Local Tissue Renin-angiotensin System Research Articles

Elucidating the Role of Pro-renin Receptors in Pancreatic Ductal Adenocarcinoma Progression: A Novel Therapeutic Target in Cancer Therapy.

Pancreatic cancer is a highly aggressive malignancy with a very poor prognosis. The 5- year survival in these patients is very low, and most patients develop drug resistance to current therapies, so additional studies are needed to identify the potential role of new drug targets for the treatment of pancreatic cancer. Recent investigations have been performed regarding the roles of pro-renin receptors (PRR) in the initiation and development of cancers. PRR is a component of the local renin-angiotensin system (RAS). Local tissue RAS has been known in diverse organ systems, including the pancreas. Various investigations have implicated that PRRs are associated with the upregulation of various signaling pathways, like the renin-angiotensin system pathway, PI3K/Akt/mTOR, and the Wnt-signaling pathways, to contribute to pathological conditions, including cancer. In this review, we presented an overview of the role of PRR in the progression of pancreatic adenocarcinoma.

Diverse biological functions of the renin-angiotensin system.

The renin-angiotensin system (RAS) has been widely known as a circulating endocrine system involved in the control of blood pressure. However, components of RAS have been found to be localized in rather unexpected sites in the body including the kidneys, brain, bone marrow, immune cells, and reproductive system. These discoveries have led to steady, growing evidence of the existence of independent tissue RAS specific to several parts of the body. It is important to understand how RAS regulates these systems for a variety of reasons: It gives a better overall picture of human physiology, helps to understand and mitigate the unintended consequences of RAS-inhibiting or activating drugs, and sets the stage for potential new therapies for a variety of ailments. This review fulfills the need for an updated overview of knowledge about local tissue RAS in several bodily systems, including their components, functions, and medical implications.

The role of (pro)renin receptor and its soluble form in cardiovascular diseases.

The renin-angiotensin system (RAS) is a major classic therapeutic target for cardiovascular diseases. In addition to the circulating RAS, local tissue RAS has been identified in various tissues and plays roles in tissue inflammation and tissue fibrosis. (Pro)renin receptor (PRR) was identified as a new member of RAS in 2002. Studies have demonstrated the effects of PRR and its soluble form in local tissue RAS. Moreover, as an important part of vacuolar H+-ATPase, it also contributes to normal lysosome function and cell survival. Evidently, PRR participates in the pathogenesis of cardiovascular diseases and may be a potential therapeutic target of cardiovascular diseases. This review focuses on the effects of PRR and its soluble form on the physiological state, hypertension, myocardial ischemia reperfusion injury, heart failure, metabolic cardiomyopathy, and atherosclerosis. We aimed to investigate the possibilities and challenges of PRR and its soluble form as a new therapeutic target in cardiovascular diseases.

Tissue Renin-Angiotensin System (tRAS) Induce Intervertebral Disc Degeneration by Activating Oxidative Stress and Inflammatory Reaction.

Lumbar intervertebral disc degeneration (IDD) has been the major contributor to low back pain (LBP). IDD is an chronic inflammation process, with the activation of plentiful inflammation-related cytokines and ECM degradation-related enzymes. In the past few years, hypertension has been reported to correlate with LBP. In addition, the local tissue renin-angiotensin system (tRAS) has been identified in multiple tissues, including the spinal cord, skin, kidney, heart, and bone. Recently, tRAS has also been established in both bovine and human intervertebral disc tissues, especially in the degenerated disc tissue. However, the exact of tRAS and IDD remains unknown. In this present study, proteomic analysis, molecular biology analysis, and animal model were all used. Firstly, we revealed that tRAS was excessively activated in the human degenerated intervertebral disc tissue via proteomic analysis and molecular biology analysis. Then, in vitro experiment suggested that Ang II could decrease the cell viability of human NP cells and promote NP cell apoptosis, senescence, oxidative stress, and NLRP3 activation in human NP cells. In addition, Ang II could also trigger degeneration and fibrosis phenotype in human NP cells. Finally, the animal model demonstrated that the local activated ACE/Ang II axis in the NP tissue could accelerate IDD in aging spontaneously hypertensive rats (SHR). Collectively, the degenerated intervertebral disc tissue showed excessively activated tRAS, and local activated tRAS could induce NP cell senescence, apoptosis, oxidative stress, and inflammatory reaction to promote IDD. These biological effects of Ang II on human NP cells may provide novel insight into further treatment of IDD.

Effects of (Pro)renin Receptor on Diabetic Cardiomyopathy Pathological Processes in Rats via the PRR-AMPK-YAP Pathway.

Diabetic cardiomyopathy (DCM) is a common complication associated with diabetes. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. Yes-associated protein (YAP) also plays a crucial role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR and YAP on DCM remains unclear. The purpose of this study was to determine the role of PRR in the pathological progression of DCM and whether PRR influences the pathological processes of diabetic cardiomyopathy through YAP. We first established diabetic cardiomyopathy rats model, downregulated the expression of PRR, and upregulated and downregulated the expression of YAP. The levels of myocardial inflammation and fibrosis were then measured and cardiac function was evaluated. In vitro, primary rat cardiac fibroblasts (CFs) were cultured with high glucose, with or without transfection with recombinant adenovirus expressing PRR, and GSK621 was used to observe the effect of AMPK. The levels of inflammation and fibrosis were measured in vitro. The results showed that PRR and YAP silencing alleviated myocardial inflammation and fibrosis. GSK621 blocked the effect of PRR on AMPK and YAP and improved CF inflammation and fibrosis. The inhibition of PRR expression offers a new therapeutic strategy for the treatment of DCM. The effects of PRR on the pathological process of DCM in rats may be mediated via the PRR-AMPK-YAP pathway.

PLASMA ACE2 ACTIVITY IS INCREASED IN PATIENTS RECOVERED FROM SARS-COV-2 INFECTION: IMPLICATIONS FOR THE PROLONGED CONSEQUENCES OF COVID-19

Objective: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilises the catalytic site of membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry. It is thought that endocytosis of ACE2 results in a decrease in membrane bound ACE2 expression, and disruption of the local tissue renin angiotensin system protection. In this study, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 leading to increased plasma ACE2 activity. Design and method: Australians aged >18 years (n=66) who had recovered from SARS-CoV-2 infection (positive result by PCR testing) and uninfected controls (n=70) were recruited. Serial samples were available in 23 recovered SARS-CoV-2 patients. Plasma ACE2 activity was measured using a fluorescent substrate-based assay and levels were compared using the Mann-Whitney or Kruskal-Wallis test. Serial ACE2 activity were analysed using the Friedman test for repeated measures. Post-hoc analysis was performed with a Bonferroni correction. Two-tailed P-values 0.05) in the proportion of hypertension, obesity, diabetes, cardiovascular disease, or use of anti hypertensive, lipid lowering, and anti-platelet medications between the controls and SARSCoV-2 patients. Plasma ACE2 activity at median 35 days post-infection [interquartile range 30-38 days] was 97-fold higher in SARS-CoV-2 patients compared to controls (5.8 [2-11.3] vs. 0.06 [0.02-2.2] pmol/min/ml, p 0.05). Conclusions: Plasma ACE2 activity is elevated after SARS-CoV-2 infection and remains elevated post-infection. Our findings indicate the need for ongoing investigation to determine if ACE2 levels identify people at risk of prolonged illness following COVID-19.

