Angiotensin-converting Enzyme mRNA Levels Research Articles

Apelin from Perivascular Adipose Tissue Is Involved in the Regulation of Vasorelaxation and Renal Function in Metabolic Syndrome

The perivascular adipose tissue (PVAT) regulates the arterial tone by releasing vasoactive molecules. PVAT dysfunction favoring the vasorelaxation response could contribute to the development of kidney disease in metabolic syndrome (MetS). Previously, we demonstrated that overactivation of angiotensin II signaling in the PVAT deteriorates the compensatory PVAT effects in rats with MetS (SHRSP.Z-Leprfa/IzmDmcr (SPZF) and SHR/NDmcr-cp (CP) rats). Apelin is an endogenous regulator of angiotensin II. Therefore, we investigated whether changes in apelin levels in the PVAT alter PVAT function and impair kidney function in MetS. Twenty-three-week-old male and female SPZF and CP rats were used. In the female CP rats, apelin mRNA levels in renal arterial PVAT, enhancing effects of the PVAT on acetylcholine-induced relaxation in renal arteries, and estimated glomerular filtration rate (eGFR) were the highest, and urine protein levels and homeostasis model assessment of insulin resistance (HOMA-IR) were the lowest. Apelin mRNA levels were positively correlated with the enhancing effects of the PVAT on vasorelaxation and eGFR but negatively correlated with urine protein levels and HOMA-IR. Moreover, apelin levels positively correlated with mRNA levels of angiotensin-converting enzyme 2 and angiotensin II type 1 receptor-associated protein, which are negative regulators of angiotensin II. This study suggests that a decline in apelin levels in the PVAT, probably owing to angiotensin II, is associated with PVAT dysfunction on vascular tone, resulting in impaired kidney function in MetS.

The metalloproteinase ADAM10 sheds angiotensin-converting enzyme (ACE) from the pulmonary endothelium as a soluble, functionally active convertase.

The renin-angiotensin-aldosterone system (RAAS) plays a critical role in the regulation of blood pressure and fluid balance, with angiotensin-converting enzyme (ACE) being a key transmembrane enzyme that converts angiotensin I to angiotensin II. Hence, ACE activity is an important drug target in cardiovascular pathologies such as hypertension. Our study demonstrates that human pulmonary microvascular endothelial cells (HPMECs) are an important source of proteolytically released ACE. The proteolytic release of transmembrane proteins, a process known as ectodomain shedding, is facilitated by membrane proteases called sheddases. By knockout and inhibition studies, we identified ADAM10 (A disintegrin and metalloprotease 10) as a primary sheddase responsible for ACE release in HEK293 cells. The function of ADAM10 as primary, constitutive sheddase of ACE was confirmed in HPMECs. Moreover, we demonstrated the physiological relevance of ADAM10 for ACE shedding in exvivo precision cut lung slices (PCLS) from human and mouse lungs. Notably, ADAM17 activity is not directly involved in ACE shedding but indirectly by regulating ACE mRNA and protein levels, leading to increased ADAM10-mediated ACE shedding. Importantly, soluble ACE generated by shedding is enzymatically active and can thereby participate in systemic RAAS functions. Taken together, our findings highlight the critical role of ADAM10 (directly) and ADAM17 (indirectly) in ACE shedding and RAAS modulation.

O48 CHRONIC EXPOSURE TO POLLUTED URBAN AIR ALTERS MOUSE SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM (RAS)

