Increased Angiotensin-converting Enzyme Activity Research Articles

Transgenic angiotensin-converting enzyme 2 overexpression in the rat vasculature protects kidneys from ageing-induced injury

Chronic kidney disease is one of the leading causes of morbidity and mortality especially among the aged population. A decline in kidney function with ageing comparable to ageing-related processes in human kidneys has also been described in Sprague-Dawley (SD) rats. The renin-angiotensin-system (RAS) plays a pivotal role in the pathophysiology of cardiovascular and kidney disease and is a successful therapeutic target. The discovery of angiotensin-(1-7) (Ang(1-7)), mainly produced by angiotensin-converting enzyme 2 (ACE2), and its receptor MAS offered a new view on the RAS. This ACE2/Ang(1-7)/MAS axis counteracts most deleterious actions of the RAS in the kidney. In order to evaluate if activation of this axis has a protective effect in ageing-induced kidney disease we generated a transgenic rat model (TGR(SM22hACE2)) overexpressing human ACE2 in vascular smooth muscle cells. These animals showed a specific transgene expression pattern and increased ACE2 activity in the kidney. Telemetric recording of cardiovascular parameters and evaluation of kidney function by histology and urine analysis revealed no alterations in blood pressure regulation and basal kidney function in young transgenic rats when compared to young SD rats. However, with ageing, SD rats developed a decline in kidney function characterized by severe albuminuria which was significantly less pronounced in TGR(SM22hACE2) rats. Concomitantly, we detected lower mRNA expression levels of kidney damage markers in aged transgenic animals. Thus, our results indicate that vascular ACE2-overexpression protects the kidney against ageing-induced decline in kidney function, supporting the kidney-protective role of the ACE2/Ang(1-7)/MAS axis.

Effects of Two Soluble ACE2-Fc Variants on Blood Pressure and Albuminuria in Hypertensive Mice: Research Letter.

Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity. Furthermore, soluble ACE2 variants have been proposed as potential therapeutics for kidney disease and hypertensive disorders. Soluble ACE2 variants conjugated to human Fc domains and selected for high-potency viral SARS-CoV-2 neutralization were prepared and evaluated for ACE2 activity in vitro. Lead candidates were then tested for systemic ACE2 activity, stability, and effects on blood pressure and albuminuria in mice with Ang II-induced hypertension. ACE2 activity of 10 soluble ACE2 variants was first assessed in cell-free conditions using a fluorogenic substrate, or by Ang II hydrolysis to Ang-(1-7). Hypertension was induced in male or female mice by implantation of osmotic minipumps containing Ang II. Two lead ACE2 variants were injected intravenously (i.v.) into hypertensive mice, followed by measurements of blood pressure (tail-cuff plethysmography), albuminuria, and tissue ACE2 activity and protein (immunoblots). Soluble ACE2-Fc variants demonstrated significant ACE2 enzymatic activity, with kinetics comparable with human recombinant ACE2. In hypertensive mice, single dose i.v. injection of ACE2-Fc variant K (10 mg/kg) significantly decreased systolic blood pressure at 24 hours, with partial lowering sustained to 48 hours, and tendency to reduce albuminuria at 72 hours. By contrast, ACE2-Fc variant I had no effect on blood pressure or albuminuria in hypertensive mice; ACE2-Fc variant K was detected by immunoblotting in plasma, kidney, heart, lung, liver, and spleen lysates 72 hours after injection, associated with significantly increased ACE2 activity in all tissues except kidney and spleen. Angiotensin-converting enzyme 2-Fc variant I had no effect on plasma ACE2 activity. Soluble ACE2-Fc variant K reduces blood pressure and tends to lower albuminuria in hypertensive mice. Furthermore, soluble ACE2-Fc variant K has prolonged tissue retention, associated with increased tissue ACE2 activity. The results support further studies directed at the therapeutic potential of soluble ACE2-Fc variant K for cardiovascular and kidney protection.

