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Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

Marcone S, Haughton K, Simpson PJ, Belton O, Fitzgerald DJ - J Inflamm (Lond) (2015)

Bottom Line: Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis.The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ).We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: FHI, Food for Health Ireland, University College Dublin, Dublin, Ireland ; School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin, 4 Ireland.

ABSTRACT

Background: Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells.

Results: Treatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells.

Conclusions: These results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.

No MeSH data available.


Related in: MedlinePlus

TNF-α-induced adhesion molecules expression is reduced by sodium casein-derived peptides in EC. (A) VCAM-1, ICAM-1 and E-selectin gene expression was performed on EC treated with casein hydrolysate or control sample for 18 h, followed by 6 h stimulation with TNF-α (0.5 ng/ml). Cells were harvested in RLT buffer, RNA was extracted and reverse transcription was performed for RT-PCR analysis. (B) For the quantification of protein surface expression of adhesion molecules, flow cytometric analysis was performed on TNF-α activated EC (6 h, 0.5 ng/ml) pre-incubated with casein hydrosylate. Data are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was carried out using one-way ANOVA employing Dunnett correction for multiple comparisons. A statistical value of *P < 0.05 or greater was considered significant; $$$$ (p < 0.0001) vehicle vs. control; ****P < 0.0001, ***P < 0.001 and **P < 0.01 treatments vs. control (TNF-α activated EC).
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Fig2: TNF-α-induced adhesion molecules expression is reduced by sodium casein-derived peptides in EC. (A) VCAM-1, ICAM-1 and E-selectin gene expression was performed on EC treated with casein hydrolysate or control sample for 18 h, followed by 6 h stimulation with TNF-α (0.5 ng/ml). Cells were harvested in RLT buffer, RNA was extracted and reverse transcription was performed for RT-PCR analysis. (B) For the quantification of protein surface expression of adhesion molecules, flow cytometric analysis was performed on TNF-α activated EC (6 h, 0.5 ng/ml) pre-incubated with casein hydrosylate. Data are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was carried out using one-way ANOVA employing Dunnett correction for multiple comparisons. A statistical value of *P < 0.05 or greater was considered significant; $$$$ (p < 0.0001) vehicle vs. control; ****P < 0.0001, ***P < 0.001 and **P < 0.01 treatments vs. control (TNF-α activated EC).

Mentions: To examine the effect of the hydrolysate on adhesion molecule expression in EC stimulated by TNF-α, EC were pre-treated with the hydrolysate or with the control sample for 18 h before adding TNF-α (0.5 ng/ml) for 6 h. As shown in Figure 2A, TNF-α enhanced VCAM-1, ICAM-1 and E-sel mRNA while the pre-treatment with the hydrolysate significantly reduced the expression of VCAM-1 (by 23% ±3%, 51% ±7% and 75% ±2%), ICAM-1 (by 0% ±4%, 18% ±3% and 43% ±5%) and E-sel (by 30% ±2%, 62% ±1% and 87% ±4%) in a dose dependent manner (with 30, 150 and 300 μg/ml of hydrolysate, respectively). The control sample had no effect on the gene expression of the adhesion molecules (dose–response experiments using the control sample are reported in Additional file 2: Figure S2). To confirm the gene expression results, we analysed the cell surface expression of adhesion molecules in EC pre-treated with the hydrolysate or with the control sample for 18 h and subsequently with TNF-α (0.5 ng/ml) for 6 h. As shown in Figure 2B, flow cytometric analysis showed a significant dose dependent (30, 150, nand 300 μg/ml) decrease of EC surface protein levels of VCAM-1 (by 15% ±8%, 37% ±11% and 82% ±8%), ICAM-1 (by 5% ±4%, 9% ±3% and 61% ±13%) and E-sel (by 22% ±7%, 31% ±8% and 77% ±6%). The control sample had no significant effect on the protein expression of the adhesion molecules. Taken together, these results suggest that the ability of the sample to modulate the gene and the cell surface expression of adhesion molecules is due to the bioactive peptides contained in the hydrolysate generated by the bacterial hydrolysis of sodium caseinate (Figure 2A).Figure 2


Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

Marcone S, Haughton K, Simpson PJ, Belton O, Fitzgerald DJ - J Inflamm (Lond) (2015)

TNF-α-induced adhesion molecules expression is reduced by sodium casein-derived peptides in EC. (A) VCAM-1, ICAM-1 and E-selectin gene expression was performed on EC treated with casein hydrolysate or control sample for 18 h, followed by 6 h stimulation with TNF-α (0.5 ng/ml). Cells were harvested in RLT buffer, RNA was extracted and reverse transcription was performed for RT-PCR analysis. (B) For the quantification of protein surface expression of adhesion molecules, flow cytometric analysis was performed on TNF-α activated EC (6 h, 0.5 ng/ml) pre-incubated with casein hydrosylate. Data are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was carried out using one-way ANOVA employing Dunnett correction for multiple comparisons. A statistical value of *P < 0.05 or greater was considered significant; $$$$ (p < 0.0001) vehicle vs. control; ****P < 0.0001, ***P < 0.001 and **P < 0.01 treatments vs. control (TNF-α activated EC).
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Related In: Results  -  Collection

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Fig2: TNF-α-induced adhesion molecules expression is reduced by sodium casein-derived peptides in EC. (A) VCAM-1, ICAM-1 and E-selectin gene expression was performed on EC treated with casein hydrolysate or control sample for 18 h, followed by 6 h stimulation with TNF-α (0.5 ng/ml). Cells were harvested in RLT buffer, RNA was extracted and reverse transcription was performed for RT-PCR analysis. (B) For the quantification of protein surface expression of adhesion molecules, flow cytometric analysis was performed on TNF-α activated EC (6 h, 0.5 ng/ml) pre-incubated with casein hydrosylate. Data are expressed as mean ± SEM of 3 independent experiments. Statistical analysis was carried out using one-way ANOVA employing Dunnett correction for multiple comparisons. A statistical value of *P < 0.05 or greater was considered significant; $$$$ (p < 0.0001) vehicle vs. control; ****P < 0.0001, ***P < 0.001 and **P < 0.01 treatments vs. control (TNF-α activated EC).
Mentions: To examine the effect of the hydrolysate on adhesion molecule expression in EC stimulated by TNF-α, EC were pre-treated with the hydrolysate or with the control sample for 18 h before adding TNF-α (0.5 ng/ml) for 6 h. As shown in Figure 2A, TNF-α enhanced VCAM-1, ICAM-1 and E-sel mRNA while the pre-treatment with the hydrolysate significantly reduced the expression of VCAM-1 (by 23% ±3%, 51% ±7% and 75% ±2%), ICAM-1 (by 0% ±4%, 18% ±3% and 43% ±5%) and E-sel (by 30% ±2%, 62% ±1% and 87% ±4%) in a dose dependent manner (with 30, 150 and 300 μg/ml of hydrolysate, respectively). The control sample had no effect on the gene expression of the adhesion molecules (dose–response experiments using the control sample are reported in Additional file 2: Figure S2). To confirm the gene expression results, we analysed the cell surface expression of adhesion molecules in EC pre-treated with the hydrolysate or with the control sample for 18 h and subsequently with TNF-α (0.5 ng/ml) for 6 h. As shown in Figure 2B, flow cytometric analysis showed a significant dose dependent (30, 150, nand 300 μg/ml) decrease of EC surface protein levels of VCAM-1 (by 15% ±8%, 37% ±11% and 82% ±8%), ICAM-1 (by 5% ±4%, 9% ±3% and 61% ±13%) and E-sel (by 22% ±7%, 31% ±8% and 77% ±6%). The control sample had no significant effect on the protein expression of the adhesion molecules. Taken together, these results suggest that the ability of the sample to modulate the gene and the cell surface expression of adhesion molecules is due to the bioactive peptides contained in the hydrolysate generated by the bacterial hydrolysis of sodium caseinate (Figure 2A).Figure 2

Bottom Line: Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis.The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ).We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: FHI, Food for Health Ireland, University College Dublin, Dublin, Ireland ; School of Medicine and Medical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin, 4 Ireland.

ABSTRACT

Background: Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells.

Results: Treatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells.

Conclusions: These results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.

No MeSH data available.


Related in: MedlinePlus