DOI: 10.5061/dryad.z8w9ghxpp
title: ApoM-bound S1P acts via endothelial S1PR1 to suppress choroidal neovascularization and vascular leak

description: Neovascular age-related macular degeneration (nAMD) is a major cause of
 vision loss in the elderly. Vascular endothelial growth factor (VEGF)
 inhibitors, which are approved for the treatment of nAMD, require
 repetitive intraocular injections and suffer from partial and non-durable
 treatment response. We report here that circulating sphingosine
 1-phosphate (S1P) carried by apolipoprotein M (ApoM) acts through the
 endothelial S1P receptor 1 (S1PR1) to suppress choroidal
 neovascularization (CNV) in laser-induced mouse nAMD model. Low plasma
 ApoM increased whereas high plasma ApoM reduced laser-induced CNV. In
 addition, endothelial S1pr1 knockout and overexpressing transgenic mice
 showed larger and reduced CNV lesion size, respectively. Systemic
 administration of ApoM-Fc, an engineered S1P chaperone protein, not only
 attenuated CNV to an equivalent degree as anti-VEGF antibody treatment but
 also suppressed vascular permeability. We suggest that the circulating
 ApoM-bound S1P action on endothelial S1PR1 provides a novel therapeutic
 strategy to treat nAMD.
description: # ApoM-bound S1P acts via endothelial S1PR1 to suppress choroidal
 neovascularization and vascular leak Dataset DOI:
 [10.5061/dryad.z8w9ghxpp](10.5061/dryad.z8w9ghxpp) ## Description of the
 data and file structure Neovascular age-related macular degeneration
 (nAMD) is a major cause of vision loss in the elderly. We investigated the
 role of Shingosine 1-phosphate (S1P) signaling via endothelial S1PR1
 action in nAMD. Laser-induced choroidal neovascularization (CNV) mouse
 model which mimics human nAMD was employed on genetic mouse models that
 modulate *S1pr1* gene in endothelium (*S1pr1* ECKO and *S1pr1* ECTG) as
 well as  *Apom* gene (*Apom* KO and *Apom* TG). Two delivery routes of
 ApoM-Fc, an engineered S1P chaperone protein, were tested to determine
 pharmacokinetics and plasma S1P levels in vivo. ApoM-Fc administration
 alone or in combination with anti-VEGF treatment were examined in mice
 after laser photocoagulation. Mouse RPE/ Choroid flat mount was prepared
 10 to 14 days post-laser photocoagulation, stained for endothelial cell
 staining followed by confocal microscopy to analyze CNV. FFA and acute
 vascular hyperpermeability assay were carried out. Mouse plasma was
 collected and prepared for S1P measurement. Western blot was performed for
 ApoM-Fc biologics and endogenous ApoM detection in collected mouse
 plasma.  ### Files and variables #### File:
 FIG1b\_011024\_CNV\_APOM\_KO\_lesion\_pooled.pzfx
 **Description:** FIG1B_quantification of CNV lesion size in ApoM wt, het,
 and KO #### File: FIG1f\_SET5\_SET11\_092023\_s1p1ecko\_cnv\_lesion.pzfx
 **Description:** FIG1F_quantification of CNV lesion size in S1pr1 ECKO and
 littermate control #### File: FIG1h\_SET13\_011624\_ectg\_cnv\_quant.pzfx
 **Description:** FIG1H_quantification of CNV lesion size in S1pr1 ECTG and
 littermate control #### File: FIG1d\_030524\_set14\_apomtg\_cnv.pzfx
 **Description:** FIG1D_quantification of CNV lesion size in ApoM TG and
 littermate control #### File: FIG2b\_WB\_QUANT\_APOMFC.pzfx
 **Description:** FIG2B_quantification of plasma ApoM-Fc band intensity 72
 hours after IP injection detected by western blot #### File:
 FIG2c\_WB\_endogenous\_APOM\_QUANT.pzfx
 **Description:** FIG2C_quantification of plasma endogenous ApoM band
 intensity after IP injection detected by western blot #### File:
 FIG2d\_091224\_apom\_intensity\_wt\_IP.pzfx
 **Description:** FIG2D_quantification of plasma ApoM-Fc band intensity
 upon IP injection at various time points detected by western blot ####
 File: FIG2h\_APOM-Fc.pzfx **Description:** FIG2H_quantification of CNV
 lesion size in vehicle control and ApoM-Fc treated group at day 10 upon