ACE2 Ubiquitination in the Paraventricular Nucleus of the Hypothalamus Contributes to Angiotensin‐II‐Induced Hypertension in Mice

Ubiquitination is a post‐translational modification of proteins where the ubiquitin moiety is covalently attached at the C‐terminus to specific lysine residues on the target protein, leading to degradation in proteasome or lysosomes. Angiotensin converting enzyme 2 (ACE2) is a transmembrane protein with a primary role in converting Angiotensin (Ang)‐II into Ang‐(1‐7) and thereby reducing blood pressure (BP). We previously reported that ACE2 undergoes internalization and degradation via ubiquitination in response to Ang‐II. Here we investigated whether ACE2 ubiquitination in the paraventricular nucleus of the hypothalamus (PVN), contributes to Ang‐II mediated hypertension in mice. Wildtype C57BL/6J (WT, n=6) and ACE2 KO (n=6) male mice were bilaterally injected into the PVN with an adeno‐associated virus vector expressing a ubiquitin resistant ACE2 mutant in which 5 lysine residues from the C‐terminus of the human ACE2 protein were replaced with arginine (hACE2‐5R). Six‐weeks later, anesthetized mice were connected to a stereotaxic setup and BP was recorded from the common carotid artery. Focal microinjections of Ang‐II (100 ng in 100 nL) were administered into PVN, and BP responses were analyzed. In another group, mice were chronically implanted subcutaneously with BP telemetry and administered Ang‐II via osmotic minipump (600 ng/kg/day) for 2‐weeks. In comparison to WT mice (n=6), acute microinjections of Ang‐II into PVN resulted in a significantly attenuated BP response in hACE2‐5R transfected WT mice (9±1 vs. 14±2 mmHg; n=6, p<0.05). A similarly attenuated response was observed in ACE2 KO mice (n=8) expressing hACE2‐5R when compared to ACE2 KO mice (7±2 vs. 16±3, n=5, p<0.05). Furthermore, chronic Ang‐II‐infused WT and ACE2 KO mice expressing hACE2‐5R exhibited a similar reduction of hypertension (125±2 and 121±1 mmHg, respectively) in comparison to their naïve WT and ACE2 KO controls (137±1 and 148±2 mmHg, respectively, p<0.05). Protein analysis revealed a significant attenuation of AT1receptor expression and increased ACE2 activity in the hypothalamus of mice transfected with hACE2‐5R in the PVN. Our results demonstrate that ACE2 is ubiquitinated by circulating Ang‐II and overexpression of ubiquitination resistant ACE2 enhances the enzyme’s activity and decreases BP in hypertensive mice.

Pharmacologic ACE-Inhibition Mitigates Radiation-Induced Pneumonitis by Suppressing ACE-Expressing Lung Myeloid Cells

Radiation-induced lung injury is a major dose-limiting toxicity for thoracic radiation therapy patients. In experimental models, treatment with angiotensin converting enzyme (ACE) inhibitors mitigates radiation pneumonitis; however, the mechanism of action is not well understood. Here, we evaluate the direct role of ACE inhibition on lung immune cells. ACE expression and activity were determined in the lung immune cell compartment of irradiated adult rats after either high dose fractionated radiation therapy to the right lung (5 fractions×9 Gy) or a single dose of 13.5 Gy partial body irradiation. Mitigation of radiation-induced pneumonitis with the ACE-inhibitor lisinopril was evaluated in the 13.5 Gy rat partial body irradiation model. During pneumonitis, we characterized inflammation and immune cell content in the lungs and bronchoalveolar lavage fluid. In vitro mechanistic studies were performed using primary human monocytes and the human monocytic THP-1 cell line. In both the partial body irradiation and fractionated radiation therapy models, radiation increased ACE activity in lung immune cells. Treatment with lisinopril improved survival during radiation pneumonitis (P=.0004). Lisinopril abrogated radiation-induced increases in bronchoalveolar lavage fluid monocyte chemoattractant protein 1 (chemokine ligand 2) and MIP-1a cytokine levels (P < .0001). Treatment with lisinopril reduced both ACE expression (P=.006) and frequency of CD45+ CD11b+ lung myeloid cells (P=.004). In vitro, radiation injury acutely increased ACE activity (P=.045) and reactive oxygen species (ROS) generation (P=.004) in human monocytes, whereas treatment with lisinopril blocked radiation-induced increases in both ACE and ROS. Radiation-induced ROS generation was blocked by pharmacologic inhibition of either NADPH oxidase 2 (P=.012) or the type 1 angiotensin receptor (P=.013). These data demonstrate radiation-induced ACE activation within the immune compartment promotes the pathogenesis of radiation pneumonitis, while ACE inhibition suppresses activation of proinflammatory immune cell subsets. Mechanistically, our in vitro data demonstrate radiation directly activates the ACE/type 1 angiotensin receptor pathway in immune cells and promotes generation of ROS via NADPH oxidase 2.

