Background/Purpose:

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid, is important for vascular homeostasis via signaling through S1P receptors. HDL-bound apolipoprotein M (ApoM) is a physiological S1P carrier that activates endothelial S1P₁ receptors, thereby increasing barrier function. ApoM-S1P also limits endothelial cell expression of adhesion molecules and activation of NF-κB in response to TNF-α. We tested a novel biologic S1P₁ receptor agonist, ApoM-Fc loaded with S1P, for its ability to protect the endothelial barrier in response to immune complex (IC) and neutrophil (PMN)-mediated injury in vitro and in vivo.

Methods:

Barrier function of human umbilical vein endothelial cells (HUVECs) in response to IC and C5a activated PMNs +/- ApoM-Fc was assessed by Electric Cell-substrate Impedance Sensing (ECIS). Phosphorylation of myosin light chain2 (p-MLC2) and VE-Cadherin staining in HUVECs after treatment with activated PMNs +/- ApoM-Fc was assessed by immunofluorescence (IF). The reverse arthus reaction (RAR) was performed in skin in mice treated locally with S1P₁ agonists and antagonists. Lung RAR was performed in WT and Apom^-/- mice and mice with an inducible endothelial cell deletion of S1P₁ (ECKO). ApoM-Fc (100 μg, IP) or PBS was administered to WT mice 2 hrs before lung RAR. PMNs and red blood cells in bronchoalveolar lavage fluid (BALF) were quantified. Lung weights and Evans blue (EB) were measured.

Results:

Activated PMNs decreased HUVEC resistance in ECIS and increased p-MLC2 and VE-Cadherin junction disassembly as assessed by IF. ApoM-Fc prevented the loss of barrier function (mean increase 224±28 Ohms, n=4) in a sustained manner (>8 hrs) and markedly reduced PMN-induced p-MLC2 and loss of VE-Cadherin. In contrast, a mutated ApoM-Fc construct that does not bind S1P was not protective. S1P also attenuated IC-mediated injury in vivo. S1P₁ agonist CYM-5442 decreased EB leak and skin weights after RAR compared to PBS treated controls (20 vs 27 mg, n=15-20, p <0.0001) and W146, a S1P₁ antagonist, increased EB leak and skin weights after RAR (24 vs 21 mg, n=12; p= 0.02). S1P₁ ECKO mice showed markedly increased lung RAR compared to controls: EB extravasation (157 vs 95 µg/g, n=3-5 mice; p=0.04), BAL WBCs (1.0 vs 0.6 X10³/µl, n=8-11; p=0.03), and lung mass (330 vs 265 mg, n=5-6; p=0.0007). Apom^-/- treated with local administration of W146 to partially block S1P₁ also showed more intense lung RAR than WT controls treated with W146, consistent with increased vascular vulnerability to injury. Importantly, delivery of S1P in vivo with ApoM-Fc was able to limit lung RAR with reduction in BALF WBCs and RBCs and decreased extravasated EB compared to PBS treated controls.

Conclusion:

These data demonstrate that stimulation of endothelial S1P₁ receptor by ApoM-HDL-S1P protects barrier integrity of the microvessels when challenged by IC-mediated vascular injury. S1P₁ receptor agonism represents a novel target to limit inflammation-induced injury in SLE and other IC mediated diseases. Moreover, because ApoM-Fc does not induce lymphopenia, unlike most other S1P₁ agonists, it could be used concomitantly with immunosuppressive therapies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/sphingosine-1-phosphate-receptor-1-mediated-endothelial-cell-barrier-function-protects-against-immune-complex-induced-vascular-injury-a-potential-novel-therapeutic-target-for-sle/