 laser injury #### File: Suppl\_FIG1d\_092223\_CNV\_Sphkko\_quant.pzfx
 **Description:** SF1D_quantification of CNV lesion size in littermate
 control and SphK RBCKO mice #### File:
 Suppl\_Fig1b\_091923\_cnv\_set10\_erg\_cnv\_lesions.pzfx
 **Description:** SF1B_quantification of ERG-positive cells per lesion in
 littermate control and ApoM KO mice after photocoagulation #### File:
 Suppl\_FIG3d\_SET18\_S1P\_CONC.pzfx **Description:** SF3D_S1P measurement
 in plasma collected from vehicle control and ApoM-Fc treated group with or
 without anti-VEGF treatment #### File:
 SupFig4b\_apom\_fc\_tm\_wb\_exp\_quant.pzfx
 **Description:** SF4B_quantification of ApoM-Fc or ApoM-TM band intensity
 in plasma detected by western blot #### File:
 Suppl\_FIG3c\_081424\_CNV\_SET18.pzfx **Description:** SF3C_quantification
 of CNV lesion size in IgG control and ApoM-Fc treated group with or
 without high dose of anti-VEGF treatment (5mg/Kg) 10 days after
 photocoagulation #### File: suppl\_fig4\_a\_raw\_wb.tif
 **Description:** SF4A_original western blot data showing ApoM-Fc, ApoM-TM,
 and endogenous ApoM detection and used for Suppl Fig4a #### File:
 FIG1a\_SET10\_SET12\_CNV\_APOMKO\_10x.zip **Description:** FIG1A_original
 confocal image data used for representative Fig1a and CNV quantification
 Fig1b #### File: FIG1c\_030424\_SET14\_ApomTG\_CNV\_10X.zip
 **Description:** FIG1C_original confocal image data used for
 representative Fig1c and CNV quantification Fig1d #### File:
 FIG1e\_112822\_SET5\_SET11\_ECKO\_10X.zip **Description:** FIG1E_original
 confocal image data used for representative Fig1e and CNV quantification
 Fig1f #### File: Suppl\_FIG1c\_SET3\_SET14\_CNV\_SKRBCKO\_10X.zip
 **Description:** SF1C_original confocal image data used for representative
 SupplFig1c and CNV quantification SupplFig1d #### File:
 Suppl\_FIG1a\_091823\_apomko\_cnv\_erg\_20x.zip
 **Description:** SF1A_original confocal image data stained for ERG and
 IB4, and used for representative SupplFig1a and CNV lesion quantification
 shown in SupplFig1b #### File: Suppl\_Fig2a.zip
 **Description:** SF2A_original confocal image data collected from S1pr1
 ECTKO and littermate control eyes after photocoagulation, stained for IB4
 and used for representative SupplFig2a and CNV quantification shown in
 SupplFig2b #### File: Suppl\_FIG3b\_CNV\_SET18\_CNV\_10X.zip
 **Description:** SF3B_original confocal image data collected from mice
 which received ApoM-Fc in combination with anti-VEGF agent and used for
 representative SupplFig3b and CNV quantification shown in SupplFig3c ####
 File: FIF2e\_3e\_S1P\_conc\_Apom-fc\_kinetics\_IP(left)\_SC(right).pzfx
 **Description:**  FIG2E_FIG3E_quantification of plasma S1P measurement
 after ApoM-Fc administration via intraperitoneal (IP, FIG2E) or
 subcutaneous (SC, FIG3E) route #### File: FIG2a\_wb\_ip\_72hr.tif
 **Description:** FIG2A_cropped western blot data for ApoM-Fc and
 endogenous ApoM detection in plasma 72 hours after intraperitoneal ApoM-Fc
 administration  #### File:
 Fig3d\_090424\_Apomfc\_intensity\_plasma\_sc.pzfx
 **Description:** FIG3D_quantification of plasma ApoM-Fc band intensity at
 various time points upon subcutaneous ApoM-Fc injection #### File:
 FIG2a\_\_raw\_WB\_IP\_veh(3\_lanes\_from\_left)\_apom-fc(4\_lanes\_right)\_one\_fc\_control\_24.01.09\_11.21.03\_PUB\_600.tif **Description:** FIG2A_original western blot data for endogenous and ApoM-Fc detection using collected plasma 72 hours after ApoM-Fc or vehicle injection, i.p. and used for quantification for Fig2b and 2c #### File: Fig3d\_wb\_SC(left)\_IP(right).tif **Description:** FIG3D_original western blot data for ApoM-Fc detection using collected plasma at various time points after ApoM-Fc or vehicle injection, s.c. and used for quantification for Fig3b and 3c #### File: Fig2d\_SC(left)\_IP(right)\_law\_wb\_data\_24.08.13\_11.17.29\_S2\_F01\_PUB\_600.tif **Description:** FIG3D_original western blot data for ApoM-Fc detection using collected plasma at various time points after ApoM-Fc or vehicle injection, s.c. and used for quantification for Fig3b and 3c #### File: Fig3h\_apom-fc\_sc\_cnv\_quant.pzfx **Description:** FIG3H_quantification of CNV lesions 14 days after subcutaneous injection of ApoM-Fc or vehicle controls upon laser injury #### File: Fig3b\_111824\_sc\_apom-fc\_wb\_quant.pzfx **Description:**  FIG3B_quantification of ApoM-Fc band intensity in plasma 72 hours after subcutaneous injection of ApoM-Fc detected by western blot #### File: Fig3c\_111824\_sc\_apom\_exp\_endo\_wb\_quant.pzfx **Description:**  FIG3C_quantification of endogenous ApoM band intensity in plasma after subcutaneous injection of ApoM-Fc detected by western blot #### File: Fig3a\_raw\_wb\_4veh(lane1-4)\_4ApoMFc(lane5-8)\_positive\_control.tif **Description:** FIG3A_original western blot data for endogenous and ApoM-Fc detection using collected plasma 72 hours after ApoM-Fc or vehicle injection, s.c. and used for quantification for Fig3b and 3c #### File: FIG2h.zip **Description:** FIG2h_original confocal image data used for quantification of CNV lesion size in vehicle control and ApoM-Fc treated group at day 10 upon laser injury shown in FIG2H #### File: FIG4j\_VASCULAR\_LEAK\_TD.pzfx **Description:** FIG4J_quantification of FITC-albumin leakage by VEGF-induced, acute vascular hyperpermeability assay #### File: FIG4g\_FFA\_SET19\_CNV.pzfx **Description:** FIG4G_quantification of Fluorescein leakage in vehicle control and ApoM-Fc treated group with or without anti-VEGF treatment at day 10 #### File: FIG4c\_100224\_cnv16\_19\_pooled\_apomFC\_vegf\_quant.pzfx **Description:** FIG4C_quantification of CNV lesion size in IgG control and ApoM-Fc treated group with or without low dose of anti-VEGF treatment (0.5mg/Kg) 10 days after photocoagulation #### File: FIG4d\_100724\_SET16\_19\_S1P\_CONC.pzfx **Description:** FIG4D_plasma S1P measurement in isotype control and ApoM-Fc treated group with or without anti-VEGF treatment at day 10 after laser injury #### File: FIG4i\_image.zip **Description:** FIG4I_original confocal image data used for quantification Fig4j #### File: Fig3g.zip **Description:** FIG3G_Original confocal images after s.c. ApoM-Fc or vehicle treatment upon laser injury, shown in Fig3g and used for CNV lesion quantification (Fig3h) #### File: Fig4f.zip **Description:** FIG4F_Original images of fluorescein leakage after FFA in vehicle control and ApoM-Fc treated group with or without anti-VEGF treatment and used in Fig4f and fluorescein leakage quantification (Fig4g) #### File: Fig4j\_Quant.zip **Description:** FIG4J_quantification of FITC-albumin leakage after acute vascular hyperpermeability assay #### File: FIG4b\_061224\_CNV\_SET16\_SET19\_10X.zip **Description:** FIG4b_original confocal images used in Fig4b representative images and quantification of CNV lesions shown in Fig4c. Samples are collected from isotype control and ApoM-Fc treated group with or without anti-VEGF treatment at day 10 after laser injury #### File: fig2g\_raw\_data\_VEH\_sma\_ib4\_hoechst.tif **Description:** FIG2G_representative confocal image of vehicle group upon SMA and iB4 staining and used for Fig2g #### File: Fig2g\_raw\_data\_ApoM-Fc\_sma\_ib4\_hoechst.20x\_070\_5L\_2\_c1-3c1\_2\_3.tif **Description:** FIG2G_representative confocal image of ApoM-Fc group upon SMA and iB4 staining and used for Fig2g #### File: FIG1g\_010324\_CNV\_SET13\_ECTG\_10x.zip **Description:** FIG1G_original confocal images of S1pr1 ECTG and littermate control mice upon laser injury used for Fig1g and used for CNV quantification in Fig1h #### File: Suppl\_FIG2b\_SET2\_ECTKO\_22-09-22.pzfx **Description:** SF2B_quantification of CNV lesion size in littermate control and S1pr ECTKO mice #### File: Fig3a\_wb\_sc\_72hr.tif **Description:** FIG3A_original/ raw western blot data for Fig3a ## Code/software ImageJ software Prism9 Adobe illustrator CS6