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Abstract: Hypertension is a worldwide health challenge, afflicting about 25% of adults. Antihypertensive drugs are available, but prolonged use is associated with side-effects. Food protein-derived antihypertensive peptides have gained substantial interests during the past decades as a natural substitute to antihypertensive drugs. Given the central role of angiotensin converting enzyme (ACE) in generating angiotensin II (Ang II), a potent vasoconstrictor, most antihypertensive peptides have been characterized as ACE-inhibitory peptides by in vitro chemical assay. However, results from a mechanistic study indicated ACE-inhibitory peptides might have other targets in addition to ACE in vivo.Notably, our previous study found that egg white ovotransferrin-derived ACE-inhibitory peptide IRW (Ile-Arg-Trp) could up-regulate mRNA level of angiotensin converting enzyme 2 (ACE2) in mesenteric artery of spontaneously hypertensive rats (SHRs). ACE2 is a homolog of ACE, but functions in an opposite way. Ang II could be cleaved by ACE2 into angiotensin (1-7) (Ang (1-7)) and protective effects could be further exerted via mas receptor (MasR). ACE2 has been considered as a novel target for cardiovascular therapy.With the discovery of the potential of IRW in targeting ACE2, the specific objectives of this thesis were to 1) evaluate the ACE2-activating potential of IRW in different systems. 2) to test the beneficial effects of IRW-mediated ACE2-activation and up-regulation in VSMCs and SHRs. 3) to identify peptides which can up-regulate ACE2 from food protein sources.IRW could increase activity of human recombinant ACE2 in an in vitro chemical assay. ACE2 activity and expressions in A7r5 cells, a VSMC cell-line, were increased by IRW treatment. Protein expressions of ACE2 in aorta and kidney of SHRs were also enhanced by IRW. Beneficial effects of IRW on intracellular events of VSMCs and the relationship with ACE2 up-regulation were studied using Ang II-stimulated A7r5 cells. Results suggested that IRW treatment could inhibit aberrant proliferation and abrogate oxidative stress in Ang II-stimulated A7r5 cells. Expressions of cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS) and metalloproteinase 9 (MMP9) were down-regulated by IRW treatment in Ang II-stimulated A7r5 cells, indicating anti-inflammatory and anti-migrant activities of IRW in VSMCs. Signaling pathways including nuclear factor κB (NF- κB) and p38 mitogen-activated protein kinases (P38 MAPK) were involved in the modulatory effects of IRW in Ang II-stimulated A7r5 cells. Further study found that IRW treatment increased the levels of ACE2 and MasR in Ang II-stimulated cells. While, ACE2 knockdown and the addition of MasR antagonist abolished the antioxidant and anti-inflammatory activities of IRW, demonstrating beneficial effects from IRW-mediated ACE2 up-regulation at least in cellular level.The contribution of ACE2-activation by IRW treatment to blood pressure reduction was evaluated in SHRs. Infusion of MasR antagonist A779 (48 µg/kg body weight/h) in SHRs abolished blood pressure-reducing effect of IRW, indicating the hypotensive effect of IRW in SHRs was via the ACE2/Ang (1-7)/MasR axis. Further analysis found that IRW oral administration (15 mg/kg body weight) increased circulating levels of ACE2 and Ang (1-7) as well as decreased circulating Ang II level. However, ACE level was unaffected. Infusion of MasR antagonist also affected the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signaling and expressions of inhibitory κB α (IκBα) and COX2 in aorta of SHRs, suggesting that enhanced endothelium-dependent vasorelaxation and reduced vascular inflammation were underlying the mechanisms of IRW as an ACE2 activator in SHRs.Pea protein was used for identification of ACE2 up-regulating peptide after thermoase and pepsin digestion. The peptide was characterized based on activity-guided fractionation. A peptide with the sequence of AKSLSDRFSY was characterized by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Activity of AKSLSDRFSY in up-regulating ACE2 expression was validated in A7r5 cells. It was found that AKSLSDRFSY up-regulated ACE2 and MasR expressions, but down-regulated COX2 expression in Ang II-stimulated A7r5 cells. While, the addition of MasR antagonist could restore the expression of COX2, suggesting the AKSLSDRFSY-mediated ACE2/MasR up-regulations in A7r5 cells could further contribute to anti-inflammatory potential of this peptide.Taken together, the present study demonstrated the ACE2-activating effect of egg white-derived tripeptide IRW. Besides, a peptide with the sequence of AKSLSDRFSY was identified from pea protein, which could increase the level of ACE2. Findings from this study advance the knowledge on molecular targets of food protein-derived antihypertensive peptides.