Анализ связи I/D полиморфизма гена ангиотензинпревращающего фермента с обменом мочевой кислоты у больных сахарным диабетом 2 типа (жителей Чувашии)

It is known that metabolic disorders of uric acid (UA), in particular hyperuricemia (HU), are associated with vascular complications of type 2 diabetes mellitus (DM 2). Previously, the authors established a relationship between the I/D polymorphism of the angiotensin-converting enzyme (ACE) gene and vascular lesions in patients with DM 2 living in Chuvashia. It was of interest to find out how the I/D polymorphism of the ACE gene and metabolic disorders of MC in patients with diabetes mellitus 2 (residents of Chuvashia) are related. ACE gene polymorphism was determined by the I/D polymerase reaction method in 104 patients with diabetes mellitus 2, in 46 of whom UA metabolism was studied in depth. The UA was determined spectrophotometrically in the blood and in daily urine, and the total and fractional renal clearance of the UA was calculated. There were no significant differences in the parameters of UA metabolism depending on the I/D polymorphism. A steady tendency towards a decrease in renal UA clearance was established in the DD genotype. In patients with the DD genotype in comparison with carriers of the I allele (genotypes ID and DD), a lower mean value of UA clearance was recorded (M 10.0, Me 8.4 ml/min versus M 12.4, Me 11.7 ml/min; Z=1.1, p=0.28) and a higher prevalence of its low (&lt;9 ml/min) values: 60% (9 out of 15) versus 34.6% (9 out of 26) (χ2=2, 18, p=0.14). It should be assumed that with an increase in the number of examined patients, the relationship of the DD genotype with a reduced UA clearance will acquire statistical significance. With the DD genotype, according to many authors, the maximum ACE activity is rectified. The authors suggest that an increase in ACE activity contributes to a decrease in UA transit through the kidneys in the DD genotype

I/D Polymorphism Gene ACE and Risk of Preeclampsia in Women with Gestational Diabetes Mellitus.

Preeclampsia (PE) and gestational diabetes mellitus (GDM) are the most common complications of pregnancy, which result in adverse outcomes for the mother and the fetus. GDM is regarded as a separate independent risk factor for PE development, as evidenced by a higher preeclampsia rate in gestational diabetes mellitus than in the general population. The role the endothelial cell dysfunction plays is considered to be the most reasonable one in the origin of these diseases. The activity of plasma and tissue angiotensin converting enzyme (ACE) is believed to be genetically controlled. The available data suggests that increased ACE activity due to deletion (D)/insertion (I) in the 16th intron of ACE gene, which is called ACE gene I/D polymorphism, is associated with preeclampsia and varies depending on the studied population and the geography. We did not find any literature data that estimates the influence of ACE gene I/D polymorphism on PE rate in pregnant women with GDM. Therefore, the present study aimed to investigate a relationship between ACE gene I/D polymorphism and preeclampsia development in the case of GDM in the Russian population. The study used the genomic DNA derived by phenol-chloroform extraction method from venous blood samples in 137 pregnant women, including samples of 74 women with GDM accompanied with PE and the blood samples of 63 women with GDM w/o preeclampsia. Genotyping of insertion/deletion in the I/D region (16 intron of АСЕ gene) was conducted by real-time PCR using the TaqMan competing probe technology. The particular features in the frequency array of alleles and genotypes of the ACE gen I/D polymorphism under review, as associated with preeclampsia development risk in pregnant women with GDM, were identified. The acquired data testify to the need to further study of ACE gene I/D region polymorphism association in a large patient sample taking into account the PE and GDM risk factors estimated in the clinical practice.

ACE2/ACE imbalance and impaired vasoreparative functions of stem/progenitor cells in aging.

Aging increases risk for ischemic vascular diseases. Bone marrow–derived hematopoietic stem/progenitor cells (HSPCs) are known to stimulate vascular regeneration. Activation of either the Mas receptor (MasR) by angiotensin-(1-7) (Ang-(1-7)) or angiotensin-converting enzyme-2 (ACE2) stimulates vasoreparative functions in HSPCs. This study tested if aging is associated with decreased ACE2 expression in HSPCs and if Ang-(1-7) restores vasoreparative functions. Flow cytometric enumeration of Lin−CD45lowCD34+ cells was carried out in peripheral blood of male or female individuals (22–83 years of age). Activity of ACE2 or the classical angiotensin-converting enzyme (ACE) was determined in lysates of HSPCs. Lin−Sca-1+cKit+ (LSK) cells were isolated from young (3–5 months) or old (20–22 months) mice, and migration and proliferation were evaluated. Old mice were treated with Ang-(1-7), and mobilization of HSPCs was determined following ischemia induced by femoral ligation. A laser Doppler blood flow meter was used to determine blood flow. Aging was associated with decreased number (Spearman r = − 0.598, P < 0.0001, n = 56), decreased ACE2 (r = − 0.677, P < 0.0004), and increased ACE activity (r = 0.872, P < 0.0001) (n = 23) in HSPCs. Migration or proliferation of LSK cells in basal or in response to stromal-derived factor-1α in old cells is attenuated compared to young, and these dysfunctions were reversed by Ang-(1-7). Ischemia increased the number of circulating LSK cells in young mice, and blood flow to ischemic areas was recovered. These responses were impaired in old mice but were restored by treatment with Ang-(1-7). These results suggest that activation of ACE2 or MasR would be a promising approach for enhancing ischemic vascular repair in aging.

