Increased level of chronic inflammation in the blood vessels, especially in the vascular endothelial cells contributes to the pathogenesis of various cardiovascular diseases such as atherosclerosis. Dietary peptide, γ-glutamyl valine (γ-EV) exhibited an anti-inflammatory effect via the allosteric activation of the calcium-sensing receptor (CaSR). CaSR is a G-protein coupled trans-membrane receptor, widely present across various mammalian tissues, including blood vessels. Hence, the present study investigates the role of γ-EV in modulating vascular inflammation via CaSR dependent pathways and identifies the key signaling molecules involved in the anti-inflammatory effect. Additionally, the study also explores the transport efficiency and route of transport of γ-EV across the intestinal epithelial cells. Human tumor necrosis factor-alpha (TNF-alpha: 5ng/mL, 6h) was used to induce inflammation in human aortic endothelial cells (HAECs), pre-treated with γ-EV at 0.01 mM, 0.1 mM, and 1 mM for 2 h, to elucidate the efficacy of γ-EV in reducing vascular inflammation. The intervention of a CaSR specific antagonist, NPS-2143, was used to confirm the role of CaSR in the anti-inflammatory effect. The transport efficiency of γ-EV was evaluated through a monolayer of intestinal epithelial cells (Caco-2) and the route of transport was evaluated by using specific transport inhibitors such as wortmannin (transcytosis), theaflavin 3’-Gallate (paracellular), and Gly-Sar (PepT1 transporter). TNF-alpha-induced increased expression of inflammatory adhesion molecules (VCAM-1, E-Selectin), chemokine (MCP-1), and cytokines (IL-6 and IL-8), was significantly downregulated by 1 mM γ-EV. Additionally, the anti-inflammatory effect of γ-EV was attenuated entirely by the intervention of NPS-2143. Further investigation identified the role of β-arrestin2, CREB, and p65, in the CaSR-dependent anti-inflammatory effect of γ-EV. Furthermore, the apparent permeability coefficient of γ-EV, across Caco-2 cells, was found to be 1.56 x 10-6 cm/sec and the transport was both paracellular and PepT1-mediated. Hence, γ-EV can be potentially used to develop therapeutics for the prevention and management of CVDs.

Biological activity of dietary γ-glutamyl valine (γ-EV) in reducing vascular inflammation and its molecular mechanisms of action

ACS Fall 2021

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