Rho GTPases are involved in S1P-enhanced glomerular endothelial cells activation with anti-myeloperoxidase antibody positive IgG

Abstract

Sphingosine-1-phosphate (S1P) is a crucial regulator in vascular inflammation. Our recent study found that under pathophysiological concentration in active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), S1P participated in MPO-ANCA-positive IgG-induced glomerular endothelial cell (GEnC) activation via a S1P receptor (S1PR)-dependent way. However, the downstream signalling pathways are not fully clear yet. In this study, we demonstrated that Rho guanosine triphosphatases (GTPases) signalling pathways, RhoA and Rac1 in particular, were implicated in MPO-ANCA-positive IgG-mediated GEnCs activation enhanced by pathophysiological concentration of S1P in AAV. These results provide mechanistic insights into vascular barrier dysfunction in AAV, which may facilitate the development of effective therapies.

Keywords: ANCA; Rho GTPases; sphingosine-1-phosphate; vasculitis.

Figure 1

RhoA and Rac1 signalling pathways were implicated in S1P‐enhanced GEnCs activation with MPO‐ANCA‐positive IgG. A, Activation of Rac1 in MPO‐ANCA‐positive IgG‐treated GEnCs upon stimulation by different concentrations of S1P. B, Activation of RhoA in MPO‐ANCA‐positive IgG‐treated GEnCs upon stimulation by different concentrations of S1P. C, The S1PR1‐dependent activation of Rac1 in GEnCs stimulated by S1P plus MPO‐ANCA‐positive IgG. D, The S1PR2‐5‐dependent activation of RhoA in GEnCs stimulated by S1P plus MPO‐ANCA‐positive IgG. E, RhoA antagonist CCG significantly down‐regulated ICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. F, RhoA antagonist CCG significantly down‐regulated VCAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. G, Rac1 antagonist NSC significantly up‐regulated ICAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. H, Rac1 antagonist NSC significantly up‐regulated VCAM‐1 level in the supernatants of GEnC stimulated by S1P plus MPO‐ANCA‐positive IgG. For the experimental groups which require IgGs, bars represent mean ± SD of MPO‐ANCA‐positive IgGs from plasma of 5 active MPO‐ANCA‐positive primary small vessel vasculitis patients or normal IgGs from plasma of five healthy donors individually. As for the experimental groups which did not require IgGs (eg, GEnCs treated with S1P alone), bars represent mean ± SD of repeated measurements of five independent experiments

Figure 2

Proposed working model for the role of Rho GTPases in S1P‐induced GEnC activation in the presence of MPO‐ANCA‐positive IgG. Under pathophysiological concentration of S1P in active AAV patients, the activation of S1PR2‐5 and their downstream RhoA signalling pathway dominates the S1P‐induced MPO‐ANCA‐positive IgG‐mediated endothelial activation, whereas the activation of S1PR1 and Rac1 signalling pathway exerts opposite effect during this process. The imbalance between different S1PRs and Rho GTPases activation might participate in the development of AAV. S1P, sphingosine‐1‐phosphate; S1PR, sphingosine‐1‐phosphate receptor; ICAM‐1, intercellular cell adhesion molecule‐1; VCAM‐1, vascular cell adhesion molecule‐1; ZO‐1, zonula occluden‐1