Tripeptide IRW Protects MC3T3-E1 Cells against Ang II Stress in an AT2R Dependent Manner.

Multiple strategies including the use of bioactive peptides and other nutraceuticals are being adopted to maintain bone health. This study provides an improved and deeper understanding of the pharmacological effects that a bioactive peptide IRW (Ile-Arg-Trp) extends on bone health. Our results showed that IRW treatment protects osteoblasts against Ang II induced decline in cell proliferation and restores protein levels of collagen type I alpha 2 chain (COL1A2) and alkaline phosphatase (ALP) levels in MC3T3-E1 cells (p < 0.05). Apart from augmentation of these mineralization factors, the angiotensin II (Ang II) induced apoptotic stress in osteoblasts was mitigated by IRW as well. At the molecular level, IRW abolished the cytochrome-c release via modulation of pro-and anti-apoptotic genes in MC3T3-E1 cells (p < 0.05). Interestingly, IRW also increased cellular levels of cytoprotective local RAAS factors such as MasR, Ang (1−7), ACE2, and AT2R, and lowered the levels of Ang II effector receptor (AT1R). Further, our results indicated a lower content of inflammation and osteoclastogenesis biomarkers such as cyclooxygenase 2 (COX2), nuclear factor kappa B (NF-κB), and receptor activator of nuclear factor kappa-B ligand (RANKL) following IRW treatment in MC3T3-E1 cells (p < 0.05). The use of an antagonist-guided cell study indicated that IRW contributed to the process of cytoprotection and proliferation of osteoblasts via Runt-related transcription factor 2 (RUNX2) in face of Ang II stress in an AT2R dependent manner. The key findings of our study showed that IRW could potentially have a therapeutic role in the treatment and/or prevention of bone disorders.

Nicotinamide Phosphoribosyltransferase as a Key Molecule of the Aging/Senescence Process.

Aging is a phenomenon underlined by complex molecular and biochemical changes that occur over time. One of the metabolites that is gaining strong research interest is nicotinamide adenine dinucleotide, NAD+, whose cellular level has been shown to decrease with age in various tissues of model animals and humans. Administration of NAD+ precursors, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), to supplement NAD+ production through the NAD+ salvage pathway has been demonstrated to slow down aging processes in mice. Therefore, NAD+ is a critical metabolite now understood to mitigate age-related tissue function decline and prevent age-related diseases in aging animals. In human clinical trials, administration of NAD+ precursors to the elderly is being used to address systemic age-associated physiological decline. Among NAD+ biosynthesis pathways in mammals, the NAD+ salvage pathway is the dominant pathway in most of tissues, and NAMPT is the rate limiting enzyme of this pathway. However, only a few activators of NAMPT, which are supposed to increase NAD+, have been developed so far. In this review, we will focus on the importance of NAD+ and the possible application of an activator of NAMPT to promote successive aging.

Egg white hydrolysate and peptide reverse insulin resistance associated with tumor necrosis factor-α (TNF-α) stimulated mitogen-activated protein kinase (MAPK) pathway in skeletal muscle cells.

PURPOSE: Excessive formation of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, has been implicated in the development of insulin resistance in obesity and type-2 diabetes. In skeletal muscle, chronic exposure to TNF-α impairs insulin-stimulated glucose uptake and insulin signaling. The aim of this study is to investigate the effects of enzymatic egg white hydrolysate (EWH) and its responsible peptide, IRW, on TNF-α-induced insulin resistance and the underlying molecular mechanisms using rat skeletal muscle cells (L6 cells). METHODS: Insulin resistance was induced by treating L6 cells with 5 ng/ml TNF-α for 24 h. Effects of EWH and IRW on glucose uptake were detected by glucose uptake assay, glucose transporter 4 (GLUT4) translocation by immunofluorescence, and western blot, while insulin-signaling pathway and mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. RESULTS: Adding both EWH and IRW significantly improved glucose uptake in TNF-α-treated cells, increased activation of insulin receptor substrate (IRS-1) tyrosine residue and protein kinase B (Akt), whereas decreased activation of IRS-1 serine residue. In addition, TNF-α-induced activation of p38-mitogen-activated protein kinase (p38) and c-Jun N-terminal kinases (JNK) 1/2 were decreased by either EWH or IRW treatment. CONCLUSION: EWH and IRW improve impaired insulin sensitivity by down-regulating the activation of p38 and JNK1/2 in TNF-α-treated skeletal muscle cells.

