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Institute of Clinical Pharmacology and Toxicology, Campus Benjamin Franklin, Charité-University Medicine Berlin, Berlin, Germany
ABSTRACT I. LOCAL RENIN-ANGIOTENSIN SYSTEMS A. Definition of Local RAS B. Components of the Local RAS 1. Prorenin and renin binding and receptors 2. ANG-(1 —7) 3. ACE and ACE2 4. N-acetyl-Ser-Asp-Lys-Pro 5. Chymase 6. Angiotensin receptors 6. Mas II. LOCALIZATION AND FUNCTIONAL ASPECTS A. Heart 1. Renin 2. ACE 3. Chymase 4. Angiotensinogen, ANG I, and ANG II 5. Angiotensin receptors 6. Function A) INOTROPIC EFFECTS. B) HYPERTROPHIC EFFECTS. C) MECHANICAL STRETCH. D) REMODELING. E) APOPTOSIS. B. Vasculature 1. Renin 2. ACE 3. Angiotensinogen, ANG I, and ANG II 4. Angiotensin receptors 5. Function A) VASCULAR TONE AND ENDOTHELIAL FUNCTION. 6. Tissue remodeling 7. Angiogenesis 8. Gender difference in cardiovascular RAS C. Nervous System 1. Central nervous system A) RENIN. B) ACE. C) OTHER ANG II FORMING ENZYMES. D) ANGIOTENSINOGEN, ANG I, AND ANG II. E) ANGIOTENSIN RECEPTORS. F) FUNCTION. 2. Peripheral nervous system A) SYMPATHETIC NERVOUS SYSTEM. B) PARASYMPATHETIC NERVOUS SYSTEM. C) SENSORY NEURONS. D. Reproductive Tract 1. Female reproductive tract A) OVARY. B) UTERUS. C) OVIDUCT. D) FUNCTION. 2. Male reproductive tract A) TESTES. B) EPIDIDYMIS. C) SEMINAL PLASMA AND SPERMATOZOA. D) GLANDULA VESICALIS. E) PROSTATE. F) VAS DEFERENS. G) SEMINAL VESICLES. H) FUNCTION. E. Skin 1. Renin 2. ACE 3. Angiotensinogen, ANG I, and ANG II 4. Angiotensin receptors 5. Function A) WOUND HEALING AND REPAIR. F. Digestive Organs 1. Salivary glands A) RENIN. B) ANGIOTENSINOGEN. C) RECEPTORS. D) FUNCTION. 2. Pancreas A) RENIN. B) ANGIOTENSINOGEN, ANG I, AND ANG II. C) ANGIOTENSIN RECEPTORS. D) FUNCTION. 3. Stomach A) ANGIOTENSINOGEN, ANG I, AND ANG II. B) FUNCTION. 4. Intestine A) RENIN. B) ACE. C) ANGIOTENSINOGEN. D) ANGIOTENSIN RECEPTORS. E) FUNCTION. 5. Colon A) ANGIOTENSINOGEN. B) FUNCTION. G. Sensory Organs 1. Eye A) FUNCTION. 2. Cochlea H. Lymphatic Tissue 1. White blood cells A) MONOCYTES. B) GRANULOCYTES. 2. Spleen A) RENIN. B) ANGIOTENSINOGEN. C) ANGIOTENSIN RECEPTORS. D) FUNCTION. 3. Thymus A) RENIN. B) ANGIOTENSIN RECEPTORS. I. Adipose Tissue 1. Renin 2. ACE 3. Angiotensinogen, ANG I, and ANG II 4. Angiotensin receptors 5. Function III. PRENATAL DEVELOPMENT 1. Renin 2. ACE 3. Angiotensinogen 4. Angiotensin receptors 5. Kidney A) MESONEPHROS. B) METANEPHROS. 6. Adrenal gland 7. Heart 8. Vasculature 9. Liver 10. Brain 11. Extraembryonic fetal tissues A) RENIN. B) ACE. C) ANGIOTENSINOGEN, ANG I, AND ANG II. D) ANGIOTENSIN RECEPTORS. E) ANGIOTENSINASES. IV. SUMMARY GRANTS ACKNOWLEDGMENTS REFERENCES
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I. LOCAL RENIN-ANGIOTENSIN SYSTEMS |
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In its "textbook" definition, the renin-angiotensin system (RAS) is a peptidergic system with endocrine characteristics. The substrate of the system, angiotensinogen, an 
-glycoprotein, is released from the liver (152, 250, 444) and is cleaved in the circulation by the enzyme renin that is secreted from the juxtaglomerular apparatus of the kidney (245, 250, 540, 631) to form the decapeptide angiotensin (ANG) I. ANG I is then activated to the octapeptide ANG II by angiotensin converting enzyme (ACE), a membrane-bound metalloproteinase, which is predominantly expressed in high concentrations on the surface of endothelial cells in the pulmonary circulation (109, 111, 250, 486, 664, 665, 767). ANG II, considered the main effector peptide of the RAS, is then acting on specific receptors, for example, to induce vasoconstriction by interacting with ANG receptors on vascular smooth muscle cells or by stimulating the release of aldosterone from the adrenal cortex (250, 285, 562).
This view of the RAS, which has been generated by accumulated evidence over decades, had to be expanded significantly by more recent findings that increased the complexity of the system. Different ANG receptors (AT1, AT2, AT4) and signal transduction pathways involved have been characterized (142, 306, 462, 505, 604, 639, 681, 691, 712, 724, 785). Moreover, additional truncated peptides such as ANG-(1 —7) have been identified (200, 310, 420, 595, 769), and alternative pathways of ANG II formation, for example, by the serine protease chymase (which can also cleave ANG I to form ANG II), have been proposed (20, 728, 730, 781).
The resulting change of our view of the RAS has been the introduction of the concept of "local" or "tissue" renin angiotensin systems (178, 415, 527). This concept was based on findings of RAS components in "unlikely" places (such as the "kidney enzyme" renin in the brain) where the endocrine actions of the system could not explain the findings (219, 220). This in turn led to new hypotheses and functional concepts of local RAS actions based on the tissue-based synthesis of ANG II. It is not surprising that the notion of tissue-based RAS with independent actions was not received with great enthusiasm, but rather led to significant controversies on the subject. Nevertheless, the database on local RAS accumulated so far has by and large been convincing and strengthened by two major technical advances, namely, the use of molecular biology and the availability of transgenic and knock-out models with altered expression of RAS components (28, 31, 106, 370, 377, 534, 537, 696).
The genes for all components of the RAS have been cloned, and gene expression studies could verify their mRNA expression and regulation in many tissues, demonstrating the possibility of local ANG II synthesis. Overexpression of RAS genes in transgenic mice and rats as well as the knock-out of these genes in mice have allowed detailed studies on the function of local RAS (28, 31, 106, 370, 376, 534, 537, 696). It has also become increasingly clear that these systems are not isolated entities but can interact with the endocrine RAS as well as other peptide systems (such as the endothelin system) on multiple levels (538, 582, 585, 614).
The early controversy on the novel concept of tissue RAS has been based on the question of local synthesis versus uptake from the circulation. A case in point represents the controversy between investigators after the demonstration of renin expression in the heart (178, 415) and those questioning local renin synthesis (751). This controversy was based on the fact that detection of renin mRNA in the heart could only be demonstrated inconsistently, leading to the suggestion that studies measuring cardiac renin were based on artifacts due to contamination with plasma renin or active renin uptake from the circulation. This issue should not threaten the concept of local RAS, since either mechanism could contribute to local ANG synthesis and actions. Modern concepts of the tissue RAS, therefore, are function oriented.
B. Components of the Local RAS
The characteristics and regulatory significance of long-known components of the tissue RAS such as renin, angiotensinogen, ACE, and ANG peptides (ANG I and II) have been reviewed extensively (27, 172, 177, 381, 415, 482, 527, 727) and will not be addressed further in this section. The focus of this section is set rather on recent discoveries concerning new factors and pathways involved in ANG biosynthesis and function.
