Signatures of anthocyanin metabolites identified in humans inhibit biomarkers of vascular inflammation in human endothelial cells

Abstract

Scope: The physiological relevance of contemporary cell culture studies is often perplexing, given the use of unmetabolized phytochemicals at supraphysiological concentrations. We investigated the activity of physiologically relevant anthocyanin metabolite signatures, derived from a previous pharmacokinetics study of 500 mg ¹³ C₅ -cyanidin-3-glucoside in eight healthy participants, on soluble vascular adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6) in human endothelial cells.

Methods and results: Signatures of peak metabolites (previously identified at 1, 6, and 24 h post-bolus) were reproduced using pure standards and effects were investigated across concentrations ten-fold lower and higher than observed mean (<5 μM) serum levels. Tumor necrosis factor-α (TNF-α)-stimulated VCAM-1 was reduced in response to all treatments, with maximal effects observed for the 6 and 24 h profiles. Profiles tested at ten-fold below mean serum concentrations (0.19-0.44 μM) remained active. IL-6 was reduced in response to 1, 6, and 24 h profiles, with maximal effects observed for 6 h and 24 h profiles at concentrations above 2 μM. Protein responses were reflected by reductions in VCAM-1 and IL-6 mRNA, however there was no effect on phosphorylated NFκB-p65 expression.

Conclusion: Signatures of anthocyanin metabolites following dietary consumption reduce VCAM-1 and IL-6 production, providing evidence of physiologically relevant biological activity.

Keywords: Adhesion; Anthocyanin; Inflammation; Metabolism.

Figure 1

Serum pharmacokinetic signatures of C3G and its metabolites in humans after the consumption of 500 mg ¹³C₅‐C3G in eight healthy male participants. Data represent mean concentration of specified metabolites from eight participants. Peak signatures (at 1, 6, and 24 h) are indicated by the dashed‐line boxes. “/” indicates isomers quantified together. Common name (chemical name): 4‐hydroxybenzyldehyde (4‐hydroxybenzoicaldehyde); Benzoic acid‐4‐glucuronide (benzoic acid‐4‐O‐glucuronide); Cyanidin‐3‐glucoside (2‐(3,4‐dihydroxyphenyl)‐5,7‐dihydroxy‐3‐chromeniumyl‐β‐D‐glucopyranoside); Ferulic acid (4‐hydroxy‐3‐methoxycinnamic acid); Hippuric acid (N‐benzoylglycine); Isovanillic acid (3‐hydroxy‐4‐methoxybenzoic acid); Isovanillic acid‐3‐glucuronide (4‐methoxybenzoic acid‐3‐O‐glucuronide); Isovanillic acid‐3‐sulfate (4‐methoxybenzoic acid‐3‐sulfate); Phloroglucinaldehyde (2,4,6‐trihydroxybenzaldehyde); Protocatechuic acid (3,4‐dihydroxybenzoic acid); Protocatechuic acid‐3‐sulfate (4‐hydroxybenzoic acid‐3‐sulfate); Protocatechuic acid‐4‐glucuronide (3‐hydroxybenzoic acid‐4‐O‐glucuronide); Protocatechuic acid‐4‐sulfate (3‐hydroxybenzoic acid‐4‐sulfate); Vanillic acid (3‐methoxy‐4‐hydroxybenzoic acid); Vanillic acid‐4‐glucuronide (3‐methoxybenzoic acid‐4‐O‐glucuronide); Vanillic acid‐4‐sulfate (3‐methoxybenzoic acid‐4‐sulfate). Adapted from de Ferrars et al. 11.

Figure 2

Effect of peak metabolite signatures on TNF‐α stimulated VCAM‐1 secretion by A) HUVEC, B) HCAEC. Cells were treated with three concentrations of three serum metabolite profiles (representing ten‐fold lower and ten‐fold higher concentrations than the mean concentrations observed by Czank et al. 8; Table 1) prior to the addition of 10 or 0.1  ng/mL TNF‐α for 24 h. Data were normalized to a TNF‐α control (no DMSO) and columns represent the mean ± SD, n = 3 biological replicates. Labeled means without a common letter differ, p ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, p ≤ 0.05 (t‐test). Abbreviations: HUVEC, human umbilical vein endothelial cells; TNF‐α, tumor necrosis factor‐α; VCAM‐1, soluble vascular adhesion molecule‐1.

Figure 3

Effect of peak metabolite signatures on TNF‐α stimulated IL‐6 secretion by A) HUVEC, B) HCAEC. Cells were treated with three concentrations of three serum metabolite profiles (representing ten‐fold lower and ten‐fold higher concentrations than the mean concentrations observed by Czank et al. 8; Table 1) prior to the addition of 10 or 0.1  ng/mL TNF‐α for 24 h. Data were normalized to a TNF‐α control (no DMSO) and columns represent the mean ± SD, n = 3 biological replicates. Labeled means without a common letter differ, p ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, p ≤ 0.05 (t‐test). Abbreviations: HCAEC, human coronary artery endothelial cells; TNF‐α, tumor necrosis factor‐α; IL‐6, interleukin‐6.

Figure 4

Effect of peak metabolite signatures on TNF‐α stimulated VCAM‐1 and IL‐6 mRNA expression in HCAEC (A) VCAM‐1, (B) IL‐6. Cells were treated with the highest concentration signature metabolites (19, 20, 44 μM reflecting 1, 6, and 24 h serum profiles respectively; Czank et al. 8) and stimulated with 0.1  ng/mL TNF‐α for 4 h. Amplification values were normalized to the geometric mean of two stable reference genes, VIPAS39 and PRDM4. Data were normalized to a TNF‐α control (no DMSO) and columns represent the mean ± SD, n = 3 biological replicates. Labeled means without a common letter differ significantly, p ≤ 0.05 (ANOVA with post hoc LSD). *Different from DMSO, p ≤ 0.05 (t‐test). Abbreviations: HCAEC, human coronary artery endothelial cells; TNF‐α, tumor necrosis factor‐α; IL‐6, interleukin‐6; VCAM‐1, vascular adhesion molecule‐1.

Figure 5

Effect of peak metabolite signatures on TNF‐α stimulated phosphor‐NFκB p65 expression in HCAEC. Cells were treated with the highest concentration signature metabolites (19, 20, 44 reflecting 1, 6, and 24 h serum profiles respectively; Czank et al. 8), and stimulated with 10  ng/mL TNF‐α for 15 min. Data were normalized to the vehicle control (DMSO) and columns represent the mean ± SD, n = 3 biological replicates. Blots are representative of one of three replicates. Density values were normalized to reference protein, GAPDH. Labeled means without a common letter differ significantly, p ≤ 0.05 (ANOVA with post hoc LSD). Comparisons of untreated cells to vehicle control (DMSO) were established via Student's t‐test, ^* p ≤ 0.05. Abbreviations: HCAEC, human coronary artery endothelial cells; TNF‐α, tumor necrosis factor‐α.