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• W2999329884 abstract "•Acidosis/GPR4 regulates endothelial paracellular gap formation and permeability•GPR4 exacerbates inflammation by increasing tissue edema and leukocyte infiltration•Pharmacological inhibition of GPR4 reduces inflammatory responses GPR4 is a pH-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and can be activated by protons in the inflamed tissue microenvironment. Herein, we report that acidosis-induced GPR4 activation increases paracellular gap formation and permeability of vascular endothelial cells through the Gα12/13/Rho GTPase signaling pathway. Evaluation of GPR4 in the inflammatory response using the acute hindlimb ischemia-reperfusion mouse model revealed that GPR4 mediates tissue edema, inflammatory exudate formation, endothelial adhesion molecule expression, and leukocyte infiltration in the inflamed tissue. Genetic knockout and pharmacologic inhibition of GPR4 alleviate tissue inflammation. These results suggest GPR4 is a pro-inflammatory receptor and could be targeted for therapeutic intervention. GPR4 is a pH-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and can be activated by protons in the inflamed tissue microenvironment. Herein, we report that acidosis-induced GPR4 activation increases paracellular gap formation and permeability of vascular endothelial cells through the Gα12/13/Rho GTPase signaling pathway. Evaluation of GPR4 in the inflammatory response using the acute hindlimb ischemia-reperfusion mouse model revealed that GPR4 mediates tissue edema, inflammatory exudate formation, endothelial adhesion molecule expression, and leukocyte infiltration in the inflamed tissue. Genetic knockout and pharmacologic inhibition of GPR4 alleviate tissue inflammation. These results suggest GPR4 is a pro-inflammatory receptor and could be targeted for therapeutic intervention. The endothelium is a dynamic barrier that can mediate the transvascular movement of fluids and immune cells between the peripheral blood and interstitial tissues. During active inflammation, the production of numerous inflammatory mediators within the inflammatory loci can increase endothelial gap formation and vascular permeability, which facilitate leukocyte infiltration and exudate formation in the inflamed tissues (Edlow and Sheldon, 1971Edlow D.W. Sheldon W.H. The pH of inflammatory exudates.Proc. Soc. Exp. Biol. Med. 1971; 137: 1328-1332Crossref PubMed Scopus (80) Google Scholar, Muller, 2003Muller W.A. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response.Trends Immunol. 2003; 24: 327-334Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Pate et al., 2010Pate M. Damarla V. Chi D.S. Negi S. Krishnaswamy G. Endothelial cell biology: role in the inflammatory response.Adv. Clin. Chem. 2010; 52: 109-130Crossref PubMed Scopus (77) Google Scholar). Accumulating evidence suggests that microenvironmental factors, such as acidic pH, can stimulate endothelial cell inflammation (Chen et al., 2011Chen A. Dong L. Leffler N.R. Asch A.S. Witte O.N. Yang L.V. Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway.PLoS One. 2011; 6: e27586Crossref PubMed Scopus (72) Google Scholar, Dong et al., 2013Dong L. Li Z. Leffler N.R. Asch A.S. Chi J.T. Yang L.V. Acidosis activation of the proton-sensing GPR4 receptor stimulates vascular endothelial cell inflammatory responses revealed by transcriptome analysis.PLoS One. 2013; 8: e61991Crossref PubMed Scopus (74) Google Scholar, Dong et al., 2017bDong L. Krewson E.A. Yang L.V. Acidosis activates endoplasmic reticulum stress pathways through GPR4 in human vascular endothelial cells.Int. J. Mol. Sci. 2017; 18: 278Crossref PubMed Scopus (38) Google Scholar, Okajima, 2013Okajima F. Regulation of inflammation by extracellular acidification and proton-sensing GPCRs.Cell. Signal. 