Effects of IRW and IQW on Oxidative Stress and Gut Microbiota in Dextran Sodium Sulfate-Induced Colitis.

BACKGROUND/AIMS: There are known links between inflammatory bowel disease (IBD) and changes in the microbiota of the gut and inflammation and oxidative stress. In this study, a colitis model induced by dextran sodium sulfate (DSS) in mice is used to evaluate whether the presence of bioactive peptides IRW (Ile-Arg-Trp) and IQW (Ile-Gln-Trp) peptides is advantageous. METHODS: The mice were arbitrarily assigned to the following four groups: (i) control (untreated), (ii) dextran sodium sulfate (DSS) treated, (iii) IRW-DSS treated, and (iv) IQW-DSS treated. For 7 days, the control group subjects had unrestricted access to untreated drinking water, whereas the drinking water supplied to the subjects in the DSS, IRW-DSS, and IQW-DSS groups during this period consisted of 5% DSS solution. The colonic lesions were scored after hematoxylin and eosin staining. Serum antioxidant capacity was analyzed by 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation decolorization test and the microbiota in the colonic contents were sequenced by HiSeq2500 PE250. RESULTS: The presence of DSS reduced daily weight gain, enhanced histopathology scores, and inhibited antioxidant enzyme expression. Superoxide dismutase, catalase, and glutathione peroxidase activities in the DSS-induced colitis model were significantly enhanced (P < 0.05) in the presence of dietary IRW and IQW. Furthermore, the Simpson index was significantly increased (P < 0.05) in the presence of dietary IRW and IQW compared to the control group. IRW and IQW increased the abundance of Coprococcus_1, Ruminococcaceae_UCG-014, and Desulfovibrio compared to the control group and DSS group. Furthermore, IQW decreased the abundance of Bacteroides in relation to the control group, but increased Parabacteroides. In addition, IRW increased the level of Anaerotruncus, Oscillibacter, and Ruminiclostridium_9 compared to the control group. CONCLUSION: This study concludes that the presence of IRW or IQW can mitigate DSS-induced oxidative stress by improving the activities of antioxidant enzymes, increasing intestinal microbial diversity and enhancing the abundance of gut microbiota, which may help maintain the homeostasis of host health and microenvironment in a DSS-induced mouse model, thus providing a potential further treatment for IBD patients.

Bioavailability and Metabolism of Bioactive Peptide IRW with Angiotensin-Converting Enzyme 2 (ACE2) Upregulatory Activity in Spontaneously Hypertensive Rats.

Peptide IRW is the first food-derived angiotensin-converting enzyme 2 (ACE2) upregulator. This study aimed to investigate the pharmacokinetic characteristics of IRW and identify the metabolites contributing to its antihypertensive activity in spontaneously hypertensive rats (SHRs). Rats were administered 100 mg of IRW/kg of the body weight via an intragastric or intravenous route. 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 metabolites are likely responsible for the blood pressure-lowering activity. Time-course metabolomics revealed a significant increase in the level of kynurenine, a tryptophan metabolite, in blood after IRW administration. Kynurenine increased the level of ACE2 in cells. Oral administration of tryptophan (W), but not dipeptide IR, lowered the blood pressure and upregulated aortic ACE2 in SHRs. Our study supports the key role of tryptophan and its metabolite, kynurenine, in IRW's blood pressure-lowering effects.

IRW (Ile-Arg-Trp) Alleviates DSS-Induced Intestinal Injury by Remodeling Gut Microbiota and Regulating Fecal SCFA Levels.