Mas Receptor Agonist AVE0991 increases surfactant protein expression under hyperoxic conditions in human lung epithelial cells

Hyperoxia in pre-term neonates is a known risk factor of bronchopulmonary dysplasia (BPD). Hyperoxia is known to cause oxidative stress, inflammatory changes that leads to surfactant deactivation, and decreased surfactant expression. The previous research has shown short term exposure to hyperoxia increases surfactant protein expression but decreased expression in long term exposure. Local tissue renin-angiotensin system (RAS) is associated with tissue injury and repair and it may play a role in BPD. Endogenous peptide angiotensin 1-7 acts on the MAS receptor. The activation of the MAS receptor was previously shown to have protective pulmonary responses. However, the effect of MAS receptor activation on surfactant proteins in hyperoxic conditions has not been tested. To determine the effects of hyperoxia with or without MAS receptor activation on Surfactant proteins. Human epithelial cell line A549 and human primary alveolar epithelial cells (AECs) were cultured to sub-confluence (60-75%) and treated with hyperoxia (95% oxygen) and normoxia (21% oxygen) for 72 hours with or without the MAS receptor agonist (AVE0991) in serum-free F-12 nutrient media. Cells were lysed and cell lysates were collected for western blot. The statistical analysis was done using Student-Newman-Keuls Multiple comparison test. Surfactant protein concentration increased in AVE treated group under the hyperoxic condition when compared to the control group in both A549 cells and human primary AECs. Surfactant protein was in higher concentration in AVE0991 treated cells in both hyperoxic and normoxic conditions when compared to the non-treated control group. MAS receptor activation via AVE0991 causes an increase in Surfactant protein concentration in both hyperoxic and normoxic conditions. As per our experiments, hyperoxic conditions decrease the production of surfactant protein when compared to normoxic conditions. These results may reveal a novel potential drug for BPD treatment and decrease its severity.

Potential of renin-angiotensin system inhibition to improve metabolic bone disorders

Metabolic bone disorder is an abnormality of bones indicated by reduced bone mass and high risk of fractures. Several lines of evidence have demonstrated that the local bone tissue renin-angiotensin system (RAS) is directly involved in bone metabolism and influences the bone health. This review aimed to assess the role of RAS in bone metabolism and comparative effectiveness of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) in reducing the bone fractures. In summary, the clinical trials, in vivo studies, and functional - pharmacological experiments suggested that the RAS regulates bone marrow metabolism and influences the bone health. Hence, it warrants further investigation on the role of ACEIs and ARBs in reducing risk fractures.

Pathobiological Interactions of Local Bone Marrow Renin-Angiotensin System and Central Nervous System in Systemic Arterial Hypertension.

Circulating renin-angiotensin system (RAS) and local paracrin-autocrin-intracrin tissue-based RAS participate in numerous pathobiological events. Pro-inflammatory, pro-fibrotic, and pro-thrombotic consequences associated with local RAS activation have been detected at cellular and molecular level. Regenerative progenitor cell therapy in response to RAS modulating pharmacotherapy has emerged as an adjunct in the context of endothelial cell injury and regeneration to improve regeneration of the vascular endothelium. Local hematopoietic bone marrow (BM) RAS symbolizes the place of cross-interaction between vascular biology and cellular events from embryogenesis to definitive hematopoiesis underlying vascular atherosclerosis. The BM microenvironment also contains Mas receptors, which control the proliferative role of Ang 1-7 on hematopoietic stem cells. Ang 1-7 is produced from Ang-II or Ang-I with the help of ACE2. Various tissues and organs also have an effect on the RAS system. The leukocytes contain and synthesize immunoreactive angiotensinogen species capable of producing angiotensin in the basal state or after incubation with renin. The significance of RAS employment in atherosclerosis and hypertension was indicated by novel bidirectional Central Nervous System (CNS) RAS–BM RAS communications. Myeloid cells generated within the context of hematopoietic BM RAS are considered as the initiators and decision shapers in atherosclerosis. Macrophages in the atherosclerotic lesions contain angiotensin peptides by which RAS blockers inhibit monocyte activation and adherence. Furthermore, vascular biology in relation to inflammation and neoplasia is also affected by local tissue RAS. The purpose of this article is to outline interactions of circulating and local angiotensin systems, especially local bone marrow RAS, in the vascular pathobiological microenvironment of CNS.

