Endothelial cell sphingosine 1-phosphate receptor 1 restrains VE-cadherin cleavage and attenuates experimental inflammatory arthritis

Abstract

In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.

Keywords: Arthritis; Endothelial cells; Inflammation; Vascular biology.

Figure 1. Vascular permeability in SIA is associated with clinical score, and genetic or pharmacological blockade of S1PR1 signaling on ECs worsens SIA.

(A) Scheme for experiment shown in (B): Clinical scores and vascular leakage, as measured by Evans blue extravasation in paws of SIA-treated mice injected with IV Evans blue (0.5% in PBS) 1 hour prior to sacrifice on days 0, 2, 3, and 8 after K/BxN serum injection; n = 5–9 mice/group for Evans blue and n = 3 mice/group for clinical score. (C) Clinical scores from K/BxN serum–treated ApoM^–/– mice and WT controls; n = 9 mice/group. (D–G) Clinical scores, representative H&E-stained paraffin sections of ankle joints, and quantification of histological scores from K/BxN serum–treated mice. Images were scanned at 5× original magnification. (D and E) S1PR1-ECKO and control mice; n = 7–12 mice/group for clinical score; n = 5–7 mice for histological score. (F and G) NIBR-0213– and vehicle control–treated C57BL/6 mice; n = 5–7 mice/group for clinical score, n = 4 mice for histological score. Significance was calculated using the unpaired 2-tailed Student’s t test. Values are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Pharmacologic and genetic enhancement of EC barrier function delays onset and attenuates severity of SIA.

(A) Clinical scores of WT C57BL/6 mice subjected to SIA, treated with CYM-5442 (0.25 mg/kg IP daily) or vehicle for 7 days; n = 5 mice/group. (B) Clinical scores of mice with a myeloid-specific KO of S1PR1 (LysM S1PR1 KO) or littermate controls treated with CYM-5442 or vehicle for 7 days; n = 7–8 mice/group. (C) Clinical scores of mice with tamoxifen-inducible EC gain of function (GOF) of S1PR1 versus tamoxifen-treated controls; n = 5–7 mice/group. Significance was calculated using the unpaired 2-tailed Student’s t test. Clinical score values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. VE-cadherin is shed from ECs and released into synovial fluids during SIA, and levels are increased in S1PR1-ECKO mice.

(A) Synovial fluid VE-cadherin in SIA-treated mice on day 7 versus untreated controls; n = 5–6 mice/group. Each dot corresponds to a sample from an individual mouse. (B) Image of synovial lavage fluids from mice injected with Evans blue IV from panel A; n = 5 mice/group. (C) Synovial VE-cadherin at 2–3 days after SIA in S1PR1-ECKO mice versus controls; n = 7–8 mice/group. (D) Clinical scores of S1PR1-ECKO mice and littermate controls at days 2–3 after SIA; n = 7–8 mice/group. (E) EB extravasation in synovial tissues on days 2–3 after SIA; n = 7–8 mice/group. (F) Model of metalloproteinase-mediated VE-cadherin cleavage resulting in the release of a 90 kDa fragment. MP, metalloproteinase. (G) Representative Western blot of synovial fluids isolated from S1PR1-ECKO mice and controls probed with an antibody targeting the N-terminal portion of VE-cadherin. Significance was calculated using the unpaired Student’s t test. Bars represent means ± SEM. **P < 0.01 or as indicated.

Figure 4. S1PR1 signaling restrains VE-cadherin shedding.

(A) Proposed model: S1PR1 signaling restrains the metalloproteinase-mediated cleavage of VE-cadherin. (B) Western blot of lysates from HUVECs treated with NIBR-0213 (10 μM) or vehicle and probed with a C-terminal–specific anti–VE-cadherin antibody (top panel) or β-actin (bottom panel). Representative image of n = 3. MM, marimastat. (C) Western blot of supernatants from HUVECs treated with 10 μM NIBR-0213 with or without MM (1 μM) for indicated times; glycosylated proteins were concentrated with concanavalin A beads, and eluates were probed with an N-terminal–specific antibody to VE-cadherin. (D) HUVECs treated with NIBR-0213 (0.5 μM) in the presence or absence of MM (1 μM) were subjected to electric cell-substrate impedance sensing (ECIS). Values are the mean ± SEM; n = 4 independent experiments. Significance was calculated using the unpaired 2-tailed Student’s t test. **P < 0.01.

