Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces expression of proinflammatory cell adhesion molecules and monocyte chemoattractant protein-1 in human endothelial cells in a dose-dependent manner. (A) Representative Coomassie blue staining and Western blot analysis of purified recombinant human holo-RBP4 (1 μg per lane). Lane 1, Coomassie blue-stained gel; lane 2, Western blot with RBP4 antibody. (B) UV spectrum analysis of purified recombinant holo-RBP4 demonstrates a retinol/RBP4 ratio of approximately 0.9, indicating that the majority of the purified RBP4 consists of holo-RBP4. (C to F) Quantitative RT-PCR analysis for mRNA expression of VCAM-1, ICAM-1, E-selectin, and MCP-1 in human retinal capillary endothelial cells (HRCEC) treated with increasing concentrations of holo-RBP4 (10 to 100 μg/ml) or BSA or left untreated (Unt.). Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance (ANOVA) with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Staining, Western Blot, Purification, Recombinant, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NADPH oxidase protein expression and stimulates proinflammatory protein expression via an NADPH oxidase-dependent mechanism in HRCEC. (A and B) HRCEC were treated with BSA or increasing concentrations of holo-RBP4 for 24 h, and cell lysates were analyzed by Western blotting for Nox2 (A) and Nox4 (B). (C through H) HRCEC were pretreated with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (C and D) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of DPI (C) or apocynin (D). (E through H) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (E), sICAM-1 (F), MCP-1 (G), and E-selectin (H) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases cellular and extracellular protein levels of proinflammatory molecules in HRCEC in a dose- and time-dependent manner. (A to C) Western blots of VCAM-1 (A), E-selectin (B), and ICAM-1(C) from HRCEC treated with BSA or increasing concentrations of RBP4 for 24 h. (D to F) Western blots of VCAM-1 (D), E-selectin (E), and ICAM-1 (F) from HRCEC treated with RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. (G to J) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (G), E-selectin (H), sICAM-1 (I), and MCP-1 (J) from HRCEC media following treatment with increasing concentrations of holo-RBP4 or BSA or no treatment (Unt.). (K to N) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (K), E-selectin (L), sICAM-1 (M), and MCP-1 (N) in HRCEC media following treatment with holo-RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases leukocyte adherence to human endothelial cells. Confluent monolayers of HRCEC were treated with either holo-RBP4 (100 μg/ml), BSA, or TNF-α (100 ng/ml) for 18 h. THP-1 monocytes were then added and cocultured for 3 h. (A) Representative phase-contrast images (magnification, ×20) of monocyte adherence to HRCEC after the indicated treatment. Adherent monocytes are apparent as small bright circular bodies above the HRCEC layer. (B) Adherent monocytes were counted per visual field at magnification of ×20. The graph shows the means ± standard deviations from 4 different visual fields for each treatment group (adherent leukocytes in TNF-α positive control were 205 ± 25 per visual field). ***, P < 0.001 versus BSA treatment by one-way ANOVA with Tukey's post hoc test. Untreat, untreated cells.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Positive Control

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NF-κB and stimulates proinflammatory protein expression at least partially through an NF-κB-dependent mechanism in HRCEC. (A) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HRCEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h; untreat, untreated cells. (B) The relative nuclear transcriptional activity of NF-κB p65 was quantified using the ELISA-based TransAM NF-κB assay. HRCEC were treated with BSA or holo-RBP4 (100 μg/ml) for 6 h, and nuclear extracts were prepared and analyzed. Western blots of beta-actin, GAPDH, and fibrillarin demonstrate the enrichment and quality of nuclear extract preparations. Lane C, cytosolic extract; lane N, nuclear extract (10 μg protein/lane). (C) HRCEC were treated with either BSA or holo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (D through I) HRCEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (D through E) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of PDTC (D) or JSH-23 (E). (F through I) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (F), sICAM-1 (G), MCP-1 (H), and E-selectin (I) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. Significant difference compared to BSA treatment (panels B and C): †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Translocation Assay, Immunocytochemistry, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated activation of NF-κB, NADPH oxidase, and proinflammatory molecules is retinol independent. (A) Quantitative RT-PCR analysis of STRA6 mRNA expression in HRCEC following 24 h of treatment with RBP4 (100 μg/ml) or BSA or no treatment (Untr.). STRA6 mRNA expression is shown relative to HPRT housekeeping gene expression and is 20-fold less abundant than HPRT. (B) STRA6 protein expression was undetectable by Western blotting of HRCEC. HEK-293A cells stably expressing STRA6 were also analyzed as a positive control. (C) HRCEC intracellular retinoid content following treatment with holo-RBP4 (100 μg/ml) or an equimolar amount (4.75 μM) of retinol, retinal, or retinoic acid. HRCEC did not uptake a significant amount of retinol from holo-RBP4. (D and E) Western blots of VCAM-1 and Nox2 (D) or Nox4 (E) in HRCEC treated with increasing concentrations of apo-RBP4 for 24 h. (F) HRCEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (G through J) ELISA-based quantification of soluble extracellular levels of MCP-1 (G), sVCAM-1 (H), sICAM-1 (I), and E-selectin (J) in HRCEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Gene Expression, Western Blot, Stable Transfection, Positive Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces NF-κB, NADPH oxidase, and proinflammatory molecules in human umbilical vein endothelial cells. (A through C) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (A), sICAM-1 (B), and MCP-1 (C) in HUVEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. (D) Western blots of E-selectin and Nox2, VCAM-1, and Nox4 in HUVEC treated with increasing concentrations of holo-RBP4 for 24 h. (E) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HUVEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h. Untreat, untreated cells. (F) HUVEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test; †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Translocation Assay, Immunocytochemistry, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated induction of proinflammatory proteins in HUVEC is via activation of NADPH oxidase and NF-κB. (A through C) HUVEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) or with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. ELISA-based quantification of soluble extracellular levels of sICAM-1 (A), sVCAM-1 (B), and MCP-1 (C) in HUVEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA; †, P < 0.01 by Student's t test. (D and E) Western blots of VCAM-1 in HUVEC following RBP4 treatment in the presence or absence of PDTC (D) or DPI (E).

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot