ApoM-bound S1P acts via endothelial S1PR1 to suppress choroidal neovascularization and vascular leak
Data files
Apr 15, 2025 version files 4.16 GB
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FIF2e_3e_S1P_conc_Apom-fc_kinetics_IP(left)_SC(right).pzfx
171.19 KB
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FIG1a_SET10_SET12_CNV_APOMKO_10x.zip
319.45 MB
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FIG1b_011024_CNV_APOM_KO_lesion_pooled.pzfx
101.88 KB
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FIG1c_030424_SET14_ApomTG_CNV_10X.zip
268.91 MB
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FIG1d_030524_set14_apomtg_cnv.pzfx
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FIG1e_112822_SET5_SET11_ECKO_10X.zip
245.74 MB
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FIG1f_SET5_SET11_092023_s1p1ecko_cnv_lesion.pzfx
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FIG1g_010324_CNV_SET13_ECTG_10x.zip
388.49 MB
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FIG1h_SET13_011624_ectg_cnv_quant.pzfx
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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
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FIG2a_wb_ip_72hr.tif
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FIG2b_WB_QUANT_APOMFC.pzfx
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FIG2c_WB_endogenous_APOM_QUANT.pzfx
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FIG2d_091224_apom_intensity_wt_IP.pzfx
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Fig2d_SC(left)_IP(right)_law_wb_data_24.08.13_11.17.29_S2_F01_PUB_600.tif
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Fig2g_raw_data_ApoM-Fc_sma_ib4_hoechst.20x_070_5L_2_c1-3c1_2_3.tif
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fig2g_raw_data_VEH_sma_ib4_hoechst.tif
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FIG2h_APOM-Fc.pzfx
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FIG2h.zip
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Fig3a_raw_wb_4veh(lane1-4)_4ApoMFc(lane5-8)_positive_control.tif
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Fig3a_wb_sc_72hr.tif
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Fig3b_111824_sc_apom-fc_wb_quant.pzfx
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Fig3c_111824_sc_apom_exp_endo_wb_quant.pzfx
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Fig3d_090424_Apomfc_intensity_plasma_sc.pzfx
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Fig3d_wb_SC(left)_IP(right).tif
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Fig3g.zip
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Fig3h_apom-fc_sc_cnv_quant.pzfx
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FIG4b_061224_CNV_SET16_SET19_10X.zip
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FIG4c_100224_cnv16_19_pooled_apomFC_vegf_quant.pzfx
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FIG4d_100724_SET16_19_S1P_CONC.pzfx
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Fig4f.zip
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FIG4g_FFA_SET19_CNV.pzfx
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FIG4i_image.zip
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Fig4j_Quant.zip
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FIG4j_VASCULAR_LEAK_TD.pzfx
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README.md
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SupFig4b_apom_fc_tm_wb_exp_quant.pzfx
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Suppl_FIG1a_091823_apomko_cnv_erg_20x.zip
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Suppl_Fig1b_091923_cnv_set10_erg_cnv_lesions.pzfx
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Suppl_FIG1c_SET3_SET14_CNV_SKRBCKO_10X.zip
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Suppl_FIG1d_092223_CNV_Sphkko_quant.pzfx
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Suppl_Fig2a.zip
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Suppl_FIG2b_SET2_ECTKO_22-09-22.pzfx
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Suppl_FIG3b_CNV_SET18_CNV_10X.zip
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Suppl_FIG3c_081424_CNV_SET18.pzfx
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Suppl_FIG3d_SET18_S1P_CONC.pzfx
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suppl_fig4_a_raw_wb.tif
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Abstract
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.
Dataset DOI: 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