Figure 5. S1PR1 blockade induces VE-cadherin shedding and vascular leak in the lung that does not depend on neutrophils.

(A) Representative image of perfused lungs from mice injected with Evans blue in the presence of NIBR-0213 or vehicle control. (B) Representative Western blot of (lane 1) plasma or (lane 2) BAL fluid from an untreated mouse, (lane 3) BAL fluid from an NIBR-0213–treated mouse, and (lane 4) HUVEC lysate indicating that in both plasma and BAL fluids, VE-cadherin is present as the cleaved 90 kDa fragment. Data represent at least 2 independent experiments. (C) Flow cytometry verification that anti-Ly6G antibodies depleted CD11b⁺ neutrophils. Left panel: isotype control–treated mice. Right panel: anti-Ly6G–treated mice. (D) Soluble VE-cadherin in BAL fluid from mice treated with isotype control antibody or anti-Ly6G 1 day prior to treatment with NIBR-0213; n = 4–5 mice/group. (E) Evans blue in BAL fluids of mice treated with isotype control antibody or anti-Ly6G 1 day prior to treatment with NIBR-0213. Each point represents an individual mouse. Statistical test was 1-way ANOVA with Tukey’s post hoc test; *P < 0.05; ***P < 0.001; or as indicated.

Figure 6. Mice with a stabilized VE-cadherin construct resist vascular leakage and VE-cadherin shedding and have attenuated SIA.

(A) Evans blue extravasation in skin 30 minutes after subcutaneous histamine injection in VE-cad-α-cat mice and controls; n = 4–5 mice/group. (B) BAL protein, n = 4–10 mice/group; and (C) BAL soluble VE-cadherin, n = 3–11 mice/group from mice treated with NIBR-0213 or vehicle control. (D) Clinical scores of VE-cad-α-cat mice and controls subjected to SIA; n = 8–9 mice/group. *P < 0.05; **P < 0.01; ns, not significant; or as indicated. Significance was calculated using the unpaired 2-tailed Student’s t test (A and D) or 1-way ANOVA and Tukey’s post hoc test (B and C).

Figure 7. S1PR1 signaling on ECs prevents ADAM10-mediated cleavage of VE-cadherin to maintain barrier function.

(A) HUVECs were transfected with control or ADAM10 siRNA 2–3 days prior to treatment with NIBR-0213 for 1 hour. Supernatants were collected and soluble VE-cadherin was quantified by ELISA; n = 3. (B) Synovial endothelial cells were treated with the ADAM10 inhibitor GI254023X (GI) (1 μM) or vehicle (DMSO) for 30–60 minutes prior to treatment with NIBR-0213, and resistance across confluent ECs was measured by ECIS. Values are the mean ± SEM; n = 3 independent experiments; ****P ≤ 0.0001. Mice were treated with GI254023X (50 mg/kg IV or DMSO) 30 minutes prior to challenge with NIBR-0213 (10 mg/kg or polyethylene glycol 200, PEG) and BAL fluids were collected. (C) Soluble VE-cadherin; n = 3–7 mice/group. (D) Evans blue; n = 3–9 mice/group. Statistical test was 1-way ANOVA with Tukey’s post hoc test; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 8. Patients with moderately to severely active RA have lower circulating S1P and decreased S1PR1 expression in synovial capillary ECs.

(A) Diagram of sphingolipid biosynthesis. (B) Sphinganine 1-phosphate (Sa1P) and S1P levels in RA and OA sera. Significance was calculated using the unpaired 2-tailed Student’s t test. Values represent mean ± SEM. (C) Violin plots indicating normalized S1PR1 expression in capillaries derived from RA and healthy synovial tissues as determined by single-cell RNA-Seq. P values were calculated using pairwise Wilcoxon rank-sum tests. *P < 0.05; ***P ≤ 0.001; or as indicated.