Mitochondrial angiotensin receptors and cardioprotective pathways.

A growing body of data provides strong evidence that intracellular angiotensin II (ANG II) plays an important role in mammalian cell function and is involved in the pathogenesis of human diseases such as hypertension, diabetes, inflammation, fibrosis, arrhythmias, and kidney disease, among others. Recent studies also suggest that intracellular ANG II exerts protective effects in cells during high extracellular levels of the hormone or during chronic stimulation of the local tissue renin-angiotensin system (RAS). Notably, the intracellular RAS (iRAS) described in neurons, fibroblasts, renal cells, and cardiomyocytes provided new insights into regulatory mechanisms mediated by intracellular ANG II type 1 (AT1Rs) and 2 (AT2Rs) receptors, particularly, in mitochondria and nucleus. For instance, ANG II through nuclear AT1Rs promotes protective mechanisms by stimulating the AT2R signaling cascade, which involves mitochondrial AT2Rs and Mas receptors. The stimulation of nuclear ANG II receptors enhances mitochondrial biogenesis through peroxisome proliferator-activated receptor-γ coactivator-1α and increases sirtuins activity, thus protecting the cell against oxidative stress. Recent studies in ANG II-induced preconditioning suggest that plasma membrane AT2R stimulation exerts protective effects against cardiac ischemia-reperfusion by modulating mitochondrial AT1R and AT2R signaling. These studies indicate that iRAS promotes the protection of cells through nuclear AT1R signaling, which, in turn, promotes AT2R-dependent processes in mitochondria. Thus, despite abundant data on the deleterious effects of intracellular ANG II, a growing body of studies also supports a protective role for iRAS that could be of relevance to developing new therapeutic strategies. This review summarizes and discusses previous studies on the role of iRAS, particularly emphasizing the protective and counterbalancing actions of iRAS, mitochondrial ANG II receptors, and their implications for organ protection.

Effects of the (Pro)renin Receptor on Cardiac Remodeling and Function in a Rat Alcoholic Cardiomyopathy Model via the PRR-ERK1/2-NOX4 Pathway.

Alcoholic cardiomyopathy (ACM) caused by alcohol consumption manifests mainly as by maladaptive myocardial function, which eventually leads to heart failure and causes serious public health problems. The (pro)renin receptor (PRR) is an important member of the local tissue renin-angiotensin system and plays a vital role in many cardiovascular diseases. However, the mechanism responsible for the effects of PRR on ACM remains unclear. The purpose of this study was to determine the role of PRR in myocardial fibrosis and the deterioration of cardiac function in alcoholic cardiomyopathy. Wistar rats were fed a liquid diet containing 9% v/v alcohol to establish an alcoholic cardiomyopathy model. Eight weeks later, rats were injected with 1 × 109v.g./100 μl of recombinant adenovirus containing EGFP (scramble-shRNA), PRR, and PRR-shRNA via the tail vein. Cardiac function was assessed by echocardiography. Cardiac histopathology was measured by Masson's trichrome staining, immunohistochemical staining, and dihydroethidium staining. In addition, cardiac fibroblasts (CFs) were cultured to evaluate the effects of alcohol stimulation on the production of the extracellular matrix and their underlying mechanisms. Our results indicated that overexpression of PRR in rats with alcoholic cardiomyopathy exacerbates myocardial oxidative stress and myocardial fibrosis. Silencing of PRR expression with short hairpin RNA (shRNA) technology reversed the myocardial damage mediated by PRR. Additionally, PRR activated phosphorylation of ERK1/2 and increased NOX4-derived reactive oxygen species and collagen expression in CFs with alcohol stimulation. Administration of the ERK kinase inhibitor (PD98059) significantly reduced NOX4 protein expression and collagen production, which indicated that PRR increases collagen production primarily through the PRR-ERK1/2-NOX4 pathway in CFs. In conclusion, our study demonstrated that PRR induces myocardial fibrosis and deteriorates cardiac function through ROS from the PRR-ERK1/2-NOX4 pathway during ACM development.