Inflammatory bowel disease (IBD) is a chronic disease of unknown etiology with a progressive and destructive course and an increasing incidence worldwide. Dietary peptides have a variety of biological functions and are effective anti-inflammatories and antioxidants, making them a prospective class of material for treating intestinal inflammation. Our study investigated the association between Ile-Arg-Trp (IRW), a dietary oligopeptide, and intestinal microbial changes during the relief of colitis using different concentrations of IRW. We found that IRW can significantly alleviate mouse colonic barrier damage caused by dextran sulphate sodium salt (DSS) and promote intestinal health. The results of microbial community composition showed that the relative abundance of Bacillota and Lactobacillus in the gut microbiota at different concentrations of IRW was significantly increased and that the abundance of Bacteroides was suppressed. Surprisingly, the relative abundance of Odoribacter also received regulation by IRW concentration and had a positive correlation with acetic acid. IRW at 0.02 mg/mL and 0.04 mg/mL significantly altered the abundance of Bacillota, Odoribacter, and Lactobacillus.

Tripeptide IRW Improves AMPK/eNOS Signaling Pathway via Activating ACE2 in the Aorta of High-Fat-Diet-Fed C57BL/6 Mice.

This study aims to investigate the effect of tripeptide IRW on the local renin-angiotensin system (RAS), particularly angiotensin-converting enzyme 2 (ACE2), and their association with signaling pathways in the aorta of a high-fat-diet (HFD)-induced insulin-resistant mouse model. C57BL/6 mice were fed HFD (45% of the total calories) for six weeks, and then IRW was added to the diet (45 mg/kg body weight (BW)) for another eight weeks. ACE2 mRNA expression and protein level(s) were increased (p < 0.05), while angiotensin II receptor (AT1R) and angiotensin-converting enzyme (ACE) protein abundance was significantly reduced (p < 0.05) in the aorta of HFD mice treated by IRW. IRW supplementation also improved glucose transporter 4 (GLUT4) abundance (p < 0.05) alongside AMP-activated protein kinase (AMPK) (p < 0.05), Sirtuin 1 (SIRT1) (p < 0.05), and endothelial nitric oxide synthase (eNOS) (p < 0.05) expression. IRW downregulated the levels of endothelin 1 (ET-1) and p38 mitogen-activated protein kinases (p38 MAPK, p < 0.05). Furthermore, the levels of AMPK and eNOS in vascular smooth muscle cells (VSMCs) were significantly reduced in ACE2 knockdown cells treated with or without IRW (p < 0.01). In conclusion, this study provided new evidence of the regulatory role of IRW on the aortic ACE2 against metabolic syndrome (MetS) in an HFD-induced insulin-resistant model.

ACE2 Activation by Tripeptide IRW (Ile-Arg-Trp) Depends on the G Protein-Coupled Receptor 30 Signaling Cascade.

This study aimed to understand how specific cell-bound receptors influence ACE2 activation by IRW. Our results showed that G protein-coupled receptor 30 (GPR30), a 7-transmembrane domain protein, was involved in IRW-mediated ACE2 increase. IRW treatment (50 µM) significantly increased the GPR30 pool levels (3.2 ± 0.5 folds) (p < 0.001). IRW treatment also boosted the consecutive GEF (guanine nucleotide exchange factor) activity (2.2 ± 0.2 folds) (p < 0.001), and GNB1 levels (2.0 ± 0.5 folds) (p < 0.05), associated with the functional subunits of G proteins, in cells. These results were translated in hypertensive animal studies as well (p < 0.05), indicated by an increase in the aortal levels of GPR30 (p < 0.01); further experiments showed an increase in downstream PIP3/PI3K/Akt pathway activation following IRW treatment. The blockade of GPR30 by an antagonist and siRNA in cells abolished the ACE2-activating ability of IRW, as shown by the depleted levels of ACE2 mRNA (p < 0.001), protein levels in whole cells and membrane, angiotensin (1-7) (p < 0.01), and ACE2 promoter HNF1α (p < 0.05). Finally, the GPR30 blockade in ACE2-overexpressing cells using the antagonist (p < 0.01) and siRNA (p < 0.05) significantly depleted the innate cellular pool of ACE2, thus confirming the relationship between the membrane-bound GPR30 and ACE2. Overall, these results showed that the vasodilatory peptide IRW could activate ACE2 via the membrane-bound receptor GPR30.