Background and Objective: Chronic exposure to air pollution induces pulmonary, cardiovascular, and renal diseases. In the current study, we investigated the hypothesis that postulates that a dysregulation of the RAS may contribute to these pathologies. Methods: Male Balb/C mice were exposed to urban air from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Control animals were exposed to filtered air. The levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2; AT1, AT2, and Mas receptors (R) were determined in mouse lungs, kidneys, and heart, through western blotting (WB) and RT-qPCR, respectively. The tissue content of cytokine mARN was determined by RT-qPCR. The tissue content of nitrotyrosine and 4-hydroxynonenal (4-HNE) levels, were determined by WB. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Systolic blood pressure was measured by the tail-cuff method. Results: Exposure to air pollution resulted in a) Increased levels of ACE and AT1R mRNA in the lungs and augmented the abundance markers of inflammation (TNF-α, IL-6, and IL-1β) and oxidative stress (nitrotyrosine and 4-HNE) in this tissue, b) increased levels of MasR mRNA (all analyzed tissues) and MasR protein levels (lungs and heart), c) upregulated mRNA and protein abundance of cardiac and renal AT2Rs, and d) Increased levels of circulating Ang II and e) augmented systolic blood pressure. Conclusions: Our findings indicate that chronic exposure to air pollution alters the expression of both tissue and systemic components of the RAS. Since both AT2R and MasRs have been shown to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse lungs, heart, and kidney after chronic exposure to polluted urban air could represent a mechanism of tissue protection against damage induced by air pollution.

EXPOSURE TO AIR POLLUTION FINE PARTICULATE MATTER (PM2.5) ALTERS SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM

Objective: Previous studies have implicated air pollution fine particulate matter (PM2.5) in various cardiovascular cardiometabolic and renal disease states. However, the molecular mechanisms by which these pollutants mediate these comorbidities have not been fully elucidated. Dysregulation of the renin-angiotensin system (RAS) may be one potential mechanism. This study aimed to investigate the participation of the RAS in air pollution-associated pathologies. Design and method: To study the impact of PM2.5 on systemic and tissue components of the RAS, male 8-week-old Balb/C mice were exposed to filtered air (FA) or urban air (UA) from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2, as well as AT1, AT2, and Mas receptors (R) abundance, were determined in kidney, heart, and lung tissue by Western Blotting (WB) and their corresponding mRNA expression was detected by RT-qPCR. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Results: Exposure to air pollution resulted in increased mRNA levels of ACE and MasR, and increased protein levels of ACE in the kidney; upregulated mRNA and protein abundance of cardiac and pulmonary AT2R and MasR, together with increased levels of proteins nitrated at Tyr residues in both kidney and lung homogenates, indicative of nitrosative stress in these tissues. In addition, exposure to PM2.5 was associated with increased levels of circulating Ang II, indicative of an exacerbation of the RAS. Conclusions: Our findings indicate that chronic exposure to air pollution induces an altered expression of both tissue and systemic components of the RAS. Given that both the AT2R and the MasR have been ascribed to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse heart and lung after chronic exposure to polluted air could represent a mechanism of tissue protection against damage induced by PM2.5.

Piper sarmentosum Roxb. Inhibits Angiotensin-Converting Enzyme Activity in Phorbol 12-Myristate-13-Acetate-Induced Endothelial Cells.

Angiotensin-converting enzyme (ACE) plays a crucial role in the pathogenesis of hypertension. Piper sarmentosum Roxb., an herb known for its antihypertensive effect, lacks a comprehensive understanding of the mechanism underlying its antihypertensive action. This study aimed to elucidate the antihypertensive mechanism of aqueous extract of P. sarmentosum leaves (AEPS) via its modulation of the ACE pathway in phorbol 12-myristate-13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were divided into five groups: control, treatment with 200 µg/mL AEPS, induction 200 nM PMA, concomitant treatment with 200 nM PMA and 200 µg/mL AEPS, and treatment with 200 nM PMA and 0.06 μM captopril. Subsequently, ACE mRNA expression, protein level and activity, angiotensin II (Ang II) levels, and angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression in HUVECs were determined. AEPS successfully inhibited ACE mRNA expression, protein and activity, and angiotensin II levels in PMA-induced HUVECs. Additionally, AT1R expression was downregulated, whereas AT2R expression was upregulated. In conclusion, AEPS reduces the levels of ACE mRNA, protein and activity, Ang II, and AT1R expression in PMA-induced HUVECs. Thus, AEPS has the potential to be developed as an ACE inhibitor in the future.