1. Prorenin and renin binding and receptors
The binding of prorenin and renin to the cell surface in tissues is of pivotal importance with regard to the physiology of local RAS at the tissue level in organs, since it provides a mechanism to generate ANG II locally in excess of the ANG II that is produced in plasma. Previously, it was suggested that binding of prorenin and/or renin is responsible for uptake into tissues (5). It was postulated that this mechanism (which is known also for other enzymes) rather than de novo renin biosynthesis at obscure local sites is responsible for local actions of ANG II. Our understanding of the potential role of prorenin and renin binding has been significantly expanded by the characterization of several proteins capable of binding prorenin and/or renin (87, 315, 488, 490).
The mannose-6-phosphate receptor (M6P) has been shown to be involved in renin and prorenin uptake into cells (132, 602, 733, 737). The cation-independent M6P is directly coupled to G proteins (225, 603) and binds not only prorenin and renin but is in general involved in transport of proteins containing M6P residues; it leads also to insulin-like growth factor II internalization and inactivation (225). M6P was shown to bind renin and prorenin on neonatal rat cardiac myocytes (601) and on human endothelial cells (5). Only glycosylated prorenin and renin are bound by this receptor, and binding is followed by internalization and activation by proteolytic cleavage and subsequent immediate degradation of renin as demonstrated on cardiac cells (488, 601). Particularly studies in endothelial cells indicated that uptake of prorenin by M6P represents a clearance mechanism (733). The role as a clearance receptor on cardiac myocytes is also supported by the finding that intracellular ANG generation could not be demonstrated following (pro)renin uptake in these cells (315, 603). In addition, Peters et al. (542) provided evidence for the existence for a prorenin binding protein on cardiac cells that is different from the M6P receptor. The functional significance of prorenin internalization in cardiomyocytes was shown in vitro and in vivo, supporting the concept of an intracrine RAS in these cells (488, 542).
It has also been suggested that proteins interacting with renin could act as renin inhibitors in vivo, such as a renin binding protein (RnBP) which was isolated from the porcine kidney (698) and identified in porcine, rat, and human tissues (693, 697, 698). Originally, RnBP was identified as a protein in the kidney that was capable of binding renin in renal homogenates giving rise to a complex designated high-molecular-weight renin (698). Subsequently, RnBP was shown to be identical to the N-acyl-D-glucosamine 2-epimerase (437, 699). The gene encoding RnBP has been mapped on chromosome X (697), and chromosomal fine-mapping in the rat indicated that this locus does not fall within a blood pressure quantitative trait locus previously identified in rat models of hypertension (812). Moreover, knock-out of the gene encoding for this protein in mice did not show any effects on RAS activity or blood pressure (621), suggesting that RnBP is of little functional significance in this context.
In contrast to these negative studies, a specific human renin receptor has been recently identified by expression cloning (490). The complementary DNA of this renin receptor encodes a 350-amino acid protein with a single transmembrane domain and no homology to any known membrane protein (490). High expression levels are detected in the heart, brain, and placenta at the mRNA level, while lower levels are observed in the kidney and liver (488, 490).
This 45-kDa membrane protein binds both prorenin and renin and shows a dual function (488). First, binding of prorenin activates cellular effects that are independent from ANG II generation by activating the mitogen-activated protein (MAP) kinases p(42)/p(44) and extracellular signal-regulated kinases (ERK) 1/2 (488, 490). Second, this receptor acts as a cofactor by increasing the efficiency of ANG I generation on the cell surface by receptor-bound prorenin and renin (488, 490). Additional studies showed that the receptor is localized in the kidney in the mesangium and in the subendothelium of renal, uterine, and cardiac blood vessels (488, 490). Nguyen and co-workers (487, 489) demonstrated that renin can bind to this receptor in human mesangial cells in culture and that binding induces hypertrophic effects and an increase of plasminogen activator inhibitor-1. Renin bound to this receptor was neither internalized nor degraded (487, 489). The gene encoding for this renin receptor, i.e., ATP6AP2, maps to the X chromosome and was recently linked to familial X-linked mental retardation and epilepsy syndrome (568). This clinical phenotype is caused by a unique exonic splice enhancer mutation in ATP6AP2, suggesting that the interaction between renin and its receptor is not only important for regulating the activity of the system but also in the mediation of signal transduction processes, for example, in brain development and cognitive function (568).