Tripeptide IRW (Ile-Arg-Trp) as a Potential Nutraceutical Intervention in Osteoporosis.

Bone health is an important medical concern in rapidly aging demographics worldwide. Excessive bone resorption, due to enhanced activity of osteoclasts, is a major underlying cause of bone disorders such as osteoporosis. Inflammation and oxidative stress are key factors contributing to increased osteoclastic activity. Like increased activity of osteoclasts, depletion of osteoblasts also contributes to weakened structural integrity of bone. Considering the epidemiology of bone disorders and aging demographics there is a substantial need for novel bone health therapeutics. IRW (Ile-Arg-Trp), an egg-derived tripeptide, exhibits a spectrum of pharmacological activity. In our recent work, we have shown that IRW inhibits osteoclastogenesis and promotes osteogenesis in the mouse macrophage RAW 264.7 and MC3T3-E1 cells. IRW treatment (25 and 50 µM) significantly inhibited osteoclastogenesis-associated factors [TRAF6 (TNF Receptor Associated Factor 6), Fos Proto-Oncogene (c-Fos), Nuclear Factor of Activated T Cells 1 (NFATc1), and cathepsin K] and upregulated osteogenesis-associated factors [RUNX2 (Runt-related transcription factor 2) and RANKL (Receptor activator of nuclear factor kappa-B ligand)] in the two cell lines. Currently, we are conducting studies to analyze the impact of IRW on Angiotensin II (Ang II)-induced stress in vitro and in vivo. In summary, our recent work presents the ability of IRW to prevent LPS-induced inflammatory bone resorption and activation of osteogenesis activity via multiple signaling pathways.

IRW (Isoleucine-Arginine-Tryptophan) Improves Glucose Tolerance in High Fat Diet Fed C57BL/6 Mice via Activation of Insulin Signaling and AMPK Pathways in Skeletal Muscle.

IRW (Isoleucine−Arginine−Tryptophan), has antihypertensive and anti-inflammatory properties in cells and animal models and prevents angiotensin-II- and tumor necrosis factor (TNF)-α-induced insulin resistance (IR) in vitro. We investigated the effects of IRW on body composition, glucose homeostasis and insulin sensitivity in a high-fat diet (HFD) induced insulin resistant (IR) model. C57BL/6 mice were fed HFD for 6 weeks, after which IRW was incorporated into the diet (45 or 15 mg/kg body weight (BW)) until week 14. IRW45 (at a dose of 45 mg/kg BW) reduced BW (p = 0.0327), fat mass gain (p = 0.0085), and preserved lean mass of HFD mice (p = 0.0065), concomitant with enhanced glucose tolerance and reduced fasting glucose (p < 0.001). In skeletal muscle, IRW45 increased insulin-stimulated protein kinase B (AKT) phosphorylation (p = 0.0132) and glucose transporter 4 (GLUT4) translocation (p < 0.001). Angiotensin 2 receptor (AT2R) (p = 0.0024), phosphorylated 5'-AMP-activated protein kinase (AMPKα) (p < 0.0124) and peroxisome proliferator-activated receptor gamma (PPARγ) (p < 0.001) were enhanced in skeletal muscle of IRW45-treated mice, as was the expression of genes involved in myogenesis. Plasma angiotensin converting enzyme-2 (ACE2) activity was increased (p = 0.0016). Uncoupling protein-1 in white adipose tissue (WAT) was partially restored after IRW supplementation. IRW improves glucose tolerance and body composition in HFD-fed mice and promotes glucose uptake in skeletal muscle via multiple signaling pathways, independent of angiotensin converting enzyme (ACE) inhibition.

Egg White Protein Ovotransferrin-Derived IRW (Ile-Arg-Trp) Inhibits LPS-Induced Barrier Integrity Dysfunction and Inflammation in Caco-2 Cells.

