DOI: 10.5061/dryad.z8w9ghxm4
title: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

description: High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC)
 function and suppress inflammation, but their utility in treating EC
 dysfunction has not been fully explored.  Here, we describe a
 fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1
 (ApoA1), the principal structural protein of HDL that forms lipid
 nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine
 1-phosphate (S1P). A1M forms HDL-like particles, binds S1P, and is
 signaling competent. Molecular dynamic simulations showed that the
 S1P-bound ApoM moiety in A1M efficiently activated the EC surface
 receptors. Treatment of human umbilical vein endothelial cells (HUVECs)
 with A1M-S1P stimulated barrier function either alone or cooperatively
 with other barrier-enhancing molecules, including the stable prostacyclin
 analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P
 injection into mice during sterile inflammation suppressed neutrophil
 influx and inflammatory mediator secretion. Moreover, systemic A1M
 administration led to a sustained increase in circulating HDL-bound S1P
 and suppressed inflammation in a murine model of LPS-induced endotoxemia.
 We propose that A1M administration may enhance vascular endothelial
 barrier function, suppress cytokine storm, and promote resilience of the
 vascular endothelium.
description: # Designer high-density lipoprotein particles enhance endothelial barrier
 function and suppress inflammation
 [https://doi.org/10.5061/dryad.z8w9ghxm4](https://doi.org/10.5061/dryad.z8w9ghxm4) All the datasets, western blot images, Excel files, Prism files, and statistical analyses related to the figures presented in this paper were stored. Empty cells in the Prism files indicate null values, and they can not be replaced by "null" or "n/a" because it interfere with the Prism analysis. If anyone has a question after reviewing the raw data and analysis, please contact the first and corresponding authors.  ## FILE LIST AND DATA-SPECIFIC INFORMATION 1. **Fig 1A Purified A1M 4ug 10ug ApoMFc ApoMFc-TM (Original gel).tif** Purified A1M (4 μg and 10 μg), ApoM-Fc (4 μg), and ApoM-FC-TM (4 μg) from CHO cell-conditioned media were separated by reducing 10% SDS-PAGE and stained with Coomassie Brilliant Blue.  2. **Fig 1B_S1P content in A1M-S1P.pzf** S1P content of A1M-S1P was analyzed by electrospray ionization-MS/MS. Purified A1M was incubated with S1P for 24 hours, lipidated, and purified by gel filtration chromatography.  3. **Fig 1C_A1M-S1P_FPLC.xlsx** FPLC elution profiles (at OD 280 nm) of 200 μl of mouse plasma, purified recombinant A1M from CHO cells, and lipidated A1M with S1P loading. 4. **Fig 3A_S1P1-Gi dissociation_Time course.prism** Temporal analysis of S1P-induced S1PR1 activation through a NanoBiT-G-protein dissociation assay. Cells were treated with 100 nM of S1P complexed with various chaperones (BSA-S1P, ApoM-Fc-S1P, or A1M-S1P). 5. **Fig 3B_S1P1-Gi_dose.prism** Dose-response analysis of S1PR1-dependent GaI activation by bovine serum albumin-S1P (BSA-S1P), ApoM-Fc-S1P, or A1M-S1P in NanoBiT-G-protein dissociation assay. The bar graphs show the responses at 1 μM S1P. 6. **Fig 3C_S1P1-b arrestin_Time.pzf** Temporal analysis of S1P-induced S1PR1 activation through a NanoBit-β-arrestin association assay 7. **Fig 3D_S1P1-b arrestin_dose.pzf** Dose analysis of S1PR1-β-arrestin coupling with BSA-S1P, ApoM-Fc-S1P, or A1M-S1P in NanoBiT assay. The bar graphs show the responses at 1 μM S1P. 8. **Fig 3E_S1P2-b arrestin_dose.pzf** Dose analysis of S1PR2-β-arrestin coupling with BSA-S1P, ApoM-Fc-S1P, or A1M-S1P in NanoBiT assay. The bar graphs show the responses at 1 μM S1P.  9. **Fig 3F_S1P3-b arrestin_dose.pzf** Dose analysis of S1PR3-β-arrestin coupling with BSA-S1P, ApoM-Fc-S1P, or A1M-S1P in NanoBiT assay. The bar graphs show the responses at 1 μM S1P.  10. **Fig 4A_TEER_ApoM-Fc-S1P and A1M-S1P.prism** TEER analysis to measure barrier function was performed on HUVECs treated with A1M-S1P, ApoM-Fc-S1P, or chaperone only (ApoM-Fc or A1M). 11. **Fig 4B_A1M Ang1 Teer 3a.prism** TEER analysis to measure barrier function was performed on HUVECs treated with vehicle, Ang1 (300 ng/mL), A1M-S1P (30 nM), or Ang1+A1M-S1P.  12. **Fig 4C_APC A1M Thrombin.pzfx** TEER analysis was performed to measure the barrier function on HUVECs, which were pretreated with media, APC (5 μg/mL), A1M-S1P (30 nM), or A1M-S1P+APC for 1 hour. Then, thrombin was added, and cells were monitored for an additional 2 hours. 13. **Fig 4D_APC AMFC Thrombin TEER.pzfx** TEER analysis was performed to measure the barrier function on HUVECs, which were pretreated with media, APC (5 μg/mL), ApoM-Fc-S1P (30 nM), or ApoM-Fc-S1P+APC for 1 hour. Then, thrombin was added, and cells were monitored for an additional 2 hours. 