The proton-sensing GPR4 receptor regulates paracellular gap formation and permeability of vascular endothelial cells

Abstract

Tissue acidosis can be a consequence of numerous disease states including stroke, myocardial infarction, limb ischemia, and inflammation. Blood vessels existing in the affected tissues are associated with the progression of acidosis-related diseases. However, the mechanisms by which endothelial cells (ECs) lining the affected blood vessels sense and respond to an acidic microenvironment remain largely unclear. We investigated the functional effects of the proton-sensing G protein-coupled receptor GPR4 in acidosisinduced endothelial inflammation. GPR4 is highly expressed in ECs and known to regulate EC inflammation and endoplasmic reticulum stress responses within acidic microenvironments. Using genetic and pharmacological approaches, we demonstrate that GPR4 activation by acidosis increases EC paracellular gap formation and permeability.We further demonstrate that GPR4-mediated paracellular gap formation is dependent on the Gα12/13 signaling pathway. To assess the functional role of GPR4 in the inflammatory response in vivo, we utilized an acute hindlimb ischemia-reperfusion mouse model. We demonstrate that both genetic deletion and pharmacological inhibition of GPR4 reduce tissue edema, exudate formation, endothelial adhesion molecule expression, and leukocyte infiltration in the inflamed tissue. Collectively, these data suggest GPR4/Gα12/13 signaling mediates acidosis-induced endothelial paracellular gap formation and permeability. This study implicates GPR4 as a candidate therapeutic target for the remediation of inflammation and tissue edema. tissue microenvironment is associated with a wide range of inflammation-associated disease states such as arthritis, inflammatory bowel disease, myocardial infarction, stroke, and limb ischemia. Previous reports note that local pH ranging from 6.0 to 7.0 is common in the microenvironments of inflamed tissues, solid tumors, and ischemic tissues [9][10][11][12].In ischemic disease, one study demonstrated that within 50 minutes of coronary artery occlusion, local tissue extracellular pH decreased from 7.4 to 5.5 in domestic pigs [13].Furthermore, in the tourniquet-induced rabbit limb ischemia model, local tissue pH decreased rapidly within 1 hour and dropped from 7.30 to 6.36 during the 4 hour course of limb ischemia [14]. In summary, an acidic interstitial pH is an inflammatory microenvironmental factor in many pathological conditions and has been demonstrated to modulate tissue, blood vessel, and immune cell functions [9][10][11][12].Cells can sense extracellular acidification through multiple molecular sensors such as acid-sensing ion channels (ASICs), transient receptor potential (TRP), and proton-sensing G protein-coupled receptors (GPCRs) [7,11,15,16]. GPR4 is a member of the proton-sensing GPCR family, which also includes GPR65 (TDAG8) and GPR68 (OGR1) [7,11,[16][17][18]. GPR4 is highly expressed in vascular endothelial cells (ECs) and has been shown to increase the expression of inflammatory cytokines, chemokines, adhesion molecules, and ER stress related genes upon...

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