S-51-6: RENOVASCULAR HYPERTENSION AND BRAIN: ROLE OF THE RENIN-ANGIOTENSIN SYSTEM AND SODIUM

Renovascular hypertension is a type of secondary hypertension. Renovascular hypertensive 2-kidney, 1-clip (2K1C) rats have an increased activity of the renin-angiotensin system, and it is an experimental model to study renovascular hypertension. Renovascular hypertensive 2K1C rats have an imbalance of RAS components in the brain, an impairment of the baroreflex and they present an increase in daily intake of hypertonic NaCl solution. Therefore, we focused on studying: 1) the effects of overexpression of RAS components in the in the nucleus of the solitary tract (NTS), a brainstem area involved with cardiovascular control, 2) the brain mechanisms involved with the sodium appetite (operationally defined as an ingestion of hypertonic NaCl solution) in 2K1C rats. In adult Holtzman rats, the left renal artery was partially occluded by a silver clip with an opening of 0.2 mm to induce 2K1C. The experiments started after 6 weeks (hypertension plateau). We observed that increasing angiotensin type 2 receptor (AT2R) expression in the NTS attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex observed in 2K1C rats. Furthermore, an observed decrease in mRNA levels of angiotensin-converting enzyme 2 in the NTS of 2K1C rats was restored to control levels after viral-mediated increases in AT2R at this site. Virally mediated blockade of an intracellular pathway activated by angiotensin II (ANG II) acting in type 1 receptor (AT1R) in the NTS attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex. Daily 0.3 M NaCl intake transiently increased from the 2nd to the 5th week after 2K1C surgery. On the 6th week, in spite of comparable daily 0.3 M NaCl intake between 2K1C and sham rats, water deprivation (used to induce sodium appetite) produced a greater increase 0.3 M NaCl intake in 2K1C rats. Moreover, this intake is totally blocked by central AT1R and mineralocorticoid receptor antagonism. These data demonstrated that ANG II acting in the AT1R in the NTS is involved with the development of hypertension and baroreflex impairment in 2K1C and that AT2 overexpression at the same site can counteract these effects. In addition, the results demonstrated the participation of ANG II and aldosterone acting centrally in the enhanced sodium intake in water deprived 2K1C rats.

Renin-angiotensin system (RAS) enzymes and placental trophoblast syncytialisation

Placental renin-angiotensin system (RAS) components; prorenin, angiotensinogen, and angiotensin (Ang) II type 1 receptor (AT1R) are upregulated during syncytialisation. This study examined whether angiotensin-converting enzyme (ACE), ACE2 and neprilysin (NEP) are also altered during syncytialisation. Two in vitro models of syncytialisation were used: forskolin-treated BeWo cells and spontaneously syncytialising primary human trophoblast cells. Term placentae and primary trophoblasts had the highest levels of ACE, ACE2 and NEP mRNA. In primary trophoblasts, ACE mRNA levels significantly increased with syncytialisation, ACE2 and NEP mRNA levels decreased. ACE, ACE2 and NEP protein levels and ACE2 activity did not change. Syncytialisation of primary trophoblasts decreased soluble (s)ACE and sNEP but not sACE2 levels. In primary trophoblasts, the balance between the enzymes controlling the two opposing pathways of the RAS was maintained. These findings were unable to be reproduced in BeWo cells. Future studies exploring placental levels of these enzymes in pregnancies complicated by placental insufficiency are warranted.

Extract of varieties of guava (Psidium guajava L.) leaf modulate angiotensin-1-converting enzyme gene expression in cyclosporine-induced hypertensive rats

