Zihan Wang | ALES Graduate Seminar

Event details: 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 Zihan Wang. This seminar is open to the general public to attend.

Zoom Link: https://ualberta-ca.zoom.us/j/95305950240?pwd%3Df7bxg87EEqFkE9E5p2Jzmpz92XUGTN.1

PhD with Dr. Jianping Wu

Thesis Topic: Food Protein-Derived Peptides with Angiotensin-Converting Enzyme 2 (ACE2) Up-regulatory Activity

Abstract:

The renin-angiotensin system, particularly the enzymes angiotensin-converting enzyme (ACE) and ACE2, plays a central role in regulating blood pressure. ACE converts angiotensin I into angiotensin II (Ang II), which constricts blood vessels, while ACE2 counteracts this by converting Ang II into angiotensin (1-7), promoting vasodilation. Bioactive peptides, such as ovotransferrin-derived IRW, have shown in vitro ACE inhibitory effects and can lower blood pressure in spontaneously hypertensive rats (SHRs), though not through direct ACE inhibition. Transcriptomic analysis revealed a novel mechanism of ACE2 upregulation, which was further confirmed by using A779, a Mas receptor blocker that targets the ACE2 pathway. The overall objective of this thesis is to explore additional ACE2 up-regulatory peptides with blood pressure-lowering effect and to investigate the mechanisms underlying this effect. The bioavailability and metabolism of the most promising peptide were studied.

In the first study, nearly 200 ACE inhibitory (ACEi) peptides were subjected to molecular docking. Of these, 20 peptides were selected for cell-based and enzymatic assays, and 5 were administered orally to SHRs (15 mg/kg/day for 7 days). Peptides IKW and RIY showed significant antihypertensive activity, activating circulating and aortic ACE2, increasing circulating Ang (1-7) levels, and decreasing Ang II levels. Peptide IQY reduced blood pressure and increased Ang (1-7) level, but had no effect on ACE and ACE2. Peptides MAW and MRW did not affect blood pressure, ACE, or ACE2. This study showed that ACE2 activation is not a common feature of ACEi peptides.

The second study further investigated the mechanisms underlying the antihypertensive effects of IKW and RIY. Both peptides reduced vascular inflammation and oxidative stress by modulating the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, lowering aortic nitrotyrosine and intracellular calcium levels, and shifting the oxylipin profile toward anti-inflammatory species. Peptide treatments in VSMCs under Ang II stimulation reduced reactive oxygen species, intracellular calcium, IL-1β, and COX2 levels. COX2 inhibition was partially mediated by upregulation of ACE2 and sirtuin-1 (SIRT1), with SIRT1 contributing to ACE2 expression, as knocking down ACE2 or blocking SIRT1 diminished these effects. These findings support that the anti-inflammatory and antioxidative effects of IKW and RIY in SHRs and Ang II-stimulated VSMCs.

Next, the bioavailability of IRW was assessed due to its stronger ACE2 up-regulatory effect. IRW (100 mg/kg) was administered orally or intravenously to rats. The bioavailability (F%) was determined to be 11.7%, and the half-lives were 7.9 ± 0.5 and 28.5 ± 6.8 min for gavage and injection, respectively. Interestingly, significant blood pressure reduction was not observed until 1.5 h post oral administration, or 2 h post injection, indicating that the peptide’s fragments are likely responsible for the blood pressure-lowering activity. Time-course metabolomics revealed a significant increase in kynurenine, a tryptophan metabolite, in blood after IRW administration. Kynurenine increased ACE2 in cells. Oral administration of tryptophan (W), but not dipeptide IR, lowered blood pressure and upregulated ACE2 in SHRs. Our study supports the key role of tryptophan and its metabolite, kynurenine, in IRW’s blood pressure-lowering effects.

The metabolomic profile after long-term IRW treatment was analyzed to explore how metabolites influence ACE2 transcription. Transcriptomic analysis of mesenteric arteries revealed 12,764 mRNAs, with 651 co-expressed with ACE2. By combining weighted gene co-expression network analysis (WGCNA) with transcription factor predictions, 17 potential ACE2 transcription factors were identified, including nuclear receptor subfamily 4 group A member 1 (Nr4a1). Concurrent metabolomic analysis identified 421 metabolites, with dopamine showing a significant increase following IRW treatment (fold change of 3.66, p = 0.037). Additionally, 26 metabolites, including dopamine, displayed expression levels similar to ACE2, indicating their potential role in ACE2 regulation. Ingenuity pathway analysis suggested that dopamine might activate Nr4a1 transcription via the dopamine D1 receptor (D1R). This was validated in EA.hy926 endothelial cells, where dopamine (1 μM) significantly increased ACE2 and Nr4a1 expression, an effect blocked by the D1R antagonist SCH23390. Nr4a1 knockdown reduced dopamine-induced ACE2 protein expression. In SHRs, D1R dependency was further confirmed as SCH23390 infusion reduced the antihypertensive effects of IRW, as well as IRW-induced upregulation of ACE2 and D1R. Although IRW lacks tyrosine, the rise in dopamine was attributed to upregulation of DOPA decarboxylase and its cofactor pyridoxal 5’-phosphate.

This research enhances understanding of how IRW regulates ACE2 in vivo. These findings open new avenues for studying bioactive peptides in ACE2-associated chronic diseases, with the application of omics technologies offering innovative insights into their mechanisms.