Effects of dehydration on cardiovascular development in the embryonic American alligator (Alligator mississipiensis)

Effects of dehydration on reptilian embryonic cardiovascular function are unknown. Here, we present the first morphological and physiological data quantifying the cumulative effects of four acute dehydration events on the embryonic American alligator, Alligator mississipiensis. We hypothesized that dehydration would alter embryonic morphology, reduce blood volume and augment the response to angiotensin II (Ang II), a key osmotic and blood volume regulatory response element in adult vertebrates. Drying events at 30%, 40%, 50%, and 60% of embryonic incubation reduced total egg water content by 14.43 ± 0.37 g, a 3.4 fold increase relative to controls. However, embyronic blood volume was greater in the dehydration group at 70% of embryonic incubation compared to controls (0.39 ± 0.044 mLg(-1) and 0.22 ± 0.03 mLg(-1), respectively), however, both groups were similar at 90% of incubation (0.18 ± 0.02 mLg(-1) in the controls and 0.23 ± 0.03 mLg(-1) in the dehydrated group). Dehydration altered the morphological phenotype and resulted in an overall reduction in embryonic mass at both incubation time points measured. Dehydration also altered the physiological phenotype, resulting in embryonic alligators that were relatively bradycardic at 90% of incubation. Arterial Ang II injections resulted in a dose dependent hypertension, which increased in intensity over the span of incubation studied. While progressive incubation altered the Ang II response, dehydration had no impact on the cardiovascular responses to the peptide. Quantification of Ang II type-1 receptor protein using western blot analysis illustrated that dehydration condition and incubation time point did not alter protein quantity. Collectively, our results show that dehydration during embryonic development of the American alligator alters embryonic morphology and baseline heart rate without altering arterial pressure and response to Ang II.     

Effects of angiotensin II on the spontaneous activity of rostral ventrolateral medullary cardiovascular neurons and blood pressure in spontaneously hypertensive rats

The interactive role of rostral ventrolateral medulla (RVL) cardiovascular neurons and brain angiotensin II (Ang II) in regulating the arterial blood pressure was examined by recording simultaneously the spontaneous activity of these spinal projecting neurons and the arterial blood pressure in the pentobarbital-anesthetized spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). It was found that Ang II elicited dose-dependent excitatory responses in a subpopulation of RVL cardiovascular neurons, followed by a subsequent increase in blood pressure. These effects of Ang II were significantly greater in SHR than in WKY. The effects were attenuated or abolished by co-administration of Ang II antagonist, [Sar¹, Ile⁸]-Ang II. Pre-administration of [Sar¹, Ile⁸]-Ang II to RVL using bilateral microinjection attenuated the blood pressure effects of intracerebroventricularly administered Ang II by as much as 70%. These results indicated that spinal projecting RVL cardiovascular neurons are important in mediating the pressor action of Ang II. The enhanced sensitivity and responsiveness of RVL cardiovascular neurons to Ang II may be pertinent to the genesis of hypertension in adult SHR.     

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.     

Angiotensin II modulates tyr-phosphorylation of IRS-4, an insulin receptor substrate, in rat liver membranes

Angiotensin II (Ang II), a major regulator of blood pressure, is also involved in the control of cellular proliferation and hypertrophy and might exhibit additional actions in vivo by modulating the signaling of other hormones. As hypertension and Insulin (Ins) resistance often coexist and are risk factors for cardiovascular diseases, Ang II and Insulin signaling cross-talk may have an important role in hypertension development. The effect of Ins on protein tyrosine phosphorylation was assayed in rat liver membrane preparations, a rich source of Ins receptors. Following stimulation, Ins (10⁻⁷ M) induced tyr-phosphorylation of different proteins. Insulin consistently induced tyr-phosphorylation of a 160 kDa protein (pp160) with maximum effect between 1 and 3 min. The pp160 protein was identified by anti-IRS-4 but not by anti-IRS-1 antibody. Pre-stimulation with Ang II (10⁻⁷ M) diminishes tyr-phosphorylation level of pp160/IRS-4 in a dose-dependent manner. Okadaic acid, the PP1A and PP2A Ser/Thr phosphatase inhibitor, increases pp160 phosphorylation induced by Ins and prevents the inhibitory effect of Ang II pre-stimulation. Genistein, a tyrosine kinase inhibitor, diminishes tyr-phosphorylation level of IRS-4. PI3K inhibitors Wortmanin and LY294002, both increase tyr-phosphorylation of IRS-4, either in the presence of Ins alone or combined with Ang II. These results suggest that Ins and Ang II modulate IRS-4 tyr-phosphorylation in a PI3K-dependent manner. In summary, we showed that Ins induces tyr-phosphorylation of IRS-4, an effect modulated by Ang II. Assays performed in the presence of different inhibitors points toward a PI3K involvement in this signaling pathway.     