2013; 25: 2263-2271Crossref PubMed Scopus (102) Google Scholar, Tobo et al., 2015Tobo A. Tobo M. Nakakura T. Ebara M. Tomura H. Mogi C. Im D.S. Murata N. Kuwabara A. Ito S. et al.Characterization of imidazopyridine compounds as negative allosteric modulators of proton-sensing GPR4 in extracellular acidification-induced responses.PLoS One. 2015; 10: e0129334Crossref PubMed Scopus (21) Google Scholar). Inflammatory tissues are characteristically acidic, owing in part to hypoxia and increased glycolytic metabolism of cells and infiltrated leukocytes resulting in heightened proton production and accumulation. The acidic tissue microenvironment is associated with a wide range of inflammation-related 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 (Huang and McNamara, 2004Huang Y. McNamara J.O. Ischemic stroke: acidotoxicity is a perpetrator.Cell. 2004; 118: 665-666Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Justus et al., 2013Justus C.R. Dong L. Yang L.V. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.Front. Physiol. 2013; 4: 354Crossref PubMed Scopus (154) Google Scholar, Lardner, 2001Lardner A. The effects of extracellular pH on immune function.J. Leukoc. Biol. 2001; 69: 522-530PubMed Google Scholar, Siesjo et al., 1996Siesjo B.K. Katsura K.I. Kristian T. Li P.A. Siesjo P. Molecular mechanisms of acidosis-mediated damage.Acta Neurochir. Suppl. 1996; 66: 8-14PubMed Google Scholar). In ischemic disease, one study demonstrated that, within 50 min of coronary artery occlusion, local tissue extracellular pH decreased from 7.4 to 5.5 in domestic pigs (Hirche et al., 1980Hirche H. Franz C. Bos L. Bissig R. Lang R. Schramm M. Myocardial extracellular K+ and H+ increase and noradrenaline release as possible cause of early arrhythmias following acute coronary artery occlusion in pigs.J. Mol. Cell. Cardiol. 1980; 12: 579-593Abstract Full Text PDF PubMed Scopus (221) Google Scholar). Furthermore, in the tourniquet-induced rabbit limb ischemia model, local tissue pH decreased rapidly within 1 h and dropped from 7.30 to 6.36 during the 4-h course of limb ischemia (Hagberg, 1985Hagberg H. Intracellular pH during ischemia in skeletal muscle: relationship to membrane potential, extracellular pH, tissue lactic acid and ATP.Pflugers Arch. 1985; 404: 342-347Crossref PubMed Scopus (51) Google Scholar). 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 (Huang and McNamara, 2004Huang Y. McNamara J.O. Ischemic stroke: acidotoxicity is a perpetrator.Cell. 2004; 118: 665-666Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Justus et al., 2013Justus C.R. Dong L. Yang L.V. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.Front. Physiol. 2013; 4: 354Crossref PubMed Scopus (154) Google Scholar, Lardner, 2001Lardner A. The effects of extracellular pH on immune function.J. Leukoc. Biol. 2001; 69: 522-530PubMed Google Scholar, Siesjo et al., 1996Siesjo B.K. Katsura K.I. Kristian T. Li P.A. Siesjo P. Molecular mechanisms of acidosis-mediated damage.Acta Neurochir. Suppl. 1996; 66: 8-14PubMed Google Scholar). 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) (Holzer, 2009Holzer P. Acid-sensitive ion channels and receptors.Handb. Exp. Pharmacol. 2009; 194: 283-332Crossref PubMed Scopus (186) Google Scholar, Justus et al., 2013Justus C.R. Dong L. Yang L.V. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.Front. Physiol. 2013; 4: 354Crossref PubMed Scopus (154) Google Scholar, Okajima, 2013Okajima F. Regulation of inflammation by extracellular acidification and proton-sensing GPCRs.Cell. Signal. 2013; 25: 2263-2271Crossref PubMed Scopus (102) Google Scholar, Sanderlin et al., 2015Sanderlin E.J. Justus C.R. Krewson E.A. Yang L.V. Emerging roles for the pH-sensing G protein-coupled receptors in response to acidotic stress.Cell Health Cytoskelet. 2015; 7: 99-109Google Scholar). GPR4 is a member of the proton-sensing GPCR family, which also includes GPR65 (TDAG8) and GPR68 (OGR1) (Justus et al., 2013Justus C.R. Dong L. Yang L.V. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.Front. Physiol. 2013; 4: 354Crossref PubMed Scopus (154) Google Scholar, Ludwig et al., 2003Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Proton-sensing G-protein-coupled receptors.Nature. 2003; 425: 93-98Crossref PubMed Scopus (457) Google Scholar, Okajima, 2013Okajima F. Regulation of inflammation by extracellular acidification and proton-sensing GPCRs.Cell. Signal. 