In the regulation of the RAS it is well established that prorenin represents the inactive precursor of renin that does not proteolytically self-activate like, for instance, pepsinogen (689). This phenomenon has been attributed to the prosegment with its 43 residues attached to the NH2 terminus of mature renin that prevents interaction with angiotensinogen and thus cleavage of ANG I (40, 267, 650). While earlier in vitro studies have suggested that prorenin can be activated by endopeptidases such as trypsin and cathepsin B or by nonproteolytic mechanisms such as by low pH (245, 465, 689), the mechanisms involved in prorenin activation in vivo and their physiological role are still not fully characterized (689). More recently, studies using specific antibodies designed from the tertiary structure of prorenin have shown that there is an essential region in the NH2-terminal region of prorenin, which is responsible for the nonproteolytic form of activation (689). Suzuki et al. (689) have demonstrated two key segments in this region termed the "gate" and "handle" regions. Subsequent studies (296) have further shown that specific binding proteins, interacting with the "handle" region, are responsible for the nonproteolytic activation of prorenin through the induction of a conformational change. These findings shed new light on earlier observations of an independent functional role of prorenin, suggesting that prorenin is a useful marker of diabetic microvascular complications (143, 423, 481) and Wilm's tumor (379).
In the kidney it has been suggested that prorenin uptake and intrarenal activation of the kidney RAS is responsible for inducing renal damage and microvascular changes (296). Interestingly, an earlier transgenic study has come to similar conclusions using a reverse approach (740). Overexpression of prorenin, subjected to site-directed mutagenesis at its proteolytic cleavage site in transgenic rats, has led to high prorenin levels in the plasma, but also to a severe renal phenotype characterized by severe nephrosclerosis in the absence of elevated blood pressure (740). Now that the mechanisms of nonproteolytic activation of prorenin appear to be deciphered, this could finally solve the controversy of elevated prorenin levels and cardiovascular phenotypes not only in the kidney but also in other organs such as the heart (542). The proposed mechanism could be the basis for the understanding of these phenomena and provide new insights into the function of intracellular RAS and their independence from the regulation of active renin in the plasma. The identification of this receptor could play an important role for the understanding of cellular effects of prorenin/renin regulation of cell-specific ANG II formation. It is likely that this discovery could change our view of tissue RAS function by defining an independent functional role for prorenin and renin that are independent from the effects of ANG II (and other peptides) generated within the classical RAS cascade. Yet, the function, relevance, and tissue specificity of the renin receptor and other potential pro(renin) binding mechanisms are still poorly defined, and future research has to investigate whether these sites function alone, together, or in combination with other mechanisms at the tissue level (87).
Alternative cleavage products of ANG I have been suggested as functional peptides in the RAS (200, 201, 595, 596). The most extensively studied of these is ANG-(1 —7), which is generated by the action of several ACE-independent enzymes from ANG I (200). Some of these enzymes such as neprylisin are also involved in the metabolism of atrial natriuretic factors and bradykinin, and ACE has been shown to be active in metabolism and breakdown of ANG-(1 —7), suggesting a complex interaction between different cardiovascular peptide systems (769). ANG-(1—7) has multiple actions that are mostly counteracting those described for ANG II (201, 310, 420, 596).