ACE2 (Angiotensin-Converting Enzyme 2) in Cardiopulmonary Diseases

Discovery of ACE2 (angiotensin-converting enzyme 2) revealed that the renin-angiotensin system has 2 counterbalancing arms. ACE2 is a major player in the protective arm, highly expressed in lungs and gut with the ability to mitigate cardiopulmonary diseases such as inflammatory lung disease. ACE2 also exhibits activities involving gut microbiome, nutrition, and as a chaperone stabilizing the neutral amino acid transporter, B0AT1, in gut. But the current interest in ACE2 arises because it is the cell surface receptor for the novel coronavirus, severe acute respiratory syndrome coronavirus-2, to infect host cells, similar to severe acute respiratory syndrome coronavirus-2. This suggests that ACE2 be considered harmful, however, because of its important other roles, it is paradoxically a potential therapeutic target for cardiopulmonary diseases, including coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2. This review describes the discovery of ACE2, its physiological functions, and its place in the renin-angiotensin system. It illustrates new analyses of the structure of ACE2 that provides better understanding of its actions particularly in lung and gut, shedding of ACE2 by ADAM17 (a disintegrin and metallopeptidase domain 17 protein), and role of TMPRSS2 (transmembrane serine proteases 2) in severe acute respiratory syndrome coronavirus-2 entry into host cells. Cardiopulmonary diseases are associated with decreased ACE2 activity and the mitigation by increasing ACE2 activity along with its therapeutic relevance are addressed. Finally, the potential use of ACE2 as a treatment target in COVID-19, despite its role to allow viral entry into host cells, is suggested.

Impact of diminazene aceturate on renin-angiotensin system, infectious myocarditis and skeletal myositis in mice: An in vitro and in vivo study

Although renin-angiotensin system (RAS) imbalance is manifested in cardiomyopathies with different etiologies, the impact of RAS effectors on Chagas cardiomyopathy and skeletal myositis is poorly understood. Given that diminazene aceturate (DMZ) shares trypanocidal, angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1–7) stimulatory effects, we investigated the impact of DMZ on cardiomyocytes infection in vitro, renin-angiotensin system, Chagas cardiomyopathy and skeletal myositis in vivo. Cardiomyocytes and T. cruzi were used to evaluate DMZ toxicity in vitro. The impact of 20-days DMZ treatment (1 mg/kg) was also investigated in uninfected and T. cruzi-infected mice as follows: control uninfected and untreated, uninfected treated with DMZ, infected untreated and infected treated with DMZ. DMZ had low toxicity on cardiomyocytes, induced dose-dependent antiparasitic activity on T. cruzi trypomastigotes, and reduced parasite load but not infection rates in cardiomyocytes. DMZ increased ACE2 activity and angiotensin-(1–7) plasma levels but exerted no interference on angiotensin-converting enzyme (ACE) activity, ACE, ACE2 and angiotensin II levels in uninfected and infected mice. DMZ treatment also reduced IFN-γ and IL-2 circulating levels but was ineffective in attenuating parasitemia, MCP-1, IL-10, anti-T. cruzi IgG, nitrite/nitrate and malondialdehyde production, myocarditis and skeletal myositis compared to infected untreated animals. As the antiparasitic effect of DMZ in vitro did not manifest in vivo, this drug exhibited limited relevance to the treatment of Chagas disease. Although DMZ is effective in upregulating angiotensin-(1–7) levels, this molecule does not act as a potent modulator of T. cruzi infection, which can establish heart and skeletal muscle parasitism, lipid oxidation and inflammatory damage, even in the presence of high concentrations of this RAS effector.

Overexpression of angiotensin converting enzyme 2 reduces anxiety-like behavior in female mice.