BackgroundHypertension has become a serious health problem with which people have to live day by day, it sometimes results in severe organ damage with a dead end. Management with Psidium guajava L. (guava) leaf extract through modulation of angiotensin-converting enzyme (ACE) mRNA expression would give succor to patients with hypertension. However, there are several reports on medicinal efficacy of guava leaf with dearth of information on its varieties. HypothesisElevation of the ACE mRNA level in the kidney plays a primary role in the acceleration of local angiotensin II formation and thus sustain hypertension. Studies have shown the significance of investigating the molecular mechanism of ACE activities in pathophysiological states, this work sought to investigate the modulatory effect of aqueous extract of leaf from different varieties of guava on ACE mRNA gene expression in cyclosporine-induced hypertensive rats. MethodsHypertension was induced via intraperitoneal (i.p) administration of cyclosporine (25mg/kg/day); an aqueous extract of leaves from white, red, and pink guava was prepared, 250 and 500mg/kg body weight of the extracts were administered to the rats by oral gavage for 7 days. The systolic (SBP) and diastolic (DBP) blood pressure was measured, ACE mRNA expression level was determined using RT-qPCR, with kidney function markers in plasma (creatinine, urea, and uric acid), plasma nitric oxide level, the kidney histopathology, as well as phenolic characterization of the guava leaves. ResultsThe results showed a significant (p<0.05) increase in SBP, DBP, plasma creatinine, urea, and uric acid concentration, as well as nitric oxide level with up-regulation of ACE gene expression in the rats' kidneys with severe glomerular damage, necrosis, and capillary collapse. However, a significant (p<0.05) down-regulation of ACE mRNA expression level, reduced serum creatinine, urea, and uric acid concentration with mild-moderate kidney damage was observed in the treated rats. The leaves extract characterization and quantification revealed chlorogenic acid, rutin, vanillic acid, and quercetin as the most abundant phenolic compounds in the leaves. ConclusionsThe oral administration of the aqueous extract of different varieties of guava leaf (rich in phenolics), reduced SBP, DBP, kidney function markers concentration in the plasma, with increased plasma nitric oxide level, ameliorate kidney damage, and down-regulate renal ACE gene expression with no significant (p>0.05) difference in activities of extracts from different varieties of guava leaves used for this study. This could be a useful therapeutic measure for the treatment/management of hypertension.

Exercise training attenuates hypertension and cardiac hypertrophy via time‐related regulation on ACE and ACE2 axes

Appropriate exercise training (ExT) has been shown to lower high blood pressure. However, prehypertension ExT has rarely been concerned. This study tested whether prehypertension ExT protects against hypertension and cardiac remodeling in spontaneously hypertensive rats (SHR), and explored the underlying mechanisms by examining cardiac angiotensin converting enzyme (ACE) and ACE2 axes. Low-intensity ExT was started in male SHR and control Wistar-Kyoto rats prior to the onset of hypertension, and lasted for either 8 or 16 weeks. Blood pressure (BP) was measured biweekly by the tail-cuff method. Cardiac function and remodeling were assessed, and ACE and ACE2 axes were determined after the final ExT. The results showed that prehypertension ExT slowed down the onset and progression of hypertension in SHR. In parallel, the hypertrophy in hypertensive heart was attenuated, myocardial fibrosis was reduced, and the impairment of left ventricular diastolic function was relieved. In SHR myocardium, the elements of ACE-AngII-AT1 axis were homogeneously and progressively increased, whereas those of ACE2-Ang(1–7)-MAS axis were heterogeneously decreased. Different time-related responses were observed for the key effecters, Ang II and Ang(1–7). Myocardial Ang II was increased progressively in SHR, and was reduced consistently by ExT. In contrast, Ang(1–7) was decreased only after 16-week sedentariness, which was abolished by ExT. Besides, 16-week ExT increased Ang(1–7) in normotensive control rats. In summary, prehypertension ExT attenuates hypertension and cardiac remodeling. Down-regulation of Ang II seems to serve as a protective mechanism throughout ExT, while up-regulation of Ang(1–7) is involved after a relatively long period of ExT. Support or Funding Information This work was supported by the National Natural Science Foundation of China (No. 81372111, 81670295), and the Natural Science Foundation of Fujian Province of China (No. 2014J01339). mRNA and protein levels of ACE and AT1 in left ventricles of WKY and SHR after 8 or 16 weeks of exercise training. mRNA and protein Expression of ACE2 and MAS receptor in left ventricles of WKY and SHR after 8 or 16 weeks of exercise training. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapeseed protein-derived peptides, LY, RALP, and GHS, modulates key enzymes and intermediate products of renin\u2013angiotensin system pathway in spontaneously hypertensive rat