Recently, a novel functional role of ACE involving outside-in signaling has been identified by Fleming and co-workers (208, 353–355). They demonstrated that ACE inhibitor binding to ACE activates ACE-associated casein kinase 2 (CK2)-mediated phosphorylation of ACE Ser-1270 (355). Depending on initial phosphorylation, ACE-associated c-Jun NH2-terminal kinase (JNK) becomes activated, most likely via ACE-associated mitogen-activated protein kinase kinase 7 (MKK7), leading to an accumulation of phosphorylated c-Jun in the nucleus and enhancement of the DNA-binding activity of activator protein-1 (AP-1, c-Jun dimer) (353). AP-1 activation influences endothelial gene expression, i.e., increases ACE as well as cyclooxygenase-2 (COX-2) expression (354). Thus it was shown that binding of an ACE inhibitor to ACE leads to activation of signal events that are likely to affect the expression of several proteins (208). The characterization of the novel signaling pathway via ACE also suggests that some of the beneficial effects of ACE inhibitors can be attributed to the activation of a distinct ACE signaling cascade rather than to the changes in ANG II and bradykinin levels (208).
ACE2 represents a zinc metalloprotease with carboxypeptidase activity that shares 
42% identity with the catalytic site of somatic ACE and can be shed from cells by cleavage NH2-terminal to the transmembrane domain (169, 713). ACE2 is involved in the generation of alternative angiotensin peptides in particular by the conversion of ANG II to ANG-(1 —7) and ANG I to ANG-(1—9) (169, 741). Thus, while ACE generates ANG II from ANG I through cleavage of the COOH-terminal dipeptide His-Leu, ACE2 catalyzes the conversion of ANG II into ANG-(1—7) by removing the COOH-terminal amino acid phenylalanine. In addition, ACE2 can cleave the COOH-terminal residue of the decapeptide ANG I, thus generating the nonapeptide ANG-(1—9), which may be subsequently converted to ANG-(1—7) by ACE. ACE2 can also cleave des-Arg(9)-bradykinin but does not hydrolyze bradykinin and is insensitive to ACE inhibitors (169, 713, 741). The fact that ACE2 generates the vasodilator ANG-(1—7) can be viewed as a further counterbalancing tissue-specific mechanism within the activated RAS.
The expression of ACE2 is (in comparison with ACE) relatively restricted to cardiac blood vessels and tubular epithelia of the kidneys, which together with its differential enzyme activity might suggest a distinctive physiological function blood pressure and volume regulation (128, 713). A recent study (119) has mapped ACE2 to a region on the X chromosome that is thought to be involved in the genetic modulation of hypertension (271, 794, 812). However, a potential role of ACE2 for genetic hypertension appears questionable since the reported expression analysis of ACE2 in hypertensive rat strains (119) was at variance with the allelic effects at the blood pressure loci observed in mapping analysis with these strains (271, 794, 812). Crackower et al. (119) performed a number of knock-out studies in mice and Drosophila, and while there were only modest effects on blood pressure in mice, the genetic manipulation resulted in severe changes in cardiac contractility, combined with increases of cardiac ANG II levels and the induction of gene pathways mediating the response to hypoxia (119). Therefore, it has been suggested that ACE2 is important as a regulator of heart function and development. Further expression studies of ACE2 on the mRNA and protein level extended the spectrum of organs in which this enzyme is expressed (251, 251, 257) and particularly demonstrated that ACE2 is abundantly present in humans in the epithelia of the lung and small intestine (251). However, taken together, no clear picture of the tissue distribution of ACE2 expression and the physiological role assigned to ACE2 has been yet obtained. Interestingly, work in cell lines suggested that ACE2 is the functional receptor for coronavirus associated with the acute respiratory syndrome, i.e., SARS-CoV (408, 789). The functional role for ACE2 for SARS-CoV replication in vivo was subsequently confirmed in mice (364). Additional studies could demonstrate that ACE2 protects against lung injury caused by SARS-CoV and other agents (301, 364).
