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Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

ABSTRACT: Cumulating evidences suggested an important role of sphingosine-1-phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)-ANCA-positive IgG-mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO-ANCA-positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane-bound and soluble ICAM-1 and VCAM-1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO-ANCA-positive IgG-induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO-ANCA-positive IgG. S1P enhanced MPO-ANCA-positive IgG-induced membrane-bound and soluble ICAM-1/VCAM-1 up-regulation of GEnCs. Soluble ICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG increased upon pre-incubation of S1PR1 antagonist, while pre-incubation of GEnCs with the S1PR1 agonist down-regulated sICAM-1 level. Blocking S1PR2-4 reduced sICAM-1 levels in the supernatants of GEnCs stimulated by S1P and MPO-ANCA-positive IgG. Pre-incubation with S1PR5 agonist could increase sICAM-1 level in the supernatants of GEnC stimulated by S1P and MPO-ANCA-positive IgG. S1P can enhance MPO-ANCA-positive IgG-mediated GEnC activation through S1PR2-5.

SUBMITTER: Sun XJ

PROVIDER: S-EPMC5824416 | biostudies-other | 2018 Mar

REPOSITORIES: biostudies-other

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Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

Sun Xiao-Jing XJ Chen Min M Zhao Ming-Hui MH

Journal of cellular and molecular medicine 20171123 3

Cumulating evidences suggested an important role of sphingosine-1-phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)-ANCA-positive IgG-mediated glomerular endot ...[more]

PMID: 29168342

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Project description:Background: Activation of coagulation system plays an important role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. Thrombin, generated during coagulation could disrupt endothelial barrier integrity through protease-activated receptor 1 (PAR1). Our previous study found that sphingosine-1-phosphate (S1P) contributed to myeloperoxidase (MPO)-ANCA-positive IgG-induced glomerular endothelial cell (GEnC) activation through a S1P receptor (S1PR)-dependent route. In recent years, S1P signaling was reported to be involved in thrombin effects on endothelial cells. This current study investigated whether the interaction between thrombin-PAR and S1P-S1PR signaling contributed to MPO-ANCA-positive IgG-induced GEnC dysfunction. Methods: The effect of thrombin on GEnC activation was analyzed from three aspects. First, morphological alteration of GEnCs was observed. Second, permeability assay was performed to determine GEnC monolayer activation quantitatively. Third, endothelin-1 (ET-1) levels were measured. Expression levels of sphingosine kinases (SphKs) and S1PRs were detected. In addition, antagonists of PAR1 and S1PR3 were employed to determine their roles. Eventually, PAR1 and tissue factor (TF) expression levels as well as TF procoagulant activity were analyzed. Results: Thrombin induced further damage of tight junction, increase in endothelial monolayer permeability as well as upregulation of ET-1 levels in GEnCs stimulated with MPO-ANCA-positive IgG. Blocking PAR1 downregulated ET-1 levels in the supernatants of GEnCs treated by thrombin plus MPO-ANCA-positive IgG. Expression levels of SphK1, S1PR3 increased significantly in GEnCs treated with thrombin plus MPO-ANCA-positive IgG. S1P upregulated PAR1 and TF expression, and enhanced procoagulant activity of TF in MPO-ANCA-positive IgG-stimulated GEnCs. Conclusion: Thrombin synergized with SphK1-S1P-S1PR3 signaling pathway to enhance MPO-ANCA-positive IgG-mediated GEnC activation.

Project description:Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was designed to investigate the role of glycolysis in glomerular endothelial cells (GECs) in a mouse model of DN. Mouse renal cortex and isolated glomerular cells were collected for single-cell and RNA sequencing. Cultured GECs were exposed to high glucose in the presence (proangiogenic) and absence of a vascular sprouting regimen. MicroRNA-590-3p was delivered by lipofectamine in vivo and in vitro. In the present study, a subgroup of GECs with proangiogenic features was identified in diabetic kidneys by using sequencing analyses. In cultured proangiogenic GECs, high glucose increased glycolysis and phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) protein expression, which were inhibited by overexpressing miRNA-590-3p. Mimics of miRNA-590-3p also increased receptor for sphingosine 1-phosphate (S1pR1) expression, an angiogenesis regulator, in proangiogenic GECs challenged with high glucose. Inhibition of PFKFB3 by pharmacological and genetic approaches upregulated S1pR1 protein in vitro. Mimics of miRNA-590-3p significantly reduced migration and angiogenic potential in proangiogenic GECs challenged with high glucose. Ten-week-old type 2 diabetic mice had elevated urinary albumin levels, reduced renal cortex miRNA-590-3p expression, and disarrangement of glomerular endothelial cell fenestration. Overexpressing miRNA-590-3p via perirenal adipose tissue injection restored endothelial cell fenestration and reduced urinary albumin levels in diabetic mice. Therefore, the present study identifies a subgroup of GECs with proangiogenic features in mice with DN. Local administration of miRNA-590-3p mimics reduces glycolytic rate and upregulates S1pR1 protein expression in proangiogenic GECs. The protective effects of miRNA-590-3p provide therapeutic potential in DN treatment.NEW & NOTEWORTHY Proangiogenetic glomerular endothelial cells (GECs) are activated in diabetic nephropathy. High glucose upregulates glycolytic enzyme phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) in proangiogenetic cells. PFKFB3 protects the glomerular filtration barrier by targeting endothelial S1pR1. MiRNA-590-3p restores endothelial cell function and mitigates diabetic nephropathy.

Dataset Information

Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

Publications

Sphingosine-1-phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti-myeloperoxidase antibody-positive IgG.

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