Hongbing Fan | ALES Graduate Seminar

Date(s) - 20/04/2021
8:00 am - 9:00 am

A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES PhD Final Exam Seminar by Hector Perez Marquez. This seminar is open to the general public to attend.


Meeting ID: 948 2921 1857
Passcode: 312978

Thesis Topic: Developing Antihypertensive Peptides from Spent Laying Hen Muscle Proteins

PhD with Dr. Jianping Wu

Seminar Abstract:

Hypertension is the leading cause of global morbidity and mortality, afflicting > 20% of adults worldwide (~1 in 4 in Canada). Pharmaceutical drugs are the primary treatment for hypertension but are generally associated with various side effects. Recently, food-derived antihypertensive peptides have been emerging alternatives for treating hypertension. Central to the pathophysiology of hypertension is the hyperactive renin-angiotensin system, specifically, the overproduced angiotensin (Ang) II, which is regulated by two enzymes, angiotensin-converting enzyme (ACE) and ACE2. ACE converts Ang I into Ang II that elevates blood pressure (BP) via Ang II type 1 receptor (AT1R), whereas ACE2 reverts this process by degrading Ang II into Ang (1–7) followed by binding with Mas receptor (MasR).

Spent hens are laying hens that have reached the end of the egg-laying cycle; there is an average yearly production of > 30 million in the Canadian egg industry over the last decade. Despite being a byproduct, spent hen is a rich source of muscle proteins that can be biotransformed into bioactive peptides with antihypertensive properties. The overall objective of this thesis is to purify and characterize novel antihypertensive peptides from spent hen muscle proteins targeting both the ACE-Ang II-AT1R (by inhibiting ACE) and the ACE2-Ang (1-7)-MasR (by upregulating ACE2) axes.

In the first study, eighteen spent hen muscle protein hydrolysates (SPHs) were prepared and an in vitro screening process was performed using either a conventional approach based on ACE inhibitory (ACEi) activity solely, or a multiple evaluation approach that considers ACEi, ACE2 upregulating (ACE2u), antioxidant, and anti-inflammatory activities, as well as their fates in the gastrointestinal tract. Three SPHs were screened and fed to spontaneously hypertensive rats (SHR); only SPH prepared by thermoase PC10F (SPH-T) reduced BP significantly. The second study further investigated the antihypertensive effect of SPH-T at two doses (high dose, 1,000 mg/kg body weight [BW], and low dose, 250 mg/kg BW). The associated BP reduction was possibly due to increased circulating ACE2 and Ang (1-7) but decreased Ang II levels, upregulated vascular ACE2 expression, as well as ameliorated vascular inflammation, oxidative stress, and fibrosis.

The third study aimed to purify ACEi peptides and ACE2u peptides from SPH-T. Finally, five potent ACEi peptides, VRP, LKY, VRY, KYKA, and LKYKA (IC50 values of 0.034–5.77 μg/mL) and four ACE2u peptides, VAQWRTKYETDAIQRTEELEEAKKK, VHPKESF, VVHPKESF (V-F), and VKW (upregulated ACE2 expression by 0.52–0.84 folds) were identified. Among them, four with either or both highest activities and from the major muscle proteins were selected for animal study; they were categorized into 3 groups: VRP (ACEi activity), V-F (ACE2u activity), and LKY and VRY (both ACEi and ACE2u activities).

Prior to assessing the in vivo efficacies of VRP, LKY, VRY, V-F, their antioxidant and anti-inflammatory effects were evaluated in vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs), upon stimulation by Ang II and tumor necrosis factor alpha (TNFa), respectively. All four peptides showed antioxidant activity in VSMCs, whereas only V-F attenuated inflammation, manifested by inhibiting expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) in VSMCs and VCAM-1 expression in ECs. VRP, LKY, and VRY exhibited antioxidant activity by acting as direct free radical scavengers whereas V-F also activated endogenous antioxidant enzymes. The anti-inflammatory effect of V-F was likely through modulation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase pathways, and that in VSMCs was partially dependent on MasR.

After orally administering VRP, LKY, VRY, and V-F to SHRs, only V-F significantly reduced BP at the daily dose of 15 mg/kg BW. Associated with BP reduction were the increased circulating ACE2 and Ang (1-7) but reduced Ang II levels, upregulated vascular ACE2 and MasR expressions, and attenuated vascular inflammation and oxidative stress. Notably, V-F was not gastrointestinal stable and its fragment after tryptic digestion, VVHPK, was also an ACE2u peptide.

This work demonstrated the presence of antihypertensive peptides in spent hen muscle proteins, providing evidence for processing spent hens into antihypertensive functional food ingredients. Furthermore, this study reported for the first time the antihypertensive effect of an ACE2u peptide (V-F) identified using in vitro methods. The discovery and characterization of ACE2u peptides manifested their feasibilities of being isolated from food proteins and uses for hypertension management.