Rapeseed proteins are a rich source of bioactive peptides. LY, RALP and GHS were previously identified from rapeseed protein hydrolysates as potent ACE and renin inhibiting peptides. In this study, the rapeseed peptides were individually evaluated for their molecular mechanisms and regulatory effects on components of the renin–angiotensin system in spontaneously hypertensive rats (SHR), including the mRNA and/or protein levels of angiotensin-converting enzyme (ACE), renin, ACE2, angiotensin II and angiotensin-(1–7) in myocardial tissues. Oral administration of 30 mg peptides/kg body weight every 2 days for five weeks significantly decreased the systolic blood pressure and the myocardial mRNA and protein levels of ACE and renin in SHR. LY, RALP and GHS also increased the expression of ACE2, angiotensin-(1-7) and Mas receptor levels, which may have mediated their antihypertensive activity. Dipeptide LY also inhibited angiotensin II protein expression in the heart tissue. Taken together, the finding demonstrates the multi-target physiological effects of the rapeseed peptides, beyond ACE and renin inhibition, which enhances knowledge of the antihypertensive mechanisms of food protein-derived peptides.

Effects of Diets with Different Proportions of Protein/Carbohydrate on Retinal Manifestations in db Mice.

Diabetic nephropathy is aggravated by a higher intake of total protein. The effects of diets with different proportions of protein and carbohydrate on diabetic retinopathy in db mice, a type-2 diabetes animal model, were examined, as well as diabetic nephropathy. Control and db mice at 5 weeks of age were fed the diets (% energy of protein/carbohydrate/fat; L-diet: 12/71/17; H-diet: 24/59/17) under ad libitum conditions and pair-feeding conditions for 6 weeks, respectively. Mice fed the H-diet showed significantly greater retinal thickness by optical coherence tomography, and lower mRNA levels of angiotensinogen. Comparing combinations of diets and genotypes, db-H mice showed significantly higher mRNA levels of angiotensin-converting enzyme, advanced glycosylation end product-specific receptor, and cluster of differentiation molecule 11b (a microglial marker) than db-L mice. Dietary protein and carbohydrate proportions influenced retinal manifestations, including retinal thickness and gene expression in control and diabetic mice.

Beraprost Sodium, a Stable Analogue of PGI2, Inhibits the Renin-Angiotensin System in the Renal Tissues of Rats with Chronic Renal Failure

Chronic renal failure (CRF) is a prolonged kidney condition characterized by decreased kidney function that can eventually develop into total kidney failure. The renin-angiotensin system (RAS) helps to regulate the balance between human bodily fluids and electrolytes. The aim of the present study was to investigate the effects of a prostacyclin analogue (beraprost sodium [BPS]) on the expression of key factors associated with local RAS activities in the renal tissues of rats with CRF. After a CRF rat model was successfully established, the levels of BUN, SCr, phosphorus, and calcium were detected by an automatic biochemistry analyzer. Furthermore, the activities of malondialdehyde (MDA) and superoxide dismutase (SOD) in rat renal tissues were measured using a colorimetric method, while the activity of angiotensin-converting enzyme (ACE) was determined by ultraviolet (UV) spectrophotometry. In situ hybridization was employed to determine the expression of angiotensin II type 1 receptor (AT). Finally, the positive expression rates of cells expressing important apoptotic proteins (Bax and Bcl-2) were determined, and the protein and mRNA levels of phosphatidylinositol 3-kinase (AKT) and key factors involved in the RAS (AT1, AT2, angiotensin ACE and angiotensinogen [AGT]) were evaluated by RT-qPCR and western blot analysis. Initial observations revealed that treatment with BPS decreased the levels of BUN, SCr and phosphorus but increased calcium levels in the renal tissues of CRF rats. Additionally, BPS reduced the levels of MDA while increasing the levels of SOD, ACE activity, and AT1 expression in the renal tissues of CRF rats. BPS inhibited glomerular hypertension and hyperfiltration; increased the mRNA and protein levels of AKT and AT2; and decreased the mRNA and protein levels of AT1, AGT, and ACE in the renal tissues of CRF rats. The results of this study demonstrate that BPS, a PGI2 analogue, inhibits the expression of key factors involved in the local RAS, resulting in a delay in the occurrence and development of CRF. The key findings of the present study ultimately highlight the potential of this PGI2 analogue as a promising therapeutic strategy for treating CRF.