2013; 25: 2263-2271Crossref PubMed Scopus (102) Google Scholar, Sanderlin et al., 2015Sanderlin E.J. Justus C.R. Krewson E.A. Yang L.V. Emerging roles for the pH-sensing G protein-coupled receptors in response to acidotic stress.Cell Health Cytoskelet. 2015; 7: 99-109Google Scholar, Yang et al., 2007Yang L.V. Radu C.G. Roy M. Lee S. McLaughlin J. Teitell M.A. Iruela-Arispe M.L. Witte O.N. Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor.Mol. Cell. Biol. 2007; 27: 1334-1347Crossref PubMed Scopus (92) Google Scholar). 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 activation by acidic pH in ECs (Chen et al., 2011Chen A. Dong L. Leffler N.R. Asch A.S. Witte O.N. Yang L.V. Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway.PLoS One. 2011; 6: e27586Crossref PubMed Scopus (72) Google Scholar, Dong et al., 2013Dong L. Li Z. Leffler N.R. Asch A.S. Chi J.T. Yang L.V. Acidosis activation of the proton-sensing GPR4 receptor stimulates vascular endothelial cell inflammatory responses revealed by transcriptome analysis.PLoS One. 2013; 8: e61991Crossref PubMed Scopus (74) Google Scholar, Tobo et al., 2015Tobo A. Tobo M. Nakakura T. Ebara M. Tomura H. Mogi C. Im D.S. Murata N. Kuwabara A. Ito S. et al.Characterization of imidazopyridine compounds as negative allosteric modulators of proton-sensing GPR4 in extracellular acidification-induced responses.PLoS One. 2015; 10: e0129334Crossref PubMed Scopus (21) Google Scholar). GPR4 has also been shown to potentiate inflammation in vivo. Recent studies found that the genetic deletion of GPR4 in mouse colitis models decreased the expression of endothelial adhesion molecules VCAM-1 and E-Selectin in the intestinal microvasculature, which was associated with reduced mucosal leukocyte infiltration and intestinal inflammation (Sanderlin et al., 2017Sanderlin E.J. Leffler N.R. Lertpiriyapong K. Cai Q. Hong H. Bakthavatchalu V. Fox J.G. Oswald J.Z. Justus C.R. Krewson E.A. et al.GPR4 deficiency alleviates intestinal inflammation in a mouse model of acute experimental colitis.Biochim. Biophys. Acta. 2017; 1863: 569-584Crossref Scopus (17) Google Scholar, Wang et al., 2018Wang Y. de Valliere C. Imenez Silva P.H. Leonardi I. Gruber S. Gerstgrasser A. Melhem H. Weber A. Leucht K. Wolfram L. et al.The proton-activated receptor GPR4 modulates intestinal inflammation.J. Crohns Colitis. 2018; 12: 355-368Crossref PubMed Scopus (22) Google Scholar). Furthermore, GPR4 was shown to increase tissue injury in a renal ischemia-reperfusion mouse model (Dong et al., 2017aDong B. Zhang X. Fan Y. Cao S. GPR4 knockout improves renal ischemia-reperfusion injury and inhibits apoptosis via suppressing the expression of CHOP.Biochem. J. 2017; 474: 4065-4074Crossref PubMed Scopus (11) Google Scholar). Our current study focuses on the acidosis/GPR4-mediated endothelial paracellular gap formation and vessel permeability in the inflammatory response. Using genetic and pharmacological approaches, we demonstrate that activation of GPR4 by acidosis induces endothelial paracellular gap formation and permeability through the Gα12/13 signaling pathway. Furthermore, we demonstrate that the genetic deletion and pharmacological inhibition of GPR4 decrease blood vessel permeability, tissue edema, and leukocyte infiltration in the acute hindlimb ischemia-reperfusion mouse model. Our data suggest that GPR4 has a proinflammatory role in the regulation of the inflammatory response. Tissue acidosis commonly exists in inflammatory microenvironments (Dong et al., 2014bDong L. Li Z. Yang L.V. Function and signaling of the pH-sensing G protein-coupled receptors in physiology and diseases.in: Chi J.T. Molecular Genetics of Dysregulated pH Homeostasis. Springer, 2014: 45-65Crossref Scopus (3) Google Scholar, Justus et al., 2013Justus C.R. Dong L. Yang L.V. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.Front. Physiol. 2013; 4: 354Crossref PubMed Scopus (154) Google Scholar, Lardner, 2001Lardner A. The effects of extracellular pH on immune function.J. Leukoc. Biol. 2001; 69: 522-530PubMed Google Scholar, Okajima, 2013Okajima F. Regulation of inflammation by extracellular acidification and proton-sensing GPCRs.Cell. Signal. 