14. **Fig 5A_CRE assay_A1M-iloprost.prism** Cells expressing a CREB-luciferase reporter with prostacyclin receptor (IP) were stimulated with vehicle or A1M-iloprost for 8 hours, and cell lysates were assayed for luciferase activity. 0 nM was used as a reference control and normalized to 1. 15. **Fig 5B_A1M-ilo and A1M-S1P.pzf** TEER analysis to measure barrier function was performed on HUVECs treated with A1M-S1P (8 μg/mL with 100 nM S1P), A1M-iloprost (25 μg/mL with 200 nM iloprost), or A1M-S1P + A1M-iloprost. 16. **Fig 5C Platelet LTA analysis.xlsx** Human platelets were assayed for aggregation in response to the thrombin receptor mimetic peptide SFLLRN 2 μM in the presence of vehicle control, A1M-iloprost 10 nM, A1M-S1P 200 nM, or in combination. 17. **Fig 6A_Luciferase TNF ApoA1 AIM-S1Pab.pzf** A1M attenuates TNF𝝰-dependent inflammation. HMEC-1 cells expressing an NF-𝝹B-luciferase reporter were assayed for TNF𝝰-induced NF-𝝹B reporter activity in the presence of ApoA1, A1M, A1M-S1P, and ApoM-Fc-S1P. 18. \**Fig 6B_Image J of ICAM1 expression.pzfx **& **Fig 6B_TNF+A1M_ICAM-1 expression.pptx** HUVECs were starved for 1 h, pre-treated for 10 minutes with ApoM-Fc-S1P (100 nM), iloprost (200 nM), or both, or A1M (200 µg/mL), A1M-S1P (200 µg/mL), or A1M-iloprost (200 µg/mL) and induced with TNF (10 ng/mL) for 5 hours. Lysates were subjected to immunoblot analysis for ICAM-1. 19. **Fig 6C_Cholesterol Efflux_A1M-S1P.pzf** Cholesterol efflux in response to human HDL (hHDL), human ApoA1 protein (hApoA1), and bacterial A1M (bA1M) in PMA-induced THP-1 cells. 20. **Fig 7A neutrophil migration inhibition stats.pzf** A1M-S1P suppresses inflammation in a murine peritonitis model. Mice were treated intraperitoneally with thioglycolate and either PBS, A1M (0.2 mg/animal), or lipidated A1M-S1P (0.2 mg/animal). Peritoneal cells were collected at 4 h and analyzed by flow cytometry. 21. **Fig 7B_cytokine array.pzfx** & **Fig 7B_A1M-S1P_Peritoneal lavage_cytokine profile.pptx** Peritoneal lavage supernatant collected at 4 h from mice treated intraperitoneally with thioglycolate and either PBS, A1M (0.2 mg/animal), or lipidated A1M-S1P (0.2 mg/animal), and assayed by cytokine array analysis. Statistical tests are done on analytes with changes in abundance after normalization (control was set at 100%).  22. **Fig 8A-C_LPS+A1M_merge.pzfx** Mice were injected intravenously with 40 mg/Kg of bA1M or PBS (vehicle) for 1 hour to allow for A1M lipidation in vivo before being injected intraperitoneally with 10 mg/Kg of LPS or saline. Mice were assessed for sepsis score (A), body temperature (B), and plasma IL-6 amounts (C) at 12 hours post-LPS injection. 23. **Fig_8D_A1M_in_LPS_injected_mice_westerns.pptx** 0.5 μl of plasma from mice injected with LPS alone (10 mg/Kg) or LPS and bA1M (40 mg/Kg) at 12 hours post-injection were subjected to Western Blot analysis for ApoM. Each lane represents an individual mouse. 24. **Fig_8E_S1P_level.pzfx** Plasma S1P content in mice injected with saline, LPS alone, or LPS and bA1M at 12 hours post-injection were analyzed by LC-MS/MS. 25. **Fig_S3A_ApoM-Fc-S1P___Thrombin_TEER.prism** TEER analysis to determine barrier function was performed on HUVECs treated for 2 hours using ApoM-S1P (0, 3, 10, 30, and 100 nM S1P) in combination with thrombin (1U/mL).  26. **Fig_S3B_ApoM-Fc-S1P___Ang1_TEER.pzfx** TEER analysis to determine barrier function was performed on HUVECs treated for 2 hours with thrombin (1 U/ml) and ApoM-Fc-S1P (200 nM), angiopoietin (Ang-1, 300 ng/ml) or both (ApoM-Fc-S1P + Ang-1). 27. **Fig_S4A_HDL-S1P_Iloprost_TEER.prism** TEER analysis to measure barrier function was performed on HUVECs treated with HDL-S1P (100 nM S1P) **(A)** alone or in combination with iloprost (200 nM).  28. **Fig_S4B_AUY_Iloprost_TEER.prism** TEER analysis to measure barrier function was performed on HUVECs treated with the S1PR1 agonist AUY954 (1 mM) alone or in combination with iloprost (200 nM). 29. **Fig_S5_EC50 of HDL-iloprost.pzfx** IC50 of iloprost and ApoA1-iloprost. 30. **Fig_S6C_FPLC_bA1M_lipidation_and_plasma.xlsx** & **Fig_S6C_bA1M_TEER.prism** Titration analysis of bA1M-S1P-dependent enhancement of barrier function in HUVECs (8 mg/ml A1M contains ~ 100 nM S1P). 31. **Fig_S6E_FLPC_HDL_fractions_of_mouse_plasma_western_blot.key** FPLC-derived HDL fraction was analyzed by western blot with ApoM antibody. 32. **Fig_S6F_plasma_S1P_of_unloaded_A1M.pzfx** Plasma was analyzed for S1P content by electrospray ionization-MS/MS. 33. **Fig_S6G_Isolated_HDL_S1P_of_unloaded_A1M.pzfx** FPLC-derived HDL fraction was analyzed for S1P content by electrospray ionization-MS/MS.