Persistent and selective upregulation of renin-angiotensin system in circulating T lymphocytes in unstable angina.

Introduction:Unstable angina is associated with an acute systemic inflammatory reaction and circulating T lymphocytes are activated. We investigated whether in unstable angina with marked immune system activation a selective upregulation of the circulating T-cell renin–angiotensin system, modulated by angiotensin II, could occur.Methods:We studied 13 unstable angina patients, 10 patients with stable angina and 10 healthy subjects. After T-lymphocyte isolation, mRNAs for angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor (AT1-R) were quantified at baseline and after angiotensin II stimulation. ACE activity in cell pellet and supernatant and angiotensin II cell content were measured.Results:Plasma renin activity was similar in controls, stable and unstable angina patients. At baseline ACE and AT1-R mRNA levels were higher (P<0.05) in T cells from unstable angina patients than in T cells from stable angina patients and controls, and further increased after angiotensin II addition to cultured T cells. ACE activity of unstable angina T cells was significantly higher than that of T cells from controls and stable angina patients. Only in T cells from unstable angina patients did angiotensin II stimulation cause the almost complete release of ACE activity in the supernatant.Conclusions:The circulating T-cell-based renin–angiotensin system from unstable angina patients was selectively upregulated. In vivo unstable angina T cells could locally increase angiotensin II concentration in tissues where they migrate independently of the circulating renin–angiotensin system.

Captopril modifies angiotensin-converting enzyme but not choline acetyltransferase gene expression in the frontal cortex of renovascular hypertensive rats

Background: The classic renin-angiotensin system (RAS) has a role in the cardiovascular system and water homeostasis in the body, but recently, the existences of this system with all of its components including angiotensinogen, angiotensin converting enzyme (ACE), and angiotensin receptors have been shown in the mammalian brain. Recent clinical studies suggest that treatment of hypertensive patients with the RAS affecting drugs, such as captopril improves their cognitive function. Aims and Objectives: This study was conducted to evaluate the effects of captopril on the frontal cortex levels of ACE and choline acetyltransferase (ChAT) in renovascular hypertensive rats receiving captopril. Materials and Methods: The rats were randomly divided into 3 groups of 8 animals; control, Goldblatt two kidney one clip (2K1C) hypertensive rats and Goldblatt 2K1C hypertensive rats received 5 mg/kg captopril. After 8 days of treatment; the rats were sacrificed and expression of ACE and ChAT in the frontal cortex were assessed by real-time polymerase chain reaction. The results were analyzed with two-way ANOVA test using SPSS software and the level of significance was set at P < 0.05. Results: Captopril treatment decreased the levels of ACE mRNA in the frontal cortex of renovascular hypertensive rats but has no significant effect in the ChAT mRNA levels. Conclusion: Captopril as a nootropic drug has no significant effect on the ChAT gene expression in the frontal cortex of renovascular hypertensive rats and it may exert its memory enhancing effect on the frontal cortex via a non-cholinergic mechanism.

Modulation of Vascular ACE by Oxidative Stress in Young Syrian Cardiomyopathic Hamsters: Therapeutic Implications.

Increased vascular angiotensin-converting enzyme (ACE) activity and oxidative stress are present in young Syrian cardiomyopathic hamsters (SCH) before the clinical manifestation of heart failure (HF). The developmental time-course of these alterations and their potential interactions, however, are still unknown. We evaluated mRNA and protein levels of ACE, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) in the vasculature of SCH from one to four months of age. Total RNA and proteins were quantified with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The role of nitric oxide (NO) on vascular ACE activity was also assessed. ACE mRNA and protein levels were up-regulated in SCH at two months of age compared with controls (CT) (p < 0.05). At this two-month stage, eNOS protein levels were lower in SCH (87%) than in CT (100%) (p < 0.05), although iNOS protein levels increased significantly (482%) compared to CT (100%; p < 0.05). In addition, ACE mRNA expression and activity were modulated by NO at two months of age. Thus, the combination of low eNOS and high iNOS protein levels may underlie vascular renin-angiotensin system (RAS) over-activation. Altogether, these factors may contribute to the development of endothelial dysfunction and vascular hyper-reactivity in the early stages of heart failure, and eventually trigger cardiac deterioration in this animal model of HF.

Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures.

Angiotensin (Ang) II plays a critical role in cardiovascular and blood pressure regulation. Ang II is produced by angiotensin-converting enzyme (ACE) and it interacts with the Ang AT1 receptor to cause much of its well-known cardiovascular effects. Ang-(1-7) is another active peptide produced by the rennin-angiotensin system. This peptide is produced from Ang I or Ang II by the catalytic activity of ACE2. Ang-(1-7) interacts with the Mas receptor to counteract many of the effects of Ang II. Thus, the ACE2/Ang-(1-7)/Mas axis acts opposite of the ACE/Ang II/AT1 axis. In this study we investigated how Ang II regulates the key enzymes of these axes, ACE and its homolog ACE2, and determined whether they are dysregulated in the hypertensive condition. Brainstem and cerebellum astrocytes isolated from the spontaneously hypertensive rat (SHR) were used in these studies. Ang II effect on the enzymes' mRNA and protein levels was measured using quantitative PCR and western blotting techniques, respectively. Results from this study showed that Ang II up-regulated ACE protein levels, but down-regulated ACE mRNA levels in brainstem and cerebellum astrocytes in both models. Ang II also reduced ACE2 mRNA expression in SHR and Wistar astrocytes isolated from both brain regions. Ang II effects on ACE2 protein were biphasic. In SHR astrocytes, Ang II-mediated ACE2 protein initially increased then decreased at later time points. In contrast, in Wistar astrocytes, Ang II initially decreased ACE2 protein expression, but up-regulated the protein at later time points. The findings of these studies suggest that Ang II has a differential effect on ACE and ACE2 expression. Furthermore, in the SHR model there may be alteration in the ACE/ACE2 balance in a manner that favors increased Ang II generation and decreased Ang-(1-7) production contributing to the hypertensive phenotype observed in this model. The levels of angiotensin (Ang) II depend on the actions of angiotensin-converting enzyme (ACE) and ACE2. We showed in astrocytes isolated from the SHRs that Ang II differentially affects ACE and ACE2 expression. There may be an alteration in the ACE/ACE2 balance favoring Ang II generation. This imbalance may contribute to the hypertensive phenotype observed inthis SHR model.

Monocytic angiotensin-converting enzyme 2 relates to atherosclerosis in patients with chronic kidney disease.

Background: Increased levels of monocytic angiotensin-converting enzyme (ACE) found in haemodialysis (HD) patients may directly participate in the pathogenesis of atherosclerosis. We demonstrated recently that uremia triggers the development of highly pro-atherogenic monocytes via an angiotensin II (AngII)–dependent mechanism. Opposing actions of the AngII-degrading ACE2 remain largely unknown. We examined the status of both ACEs and related receptors in circulating leukocytes of HD, not-dialyzed CKD and healthy individuals. Furthermore, we tested the possible impact of monocytic ACEs on atherogenesis and behaviour of the cells under conditions mimicking chronic renal failure. Methods: Expression of ACE, ACE2, AT1R, AT2R and MASR was investigated on circulating leukocytes from 71 HD (62 ± 14 years), 24 CKD stage 3–5 (74 ± 10 years) patients and 37 healthy control subjects (53 ± 6 years) and isolated healthy monocytes treated with normal and uremic serum. Analyses of ACE, ACE2, ICAM-1, VCAM-1, MCSF and endothelial adhesion were tested on ACE-overexpressing THP-1 monocytes treated with captopril or losartan. ACE2-overexpressing monocytes were subjected to transmigration and adhesion assays and investigated for MCP-1, ICAM-1, VCAM-1, MCSF, AT1R and AT2R expression. Results: The ACE mRNA level was significantly increased in HD and CKD stage 3–5 leukocytes. Correspondingly, ACE2 was downregulated and AngII as well as MAS receptor expression was upregulated in these cells. Healthy monocytes preconditioned with uremic serum reflected the same expressional regulation of ACE/ACE2, MAS and AngII receptors as those observed in HD and CKD stage 3–5 leukocytes. Overexpression of monocytic ACE dramatically decreased levels of ACE2 and induced a pro-atherogenic phenotype, partly reversed by AngII-modifying treatments, leading to an increase in ACE2. Overexpression of ACE2 in monocytes led to reduced endothelial adhesion, transmigration and downregulation of adhesion-related molecules. Conclusions: HD and not-dialyzed CKD stage 3–5 patients show enhanced ACE and decreased ACE2 expression on monocytes. This constellation renders the cells endothelial adhesive and likely supports the development of atherosclerosis.

