Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac⁰-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac³-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9 μM⁻¹ min⁻¹ for BK and Toac³-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.     

Angiotensin-converting enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in apolipoprotein E-deficient mice

Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF-β1 and collagen I and elevated renal Ang-(1–7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100 nmol L⁻¹) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-β1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1 mg mL⁻¹) or mTOR inhibitor rapamycin (10 μmol L⁻¹). These protective effects of rhACE2 were eradicated by the Ang-(1–7)/Mas receptor antagonist A779 (1 μmol L⁻¹). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1–7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders.     

Telmisartan attenuates aortic hypertrophy in hypertensive rats by the modulation of ACE2 and profilin-1 expression

Profilin-1 has recently been linked to vascular hypertrophy and remodeling. Here, we assessed the hypothesis that angiotensin (Ang) II type I receptor antagonist telmisartan improves vascular hypertrophy by modulation of expression of profilin-1 and angiotensin-converting enzyme 2 (ACE2). Ten-week-old male spontaneously hypertensive rats (SHR) were received oral administration of telmisartan (5 or 10 mg/kg; daily) or saline for 10 weeks. Compared with Wistar–Kyoto (WKY) rats, there were marked increases in systolic blood pressure and profilin-1 expression and reduced ACE2 and peroxisome proliferator activated receptor-γ (PPARγ) levels in aorta of SHR, associated with elevated extracellular-signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) phosphorylation signaling and aortic hypertrophy characterized with increased media thickness, which were strikingly reversed by telmisartan. In cultured human umbilical artery smooth muscle cells (HUASMCs), Ang II induced a dose-dependent increase in profilin-1 expression, along with decreased ACE2 protein expression and elevated ERK1/2 and JNK phosphorylation. In addition, blockade of ERK1/2 or JNK by either specific inhibitor was able to abolish Ang II-induced ACE2 downregulation and profilin-1 upregulation in HUASMCs. Importantly, treatment with telmisartan (1 or 10 μM) or recombinant human ACE2 (2 mg/ml) largely ameliorated Ang II-induced profilin-1 expression and ERK1/2 and JNK phosphorylation and augmented PPARγ ?expression in the cultured HUASMCs. In conclusion, telmisartan treatment attenuates vascular hypertrophy in SHR by the modulation of ACE2 and profilin-1 expression with a marked reversal of ERK1/2 and JNK phosphorylation signaling pathways.     

l-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca^2 + and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.     

Melanoma incidence and exposure to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers

A reduced incidence of nonmelanoma skin cancer among users of angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARb) has been reported. A similar effect is suggested for cutaneous melanoma. We aimed to investigate the possible association between use of ACEi and ARb and the risk of cutaneous melanoma. A general population-based case control study with the PHARMO database, containing drug-dispensing records from community pharmacies and the national pathology database (PALGA) was conducted. Cases were patients with a primary cutaneous melanoma between January 1st 1991 and December 14th 2004, aged ≥18 years and having ≥3 years of follow-up prior to diagnosis. Finally, 1272 cases and 6520 matched controls were included. Multivariable conditional logistic regression showed no statistically significant associations between the incidence of melanoma and the use of ACEi (adjusted OR = 1.0, 95%CI: 0.8–1.3) or ARb (adjusted OR = 1.0, 95%CI: 0.7–1.5). Thus, in this study, the use of ACEi or ARb does not seem to protect against the development of cutaneous melanoma. However, we cannot exclude an association between ACEi and ARb exposure and an increased or decreased incidence of cutaneous melanoma.     