It is well-known that ACE is acting on several substrates such as ANG I and bradykinin. One of these, N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), is a hematopoetic factor that is a natural substrate for the NH2-terminal domain of ACE (573). The breakdown of Ac-SDKP can be blocked by ACE inhibitor treatment resulting in an increase of its plasma levels, and it has been suggested that measurement of Ac-SDKP could be a marker for the clinical efficiency of ACE inhibition (25). In the hematopoetic system, Ac-SDKP acts on the cell cycle and prevents the activation of pluripotent stem cells (26), and levels of Ac-SDKP have recently been associated with anemia in heart failure patients treated with ACE inhibitors (734). Although its relevance for cardiovascular regulation still remains unclear, accumulating data indeed support a functional role of Ac-SDKP (554, 555, 569, 761, 800).These functions might include the stimulation of angiogenesis (761) and particularly antifibrotic effects that could point to possible effects in repair and remodeling processes in the cardiovascular system (554, 555, 569, 761, 800) and kidney (88, 646, 744).
An ANG II-forming serine protease termed human heart chymase has been postulated as an activator in an alternative pathway of ANG II formation in the heart (728, 730). It is not affected by ACE inhibition and has been suggested as relevant for alternative pathways of ANG II generation (728, 730, 781). Although several other alternative enzymes involved in ANG II formation had been described previously such as cathepsins and tonin, chymase deserves special attention due to its high substrate specificity. The enzyme is also expressed in the vascular wall, where it has been suggested as a possible player in ANG II-mediated arteriosclerosis (20). Although its ultimate relevance for the cardiac RAS remains to be determined, since its cellular localization is largely restricted to mast cells (418, 728), experimental studies with selective chymase inhibitors in animal models of heart diseases have thus far generated promising results (167, 700).
The actions of ANG II are mediated predominantly by two seven transmembrane domain receptors termed AT1 and AT2 showing a complex pattern of regulation and function (142, 306, 336, 462, 604, 639, 681, 712, 724, 725, 785). In rat and mouse, two AT1 subtypes have been cloned and characterized; they are termed AT1A and AT1B (302). The AT1 and AT2 subtypes show similar properties of ANG II binding but different genomic structure and localization as well as tissue-specific expression and regulation (142). Whereas most of the well-known actions of ANG II such as vasoconstriction and aldosterone release are mediated by the AT1 receptor, the AT2 receptor has been considered to be more of an enigma (403, 724). It appears to play an important functional role in prenatal development, and in the adult, AT2-mediated actions have been shown to counteract AT1 effects such as cell proliferation in vitro (681) and in vivo (462). Increasing evidence supports a role of AT2 particularly in the regulation of growth, differentiation, and regeneration of neuronal tissue (676). The existence of an additional ANG receptor termed AT4 has been postulated, which is interacting with a truncated ANG peptide, ANG IV or ANG-(3 —8) (66, 89, 691, 785). Thus the AT4 receptor was originally defined as the specific, high-affinity binding site for the hexapeptide ANG IV. Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT4 receptor (453). The AT4 binding site has been found in heart, vascular smooth muscle, kidney, colon, adrenal gland, prostate, and many brain regions in processing sensory and motor function (66, 89, 255, 691, 785).
Two additional novel mechanisms of ANG receptor binding leading to contrasting effects have been recently demonstrated (1, 140, 141, 756). First, AbdAlla et al. (1) reported that the AT2 receptor can directly bind to the AT1 receptor and thereby antagonizes the function of AT1. It was shown that heterodimerization between both receptors led to inhibition of AT1 signaling that was independent of AT2 receptor activation (1). Second, activation of AT1 receptor by autoantibodies in women with preeclampsia was demonstrated (756). These stimulatory autoantibodies are directed against the second extracellular AT1 receptor loop and are capable of inducing PKC-mediated effects in vascular smooth muscle cells (756). Although both mechanisms have not been proven with certainty and confirmed by independent groups, they opened new arenas for future research regarding ANG receptor activation and inhibition.