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.     

Endothelin-1 modulates angiotensin II in the development of hypertension in fructose-fed rats

Two of the most potent vasoconstrictors, endothelin-1 (ET-1) and angiotensin II (Ang II), are upregulated in fructose hypertensive rats. It is unknown whether an interrelationship exists between these peptides that may contribute to the development of fructose-induced hypertension. The objective of this study was to investigate the existence of an interaction between the endothelin and renin angiotensin systems that may play a role in the development of fructose-induced hypertension. High fructose feeding and treatment with either bosentan, a dual endothelin receptor antagonist, or with L-158,809, an angiotensin type 1 receptor antagonist, were initiated simultaneously in male Wistar rats. Systolic blood pressure, fasted plasma parameters, insulin sensitivity, plasma Ang II, and vascular ET-1-immunoreactivity were determined following 6 weeks of high fructose feeding. Rats fed with a high fructose diet exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia, hypertension, and elevated plasma Ang II. Treatment with either bosentan or L-158,809 significantly attenuated the rise in blood pressure with no effect on insulin levels or insulin sensitivity in fructose-fed rats. Bosentan treatment significantly reduced plasma Ang II levels, while L-158,809 treatment significantly increased vascular ET-1-immunoreactivity in fructose-fed rats. Thus, treatment with the endothelin receptor antagonist prevented the development of fructose-induced hypertension and decreased plasma Ang II levels. These data suggest that ET-1 contributes to the development of fructose-induced hypertension through modulation of Ang II levels.     

Apoptosis induction and inhibition of cellular proliferation by angiotensin II: possible implication and perspectives

The renin-angiotensin system plays a pivotal role in the regulation of fluid, electrolyte metabolism and blood pressure. Molecular cloning and pharmacological studies have defined two major classes of Angiotensin II (Ang II) receptors, designated AT1 and AT2. Recently, it has been well recognized that Ang II, beside its classical physiological actions, is a profibrogenic peptide and displays characteristics of a growth factor. The emerging picture suggests that angiotensin receptor subtypes exert opposing features in many aspects of their biological function, most importantly in cellular growth and proliferation. Accordingly, the proliferative and/or growth-promoting effects of Ang II are thought to be mediated by AT1 receptor, whereas the AT2 receptor subtype may have growth-inhibitory properties. The novel finding that Ang II is able to induce apoptosis by AT2 receptors in diverse cell types is of great scientific interest, as recent studies revealed a role for apoptosis as a deliberate form of cell death in the pathogenesis of various cardiovascular diseases such as heart failure and vascular remodeling. Furthermore apoptotic cell death might occur during the development of progressive glomerulosclerosis. It is tempting to speculate that autocrine-paracrine vasoactive substances such as Ang II might regulate these apoptotic processes during pathogenic conditions.     

Cardiovascular regulation during hypoxia in embryos of the domestic chicken Gallus gallus