2013; 25: 2263-2271Crossref PubMed Scopus (102) Google Scholar, Sanderlin et al., 2015Sanderlin E.J. Justus C.R. Krewson E.A. Yang L.V. Emerging roles for the pH-sensing G protein-coupled receptors in response to acidotic stress.Cell Health Cytoskelet. 2015; 7: 99-109Google Scholar). However, the involvement of acidosis in endothelial cell (EC) gap formation is largely unknown. Four primary vascular ECs were cultured to a confluent monolayer and were treated with either physiological pH 7.4 or acidic pH 6.4 for 5 h to assess acidosis-induced paracellular gap formation. Under physiological pH 7.4, all ECs maintained a cellular monolayer with no gap formation over the 5-h time course. However, under acidic pH 6.4 the cellular monolayers were disrupted and paracellular gap formation was observed (Figure 1A). The gap formation of EC monolayers was determined in human umbilical vein endothelial cells (HUVECs), human pulmonary artery ECs (HPAECs), human colon microvascular ECs (HMVECs-Colon), and human lung microvascular ECs (HMVECs-Lung) by calculating the total percent area of gaps in each field of view (Figures 1B–1E). ECs treated with acidic pH 6.4 for 5 h developed approximately 4%–5% gap area relative to the total area (Figures 1B–1E). No gaps, however, could be detected within physiological pH conditions. To determine the role of the pH-sensing receptor GPR4 in acidosis-induced EC gap formation, we used genetic and pharmacological approaches to modulate GPR4 expression and activity, respectively. HUVECs were stably transduced with either control (HUVEC/vector), GPR4 overexpression (HUVEC/GPR4), or GPR4 signaling-defective mutant (HUVEC/GPR4 R115A) overexpression constructs. GPR4 knockdown was achieved with transduction of GPR4 shRNA (HUVEC/GPR4 shRNA) and compared with control shRNA (HUVEC/control shRNA). As previously described (Chen et al., 2011Chen A. Dong L. Leffler N.R. Asch A.S. Witte O.N. Yang L.V. Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway.PLoS One. 2011; 6: e27586Crossref PubMed Scopus (72) Google Scholar, Dong et al., 2013Dong L. Li Z. Leffler N.R. Asch A.S. Chi J.T. Yang L.V. Acidosis activation of the proton-sensing GPR4 receptor stimulates vascular endothelial cell inflammatory responses revealed by transcriptome analysis.PLoS One. 2013; 8: e61991Crossref PubMed Scopus (74) Google Scholar), real-time RT-PCR analysis showed that GPR4 mRNA expression was increased by ∼14-fold in the overexpression HUVEC cells and decreased by >90% in the shRNA knockdown cells (Figure S1). HUVECs were evaluated under physiological pH 7.4 or acidic pH 6.4 for 5 h. Treatment with physiological pH 7.4 resulted in no observable gap formation. However, under acidic pH 6.4, HUVEC/vector cell monolayers developed ∼4% gap formation (Figure 2A). Overexpression of GPR4 significantly increased acidosis-induced gap formation by ∼2.5 fold (∼10%–11%) under acidic conditions when compared with HUVEC/vector. Conversely, HUVEC/GPR4 R115A mutant decreased the percentage of gap area when compared with HUVEC/vector (∼1.8% versus ∼4%, respectively). Furthermore, knockdown of GPR4 by shRNA decreased the percentage of gap area to ∼2.5% in acidic pH 6.4 conditions when compared with HUVEC/control shRNA (∼4.5%) (Figure 2B). We next assessed the effects of a GPR4 inhibitor (EIDIP) on acidosis-induced paracellular gap formation in HUVEC/vector and HUVEC/GPR4 cells (Figures 2C and 2D). Pharmacological inhibition of GPR4 attenuated acidosis-induced gap formation. A dose-dependent decrease in the acidosis-induced gap development could be observed with increasing inhibitor concentrations during HUVEC/vector and HUVEC/GPR4 treatments when compared with vehicle (Figures 2C and 2D). The results indicate that acidosis-induced EC paracellular gap formation is dependent on GPR4 activation by acidic pH. Previous studies demonstrate that the Gα12/13/Rho GTPase pathway can regulate cytoskeletal dynamics, endothelial gap formation, and endothelial permeability (Thennes and Mehta, 2012Thennes T. Mehta D. Heterotrimeric G proteins, focal adhesion kinase, and endothelial barrier function.Microvasc. Res. 2012; 83: 31-44Crossref PubMed Scopus (25) Google Scholar, van Buul and Hordijk, 2004van Buul J.D. Hordijk P.L. Signaling in leukocyte transendothelial migration.