The inhibitory activity of HL-7 and HL-10 peptide from scorpion venom (Hemiscorpius lepturus) on angiotensin converting enzyme: Kinetic and docking study

The hypertension is one of the highest risk factors for stroke, myocardial infarction, vascular disease and chronic kidney disease. Angiotensin converting enzyme (ACE) has an important role in the physiological regulation of cardiovascular system. ACE inhibition is a key purpose for hypertension treatment. In this study, two peptides named HL-7 with the sequence of YLYELAR (MW: 927.07 Da) and HL-10 with the sequence of AFPYYGHHLG (MW: 1161.28 Da) were identified from scorpion venom of H. lepturus. The inhibitory activity of HL-7 and HL-10 was examined on rabbit ACE. The inhibition mechanisms were assayed by kinetic and docking studies. The IC₅₀ values for ACE inhibition of HL-7 and HL-10 were 9.37 µM and 17.22 µM, respectively. Lineweaver-Burk plots showed that two peptides inhibited rabbit ACE with competitive manner. The molecular docking conformed experimental results and showed that the two peptides interacted with N-domain and C-domain active sites. Also, docking study revealed that the two peptides can form hydrogen and hydrophobic bonds at their binding sites. Both peptides had higher affinity to N-domain. Our results showed that HL-7 exhibited more strong interactions with amino acids at active site. It seems that HL-10 peptide could occupy more space, thereby inhibiting the substrate entrance to active site.     

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor – captopril and the ANGII type 1 receptor blocker – losartan. In vitro, both ANGII and ANGI stimulation significantly (P < 0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P < 0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P < 0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness.     

Fetal betamethasone exposure attenuates angiotensin-(1-7)-Mas receptor expression in the dorsal medulla of adult sheep

Glucocorticoids including betamethasone (BM) are routinely administered to women entering into early preterm labor to facilitate fetal lung development and decrease infant mortality; however, fetal steroid exposure may lead to deleterious long term consequences. In a sheep model of fetal programming, BM-exposed (BMX) offspring exhibit elevated mean arterial pressure (MAP) and decreased baroreflex sensitivity (BRS) for control of heart rate by 0.5-years of age associated with changes in the circulating and renal renin-angiotensin systems (RAS). In the brain solitary tract nucleus, angiotensin (Ang) II actions through the AT1 receptor oppose the beneficial actions of Ang-(1-7) at the Mas receptor for BRS regulation. Therefore, we examined Ang peptides, angiotensinogen (Aogen), and receptor expression in this brain region of exposed and control offspring of 0.5- and 1.8-years of age. Mas protein expression was significantly lower (>40%) in the dorsal medulla of BMX animals at both ages; however, AT1 receptor expression was not changed. BMX offspring exhibited a higher ratio of Ang II to Ang-(1-7) (2.30 ± 0.36 versus 0.99 ± 0.28; p < 0.01) and Ang II to Ang I at 0.5-years. Although total Aogen was unchanged, Ang I-intact Aogen was lower in 0.5-year BMX animals (0.78 ± 0.06 vs. 1.94 ± 0.41; p < 0.05) suggesting a greater degree of enzymatic processing of the precursor protein in exposed animals. We conclude that in utero BM exposure promotes an imbalance in the central RAS pathways of Ang II and Ang-(1-7) that may contribute to the elevated MAP and lower BRS in this model.     

The effects of angiotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis

Otosclerosis is a complex disease characterized by an abnormal bone turnover of the otic capsule resulting in conductive hearing loss. Recent findings have shown that angiotensin II (Ang II), a major effector peptide of the renin–angiotensin system, plays an important role in the pathophysiology of otosclerosis, most likely by its proinflammatory effects on the bone cells. Because reactive oxygen species play a role both in inflammation and in the cellular signaling pathway of Ang II, the appearance of protein adducts of the “second messenger of free radicals,” the aldehyde 4-hydroxynonenal (HNE), in otosclerotic bone has been analyzed. Immunohistochemical analysis of HNE-modified proteins in tissue samples of the stapedial bones performed on 15 otosclerotic patients and 6 controls revealed regular HNE–protein adducts present in the subperiosteal parts of control bone specimens, whereas irregular areas of a pronounced HNE–protein adduct presence were found within stapedial bone in cases of otosclerosis. To study possible interference by HNE and Ang II in human bone cell proliferation, differentiation, and induction of apoptosis we used an in vitro model of osteoblast-like cells. HNE interacted with Ang II in a dose-dependent manner, both by forming HNE–Ang II adducts, as revealed by immunoblotting, and by modifying its effects on cultured cells. Namely, treatment with 0.1 nM Ang II and 2.5 μM HNE stimulated proliferation, whereas treatment with 10 μM HNE or in combination with Ang II (0.1, 0.5, and 1 nM) decreased cell proliferation. Moreover, 10 μM HNE alone and with Ang II (except if 1 nM Ang II was used) increased cellular differentiation and apoptosis. HNE at 5 μM did not affect differentiation nor significantly change apoptosis. On the other hand, when cells were treated with lower concentrations of HNE and Ang II we observed a decrease in cellular differentiation (combination of 1.0 or 2.5 μM HNE with 0.1 nM Ang II) and decrease in apoptosis (0.1 and 0.5 nM Ang II). Cellular necrosis was increased with 5 and 10 μM HNE if given alone or combined with Ang II, whereas 0.5 nM Ang II and combination of 1 μM HNE with Ang II (0.1 and 0.5 nM) reduced necrosis. These results indicate that HNE and Ang II might act mutually dependently in the regulation of bone cell growth and in the pathophysiology of otosclerosis.     