Renewed interest in the use of the embryonic chicken as a model of perinatal cardiovascular regulation has inspired new questions about the control mechanisms that respond to acute perturbations, such as hypoxia. The objectives of this study were to determine the cardiovascular responses, the regulatory mechanisms involved in those cardiovascular responses, and whether those mechanisms involved the central nervous system (CNS) of embryonic chickens. Heart rate (f(H)) and blood pressure were measured in chicken embryos of different incubation ages during exposure to different levels of hypoxia (15, 10, and 5% O(2)). At all levels of hypoxia and at all developmental ages, a depression of f(H) and arterial pressure was observed, with the exception of day 20 embryos in 15 and 10% O(2). The intensity of the embryonic f(H) and blood pressure responses were directly related to the level of hypoxia used. Muscarinic and alpha-adrenergic receptor stimulation limited the hypoxic hypotension on days 15-19 and 15-21, respectively, as indicated after blockade with atropine and phentolamine. During the final 3 days of incubation, the intensity of the hypoxic hypotension was magnified due to alpha-vasodilation caused by beta-adrenergic and muscarinic receptor stimulation. In 19- to 21-day-old embryos, the f(H) response to hypoxia was limited by alpha-adrenergic receptor stimulation as indicated by the accentuated bradycardia after blockade with phentolamine. Furthermore, on day 21, atropine limited the hypoxic bradycardia, indicating that muscarinic receptors also play a role in the f(H) response at this age. In addition, the muscarinic actions on the heart and the adrenergic effects on the vasculature appeared to occur through a hypoxic-induced direct release from chromaffin tissue and autonomic nerve terminals. Thus, in embryonic chickens, the only cardiovascular response to hypoxia that involves the CNS was the cholinergic regulation of arterial pressure after day 15 of incubation. Therefore, although embryonic chickens and fetal sheep, the standard models of perinatal cardiovascular physiology, respond to hypoxia with a similar redistribution of cardiac output, the underlying mechanisms differ between these species.     

The antihypertensive effect of peptides: a novel alternative to drugs?

Many types of bioactive peptides that inhibit angiotensin I, angiotensin I converting enzyme (ACE) and Ang II type 1 receptor (AT1) in the cardiovascular system contribute to the prevention and treatment of hypertension. These inhibitory peptides are derived from many food proteins or artificial synthetic products. Further research examining the bioavailability of ACE inhibitory peptides will lead to the development of more effective ACE inhibitory peptides and foods. Our research also demonstrates that ACE inhibitory peptide LAP may lower blood pressure with no adverse effects.     

Genetic deletion of angiotensin AT2 receptor leads to increased cell numbers in different brain structures of mice

Angiotensin II (Ang II) is a potent vasoactive peptide and displays growth factor-like properties. Different high-affinity Ang II receptor subtypes (AT1A, AT1B and AT2) have been cloned. They are expressed in various brain structures. Additionally, it has been assumed that Mas could interact directly or indirectly with the renin-angiotensin system. The AT1 receptor mediates pressor and mitogenic effects of Ang II, whereas physiological function and signaling mechanisms of the AT2 receptor remain poorly understood. Recent reports have shown that Ang II could mediate apoptosis through AT2 receptors. Since the AT1A, AT2 and Mas knockout mice provide new tools for uncovering potential actions of Ang II, the cell number in different brain structures of male adult wild-type mice and mice deficient for AT1A, AT2 or Mas was evaluated to get more insight into the role of Ang II in central nervous system development. In nearly all investigated brain structures (cortex, hippocampus, amygdala, thalamus), the cell number was significantly higher in AT2-deficient mice in comparison to wild-type mice. To the contrary, in AT1A-deficient mice the cell number was significantly less than in controls in the lateral geniculate and the medial amygdaloid nucleus. However, cell numbers were not changed in Mas-knockout mice compared to their wild-types. These results show the contrary effects of both angiotensin receptors on cell growth and represent the first demonstration of their action on neuronal cell development evidenced in the adult mouse brain.     

Activation of ERK5 in angiotensin II-induced hypertrophy of human aortic smooth muscle cells

Extracellular signal-regulated kinase 5 (ERK5), a recently discovered mitogen-activated protein kinase (MAPK), plays a key role in the development and pathogenesis of cardiovascular disease. In order to clarify the pathophysiological significance of ERK5 in vascular remodeling, we investigated ERK5 phosphorylation in hypertrophy of human aortic smooth muscle cells (HASMCs) induced by angiotensin II (Ang II). The AT1 receptor was involved in Ang II-induced ERK5 activity. Hypertrophy was detected by the measurement of protein synthesis with [³H]-Leu incorporation in cultured HASMCs. Ang II rapidly induced phosphorylation of ERK5 at Thr218/Tyr220 residues in a time- and dose-dependent manner. Activation of myocyte enhancer factor-2C (MEF2C) by ERK5 was inhibited by PD98059. Transfecting HASMCs with small interfering RNA (siRNA) to silence ERK5 inhibited Ang II-induced cell hypertrophy. Thus, ERK5 phosphorylation contributes to MEF2C activation and subsequent HASMC hypertrophy induced by Ang II, for a novel molecular mechanism in cardiovascular diseases induced by Ang II.     

Effects of angiotensin II on bone cells in vitro

Other than its known effects on the cardiovascular system, angiotensin II (Ang II) stimulates cell growth in several cell types. In this study, we examined whether it also might affect bone cell metabolism. Ang II stimulated DNA and collagen synthesis and decreased alkaline phosphatase (AP) activity in bone cell populations derived from the periosteum of fetal rat calvariae. Similar effects of Ang II were observed on human adult bone cells obtained by collagenase digestion from trabecular bone. Clonal cell analysis, autoradiographic studies, and receptor subtype analysis suggested the presence of specific Ang II receptor subtype 1 (AT₁) binding sites on AP⁺ osteoblastic precursor cells. Ang II had no direct effects on osteoblastic cells with a mature phenotype, but paracrine effects of Ang II on mature osteoblasts could be observed upon coculture with Ang II-responsive bone cell populations. Because Ang II is known to be locally generated by endothelial cells, Ang II might play an important role in coordinating capillary cell growth and osteoblastic bone formation during bone remodeling. J. Cell. Physiol. 175:89–98, 1998. © 1998 Wiley-Liss, Inc.     

Enalapril and losartan restored blood pressure and vascular reactivity in intrauterine undernourished rats

Epidemiological studies suggest that intrauterine undernutrition plays an important role in the development of arterial hypertension and endothelial dysfunction in adulthood. We have evaluated the effect of the Renin Angiotensin System inhibition on the blood pressure and the mesenteric arteriolar reactivity of the intrauterine undernourished rats. Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. In this study only the male offspring was used. At 16 weeks of age, the rats were used for the study of blood pressure, microvascular reactivity studied in vivo-in situ to Angiotensin II (Ang II), Bradykinin (Bk) and Acetylcholine (Ach) before and after either losartan (10 mg/kg/15 days) or enalapril (15 mg/kg/21 days) treatment. We also evaluated the mesenteric and plasmatic Angiotensin Converting Enzyme (ACE), renal function, lipid plasmatic content, and insulin and glucose metabolism. Intrauterine undernutrition induced hypertension and increased response of mesenteric arterioles to Ang II and decreased vasodilation to Bk and Ach. The treatments with losartan or enalapril normalized the blood pressure levels and significantly improved the arteriolar responses to Bk, Ach and reduced the response to Ang II. No differences have been detected to ACE activity, renal function, lipid content and insulin and glucose metabolism. This study shows for the first time that Renin Angiotensin System inhibitors can normalize the cardiovascular alterations induced by intrauterine undernutrition.     

Isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system

The renin-angiotensin (RA) system plays an important role in regulating blood pressure and fluid balance. In the search for bioactive peptides with an antibody binding to the N-terminal portion of angiotensin II (Ang II), we isolated a new angiotensinogen-derived peptide from the rat small intestine. Consisting of 12 amino acids, this peptide was termed proangiotensin-12 based on its possible role of an Ang II precursor. Proangiotensin-12 constricted aortic strips and, when infused intravenously, raised blood pressure in rats, while both the vasoconstrictor and pressor response to proangiotensin-12 were abolished by captopril and by CV-11974, an Ang II type I receptor blocker. Proangiotensin-12 is abundant in a wide range of organs and tissues including the small intestine, spleen, kidneys, and liver of rats. The identification of proangiotensin-12 suggests a processing cascade of the RA system, different from the cleavage of angiotensinogen to Ang I by renin.     

Prolonged Subcutaneous Administration of Oxytocin Accelerates Angiotensin II-Induced Hypertension and Renal Damage in Male Rats