Effects of IQP, VEP and Spirulinaplatensis hydrolysates on the local kidney renin angiotensin system in spontaneously hypertensive rats.

The aim of the present study was to investigate the antihypertensive effects of the bioactive Spirulinaplatensis peptides Ile‑Gln‑Pro (IQP), Val‑Glu‑Pro (VEP), as well as Spirulinaplatensis hydrolysates (SH), and assessed whether the synthesis of components of the myocardial and renal local renin angiotensin system (RAS) are regulated differentially in spontaneously hypertensive rats (SHR). The SHR were administrated with IQP, VEP and SH respectively (10mg/kg/day) for 6weeks and received continuous monitoring of blood pressure (BP) for two more weeks. During the trial, the rats' kidney tissues were removed from these rats and collected at weeks 3, 6 and 8. The expression of the main components of local kidney RAS was measured at the mRNA levels by reverse transcription‑quantitative polymerase chain reaction, and at the protein levels by ELISA or western blotting. Oral administration of IQP, VEP and SH into SHR resulted in marked antihypertensive effects. IQP, VEP and SH decreased rats' BP by affecting the expression of local kidney RAS components via downregulating the angiotensin‑converting enzyme (ACE), Ang II and angiotensin II (Ang II) and angiotensin type‑1 receptor (AT 1), while upregulating ACE2, Ang (1‑7), Mas and AT 2. The comparisons of SH effects on local tissue RAS demonstrated that local kidney RAS regulated BP via the ACE‑Ang II‑AT 1/AT 2 axis and the ACE2‑Ang (1‑7)‑Mas axis primarily at the mRNA level, while the local myocardium RAS mainly at the protein level. This preliminary study suggests that the main components of local RAS presented different expression levels in myocardium and kidney, which is important to the development of bioactive peptides targeting for lowering BP by changing the levels of some components in local RAS in specific tissues.

Effects of brain renin- angiotensin system inhibition in ISIAH rats with inherited stress-induced arterial hypertension

The renin-angiotensin system (RAS) is one of the main systems regulating arterial pressure and water-salt homeostasis of the body and is involved in the pathogenesis of cardiovascular diseases. Angiotensin peptides – products of enzymatic hydrolysis of angiotensinogen – can be synthesized not only in the blood stream, but also in tissues, including various regions of the brain. Studies of local tissue RAS in the context of arterial hypertension have been conducted for a long time. It has been shown that a steady arterial pressure increase is often associated with changes in the functioning of the central (brain) RAS in various animal models of hypertensive disease and in humans. Nevertheless, it is still not completely clear whether these changes alone are sufficient for the formation of hypertensive status, and whether the components of the central RAS can be used as targets for the treatment of hypertensive disease. Effects of prolonged inhibition of the brain RAS on blood pressure and expression of RAS genes in brain and kidney tissues in ISIAH (inherited stress-induced arterial hypertension) rats were studied. Inhibition was performed using widely used pharmacological agents, losartan and benazepril. Osmotic minipumps were used to deliver drugs to the lateral ventricle of the brain. It was shown that prolonged inhibition of the central RAS, AT1 receptors in particular, can lead to a decrease in blood pressure and significant changes in the level of expression of brain RAS genes in ISIAH rats. The mRNA level of RAS genes in the kidney does not significantly change due to this inhibition. Thus, the participation of the central RAS in the pathogenesis and maintenance of hypertensive status during stress induced form of hypertensive disease in ISIAH rats was confirmed.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Renin-angiotensin-aldosterone system in ISIAH rats with stressinduced arterial hypertension

