Thrombin Contributes to Anti-myeloperoxidase Antibody Positive IgG-Mediated Glomerular Endothelial Cells Activation Through SphK1-S1P-S1PR3 Signaling

Abstract

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.

Keywords: ANCA; endothelium; sphingosine-1-phosphate; thrombin; vasculitis.

Figure 1

Thrombin could enhance MPO-ANCA-positive IgG-mediated GEnC activation. (A) Thrombin could induce alterations in cellular morphology of GEnCs in the presence of MPO-ANCA-positive IgG. (B) Quantitive assessment of ZO-1 in GEnCs upon stimulation by thrombin and MPO-ANCA-positive IgG. (C) Quantitive assessment of VE-cadherin in GEnCs upon stimulation by thrombin and MPO-ANCA-positive IgG. Bars represent mean ± SD of repeated measurements of five independent experiments or donors. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

(A) Thrombin could induce increased endothelial permeability of GEnC monolayers in the presence of MPO-ANCA-positive IgG. (B) Thrombin could upregulate ET-1 levels in the supernatant of GEnCs in the presence of MPO-ANCA-positive IgG. (C) PAR1 mediated the thrombin-induced ET-1 upregulation in GEnCs in the presence of MPO-ANCA-positive IgG. Bars represent mean ± SD of repeated measurements of five independent experiments or donors. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

SphK1–S1P-S1PR3 signaling was involved in thrombin -induced MPO-ANCA-positive IgG-mediated GEnC activation. (A) SphK1 and S1PR3 expression levels were elevated in MPO-ANCA-positive IgG-treated GEnCs upon thrombin stimulation. (B) S1PR3 mediated the thrombin-induced ET-1 upregulation in MPO-ANCA-positive IgG-treated GEnCs. (C) FTY720 significantly downregulated ET-1 levels in the supernatants of GEnCs stimulated by thrombin and MPO-ANCA-positive IgG. Bars represent mean ± SD of repeated measurements of five independent experiments or donors. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

PAR1 expression levels were elevated in MPO-ANCA-positive IgG-treated GEnCs upon stimulation by S1P. Bars represent mean ± SD of repeated measurements of five independent experiments or donors. **P < 0.01, ***P < 0.001.

Figure 5

S1P enhanced the expression and activity of TF in GEnCs in the presence of MPO-ANCA-positive IgG. (A) S1P enhanced the expression levels of TF in GEnCs in the presence of MPO-ANCA-positive IgG. (B) S1P enhanced the procoagulant activity of TF in GEnCs in the presence of MPO-ANCA-positive IgG. Bars represent mean ± SD of repeated measurements of five independent experiments or donors. *P < 0.05, ***P < 0.001.

Figure 6

Proposed working model for the role of SphK1-S1P-S1PR3 in thrombin-induced GEnC activation in the presence of MPO-ANCA-positive IgG. Thrombin could enhance MPO-ANCA-positive IgG-induced GEnC activation and injury via PAR1. At the same time, thrombin might activate SphK1-S1P-S1PR3 axis in GEnCs in the presence of MPO-ANCA-positive IgG. Furthermore, S1P of pathophysiological concentration in active AAV patients might induce PAR1 expression as well as enhance both expression level and activity of tissue factor in MPO-ANCA-positive IgG-treated endothelial cells, which might further activate the coagulation system, thus forming a vicious loop. S1P, sphingosine-1-phosphate; S1PR, sphingosine-1-phosphate receptor; Sph, sphingosine; SphK, sphingosine kinases; PAR, protease-activated receptor; ET-1, endothelin-1; ZO-1, zonula occludens-1.