Tripeptide IRW derived from egg ovotransferrin was initially identified to be an inhibitor of angiotensin-converting enzyme. Later, IRW has been shown to possess various bioactivities, including anti-inflammatory activity and the ability to suppress colitis development. Nevertheless, its role in protecting intestinal barrier integrity has not been reported. This study aims to investigate the effect of IRW on inhibiting intestinal barrier dysfunction and inflammation in lipopolysaccharide (LPS)-treated Caco-2 cells. Pretreatment with IRW could mitigate the LPS-induced reduction of transepithelial electronic resistance values and decrease the paracellular permeation of differentiated Caco-2 cell monolayers. Meanwhile, IRW restored the expression level and cell surface distribution of the tight junction protein occludin. Furthermore, IRW showed LPS-neutralizing activity and could significantly inhibit LPS-induced activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In conclusion, our study demonstrated the ability of IRW to prevent LPS-induced intestinal barrier dysfunction and prohibit inflammatory responses.

Tripeptide IRW Upregulates NAMPT Protein Levels in Cells and Obese C57BL/6J Mice.

Nicotinamide adenine dinucleotide (NAD+) plays a vital role in cellular processes that govern human health and disease. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in NAD+ biosynthesis. Thus, boosting NAD+ level via an increase in NAMPT levels is an attractive approach for countering the effects of aging and metabolic disease. This study aimed to establish IRW (Ile-Arg-Trp), a small tripeptide derived from ovotransferrin, as a booster of NAMPT levels. Treatment of muscle (L6) cells with IRW increased intracellular NAMPT protein levels (2.2-fold, p < 0.05) and boosted NAD+ (p < 0.01). Both immunoprecipitation and recombinant NAMPT assays indicated the possible NAMPT-activating ability of IRW (p < 0.01). Similarly, IRW increased NAMPT mRNA and protein levels in the liver (2.6-fold, p < 0.01) and muscle tissues (2.3-fold, p < 0.05) of C57BL/6J mice fed with a high-fat diet (HFD). A significantly increased level of circulating NAD+ was also observed following IRW treatment (4.7 fold, p < 0.0001). Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT) and NAD+ in vivo (p < 0.05). However, IRW treatment did not boost NAMPT levels in SIRT1 KO cells, indicating a possible SIRT1 dependency for the pharmacological effect. Overall, these data indicate that IRW is a novel small peptide booster of the NAMPT pool.

Egg-Derived Tripeptide IRW Attenuates LPS-Induced Osteoclastogenesis in RAW 264.7 Macrophages via Inhibition of Inflammatory Responses and NF-κB/MAPK Activation.

Excessive bone resorption, because of increased osteoclastic activity, is a key underlying cause of osteolytic disorders. Lipopolysaccharide (LPS) is a potent factor to stimulate osteoclastic activity by inducing inflammatory stress. An egg-derived tripeptide IRW (Ile-Arg-Trp) was previously shown to exert anti-inflammatory activity. The overall objective of this study was to investigate the effect of IRW on inhibiting LPS-induced osteoclastogenesis and inflammatory bone resorption in the mouse macrophage RAW 264.7 cells. IRW (25 and 50 µM) significantly inhibited the LPS-induced osteoclast formation and resorptive activity. Meanwhile, IRW significantly suppressed the LPS-induced expression of TNF-α, IL-6, iNOS, COXII, NO, and PGE2. Furthermore, IRW regulated a group of osteoclastogenesis-associated factors (TRAF6, c-Fos, NFATc1, and cathepsin K) because of the inhibition of LPS-activated NF-κB and MAPK pathways. In conclusion, our study suggested the ability of IRW to prevent LPS-induced inflammatory bone resorption activity via the inhibition of inflammatory responses and the activation of osteoclastogenesis-associated signaling pathways.

A Novel Angiotensin Converting Enzyme 2 (ACE2) Activating Peptide: A Reflection of 10 Years of Research on a Small Peptide Ile-Arg-Trp (IRW).

