Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) Purified A1M (4 μg) from CHO cell conditioned media was separated by reducing 10% SDS-PAGE and stained with Coomassie brilliant blue. (B) Purified A1M was incubated with S1P for 24 hours, lipidated, and purified by gel filtration chromatography. S1P content of A1M-S1P was analyzed by electrospray ionization-MS/MS. The resulting data are the mean ± S.D.; N = 5 biological replicates per group. ****P < 0.0001 by unpaired t-test. (C) Representative FPLC elution profiles (OD 280 nm) of 200 μl of mouse plasma (black), purified recombinant A1M (blue), and lipidated A1M with S1P loading (A1M-S1P, red). FPLC elution of plasma standards: vLDL/LDL (21–26 mL), HDL (28–31 mL), and soluble protein (31–35 mL) fractions. Elution profiles are representative of 3 independent experiments. (D) Representative negative-stain transmission EM image of lipidated A1M-S1P complex with some particles highlighted in white circles (top) and 2D averages with potential ApoM densities indicated by arrows (bottom). Box dimension of each 2D average is 215 Å. Images are representative of 3 independent experiments. (E) Schematic model of A1M-S1P.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Purification, SDS Page, Staining, Incubation, Filtration, Chromatography, Tandem Mass Spectroscopy, Clinical Proteomics, Recombinant, Transmission Assay

Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) Side view of A1M-S1P complex with and without the phospholipids, highlighting the S1P binding pocket in both the ApoM-S1P and ApoA1-fused systems. Monomer 1 - ApoA1 in sienna, linker in brown, and ApoM in beige. Monomer 2 - ApoA1 in sage, linker in turquoise, and ApoM in teal. Detail of the binding pocket in A1M-S1P and ApoM-S1P are shown in the right panel. (B) Projection of protein atoms along the top two dominant eigenvectors (eigenvector 1 - PC1 and eigenvector 2 – PC2) is responsible for over 40% of the dominant collective variances in the structure set, generating 5 clusters for which the three-dimensional structure of the most representative member of each cluster is shown. Permanence times: cluster 1 – 9.6%, cluster 2 – 52.9%, cluster 3 – 18.7%, cluster 4 – 12.2%, cluster 5 – 6.5%. (C) Distribution of the proportion of variance between the PCs.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Binding Assay

Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) Temporal analysis of S1P-induced S1PR1 activation in cells treated with 100 nM of S1P complexed with various chaperones through a NanoBiT G-protein dissociation assay (N = 3 biological replicates per group). (B) Dose-response analysis of S1PR1-dependent Gαi activation by bovine serum albumin-S1P (BSA-S1P), ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay assessing G-protein dissociation (N = 3 biological replicates per group). (C) Temporal analysis of S1P-induced S1PR1 activation in cells treated with 100 nM of S1P complexed with various chaperones through a NanoBit β-arrestin association assay (N = 3 biological replicates per group). (D) Dose analysis of S1PR1-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (N = 3 biological replicates per group). (E) Dose analysis of S1PR2-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (N = 3 biological replicates per group). (F) Dose analysis of S1PR3-β-arrestin coupling induced by BSA-S1P, ApoM-Fc-S1P, or A1M-S1P by NanoBiT assay (N = 3 biological replicates per group). Data represent mean ± S.D. The bar graphs in (B), (D), (E), and (F) show the responses at 1 μM S1P. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Activation Assay, Histone Association Assay

Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) TEER analysis to measure barrier function was performed on HUVECs treated with A1M-S1P, ApoM-Fc-S1P or chaperone only (N = 3 biological replicates per group). (B) TEER analysis to measure barrier function was performed on HUVECs treated with Ang-1 (300 ng/mL), A1M-S1P (30 nM) or both (N = 3 biological replicates per group). (C and D) TEER analysis to measure barrier function was performed on HUVECs treated with APC (5 μg/mL) alone or with A1M-S1P (30 nM) (C) or ApoM-Fc-S1P (30 nM) (D). After 1 hour pretreatment, thrombin was added for an additional 2 hours. N = 3 biological replicates per group. The EC barrier index (above the baseline) and EC barrier degradation index (below the baseline) were analyzed by calculating the area under the curve of TEER value and are presented as means ± SD. ***P < 0.001, ****P < 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques:

Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) 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 (N = 3 biological replicates per group). Data are presented as means + S.D. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ordinary two-way ANOVA with Tukey’s multiple comparisons test. TNFα is the reference group. (B) HUVECs were starved for 1 h, pre-treated for 10 minutes with ApoM-Fc-S1P (100 nM), iloprost (200 nM), both ApoM-Fc-S1P and iloprost, A1M (200 μg/mL), A1M-S1P (200 μg/mL), or A1M-iloprost (200 μg/mL) and induced with TNFα (10 ng/mL) for 5 hours. Lysates were subjected to immunoblot analysis for ICAM-1. Quantification of immunoblots was analyzed from 3 biological replicates per group. Data are presented as means ± S.D. *P < 0.05, **P < 0.01, ****P < 0.0001 by ANOVA with post-hoc Holm-Sidak’s multiple comparisons test. (C) Cholesterol efflux in response to human HDL (hHDL), human ApoA1 protein (hApoA1), and bacterial A1M (bA1M) in PMA-induced THP-1 cells (N ≥ 5 independent experiments). Data are presented as means + S.D. *P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Expressing, Luciferase, Activity Assay, Western Blot

Journal: Science signaling

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

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A to C) 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. Groups were saline (N ≥ 6 mice), LPS alone (N ≥ 14 mice), and LPS with A1M (N ≥ 10 mice). Mice were assessed for sepsis score (A), body temperature (B), and plasma IL-6 amounts (C) at 12 hours post-LPS injection. Data are presented as means ± S.D. ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (D) 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. (E) 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 (N = 4 mice per group). Data are presented as means ± S.D. ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Creation of the A1M fusion constructs The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Injection, In Vivo, Saline, Clinical Proteomics, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Clinical Investigation

Article Title: Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway

doi: 10.1172/JCI146219

Figure Lengend Snippet: ( A ) Liver Apom gene expression in 13-week-old db/db and db/+ control mice. ( B ) Liver Apom gene expression in 25-week-old mice. ( C ) Western blot of ApoM expression in total plasma from 13-week-old and 25-week-old mice. ( D ) Western blot of ApoM and ApoA1 expression in HDL fractionated by sequential density ultracentrifugation from plasma of 13-week-old and 25-week-old mice. ( E ) Representative Western blot of ApoM and ApoA1 expression in ultracentrifuge-fractionated lipoproteins from plasma of 13-week-old mice. ( F – H ) S1P levels in 13-week-old db/db and db/+ control mice. ( F ) Total plasma S1P levels. ( G ) Plasma S1P distribution on ultracentrifuge-fractionated lipoproteins. ( H ) Percentage of plasma S1P distribution. n = 5–6/group. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by Student’s t test.

Article Snippet: For the adenovirus experiments, adult male mice were injected intravenously with murine ApoM adenovirus (Welgen) at 0.5 × 10 9 virus particles per gram of body weight, 8 days prior to euthanasia, and were fed a standard chow diet.

Techniques: Gene Expression, Control, Western Blot, Expressing, Clinical Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway

doi: 10.1172/JCI146219

Figure Lengend Snippet: ( A ) Hepatic Apom gene expression in adult male mice ( n = 5–8/group). ( B ) Representative Western blots of ApoM expression in liver lysates from adult male mice. C, littermate control mice; F, L-FoxO1,3,4 mice. ( C ) Hepatic Apom gene expression in mice of both sexes that were sacrificed on P2 ( n = 10–20/group). ( D ) Hepatic Apom gene expression following acute knockdown via AAV8.Tbg.Cre in adult male Foxo1 fl/fl , Foxo3 fl/fl , and Foxo4 fl/Y mice ( n = 5/group). Values are shown relative to littermate controls. ( E ) Schematic representation of FoxO1 protein. ( F – H ) Foxo1 , G6pc , and Apom gene expression in primary hepatocytes from WT mice that were transduced with different FoxO1 mutants. ( F ) FoxO1-ADA mutant: the 3 Akt phosphorylation sites are mutated, causing FoxO1 to be constitutively nuclear. Data indicate the mean ± SEM of triplicates of 3 independent experiments. ( G ) FoxO1-ADA-DBD mutant: contains the ADA mutation and a mutation disrupting the DNA binding domain. Data indicate the mean ± SEM of triplicates of 3 independent experiments. ( H ) FoxO1-Δ256 mutant: a dominant-negative version of FoxO1 that lacks the transactivation domain. ( I ) Chip-qPCR of Apom , Igfbp1 , and G6pc from livers of mice with a knockin allele of FoxO1-Venus. The mice were fed either chow or a HFD for 4 weeks. Data are presented as the mean ± SEM. * P < 0.05 and *** P < 0.001, by Student’s t test.

Article Snippet: For the adenovirus experiments, adult male mice were injected intravenously with murine ApoM adenovirus (Welgen) at 0.5 × 10 9 virus particles per gram of body weight, 8 days prior to euthanasia, and were fed a standard chow diet.

Techniques: Gene Expression, Western Blot, Expressing, Control, Knockdown, Transduction, Mutagenesis, Phospho-proteomics, Binding Assay, Dominant Negative Mutation, ChIP-qPCR, Knock-In

Journal: The Journal of Clinical Investigation

Article Title: Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway

doi: 10.1172/JCI146219

Figure Lengend Snippet: Chow-fed mice were injected intraperitoneally with gold-thioglucose or saline and were continued on a chow diet for 13 weeks. ( A ) Total body weight. ( B ) Plasma glucose levels after 5 hours fasting. ( C ) Plasma insulin levels after 5 hours fasting. ( D ) Hepatic Foxo1 , Apom , Scarb1 , and G6pc gene expression. ( E ) Representative Western blot of ApoM and ApoA1 expression in total plasma from chow-fed mice. CS, littermate control mice treated with saline; FS, L-FoxO1,3,4 mice treated with saline; CG, littermate control mice treated with gold-thioglucose; FG, L-FoxO1,3,4 mice treated with gold-thioglucose. * P < 0.05, ** P < 0.01, *** P < 0.001 versus control saline. ## P < 0.01 and ### P < 0.001 versus L-FoxO134-saline. && P < 0.01 versus control-GTG, by 2-way ANOVA. Data are presented as the mean ± SEM. ( n = 5–9/group for all panels)

Article Snippet: For the adenovirus experiments, adult male mice were injected intravenously with murine ApoM adenovirus (Welgen) at 0.5 × 10 9 virus particles per gram of body weight, 8 days prior to euthanasia, and were fed a standard chow diet.

Techniques: Injection, Saline, Clinical Proteomics, Gene Expression, Western Blot, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway

doi: 10.1172/JCI146219

Figure Lengend Snippet: ( A ) Representative Western blot of ApoM expression in total plasma from chow-fed mice ( n = 4/group). ( B ) Total S1P levels in plasma from chow-fed mice. Differences were evaluated by Student’s t tests. ( C ) Distribution of S1P in plasma fractionated by size-exclusion chromatography. Cholesterol levels in the control mice are shown as a reference to demonstrate the fractions in which HDL particles were eluted (gray shaded area). VLDL and LDL peaks are not shown, but were eluted in fractions approximately 25 and 35, respectively. ( D ) Distribution of S1P in lipoproteins fractions from mice fed a WTD for 3 weeks. Cholesterol levels from the control mice are shown as a reference to demonstrate the fractions in which HDL particles were eluted (gray shaded area). VLDL and LDL peaks are not shown, but were eluted in fractions approximately 21 and 31, respectively. ( E ) Western blot of ApoA1 and ApoM in lipoprotein fractions from chow diet–fed L-FoxO1,3,4 mice. ( F ) Western blot of ApoA1 and ApoM in lipoprotein fractions from Western diet–fed L-FoxO1,3,4 mice. ( G ) Plasma S1P distribution on ultracentrifuge-fractionated lipoproteins from control, CETP, L-FoxO1,3,4, and L-FoxO1,3,4:CETP mice. ( H ) Cholesterol efflux capacity of HDL isolated from Western diet–fed L-FoxO1,3,4 mice and littermate controls. Each sample is pooled HDL from 2 mice. * P < 0.05 versus control mice, by Kruskal-Wallis 1-way ANOVA; # P < 0.05 versus CETP mice, by Mann-Whitney U post hoc test. Data are presented as the mean ± SEM.

Article Snippet: For the adenovirus experiments, adult male mice were injected intravenously with murine ApoM adenovirus (Welgen) at 0.5 × 10 9 virus particles per gram of body weight, 8 days prior to euthanasia, and were fed a standard chow diet.

Techniques: Western Blot, Expressing, Clinical Proteomics, Size-exclusion Chromatography, Control, Isolation, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway

doi: 10.1172/JCI146219

Figure Lengend Snippet: Chow-fed mice were injected intravenously with murine ApoM adenovirus (0.5 × 10 9 virus particles/gram of body weight), 8 days prior to euthanasia. ( A ) Apom and Foxo1 gene expression in liver ( n = 4–8/group). ** P < 0.01 versus controls; # P < 0.05 versus L-FoxO1,3,4-GFP. ( B ) Western blot of expression of ApoM and ApoA1 from HDL fractionated by ultracentrifugation. ( C ) Total plasma S1P levels ( n = 5–6/group). ( D ) Plasma S1P distribution on ultracentrifuge-fractionated lipoproteins ( n = 3–8/group). * P < 0.05 versus control-GFP; # P < 0.05 versus L-FoxO1,3,4-GFP. ( E ) Intraperitoneal glucose tolerance test, 3 days prior to euthanasia ( n = 4–6/group). * P < 0.05, ** P < 0.01, and *** P < 0.001, control-GFP versus L-FoxO1,3,4. # P < 0.05, control-AdApoM versus L-FoxO1,3,4-AdApoM. Statistical significance was determined by Kruskal-Wallis 1-way ANOVA with the Mann-Whitney U post hoc test ( A and C – E ). Data are presented as the mean ± SEM.

Article Snippet: For the adenovirus experiments, adult male mice were injected intravenously with murine ApoM adenovirus (Welgen) at 0.5 × 10 9 virus particles per gram of body weight, 8 days prior to euthanasia, and were fed a standard chow diet.

Techniques: Injection, Virus, Gene Expression, Western Blot, Expressing, Clinical Proteomics, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Apolipoprotein M Inhibits Angiogenic and Inflammatory Response by Sphingosine 1-Phosphate on Retinal Pigment Epithelium Cells

doi: 10.3390/ijms19010112

Figure Lengend Snippet: Intravitreal ApoM injection inhibited choroidal neovascularization (CNV) growth in a murine model. ( a , b ) CNV was visualized in flat-mounted choroid by fluorescein in laser-induced CNV model mice treated with: vehicle ( a ); or 1 μM recombinant apolipoprotein M (ApoM) ( b ). The red ellipses outline the area of CNV. ( c ) CNV lesion volumes were significantly decreased in ApoM-treated mice compared with control mice. Each column represents mean SE, n = 8. Statistical analysis was performed using Unpaired Student’s t -test. p Values were labeled as ※ ( p < 0.05) vs. control (phosphate-buffered saline ).

Article Snippet: Recombinant human and murine ApoM was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Recombinant, Labeling, Saline