Because the renin-angiotensin system (RAS) has a wide range of opportunities in the regulation of fluid and electrolyte balance and arterial pressure, it is currently hypothesized that alterations in systemic circulating or local tissue RAS are some of the most important pathogenetic factors in the development of essential hypertension. The aim of the study was to investigate circulating and local tissue RAS activities in ISIAH rats with stress-induced arterial hypertension. We estimated the serum levels of renin, the angiotensin-converting enzyme, angiotensin II and aldosterone by an enzymelinked immunosorbent assay, and mRNA expression of RAS genes in kidney, adrenals and brain tissues was measured by the real-time polymerase chain reaction. The mRNA expression of the renin gene (Ren) in the ISIAH rats was significantly decreased as compared to the normotensive WAG rats, but plasma renin concentrations had no difference. At the same time, the serum levels of angiotensin II and aldosterone in the ISIAH rats were enhanced, which suggests the existence of an ectopic site of angiotensin synthesis. Expression of RAS genes in the adrenals of hypertensive rats was unchanged. By contrast, a significant increase of RAS genes expression was found in the brain tissues. The mRNA of the Ren gene was increased in the hypothalamus, and the mRNA of Ace gene was increased in the brain stem of the ISIAH rats. This may be indicative of a local increase of RAS activity in the brain tissues of ISIAH rats. Nevertheless, the results of the study define ISIAH rat strain as a model of human low-renin hypertension.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

Silver(I)/6-hydroxyiminolumazine compounds differently modify renin–angiotensin system-regulating aminopeptidases A and N in human neuroblastoma and glioma cells

We have described that local tissue renin–angiotensin-system (RAS) is involved in tumor growth in a rat model of experimental glioma in vivo, through the modification of their corresponding local proteolytic regulatory enzymes. Thus, we have found a time-dependent significant decrease in aminopeptidase N (APN) and a significant increase in aminopeptidase A (APA) activities concomitantly with tumor growth in tumor tissue whereas no changes were found in circulating aminopeptidase activities; we suggested that angiotensin peptides may play an essential step in both tumor infiltration and associated angiogenesis. Here we analyze in vitro the antiproliferative efficacy, apoptotic properties and effects of three new disilver complexes containing E-6-(hydroxyimino)ethyl-1,3,7-trimethyllumazine (lumazine=pteridine-2,4(1H,3H)-dione) on RAS-regulating APA and APN specific activities in human neuroblastoma and glioma cell lines NB69 and U373-MG. Disilver compounds showed cytotoxicity against both cell lines, although their potency was different for each cell type. Furthermore, NB69 cells need higher concentrations of silver complexes than U373-MG cells to obtain a 50% growth inhibition. All compounds showed apoptotic effects, with U373-MG cells being more susceptible. The three silver complexes tested also show a dose-dependent inhibitory effect on APA activity in NB69 and U373-MG cells, although U373-MG cells are more sensitive. On the contrary, none of them showed effects on APN activity in NB69 neuroblastoma cells whereas the three compounds showed a dose-dependent stimulatory effect on APN activity in U373-MG glioma cells with a similar potency. Disilver complexes show specific antitumor activity against brain tumor cells acting through the paracrine regulating system mediated by local tissue RAS.

Angiotensin-converting enzyme 2 is subject to post-transcriptional regulation by miR-421

ACE2 (angiotensin converting enzyme 2) plays a critical role in the local tissue RAS (renin-angiotensin system) by hydrolysing the potent hypertensive and mitogenic peptide AngII (angiotensin II). Changes in the levels of ACE2 have been observed in a number of pathologies, including cardiovascular disease, but little is known of the mechanisms regulating its expression. In the present study, therefore, the potential role of miRNAs in the regulation of ACE2 expression in primary human cardiac myofibroblasts was examined. Putative miRNA-binding sites were identified in the 3'-UTR of the ACE2 transcript using online prediction algorithms. Two of these, miR-200b and miR-421, were selected for further analysis. A reporter system using the 3'-UTR of ACE2 fused to the coding region of firefly luciferase was used to determine the functionality of the identified binding sites invitro. This identified miR-421, but not miR-200b, as a potential regulator of ACE2. The ability of miR-421, an miRNA implicated in the development of thrombosis, to down-regulate ACE2 expression was subsequently confirmed by Western blot analysis of both primary cardiac myofibroblasts and transformed cells transfected with a synthetic miR-421 precursor. Real-time PCR analysis of miR-421 revealed widespread expression in human tissues. miR-421 levels in cardiac myofibroblasts showed significant inter-patient variability, in keeping with the variability of ACE2 expression we have observed previously. In conclusion, the present study is the first to demonstrate that ACE2 may be subject to post-transcriptional regulation and reveals a novel potential therapeutic target, miR-421, which could be exploited to modulate ACE2 expression in disease.

