Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) Schematic model of ApoA1-ApoM (A1M). (B) Purified A1M (4 μg) was separated by reducing 10% SDS-PAGE and stained with Coomassie brilliant blue. (C) Purified A1M (100 μg) was incubated or not with S1P for 24 hours, purified by gel filtration chromatography and analyzed for S1P content by electrospray ionization-MS/MS. The resulting data are the mean + S.D.; N=5. (D, E, F) Representative FPLC elution profiles (OD 280) of 200μl of mouse plasma, purified recombinant A1M, and A1M after lipidation and S1P loading. (D) FPLC elution of plasma standards: LDL (22-26ml) HDL (28-32ml), Soluble protein fraction (32-38ml). (E) nascent A1M protein (100μg) (F) lipidated A1M/S1P (1 mg). G) Representative negative-stain EM image of A1M with some particles highlighted in white circle (left) and 2D averages with potential ApoM densities indicated by arrows (right). Box dimension of each 2D average is 215 Å.

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Purification, SDS Page, Staining, Incubation, Filtration, Chromatography, Tandem Mass Spectroscopy, Recombinant

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) A1M/S1P-dependent enhancement of barrier function in HUVEC (24 μg/ml A1M contains ~ 300 nM S1P). (B) Comparison of A1M/S1P and ApoM-Fc/S1P by TEER using ApoA1-ApoM/S1P (16μg/ml of A1M or ApoM-Fc ~ 200nM S1P). Unloaded chaperones were used as controls. Data are presented as area under the curve (N = 3; mean ± SD. P < 0.0001, two-way ANOVA followed by paired student t-test t test). (C) HUVECs were analyzed for barrier protection by TEER analysis using ApoM-Fc/S1P (30 nM) and Ang-1 (300 ng/ml) individually or in combination. (D) HUVECs were analyzed for barrier protection by TEER analysis in response to thrombin (1U/ml) treatment using ApoM-Fc/S1P (200nM), Ang-1 (300ng/ml) or in combination. For C and D, data were analyzed by non-parametric t-test (Mann-Whitney) (****P<0.0001). (E-H) HUVECs were analyzed for barrier protection by TEER analysis using either A1M/S1P (30nM) (E) or ApoM-Fc/S1P (30nM) (G) in conjunction with APC (5μg/ml). After 1 hour pretreatment, thrombin was added for an additional 2 hours. Area Under the Curve was analyzed (n=3) followed by non-parametric t-test (Mann-Whitney) of control or individual treatments vs combined treatments (F,G). ****P<0.0001 for (E,G) and P<0.01 for (F,G).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) HMEC NF-κB-luciferase reporter cells were assayed for TNFα-induced NF-κB luciferase reporter activity in the presence of ApoA1, A1M and A1M/S1P. (N=3; Mean + S.D.; **P< 0.01 *P<0.05 Student t-test). (B) Effect of ApoM-Fc and Albumin-S1P on TNFα-induced NF-κB luciferase reporter activity (N= 3 Mean + S.D.). (C) HUVECs were assayed for TNFα induction of ICAM-1 expression by immunoblot analysis. Cultures were pre-treated for 10 minutes with ApomFc/S1P (100nM), Iloprost (200nM), or both in combination, as well as A1M, A1M/S1P, A1M/Iloprosst (all 200μg/ml) and induced with TNFα (10ng/ml) for 5 hours. Image J was used to obtain semi-quantitative values from scans of immunoblots (N=3).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Luciferase, Activity Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) Isolated peritoneal Mouse Neutrophils were assayed for oxidative burst after fMLF stimulation. Cells were pre-incubated for 10 minutes with vehicle control, ApoM-Fc/S1P (S1P 100nM), ApoA1/Iloprost (160nM), or in combination. Data are presented as normalized luminol fluorescence (B) Composite data for N=4 independent experiments. Area under the Curve (AUC) data were analyzed by non-parametric t-test (Mann-Whitney) and P values <0.0001. (C) Mice were treated with with thioglycolate and either PBS, A1M (200μg) or A1M/S1P (200μg). Peritoneal cells (4 h) were counted and analyzed by flow cytometry. Data were analyzed by one-way ANOVA followed by student t-test P=0.0317. (D) Peritoneal lavage supernatant from (C) was assayed by cytokine array analysis. Resulting blots were analyzed on IMAGEJ and differentially expressed analytes were quantified and data are expressed after normalization (Control = 100%). Data was analyzed by 2-way ANOVA followed by multiple paired student t-test (C5/C5a P= 0.028, G-CSF P= 0.004, sICAM-1 P= 0.025, IL-6 P= 0.001, IL-16 P= 0.0007, M-CSF P= 0.049, CCL2 P=0.009).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Isolation, Incubation, Fluorescence, MANN-WHITNEY, Flow Cytometry