IRW (Ile-Arg-Trp) was identified as an inhibitor of angiotensin converting enzyme (ACE) from egg white protein ovotransferrin through an integrated in silico digestion and quantitative structure and activity relationship prediction in 2011. Oral administration of IRW to spontaneously hypertensive rats (SHRs) can significantly reduce blood pressure, via upregulation of ACE2, but not through the inhibition of ACE. ACE2 converts Ang II into Ang (1-7), thus lowering blood pressure via Mas receptor (MasR); coinfusion of Mas receptor antagonist A779 and IRW in SHRs abolished blood pressure-lowering effect of IRW, supporting a key role of ACE2/Ang (1-7)/MasR axis. Our ongoing study further established new roles of IRW as an antioxidant, an anti-inflammatory agent, an insulin sensitizer, and a bone cell anabolic. Future studies are warranted to understand the unique structure features of this peptide, its mechanisms of action at various targets, its bioavailability and metabolism, and its possible roles toward COVID-19.

Egg Protein Transferrin-Derived Peptides IRW and IQW Regulate Citrobacter rodentium-Induced, Inflammation-Related Microbial and Metabolomic Profiles.

Bioactive peptides that target the gastrointestinal tract can strongly affect the health of animals and humans. This study aimed to evaluate the abilities of two peptides derived from egg albumin transferrin, IRW and IQW, to treat enteritis in a mouse model of Citrobacter rodentium-induced colitis by evaluating serum metabolomics and gut microbes. Forty-eight mice were randomly assigned to six groups: basal diet (CTRL), intragastric administration Citrobacter rodentium (CR), basal diet with 0.03%IRW (IRW), CR with 0.03% IRW (IRW+CR), basal diet with 0.03%IQW (IQW) and CR with 0.03% IQW (IQW+CR). CR administration began on day 10 and continued for 7 days. After 14 days of IRW and IQW treatment, serum was collected and subjected to a metabolomics analysis. The length and weight of each colon were measured, and the colon contents were collected for 16srRNA sequencing. The colons were significantly longer in the CR group, compared to the CTRL group. A serum metabolomics analysis revealed no significant difference in microbial diversity between the six groups. Compared with the CTRL group, the proportions of Firmicutes and Actinobacteria species decreased significantly and the proportions of Bacteroidetes and Proteobacteria species increased in the CR group. There were no significant differences between the CTRL and other groups. The serum metabolomics analysis revealed that Infected by CR increased the levels of oxalic acid, homogentisic acid and prostaglandin but decreased the levels of L-glutamine, L-acetyl carnitine, 1-methylhistidine and gentisic acid. Therefore, treatment with IRW and IQW was shown to regulate the intestinal microorganisms associated with colonic inflammation and serum metabolite levels, thus improving intestinal health.

Tripeptide IRW initiates differentiation in osteoblasts differentiation via the RUNX2 pathway.

BACKGROUND: Osteoblasts maintain the structural integrity of bone via differentiation and mineralization; therefore, their malfunction or reduced activity can cause serious bone disorders. Although studies have demonstrated the association between nutrients and bone, research on food-derived bioactive peptides and bone health are scanty. METHODS: Osteoblasts MC3T3-E1 were treated with IRW (50 and 25 µM). Cell proliferation, cell cycle, osteoblastic differentiation, and mineralization were tested to evaluate the effects of IRW on osteogenesis promotion. The activation of PI3K-Akt-RUNX2 pathway and collagen synthesis were investigated to better understand the functions of IRW. RESULTS: IRW treatment (50 and 25 µM) in MC3T3-E1 cells caused a significant increase in cell proliferation by increasing the percentage of S and G2/M phase. Furthermore, IRW promoted mineralization in MC3T3-E1 cells. Mechanistically, we found that IRW treatment resulted in a 4-fold increase of Akt serine phosphorylation and a 2-fold increase of its downstream target RUNX2. Expression levels of RUNX2 associated proteins were concomitantly altered: ALP (2-fold increase), Col1A2 (2-fold increase), RANKL (2-fold decrease), and OPG (2-fold increase). Meanwhile, a parallel collagen synthesis pathway was found to contribute to IRW-stimulated osteogenesis. CONCLUSIONS: IRW, an egg-derived small bioactive peptide enhances osteoblastic activity and stimulates osteogenesis. The stimulation is primarily due to the activation of PI3K-Akt-RUNX2 pathway and its downstream effectors, accompanied by a secondary collagen synthesis pathway. GENERAL SIGNIFICANCE: Our results revealed the positive effects of tripeptide IRW on regulating osteogenesis and collagen synthesis, indicating its potential for the prevention or treatment of osteoporosis.