Accumulating evidence has revealed an intricate role for the renin-angiotensin system (RAS) in the progression or alleviation of stress-related disorders. Along these lines, the ‘pro-stress’ actions of angiotensin-II (Ang-II) are largely thought to be mediated by the angiotensin type-1a receptor (AT1aR). On the other hand, a counter regulatory limb of the RAS that depends on the conversion of Ang-II to angiotensin-(1–7) by angiotensin-converting enzyme 2 (ACE2) has been postulated to exert stress-dampening actions. We have previously found that augmenting ACE2 activity is potently anxiolytic and blunts stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis in male mice. Whether increasing ACE2 activity also relieves stress and anxiety in females has not yet been determined. Consequently, this series of experiments tests the hypothesis that augmenting ACE2 expression is anxiolytic and dampens the activity of the HPA axis in female mice. Using the Cre-LoxP system, we generated female mice that were homo-, heterozygous or wild-type for a mutated allele resulting in ubiquitous overexpression of ACE2. Next, we used qPCR to determine that levels of ACE2 mRNA isolated from central and peripheral tissues was dependent on genotype. That is, mice homo- and heterozygous for the ACE2 overexpression had significantly greater levels of ACE2 mRNA relative to littermate matched wild-type controls. Interestingly, anxiety-like behavior as determined by the elevated plus maze, light-dark box and novelty-induced hypophagia tests was also affected by genotype. Specifically, ACE2 overexpression significantly decreased anxiety-like behavior in paradigms dependent on approach-avoidance conflict and novelty; however, locomotor activity was similar amongst the genotypes. Final experiments measured plasma corticosterone to evaluate whether increasing ACE2 alters basal and/or stress-induced HPA axis activity. In contrast to what was previously found in males, increasing ACE2 expression had no effect on plasma corticosterone under basal conditions or subsequent to an acute restraint challenge. Collectively, these results suggest that increasing ACE2 expression potently elicits anxiolysis in female mice without altering HPA axis activity.

Effects of Catechin on Activity of Angiotensin-Converting Enzyme and Generation of Reactive Oxygen Species in Rat Aorta.

Activity of angiotensin-converting enzyme (ACE) and intensity of ROS generation in the aorta were studied in male Wistar rats intraperitoneally injected with a mixture of (+) and (-) stereoisomers of catechin. ACE activity in aortal segments was evaluated by hydrolysis of hippuryl-histidine-leucine; ROS generation was measured by oxidation of dichlorodihydrofluorescein. The dynamics of ACE activity and intensity of ROS generation in the aorta after catechin administration and their dependence on the catechin dose were studied. The effects of dihydroquercetin and fucoidin on the studied parameters were analyzed. Catechin increased ACE activity; the maximum increase was achieved in 3 h after administration. Catechin dose producing a half-maximum increase in ACE activity was 0.04 μg/kg. The effects of catechin on ACE activity was attenuated by dihydroquercetin and completely abolished by fucoidin. Catechin did not enhance ROS production in the aorta, and in a dose of 0.1 μg/kg even inhibited this process. It is hypothesized that isomers of catechin produce opposite effects on ROS generation in the aorta.

Зміни активності ангіотензинперетворюючого ферменту в структурах гіпоталамо-гіпофізарно-адренокортикальної системи та функції кори надниркових залоз щурів під дією метанандаміду

Мета роботи — дослідити вплив метанандаміду — метаболічно стійкого похідного ендоканабіноїду N-арахідоноїлетаноламіну на активність ангіотензинперетворюючого ферменту (АПФ) у центрах регуляції гіпоталамо-гіпофізарно-адренокортикальної системи (ГГАС), а також на рівень кортикостероїдів у плазмі крові щурів. Матеріал і методи. Активність АПФ у гіпоталамусі, аденогіпофізі, надниркових залозах і плазмі крові щурів визначали за допомогою флуориметричного методу, використовуючи як субстрат Benzoyl-Gly-His-Leu (Sigma, США). Вміст 11-гідроксикортикостероїдів (11-ОКС) у плазмі крові щурів встановлювали із застосуванням флуориметричного мікрометоду. Результати. Через 1 годину після одноразового введення інтактним щурам метанандаміду в дозах 0,2 мг/кг і 0,02 мг/кг активність АПФ у центральних ланках ГГАС — гіпоталамусі та аденогіпофізі підвищувалася, активність АПФ унадниркових залозах та активність циркулюючого в крові ферменту знижувалися. Після одноразового введення інтактним щурам метанандаміду в дозі 0,2 мг/кг виявлено підвищення рівня 11-ОКС у плазмі крові щурів, введення метанандаміду в низькій дозі — 0,02 мг/кг не призводило до вірогідних змін рівня кортикостероїдів у крові тварин. Висновки. Екзогенні канабіноїди модулюють активність ангіотензинової системи в різних ланках ГГАС, стимулюючи активність АПФ у гіпоталамусі й аденогіпофізі та знижуючи активність ферментув надниркових залозах і плазмі крові через 1 годину після одноразового введення метанандаміду інтактним щурам. Через кортикотропін-рилізинг активність ангіотензинів гіпоталамуса та аденогіпофіза підвищення активності АПФ у цих структурах може бути одним із важливих чинників активації ГГАС, зокрема синтезу кортикостероїдів, спричиненого одноразовим введенням метанандаміду у високих дозах.

