Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

Abstract

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.

https://doi.org/10.5061/dryad.z8w9ghxm4

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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.
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.
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.
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).
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.
Fig 3C_S1P1-b arrestin_Time.pzf

Temporal analysis of S1P-induced S1PR1 activation through a NanoBit-β-arrestin association assay
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
**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.
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.
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.
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%).
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.
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.
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.
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).
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).
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).
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).
Fig_S5_EC50 of HDL-iloprost.pzfx

IC50 of iloprost and ApoA1-iloprost.
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).
Fig_S6E_FLPC_HDL_fractions_of_mouse_plasma_western_blot.key

FPLC-derived HDL fraction was analyzed by western blot with ApoM antibody.
Fig_S6F_plasma_S1P_of_unloaded_A1M.pzfx

Plasma was analyzed for S1P content by electrospray ionization-MS/MS.
Fig_S6G_Isolated_HDL_S1P_of_unloaded_A1M.pzfx

FPLC-derived HDL fraction was analyzed for S1P content by electrospray ionization-MS/MS.

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

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Abstract

README: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

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