Egg White-Derived Antihypertensive Peptide IRW (Ile-Arg-Trp) Inhibits Angiotensin II-Stimulated Migration of Vascular Smooth Muscle Cells via Angiotensin Type I Receptor.

Excessive proliferation, inflammation, oxidative stress, and migration induced by angiotensin II (Ang II), occurring in vascular smooth muscle cells (VSMCs) during vascular remodelling, are major pathogenesis of hypertension. Antihypertensive peptides derived from food proteins are promising alternatives in preventing/treating hypertension and associated complications. In addition to reducing high blood pressure in spontaneously hypertensive rats, egg white ovotransferrin-derived antihypertensive IRW (Ile-Arg-Trp) was shown to exert antiproliferative, antioxidant, and anti-inflammatory effects in A7r5 cells (a vascular smooth muscle cell line) against Ang II stimulation, further indicating its potential in retarding vascular remodelling. Since its regulatory role in migration of VSMC is unclear, the objective of this study was to evaluate the antimigrant activity of IRW in Ang II-stimulated A7r5 cells. It was found that IRW could downregulate matrix metallopeptidase 9 (MMP9) expression and inhibit migration of Ang II-stimulated A7r5 cells, which was associated with inactivation of p38/MAPK signaling. More importantly, the antimigrant activity of IRW in Ang II-stimulated A7r5 cells was dependent on angiotensin type I receptor (AT1R). Our study provided the first evidence that egg ovotransferrin-derived antihypertensive peptide IRW inhibited migration of VSMCs.

Egg White-Derived Tripeptide IRW (Ile-Arg-Trp) Is an Activator of Angiotensin Converting Enzyme 2.

Angiotensin converting enzyme 2 (ACE2) plays beneficial roles in the renin angiotensin aldosterone system. Our previous studies indicated that egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) could upregulate ACE2 mRNA level in mesenteric artery of spontaneously hypertensive rats (SHRs), suggesting the potential of IRW as an in vivo ACE2 activator. In this study, the effects of IRW on activity and protein expression of ACE2 were investigated. Results indicated that IRW activated human recombinant ACE2 with an EC50 value of 7.24 × 10-5 M. In rat aortic vascular smooth muscle cell line A7r5 cells, IRW treatment (50 µM) significantly increased the expression and activity of ACE2. Oral administration of IRW to SHRs upregulated ACE2 protein levels in kidney and aorta. Molecular docking study suggested that IRW might activate ACE2 through interaction with the subdomain I near the active site through hydrogen bonds. Overall, this study established IRW as the first food-derived ACE2 activating peptide.

Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide IRW (Ile-Arg-Trp) on Vascular Smooth Muscle Cells against Angiotensin II Stimulation.

The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal proliferation, oxidative stress, and inflammation in vascular smooth muscle cells (VSMCs) that contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on VSMCs are still unclear. In the present study, we evaluated the antiproliferative, antioxidant, and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production, and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-κB pathway. These findings could further our understanding on the antihypertensive mechanism of IRW beyond vascular endothelium.

Characterization of Corning EPMA Standard Glasses 95IRV, 95IRW, and 95IRX.

The preparation, synthesis, and characterization of Corning trace-element glasses 95IRV, 95IRW, and 95IRX by bulk chemical and electron microprobe techniques is discussed. Working values for the doped elements in the 95-series glasses are established. Blank values have been determined by both bulk chemical and electron microprobe analysis, and important x-ray interferences are highlighted. Chemical homogeneity both within a rod cross-section, and along cane length has been documented. These glasses are standard reference materials intended for use as both primary and secondary electron microprobe standards.