Oxytocin and its receptor are synthesised in the heart and blood vessels but effects of chronic activation of this peripheral oxytocinergic system on cardiovascular function are not known. In acute studies, systemic administration of low dose oxytocin exerted a protective, preconditioning effect in experimental models of myocardial ischemia and infarction. In this study, we investigated the effects of chronic administration of low dose oxytocin following angiotensin II-induced hypertension, cardiac hypertrophy and renal damage. Angiotensin II (40 μg/Kg/h) only, oxytocin only (20 or 100 ng/Kg/h), or angiotensin II combined with oxytocin (20 or 100 ng/Kg/h) were infused subcutaneously in adult male Sprague-Dawley rats for 28 days. At day 7, oxytocin or angiotensin-II only did not change hemodynamic parameters, but animals that received a combination of oxytocin and angiotensin-II had significantly elevated systolic, diastolic and mean arterial pressure compared to controls (P < 0.01). Hemodynamic changes were accompanied by significant left ventricular cardiac hypertrophy and renal damage at day 28 in animals treated with angiotensin II (P < 0.05) or both oxytocin and angiotensin II, compared to controls (P < 0.01). Prolonged oxytocin administration did not affect plasma concentrations of renin and atrial natriuretic peptide, but was associated with the activation of calcium-dependent protein phosphatase calcineurin, a canonical signalling mechanism in pressure overload-induced cardiovascular disease. These data demonstrate that oxytocin accelerated angiotensin-II induced hypertension and end-organ renal damage, suggesting caution should be exercised in the chronic use of oxytocin in individuals with hypertension.     

Hypotensive activity of TCV-116, a newly developed angiotensin II receptor antagonist, in spontaneously hypertensive rats.

TCV-116, a recently developed angiotensin II (Ang II) receptor antagonist, was administered orally (1 mg/kg per day) to 10-week-old spontaneously hypertensive rats (SHR) for 2 weeks. Blood pressure and plasma components of the renin-angiotensin-aldosterone system were determined in these rats. TCV-116 produced a marked reduction in blood pressure without altering heart rate. Whereas plasma renin concentration (PRC), angiotensin I (Ang I) and angiotensin II (Ang II) all were significantly increased, plasma aldosterone was decreased by approximately 70% compared with control animals. These results not only indicate therapeutic efficacy of this agent in the chronic treatment of human hypertension, but support also the concept that the renin-angiotensin system plays an important role in the control of blood pressure in this animal model of human essential hypertension.     

Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monoclonal antibodies in the rat adrenal gland

Angiotensin II (Ang II), a major regulator of cardiovascular function and body fluid homeostasis, mediates its biological actions via two subtypes of G protein-coupled receptors, termed AT(1) and AT(2). The primary goal of this study was to raise monoclonal anti-peptide antibodies specific to angiotensin AT(1)- and AT(2)-receptor subtypes and to Ang II itself and using these monoclonal antibodies to determine the intraadrenal localization of AT(1) and AT(2) receptors and Ang II in male adult rats. Immunocytochemistry unambiguously demonstrates a regional colocalization of Ang II and angiotensin II receptors in the adrenal gland. The novel antibodies localized Ang II and the AT(1) receptors to the zona glomerulosa of the cortex and to the medulla whereas AT(2) receptors were limited to the medulla. The specificity of immunostaining was documented by pre-adsorption of the antibody with the immunogenic peptide. Our data underscore that AT(1) appears to mediate most of the physiological actions of Ang II in adrenal. Western blot analysis of rat adrenal protein extracts using AT(1) antibody showed a predominant 73-kDa band and a weaker 97-kDa immunoreactive band corresponding to glycosylated forms of the AT(1) receptor. Immunostaining with anti-AT(2) yielded one major immunoreactive band of 73-kDa size and one additional fainter band of 120 kDa. These antibodies may prove of value in unraveling the subcellular localization and intracellular effector pathways of AT(1) and AT(2).     

Modulation and function of extrarenal angiotensin receptors

Historically, physiological modulation of the activity of the renin-angiotension system (RAS) was thought to be mediated only by changes in renin secretion. Hence, altered dietary sodium (Na) intake, changes in renal perfusion pressure, and/or renal adrenoreceptor activity would lead to changes in renin release and plasma angiotensin II (Ang II) concentration, which in turn contribute to regulation of blood pressure and sodium balance. Later, it became apparent that angiotensinogen availability and Ang-converting enzyme activity are also rate-limiting factors that influence the activity of RAS. Finally, over the past few years, evidence has accumulated that indicates the number of Ang II receptors and their subtypes are of great importance in regulating the activity and function of RAS. Cloning of the Ang II receptor genes, development of specific receptor-antagonist ligands, and establishment of genetically mutated animal models have led to greater understanding of the role of Ang II receptors in the regulation of RAS function and activity. This review focuses on the functions and regulation of Ang II receptors in vascular tissues and in the adrenal gland. The authors suggest that identification of control elements for Ang II receptor expression, which are tissue-specific, may provide a basis for future therapeutic manipulation of Ang II receptors in cardiovascular disease states.     

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.