Renin-angiotensin and kallikrein-kinin systems: a significance in the benign prostatic hyperplasia pathogenesis

Background . Benign prostatic hyperplasia (BPH) is the most common disease in older men. BPH pathophysiology is poorly understood. Although, it is known that the transmission of androgenergic signals and the reactivity of prostate's stroma as well as inflammatory factors are known to be the main pathophysiological mechanisms. In this regard, it is of interest to study the activity of enzymes and their inhibitors of the renin-angiotensin and kallikrein-kinin systems in BPH. Objectives. The study of new molecular mechanisms of the BPH pathogenesis. Materials and methods. The activity of the angiotensin-converting enzyme (ACE), the kallikrein-like activity and the prekallikrein content were determined. The total arginine-esterase activity was the inhibitory activity of the a1-proteinase inhibitor and a2-macroglobulin in the prostate secretion in men with BPH. A sharp increase of ACE activity in BPH leads to the accumulation of angiotensin II in the prostate secretion. A consequence of the activation of ACE in prostate secretion is a decrease in the content of bradykinin. An increase of the a1-proteinase inhibitor suppressing activity in prostate secretion at BPH indicates an increase in leukocyte degranulation activity during the development of the inflammatory process. Results. A sharp increase of ACE activity in BPH leads to the accumulation of angiotensin II in the prostate secretion. A consequence of the activation of ACE in prostate secretion is a decrease in the content of bradykinin. An increase of the a1-proteinase inhibitor suppressing activity in prostate secretion at BPH indicates an increase in leukocyte degranulation activity during the development of the inflammatory process. Conclusion. Metabolic basis for the BPH development can be mediated by impaired metathesis of angiotensin II and bradykinin in the prostate.

Genetic Association of Angiotensin-Converting Enzyme (ACE) Gene I/D Polymorphism with Preterm Birth in Korean Women: Case-Control Study and Meta-Analysis.

Background and Objectives: The ACE gene encodes the angiotensin-converting enzyme (ACE), a component of the renin-angiotensin system. Increased ACE activity may cause abnormal regulation of placental circulation and angiogenesis, resulting in adverse pregnancy outcomes. Previous studies have reported that the insertion/deletion (I/D) polymorphism of the ACE gene is associated with the development of preterm birth (PTB). However, results of the association between ACE gene I/D and PTB are inconsistent in various populations. Therefore, we performed a case-control study and a meta-analysis to evaluate the association between ACE I/D polymorphism and PTB. Materials and Methods: We analyzed a total of 254 subjects (111 patients with PTB and 143 women at ≥38 weeks gestation) for the case-control study. For the meta-analysis, we searched Google Scholar, PubMed, and NCBI databases with the terms “ACE,” “angiotensin-converting enzyme,” “preterm birth,” “preterm delivery,” and their combinations. Results: Our results of the case-control study indicated that ACE I/D polymorphism is significantly associated with PTBs in the overdominant genetic model (odds ratio (OR) 0.57, 95% confidence interval (CI) 0.347–0.949, p = 0.029) and that the ID genotype of ACE I/D polymorphism has a protective effect for PTB (OR 0.57, 95% CI 0.333–0.986, p = 0.043). Similarly, the meta-analysis showed that the OR for the ACE gene ID genotype was 0.66 (95% CI 0.490–0.900, p < 0.01). Conclusion: The ACE gene ID genotype has a significant association with PTB and is a protective factor for PTB. A larger sample set and functional studies are required to further elucidate of our findings.