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 824-833Crossref PubMed Scopus (122) Google Scholar). In line with these observations, GPR4 can couple to Gα12/13 and Rho GTPase when expressed in cancer cell lines (Castellone et al., 2011Castellone R.D. Leffler N.R. Dong L. Yang L.V. Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor.Cancer Lett. 2011; 312: 197-208Crossref PubMed Scopus (53) Google Scholar, Justus and Yang, 2015Justus C.R. Yang L.V. GPR4 decreases B16F10 melanoma cell spreading and regulates focal adhesion dynamics through the G13/Rho signaling pathway.Exp. Cell Res. 2015; 334: 100-113Crossref PubMed Scopus (14) Google Scholar). For this reason, we investigated the role of the GPR4/Gα12/13/Rho GTPase pathway in acidosis-induced EC gap formation. The p115 RGS Gα12/13 inhibitory construct (Kozasa et al., 1998Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.Science. 1998; 280: 2109-2111Crossref PubMed Scopus (721) Google Scholar, Yang et al., 2005Yang L.V. Radu C.G. Wang L. Riedinger M. Witte O.N. Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.Blood. 2005; 105: 1127-1134Crossref PubMed Scopus (125) Google Scholar) was stably transduced into HUVEC/vector and HUVEC/GPR4 cells. We next performed the gap formation assay under physiological and acidic pH conditions for 5 h. HUVEC/p115 RGS cells treated with acidic pH had significantly reduced gap formation when compared with the vector control (Figures 3A and 3B ). Furthermore, thiazovivin and staurosporine, two chemical inhibitors for Gα12/13 downstream effectors Rho-associated kinase (ROCK) and myosin light-chain kinase (MLCK), respectively (Justus and Yang, 2015Justus C.R. Yang L.V. GPR4 decreases B16F10 melanoma cell spreading and regulates focal adhesion dynamics through the G13/Rho signaling pathway.Exp. Cell Res. 2015; 334: 100-113Crossref PubMed Scopus (14) Google Scholar), were used in HUVEC/vector and HUVEC/GPR4 cells under acidic conditions. Thiazovivin significantly decreased gap formation percentage in HUVEC/vector and HUVEC/GPR4 cells when compared with the vehicle controls under acidic pH. Staurosporine nearly abolished acidosis-induced gap formation in HUVEC/vector and HUVEC/GPR4 cells (Figures 3C and 3D). Collectively, the results suggest that acidosis-induced EC gap formation relies, at least in part, on the GPR4/Gα12/13 pathway. Additionally, our results demonstrated that acidosis also induced F-actin stress fiber formation and decreased VE-cadherin expression at the site of paracellular gaps in ECs (Figures S2 and S3). To assess the role of actin cytoskeleton in acidosis/GPR4-mediated EC gap formation, HUVECs were treated with an actin cytoskeleton inhibitor cytochalasin D (CytoD). Cytochalasin D significantly reduced acidosis-induced gap formation in HUVEC/vector and HUVEC/GPR4 cells (Figures 3C and 3D). Moreover, p115 RGS, thiazovivin, staurosporine, and cytochalasin D substantially decreased F-actin stress fiber formation in HUVECs (Figure S4). Taken together, these results suggest that the Gα12/13/Rho/ROCK/MLCK/actin cytoskeleton pathway is important for acidosis/GPR4-induced endothelial paracellular gap formation. We next assessed if GPR4-dependent paracellular gap formation can functionally result in increased endothelial permeability. Endothelial permeability was assessed by the fluorescein isothiocyanate conjugated-dextran (FITC-dextran) permeability assay whereby diffusion of FITC-dextran through the endothelial monolayer from the upper to lower chamber of the Transwell insert was assessed. Cells were treated with physiological pH 7.4 or acidic pH 6.4 for 5 h followed by the addition of FITC-dextran. Acidic pH treatment significantly increased FITC-dextran permeability in HUVEC/vector cells when compared with physiological pH. Moreover, when GPR4 is overexpressed and treated with acidic pH there was a further increase in acidosis-induced FITC-dextran when compared with HUVEC/vector cells. When GPR4 expression is knocked down or signaling is defective (R115A mutant) (Chen et al., 2011Chen A. Dong L. Leffler N.R. Asch A.S. Witte O.N. Yang L.V. Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway.PLoS One. 2011; 6: e27586Crossref PubMed Scopus (72) Google Scholar), FITC-dextran permeability was significantly decreased compared with controls under acidic conditions (Figure 4). These results suggest that acidosis increases EC permeability through GPR4. Next, we assessed the functional role of GPR4 in a tourniquet cuff-based acute hindlimb ischemia-reperfusion mouse model (Bonheur et al., 2004Bonheur J.A. Albadawi H. Patton G.M. Watkins M.T. A noninvasive murine model of hind limb ischemia-reperfusion injury.J. Surg. Res. 2004; 116: 55-63Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This mouse model can cause severe inflammation resulting in increased vessel permeability, tissue edema, and leukocyte infiltration in the affected tissue. The ischemia-reperfusion associated inflammation was induced in wild-type (WT) and GPR4 knockout (KO) mice. Consistent with previous report (Yang et al., 2007Yang L.V. Radu C.G. Roy M. Lee S. McLaughlin J. Teitell M.A. Iruela-Arispe M.L. Witte O.N. Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor.Mol. Cell. Biol. 2007; 27: 1334-1347Crossref PubMed Scopus (92) Google Scholar), the absence of GPR4 mRNA expression in KO mouse tissues was confirmed by RT-PCR (Figure S5). The sham and cuff limbs were measured for circumference differences between pre- and post-procedure measurements. GPR4 KO mice had less observable tissue edema in the tourniquet-affected limb following the ischemia and reperfusion event when compared with WT mice (Figure 5). Inflammatory exudates in the interstitial space between the skin and the limb muscle/body peritoneum on the tourniquet subjected side were collected and measured followed by histology (Figure 6). GPR4 KO mice had reduced inflammatory exudate when compared with the WT mice (∼6 versus ∼65 mg, respectively) (Figures 6A, 6B, and 6I). Furthermore, histological analysis of the inflammatory exudates revealed that GPR4 KO mice had reduced leukocyte infiltrates when compared with WT (Figures 6D, 6F, and 6J). No red blood cells were observed in the exudate, suggesting that the exudate is due to increased vascular permeability but not vessel hemorrhaging (Figures 6A–6F). To further assess the GPR4-mediated vessel permeability, we performed immunohistochemistry for plasma protein immunoglobulin G (IgG) and detected IgG within inflammatory exudates (Figures 6G and 6H), indicating an increased vascular permeability to plasma protein. Furthermore, less plasma IgG could be observed in the GPR4 KO cuff-affected tissues compared with WT cuff-affected tissues indicating reduced endothelial cell permeability in the KO (Figures 6G and 6H). We also performed immunohistochemistry of CD31 (a pan-endothelial marker) to assess blood vessel density in the hindlimb dermis and hypodermis tissues. No significant difference in blood vessel density was observed between WT and GPR4 KO or between sham and cuff-affected dermis and hypodermis tissues within the 24-h ischemia and reperfusion (Figure S6). To provide a molecular explanation for GPR4-mediated inflammation within vascular endothelial cells, we performed immunohistochemistry to examine the expression of VCAM-1 and E-selectin within the endothelium of the cuff and sham-affected dermis and hypodermis tissues. Overall, VCAM-1 and E-selectin protein expression was increased within the cuff-affected limb vasculature when compared with sham (Figure 7). Immunohistochemical analysis of VCAM-1 revealed expression on a variety of cell types, such as skeletal muscle, fibroblast, and vascular endothelial cells, which is consistent with the previous literature (Epperly et al., 2002Epperly M.W. Sikora C.A. DeFilippi S.J. Gretton J.E. Bar-Sagi D. Archer H. Carlos T. Guo H. Greenberger J.S. Pulmonary irradiation-induced expression of VCAM-I and ICAM-I is decreased by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy.Biol. Blood Marrow Transplant. 