Whole-body basal nitric oxide production is impaired in postprandial endothelial dysfunction in healthy rats

In healthy humans, a high-saturated-fat/high-sucrose meal induces vascular endothelial dysfunction, a hallmark of atherogenesis. This transient dysfunction indicates a loss in nitric oxide (NO) production and/or bioactivity in the vasculature but it remains unknown if this is the local manifestation of a general impairment in NO pathway in the postprandial state. Here, we studied whole-body NO production and systemic NO bioactivity in postprandial endothelial dysfunction, as induced by a high-saturated-fat, high-sucrose meal.We first developed a physiological test of endothelial function on conscious rats, based on the transient fall in blood pressure after iv acetylcholine, and showed that this response was NO-dependent. As assessed with this method in healthy rats, endothelial function decreased during the postprandial state, being 60 ± 7% lower than baseline at 6 h after the meal challenge, associated with important elevations in plasma triglycerides and hydroperoxides. Aortic superoxide anion production, as assessed by oxidative fluorescent detection, was higher 6 h after the meal challenge than after the nutrients vehicle (water). During the postprandial period, plasma cGMP, but not plasma ANP, markedly decreased, indicating a general decrease in NO bioavailability, which was numerically maximal 4 h after the meal challenge. As determined 4 h after ingestion by a tracer-based method using iv [¹⁵N₂-(guanido)]-arginine, the whole-body NO production fell by 27 ± 9% postprandially.This is the first study evidencing that a meal challenge that impairs the stimulated, NO-mediated, vascular response also reduces whole-body basal NO production and bioavailability. Postprandial pathophysiology may build on this general, fundamental alteration in NO production.     

Non-disulfide-bridged peptides from Tityus serrulatus venom: Evidence for proline-free ACE-inhibitors

The present study purifies two T. serrulatus non-disulfide-bridged peptides (NDBPs), named venom peptides 7.2 (RLRSKG) and 8 (KIWRS) and details their synthesis and biological activity, comparing to the synthetic venom peptide 7.1 (RLRSKGKK), previously identified. The synthetic replicate peptides were subjected to a range of biological assays: hemolytic, antifungal, antiviral, electrophysiological, immunological and angiotensin-converting enzyme (ACE) inhibition activities. All venom peptides neither showed to be cytolytic nor demonstrated significant antifungal or antiviral activities. Interestingly, peptides were able to modulate macrophages’ responses, increasing IL-6 production. The three venom peptides also demonstrated potential to inhibit ACE in the following order: 7.2 > 7.1 > 8. The ACE inhibition activity was unexpected, since peptides that display this function are usually proline-rich peptides. In attempt to understand the origin of such small peptides, we discovered that the isolated peptides 7.2 and 8 are fragments of the same molecule, named Pape peptide precursor. Furthermore, the study discusses that Pape fragments could be originated from a post-splitting mechanism resulting from metalloserrulases and other proteinases cleavage, which can be seen as a clever mechanism used by the scorpion to enlarge its repertoire of venom components. Scorpion venom remains as an interesting source of bioactive proteins and this study advances our knowledge about three NDBPs and their biological activities.     