Effects of Ang II on Angiotensin Converting Enzyme mRNA Levels in Cultured Rat Astrocytes

Objective To investigate Angiotensin (Ang) II effect on Angiotensin Converting Enzyme (ACE) mRNA expression in cultured rat astrocytes isolated from spontaneous hypertensive rats (SHR) as compared to Wistar controls. Background ACE is a zinc-dependent peptidase responsible for converting Ang I into the biologically active octapeptide Ang II. Pioneering studies on ACE were oriented towards understanding the etiology of hypertension, a disease in which Ang II plays a crucial role. In the current study, SHR were used as a hypertensive rat model, whereas, Wistar was used as the control. Methods: Astrocytes were isolated and cultured from the cerebellum (CB) and the brainstem (BS) of neonatal rat brains from both species. Quiescent astrocytes were treated with 100nM Ang II at different time points. mRNA was isolated and the levels of ACE mRNA were measured using real time polymerase chain reaction technique. Studies are ongoing to measure ACE protein levels. Results: Compared to basal ACE levels, Ang II significantly decreased ACE mRNA levels at most time points examined in CB and BS astrocytes isolated from SHR and Wistar rats. There was no significant difference at most time points between Ang II-induced ACE expression in SHR versus Wistar astrocytes. A comparison of the basal mRNA levels of ACE showed a significantly lower ACE mRNA expression in the SHR cerebellar brain region and no differences in the SHR BS region as compared to the Wistar counterparts. Conclusions:These findings suggest that basal levels of ACE are differentially expressed in astrocytes isolated from SHR as compared to Wistar rats. Moreover, ACE may have a key role in the auto-regulatory production of Ang II from Ang I in SHR and Wistar rat astrocytes.

Expression of genes of the cardiac and renal renin–angiotensin systems in preterm piglets: is this system a suitable target for therapeutic intervention?

The newborn circulating, cardiac and renal renin-angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs.

Increased expression of angiotensin II type 2 receptors in the solitary-vagal complex blunts renovascular hypertension.

Angiotensin II increases and decreases arterial pressure by acting at angiotensin type 1 and type 2 receptors, respectively. Renovascular hypertensive rats exhibit a high level of activity of the peripheral and central renin-angiotensin system. Therefore, in the present study, we evaluated the effect of increasing the expression of angiotensin type 2 receptors in the solitary-vagal complex (nucleus of the solitary tract/dorsal motor nucleus of the vagus), a key brain stem region for cardiovascular regulation, on the development of renovascular hypertension. Holtzman normotensive rats were implanted with a silver clip around the left renal artery to induce 2-kidney 1-clip renovascular hypertension. Three weeks later, rats were microinjected in the solitary-vagal complex with either an adenoassociated virus to increase the expression of angiotensin type 2 receptors or with a control vector. We observed that increasing angiotensin type 2 receptor expression in the solitary-vagal complex attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex and the increase in the low-frequency component of systolic blood pressure observed in renovascular hypertensive rats. Furthermore, an observed decrease in mRNA levels of angiotensin-converting enzyme 2 in the solitary-vagal complex of renovascular hypertensive rats was restored to control levels after viral-mediated increases in angiotensin type 2 receptors at this site. Collectively, these data demonstrate specific and beneficial effects of angiotensin type 2 receptors via the brain of hypertensive rats and suggest that central angiotensin type 2 receptors may be a potential target for therapeutics in renovascular hypertension.