2002; 8: 175-187Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Ulyanova et al., 2005Ulyanova T. Scott L.M. Priestley G.V. Jiang Y. Nakamoto B. Koni P.A. Papayannopoulou T. VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin.Blood. 2005; 106: 86-94Crossref PubMed Scopus (94) Google Scholar). Interestingly, we observed a decrease of VCAM-1 protein expression on vascular endothelial cells in GPR4 KO cuff-affected dermis and hypodermis of the limbs when compared with WT (Figures 7A–7D and 7I). E-selectin expression was observed on endothelial cells and fibroblasts as previously reported (Bajnok et al., 2017Bajnok A. Ivanova M. Rigo Jr., J. Toldi G. The distribution of activation markers and selectins on peripheral T lymphocytes in preeclampsia.Mediators Inflamm. 2017; 2017: 8045161Crossref PubMed Scopus (23) Google Scholar, Harashima et al., 2001Harashima S. Horiuchi T. Hatta N. Morita C. Higuchi M. Sawabe T. Tsukamoto H. Tahira T. Hayashi K. Fujita S. et al.Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells.J. Immunol. 2001; 166: 130-136Crossref PubMed Scopus (106) Google Scholar, Vainer et al., 1998Vainer B. Nielsen O.H. Horn T. Expression of E-selectin, sialyl Lewis X, and macrophage inflammatory protein-1alpha by colonic epithelial cells in ulcerative colitis.Dig. Dis. Sci. 1998; 43: 596-608Crossref PubMed Scopus (28) Google Scholar). Similar to VCAM-1 expression patterns, there is an observable decrease in endothelial E-selectin protein expression in GPR4 KO cuff-affected dermis and hypodermis of the limbs when compared with WT (Figures 7E–7H and 7J). To further assess the role of GPR4, we incorporated the use of a highly potent and selective GPR4 antagonist (referred to as GPR4 antagonist 13) (Velcicky et al., 2017Velcicky J. Miltz W. Oberhauser B. Orain D. Vaupel A. Weigand K. Dawson King J. Littlewood-Evans A. Nash M. Feifel R. et al.Development of selective, orally active GPR4 antagonists with modulatory effects on nociception, inflammation, and angiogenesis.J. Med. Chem. 2017; 60: 3672-3683Crossref PubMed Scopus (16) Google Scholar) within the acute hindlimb ischemia-reperfusion mouse model. Our results demonstrated that the GPR4 antagonist 13 significantly decreased upper and lower limb tissue edema measured by the circumference of the leg compared with limbs of the vehicle control (Figure 8). In addition, there was significantly less leukocyte infiltration within the inflammatory exudate in the GPR4 antagonist 13-treated mice when compared with the vehicle-treated mice (Figures 9A, 9B, and 9F ). Furthermore, the exudate weight was also decreased in GPR4 antagonist 13-treated mice when compared with vehicle (Figure 9E). Additionally, vascular permeability was assessed and there was a reduction in plasma IgG diffusion in the exudate by the treatment of the GPR4 antagonist 13 when compared with vehicle treatment (Figures 9C and 9D). GPR4 antagonist 13 treatment also reduced the level of an inflammatory marker, C-reactive protein (CRP), in the mouse serum (Figure S7). To further examine the role of GPR4 within ECs, we performed immunohistochemistry to analyze the expression of VCAM-1 and E-selectin within the endothelium of the cuff-affected dermis and hypodermis tissues. We observed that ECs in the GPR4 antagonist 13-treated mice had reduced VCAM-1 and E-selectin protein expression compared with the vehicle-treated mice (Figure 10). Taken together, these data suggest that GPR4 can mediate inflammation, vessel permeability, and leukocyte infiltration into inflamed tissues.Figure 9GPR4 Ant" @default.
• W2999329884 created "2020-01-23" @default.
• W2999329884 creator A5005447086 @default.
• W2999329884 creator A5018766790 @default.
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• W2999329884 date "2020-02-01" @default.
• W2999329884 modified "2023-10-15" @default.
• W2999329884 title "The Proton-Sensing GPR4 Receptor Regulates Paracellular Gap Formation and Permeability of Vascular Endothelial Cells" @default.
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