Epigallocatechin-3-gallate inhibits interleukin-6- and angiotensin II-induced production of C-reactive protein in vascular smooth muscle cells

AimsExtensive research suggests that atherosclerosis is an inflammatory disease and that epigallocatechin-3-gallate (EGCG) is able to inhibit the formation and development of atherosclerosis. However, the mechanisms of action of EGCG against atherosclerosis are still unclear. Therefore, the effect of EGCG on interleukin-6 (IL-6)- and angiotensin II (Ang II)-induced CRP production in vascular smooth muscle cells (VSMCs) was studied to provide experimental evidence for its anti-inflammatory and anti-atherosclerotic actions.Main methodsRat VSMCs were cultured, and IL-6 (10^? 7 M) and Ang II (10^? 7 M) were used as stimulants for CRP generation. The CRP concentration in the supernatant was measured with ELISA, and mRNA and protein expression of CRP was assayed with RT-qPCR and immunocytochemistry, respectively. The production of reactive oxygen species (ROS) and superoxide anion (O₂^?) was detected with ROS and O₂^? assay kits, respectively.Key findingsThe results showed that both IL-6 and Ang II increased CRP levels in the supernatant of VSMCs and induced mRNA and protein expression of CRP in VSMCs, whereas pretreatment of the cells with EGCG (1 × 10^? 6 M, 3 × 10^? 6 M, 10 × 10^? 6 M) significantly inhibited IL-6- and Ang II-induced production and expression of CRP in VSMCs in a concentration-dependent manner. Additionally, Ang II stimulated O₂^? and ROS generations in VSMCs, and EGCG decreased the Ang II-induced increase of O₂^? and ROS in a concentration-dependent fashion.SignificanceThese results suggest that EGCG plays an anti-inflammatory role via inhibiting IL-6- and Ang II-induced CRP secretion, as well as the Ang II-induced generation of O₂^? and ROS in VSMCs, which contributes to its anti-atherosclerotic action.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

Low-dose chronic lead exposure increases systolic arterial pressure and vascular reactivity of rat aortas

Chronic lead exposure induces hypertension affecting endothelial function. We investigated whether low-concentration lead exposure alters blood pressure and vascular reactivity, focusing on the roles of NO, oxidative stress, cyclooxygenase-derived vasoconstrictor prostanoids, and the local angiotensin–renin system. Aortic rings from 3-month-old Wistar rats were treated daily with lead acetate (first dose 4 mg/100 g, subsequent doses 0.05 mg/100 g, im) or vehicle for 30 days. Treatment increased lead blood levels (12 μg/dl), blood pressure, and aortic ring contractile response to phenylephrine (1 nM–100 mM). Contractile response after L-NAME administration increased in both groups but was higher after lead treatment. Lead effects on R_max decreased more after apocynin and superoxide dismutase administration compared to control. Indomethacin reduced phenylephrine response more after lead treatment than in controls. The selective COX-2 inhibitor NS398, thromboxane A₂/prostaglandin H2 receptor antagonist SQ 29,548, TXA₂ synthase inhibitor furegrelate, EP₁ receptor antagonist SC 19220, and ACE inhibitor and AT₁ receptor antagonist losartan reduced phenylephrine responses only in vessels from lead-treated rats. Basal and stimulated NO release was reduced and local O₂⁻ liberation increased in the lead-treated group compared to controls. eNOS, iNOS, and AT₁ receptor protein expression increased with lead exposure, but COX-2 protein expression decreased. This is the first demonstration that blood Pb²⁺ (12 µg/dl) concentrations below the WHO-established values increased systolic blood pressure and vascular phenylephrine reactivity. This effect was associated with reduced NO bioavailability, increased reactive oxygen species production, increased participation of COX-derived contractile prostanoids, and increased renin–angiotensin system activity.     

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration

The omega-3 polyunsaturated fatty acids (ω − 3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω − 3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.     