Anxiolytic Effects of Overexpressing Angiotensin Converting Enzyme 2 (ACE2) in Female Mice

Considerable evidence has implicated the renin‐angiotensin system (RAS) as an important target for the therapeutic alleviation of stress‐related mood disorders. Along these lines, the ‘pro‐stress’ actions of Angiotensin‐II are largely thought to be mediated by the angiotensin type‐1 receptor (AT1R). On the other hand, the protective limb of the RAS that depends on the conversion of angiotensin‐II to angiotensin‐(1–7) by angiotensin‐converting enzyme 2 (ACE2) has been postulated to dampen stress and relieve anxiety. We have previously found that augmenting ACE2 activity has potent anxiolytic and hypothalamic‐pituitary‐adrenal (HPA) axis‐dampening effects in male mice. Relative to males, females are more susceptible to stress‐related mood disorders; however, whether increasing ACE2 activity in females also blunts stress responses has not been determined. This study tests the hypothesis that ACE2 overexpression decreases anxiety‐like behavior and attenuates stress‐induced HPA axis activation in female mice. We used the Cre/Lox system to generate female mice that ubiquitously overexpress ACE2. Female ACE2 knock‐in (ACEKI) mice and their wild‐type littermate controls were tested in the elevated‐plus maze (EPM), Light‐Dark Box (LDB), and Graham Cracker Test to evaluate the impact that augmenting ACE2 expression has on anxiety‐like behavior. Female mice were also subjected to an acute 30 min restraint stress challenge and blood samples were collected in order to evaluate HPA (i.e., corticosterone) reactivity. As expected, Cre‐Lox mediated overexpression of ACE2 resulted in elevated ACE2 mRNA and enzymatic activity in all tissues examined (i.e., hypothalamus, pituitary, blood, and brain). Relative to controls, ACE2KI mice spent significantly (p &lt; 0.05) more time in the open arms of the EPM and in the light side of the LDB. When placed in a novel environment, ACEKI mice exhibited a significantly (p&lt;0.05) decreased latency to consume graham crackers relative to controls. Importantly, total distance traveled in the EPM was not different between the groups, indicating that the locomotor activity of the female mice is unchanged by ACE2 overexpression. Interestingly, basal and acute stress‐induced corticosterone levels did not differ between ACE2KI and WT female mice. These results suggest that although neuroendocrine response to acute restraint was unaffected in females, ACE2 overexpression has anxiolytic effects on both male and female ACE2KI mice. Furthermore, our findings support the idea that the RAS may be a worthy target in the search for novel therapeutic interventions for stress‐related disorders.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated Plasma Angiotensin Converting Enzyme 2 (ACE2) Activity is Associated with Embolic Stroke of Undetermined Source

Background: ACE2 activity levels correlate with adverse left atrial remodelling in patients with atrial fibrillation (AF). Several biochemical and structural markers have been associated with embolic stroke of undetermined source (ESUS). We investigated the relationship between ACE2 activity and ESUS. Methods: This prospective case control study compared patients with ESUS against a control group matched for vascular risk factors. ESUS was diagnosed following cerebral and carotid vascular imaging and 24 hours of cardiac monitoring to exclude AF. Blood samples were collected for measurement of ACE2 activity, D-Dimer and high sensitivity troponin T (hsTnT). Results: Fifty-one ESUS patients (67 ± 7 years, CHA2DS2-VASc score 2) were compared with 47 control patients (66 ± 7 years, CHA2DS2-VASc score 2). Median ACE2 activity (10 pmol/min/ml vs. 7 pmol/min/ml, p < 0.05) and D-Dimer levels (0.40 mg/L vs 0.35 mg/L, p < 0.05) were significantly higher in the ESUS group. There was a significant but weak positive correlation between increased ACE2 activity and hsTnT (r = 0.20, p < 0.05). Left atrial volume index (LAVI) on echocardiography was significantly higher in the ESUS group (40 ml/m2 vs 36 ml/m2, p < 0.05). On regression modelling, adjusting for LAVI, only ACE2 activity (OR 1.04, CI: 1.002–1.09; p < 0.05) remained significant for ESUS. Conclusion: ACE2 activity levels are associated with ESUS, independent of LAVI. Further studies are warranted to investigate whether ACE2 activity can identify ESUS patients that may benefit from oral anticoagulation.

49 - Doxycycline treatment exerts antioxidant effects and decreases angiotensin-converting enzyme activity in renovascular hypertension

Introduction doxycycline (Dox) exerts important antioxidant effects and is able to chelate divalent metals present in the angiotensin-converting enzyme (ACE) structure that are essential for its activity. Increased ACE activity results in angiotensin (Ang) II formation that triggers oxidative stress in hypertension. In this way, we hypothesized that Dox inhibits ACE activity in hypertensive rats, reduces ROS production and ameliorates hypertension. Methods Sham-operated and 2K1C hypertensive rats were treated with Dox or water for 4 week. After treatment, two catheters were inserted into femoral artery or vein to record blood pressure and AngI infusion, respectively. Reactive oxygen species (ROS) were measured by lucigenin chemiluminescence and dihydroethidium oxidation in aortas. ACE activity was evaluated using a fluorescent substrate. To evaluate whether Dox inhibits ACE activity in vitro, aortic tissues from 2K1C rats were incubated with Dox or captopril. To evaluate acute Dox effects in vivo AngI was infused after single administration of Dox or saline in normotensive rats. Data were considered significant when p Results Dox treatment reduced 21% the SBP in 2K1C rats (p Conclusion Chronic treatment with Dox inhibited ACE activity in aorta of 2K1C rats and contributed to the reduction of SBP and ROS formation. The mechanism by which Dox inhibits ACE activity was not dependent of metal chelation and needs further investigation.