Local thyroid renin–angiotensin system in experimental breast cancer

An association between breast cancer and thyroid dysfunction exists although the underlying mechanisms remain to be elucidated. Numerous studies have characterized the role of thyroid hormones in controlling the synthesis and secretion of renin–angiotensin system (RAS) components, but little information is available on the putative role of the local RAS on thyroid function. AimsHere we analyze several soluble and membrane-bound RAS-regulating aminopeptidase activities in thyroid gland from rats with mammary tumors and the relationship with the circulating levels of thyroid stimulating hormone (TSH) and free thyroxin (fT4). Main methodsWe analyze soluble and membrane-bound RAS-regulating aminopeptidase activities fluorometrically using their corresponding aminoacyl-β-naphthylamide as the substrate. Key findingsWe have found in rats with mammary tumors a concomitant change of thyroid RAS-regulating enzymes and thyroid hormone production. SignificanceWe suggest that existence of alterations in the regulatory mechanisms mediated by the angiotensins of the local tissue RAS as a consequence of the carcinogenic process which could act alone or in combination with alterations at a higher level of regulation such as the hypothalamus–pituitary axis.

Abstract 631: Big Angiotensin-25 (Bang-25): A Novel Glycosylated Angiotensin-related Peptide Isolated From Human Urine

The renin-angiotensin system (RAS) plays key roles in the regulation of blood pressure and body fluid dynamics. It is now recognized that a wide variety of tissues and organs, including the heart, vasculature, kidney and nervous system, among others, contain a local tissue RAS, which exerts autocrine/paracrine effects and acts independently of the systemic RAS. In 2006, we isolated and characterized proangiotensin-12, a component of the tissue RAS in rat small intestine. Similar angiotensin-related peptides had never been reported in humans. When we screened human urine gel filtration fractions for bioactive peptides using an antibody raised against the N-terminal portion of Angiotensin II (Ang II), we found a highly immunoreactive peptide with an apparent molecular weight of about 5,000. Therefore, we purified this peptide from 5.5 L of human urine using ion-exchange, gel-filtration, and affinity chromatography, and finally reverse-phase high-performance liquid chromatography (RP-HPLC) steps. We isolated and identified a novel Aogen-derived peptide, Big angiotensin-25 (Bang-25), which is composed of 25 amino acids and is N-glycosylated on its 14th amino acid (Asn) and has a cysteine linked to its 18th amino acid (Cys). We then assessed the distribution of Bang-25 in tissues by immunostaining with an antiserum raised against the C-terminal portion of the peptide. We found that Bang-25 was abundantly expressed in a number of human tissues, including kidney, heart, adrenal gland, pancreas and placenta. In the kidney, Bang-25 is localized predominantly to podocytes. Then, compared with angiotensinogen, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. In the present study, we have isolated and identified a novel Angiotensin-derived peptide, Bang-25, which is composed of 25 amino acids and is N-glycosylated on its 14th amino acid (Asn) and has a cysteine linked to its 18th amino acid (Cys). The identification of Bang-25 suggests the existence of a RAS processing cascade different from the renin-catalyzed cleavage of Angiotensin to Ang I, and provides a potential target for assessing tissue RAS status and for the development of new therapeutic approaches to related diseases.