Angiotensin I converting enzyme (ACE) inhibitory peptides from salmon byproduct protein hydrolysate by Alcalase hydrolysis

Angiotensin I converting enzyme (ACE) inhibitory peptides were produced from salmon byproduct proteins via enzymatic hydrolysis using Alcalase, flavourzyme, neutrase, pepsin, protamex and trypsin. Among them, Alcalase hydrolysate showed the highest ACE inhibitory activity, thus ACE inhibitory peptides were purified using consecutive chromatography. The purified ACE inhibitory peptides were identified to be Val-Trp-Asp-Pro-Pro-Lys-Phe-Asp (P1), Phe-Glu-Asp-Tyr-Val-Pro-Leu-Ser-Cys-Phe (P2), and Phe-Asn-Val-Pro-Leu-Tyr-Glu (P4) by time of flight-mass spectrometry/mass spectrometry (TOF-MS) analysis. The IC₅₀ values against ACE activity were 9.10 μM (P1), 10.77 μM (P2) and 7.72 μM (P4). The inhibition mode of P1, P2 and P4 was analyzed using the Lineweaver–Burk plots, demonstrating P1 to be a non-competitive inhibitor, P2 and P4 having a mixed inhibition mode. Taken together, the salmon byproduct protein hydrolysate and/or its active peptides can be used in foods for its benefits against hypertension and related diseases.     

Interaction with angiotensin-converting enzyme-encoding gene in female infertility: Insertion and deletion polymorphism studies

Angiotensin-converting enzyme (ACE), a key enzyme in the renin– angiotensin–aldosterone system, converts angiotensin I to angiotensin II. Ethnic origin should be carefully considered in studies pertaining to ACE I/D genotype and disease etiology. This study was evaluated between the ACE gene I/D polymorphism and female infertility in the Saudi population. Out of a A total of 300 women who participated in this study genomic DNA samples from the 150 infertile and 150 fertile women’s were isolated who has participated in this study. Genomic DNA was isolated using an Invitrogen kit according to the manufacturer’s protocol, and D allele specific primers were used for amplification by polymerase chain reaction. Electrophoresis was carried out on a 2% agarose gel. The mean age and BMI of the cases and controls were similar (p > 0.05), and a significant association was noted between the family history and female infertility (p = 0.0001). The D allele (OR: 1.67 [95% CI: 1.18–2.35], p = 0.003), DD genotype (OR: 2.46 [95% CI: 1.20–5.02], p = 0.01) and dominant model (OR: 1.97 [95% CI: 1.00–3.88], p = 0.04) were significantly associated with female infertility or fertility. The results of this study show that the ACE polymorphism plays an important role in female infertility in the Saudi population.     

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52 ± 2.44E-10 and 5.87 ± 1.3E–9 M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33 ± 1.15E-9 and 4.11 ± 1.09E–9 M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P < 0.05). They also significantly reduced the serum sodium level and increased the urine volume (P < 0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.     

Angiotensin II-accelerated vulnerability of carotid plaque in a cholesterol-fed rabbit model-assessed with magnetic resonance imaging comparing to histopathology

This study sought to reveal the effect of angiotensin II (Ang II)-induced atherosclerotic vulnerability in rabbits and to determine whether in vivo magnetic resonance imaging (MRI) can determine the effect of Ang II on atherosclerotic development over time. In total, 24 elderly male New Zealand white rabbits underwent an intravascular balloon injury in the left common carotid artery (LCCA) and were subsequently fed a high cholesterol diet for 12 weeks. At 8 weeks, rabbits were randomly assigned to receive either Ang II (1.4 mg/kg/d, Ang II group) or vehicle (phosphate-buffered saline, control) via a subcutaneous osmotic minipump for 4 weeks. The rabbits were imaged three times: at baseline and at 8 and 12 weeks. After the 12-week MRI scanning, rabbits were euthanized to obtain pathological and histological data. Atherosclerotic plaques were identified in the 21 rabbits that survived the 12-week trial. Typical feature of vulnerable plaques (VP), intraplaque hemorrhage, were observed in 6 of 10 animals (60.0%) in the Ang II group. The Cohen K value of MR imaging between the AHA classifications was 0.82 (0.73–0.91; P < 0.001). MRI revealed that the change in carotid morphology were significantly different between the Ang II and control group plaques. Our results support an important role for Ang II in plaque vulnerability by promoting intraplaque neovascularization and hemorrhage as well as inflammation. The vulnerable features induced by Ang II in rabbit carotid plaques could be accurately monitored with MRI in vivo and confirmed with histomorphology.