Cardioprotective Peptides from Dry-Cured Ham in Primary Endothelial Cells and Human Plasma: An Omics Approach

Abstract

Cardiovascular diseases are a leading cause of mortality, driving the search for alternative preventive strategies. This study investigates the antioxidant effects, among others, of a mixture of four bioactive peptides (BPs) derived from dry-cured pork ham on endothelial cells from healthy (C-HUVECs) and gestational diabetes (GD-HUVECs) pregnancies, as well as human plasma, using an integrative omics approach. Human umbilical vein endothelial cells (HUVECs) were treated with 300 μM purified BP, followed by transcriptomic and proteomic analyses. The results revealed significant alterations in mitochondrial gene expression and downregulation of genes associated with inflammation and oxidative stress in healthy HUVECs. Furthermore, BP treatment modulated key signalling pathways, including Ras and MAPK, leading to changes in the phosphorylation of ERK, AKT, and NF-κB, suggesting potential cardioprotective effects. The effects of BP were compared to those of the antioxidant hydroxytyrosol, highlighting their relative efficacy in vascular protection. The proteomic analysis of human plasma demonstrated BP-induced modulation of lipid metabolism, inflammation, and oxidative stress with notable changes in proteins such as APOA1 and MMP-8. These natural compounds demonstrate significant preventive potential in vascular health, highlighting their promise as effective tools for reducing cardiovascular risk before the progression of the pathology. These findings emphasize the importance of integrative omics in understanding the mechanisms behind BP's effects and suggest promising applications for nutraceuticals aimed at cardiovascular protection.

Keywords: ROS; bioactive peptides; cardiovascular health; endothelial dysfunction; hydroxytyrosol; omics analysis; oxidative stress.

Figure 1

Expression of inflammatory markers measured by RT-qPCR after 2, 6, and 24 h of 300 µM BP, and/or 1 ng/mL TNF-α in C-HUVECs. (A) VCAM-1 and (B) TNF-α levels. Fold induction for each gene was calculated versus the control at the corresponding time point. Results represent the average ± SD of three independent experiments. Statistical significance was determined via ANOVA followed by Tukey’s multiple comparison test. (*) Significantly different from control. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.005.

Figure 2

Protein expression levels of p-AKT, p-ERK, and p-NF-kB were analysed by Western blot in (A) C-HUVECs and (B) GD-HUVECs after 1 ng/mL TNF-α treatment for 6 h and/or 300 µM BP. The ratio of densitometries of p-AKT, p-ERK, and p-NF-kB to β-actin expresses the relative quantitative expression. The Western blot has been manually cut to show these specific bands.

Figure 3

FunRich analysis. The possible (A) biological processes and (B) biological pathways performed on plasmatic proteins identified by the proteomic analysis. FunRich: Functional Enrichment tool.

Figure 4

Protein–protein interactions and clusters of identified proteins by proteomic approaches (STRING database). The figure highlights the connections between differentially represented proteins. Proteins include collagen type XIII alpha 1 chain (COL13A1), Mitogen-activated protein kinase 12 (MAP3K12), Apolipoprotein A1 (APOA1), Epidermal Growth Factor (EGF), Phosphatidylinositol-4-Phosphate 3-Kinase (PIK3C2A), among others. Nodes represent proteins while the association between proteins are indicated by lines.