelial cells [8, 11,12,13,14]. PAR-1 is activated when thrombin cleaves its extracellular N-terminal domain between residues Arg-41 and Ser-42, which unmasks a new N-terminus that serves as a tethered ligand [8]. Drugs that limit impairment of the lung endothelial barrier by thrombin, are not available, but desirable for their potential to improve outcomes

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