Abstract P394: Ace Activity and Its Expression as a Hypertension Biomarker in the Urine of Pediatric Renal Transplant Recipients

Pediatric hypertension is a risk factor for hypertension in adults. Primary hypertension results from genetic, environmental, and behavioral factors. Renin Angiotensin System is important to control cardiovascular and renal functions. Angiotensin Converting Enzyme (ACE) has isoforms: somatic (130-190 kDa) and testicular (90-110 kDa). Casarini et al. described the N-domain isoform of 90kDa, in urine of hypertensive humans as a possible hypertension biomarker. Objective: Analyze ACE isoform in urine of pediatric renal transplant recipients before and after transplantation and urine of donor, to verify the 90 kDa isoform transmission and correlating with hypertension. Methods: 30 pediatric recipients with living donors; ACE activity was performed before, 1, 3 and 6 months post transplantation using ZPhe and HHL as substrates and its ratio. Results: 69% had pre-transplant hypertension and 85.7% family history. After 6 months 25% remained. From the pre-transplant until the sixth month after, ACE activity was reduced. In the model of linear regression between ACE activity and variables, including pre-transplantation for the substrates ZPhe-HL remained significant, time and uropathy (0.023 more); with HHL, time and uropathy with higher activity of ACE (0.019 more) and for weight, reduction of ACE activity ( 0.00038 at 1 kg increase in weight). At post-transplant only time and weight remained significant with ZPhe-HL, and for HHL uropathy, proteinuria (0.010 more ACE activity) and weight. Recipients before transplantation had higher urinary ACE activity when compared to controls. Western blotting data showed that all recipients without ACE 90KDa when received a kidney from the donor that had this isoform, started to express it. Conclusions: Increased ACE activity in uropathies may be associated with renal injury or intra-renal production. Proteinuria, a marker of renal injury, can be associated with increase on ACE activity. With nutritional recovery and a healthy kidney after transplantation, patients can reverse the degree of renal damage and decrease the production of ACE.

Flavonoids decrease the radiation-induced increase in the activity of the angiotensin-converting enzyme in rat aorta

The basic factor of cardiovascular diseases is atherosclerosis, which is due largely to an increase in the activity of the angiotensin-converting enzyme (ACE) in vessels. Flavonoids diminish the risk of cardiovascular diseases and the flavonoid taxifolin normalizes the activity of ACE. We examined the efficiency of seven flavonoids in preventing an increase in ACE activity in aorta of rats exposed to ionizing radiation. It was shown that the activity of flavones and flavonols decreases with an increase in the number of OH groups in the A and B rings, respectively. The reduction in the activity of flavonoids within the classes correlates with a decrease in their lipophilicity. Flavanonols (taxifolin) are more active than flavonols, and flavonols are more active than flavones.

Dihydroquercetin and Fucoidin Inhibit the Increase of Angiotensin-Converting Enzyme Activity in the Rat Aorta after Irradiation.

The time course of angiotensin-converting enzyme activity in the rat aorta after fractionated exposure to ionizing radiation and the effects of dihydroquercetin and fucoidin on this parameter were studied. Male Wistar rats were exposed to single or repeated (fractionated) X-ray radiation in a dose of 2.5 Gy at 200 kV. Activity of angiotensin-converting enzyme in aorta segments was evaluated 2 h after the last exposure by hydrolysis of hippuryl-histidineleucin substrate. Enzyme activity in the rat aorta was higher than normally after all the studied doses of fractionated exposure (2.5 Gy per fraction) with the maximum effect after the total dose of 7.5 Gy (3 fractions). Fucoidin, a blocker of endothelium receptors realizing the leukocyte adhesion to the endothelium, and flavonoid dihydroquercetin inhibiting expression of adhesion molecules in the endothelium abolished the increase in activity of angiotensinconverting enzyme in the rat aorta after single exposure; moreover, dihydroquercetin reduced significantly the effect of fractionated exposure. These data indicate that leukocyte adhesion to the endothelium is an important factor contributing to the increase of angiotensin-converting enzyme activity in the aorta.