The Mas protooncogene is characterized as a G protein-coupled receptor originally described as a factor involved in tumorigenesis. It has been suggested that Mas is a functional ANG receptor (311), a hypothesis which has been challenged since binding of ANG II to cells expressing Mas could not be shown, suggesting that it is only indirectly involved in ANG II signal transduction. Experiments on Mas knockout mice have indeed shown a functional interaction between Mas and the AT1 receptor (750). This interaction may be attributable to heterooligomerization between Mas and the AT1 receptor and leading to inhibition of ANG II effects mediated by AT1 (359). It remains to be determined, however, whether the proposed effects mediated by ANG-(1 —7) or other ANG peptides via Mas could indeed be of functional relevance in vivo (597).
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II. LOCALIZATION AND FUNCTIONAL ASPECTS |
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The purpose of this review is to integrate aspects of localization of local RAS components with function and will focus predominantly on the physiological rather than pathophysiological implications of these findings. In some instances, pathophysiological concepts will be used to enhance understanding of their physiological basis. We do not discuss the RAS in kidney and adrenal gland as these have been extensively reviewed elsewhere (28, 59, 76, 259, 318, 343, 373, 376, 401, 425, 450, 473, 627, 652), and we rather focus on local RAS in other organs that are not typically associated with ANG formation.
The existence and function of a specific cardiac RAS has been a matter of debate since it has been difficult to differentiate the effects of intracardiac ANG II generation from actions by plasma-borne ANG II. Nevertheless, it has become quite clear that cardiac actions of drugs inhibiting ANG II actions such as ACE inhibitors and ANG II receptor blockers are in part explained by local effects at the cellular level, for example, on cardiac remodeling. The predominant physiological role of the cardiac RAS appears to be the maintenance of an appropriate cellular milieu balancing stimuli inducing and inhibiting cell growth and proliferation as well as mediating adaptive responses to myocardial stress, for example, after myocyte stretch. A schematic representation of cardiac ANG pathways is shown in Figure 1.
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The existence and relevance of cardiac renin expression has been a matter of controversial debates, although some investigators have been able to detect renin mRNA in the heart by Northern blotting (174), solution hybridization assays (531), and RT-PCR (532) in various species. Renin mRNA expression in all of these studies, however, was rather low, and very high amounts of mRNA or total RNA had to be used to bring the renin signal to the level of detection. Other investigators challenged these findings and were unable to find proof for local mRNA expression in the literature (751), claiming that the measurement of cardiac renin mRNA was based on contamination problems and artifacts. This was supported by findings that cultured cardiomyocytes or fibroblasts did not synthesize renin. Ultimate proof of the presence of local renin synthesis in the heart was to be expected by transgenic overexpression using the native renin promoter. Transgenic mice carrying a genomic human renin construct showed no cardiac renin expression (798), whereas transgenic rats carrying a genomic construct of the mouse Ren-2 gene under control of its own promoter expressed high levels of renin mRNA in the heart (549). This suggests that at least in some species, the heart is a site of extrarenal renin production. Other authors have suggested that, whereas renin is not synthesized in the heart under physiological conditions, renin gene expression may be turned on in pathophysiological situations (147). It is interesting to note in this context that an additional truncated renin mRNA has been described in heart tissue which lacks the prefragment of preprorenin and results in the formation of a truncated prorenin protein termed exon 1A renin (103, 541). The initial characterization of this isoform in rat adrenocortical cells revealed that this alternative transcript encodes for a truncated prorenin that is imported into mitochondria (104). Subsequent studies demonstrated the expression of this truncated isoform in the rat heart and suggested that only this alternative renin transcript but not the full-length isoform is expressed in the rat heart (103, 541, 542). This conclusion was based on studies in which renin expression was analyzed by a sensitive nested RT-PCR method allowing the differentiation between the full-length transcript coding for preprorenin and the transcript coding for the truncated intracellular isoform (103, 541, 542). While the latter was detected in various rat tissues in parallel with the full-length mRNA, the exon 1A renin was the only transcript of the renin gene expressed in the heart (103, 541, 542). Moreover, it was shown that in disease states such as cardiac hypertrophy or myocardial infarction, there is no expression of the mRNA coding for the full-length preprorenin but exclusively the exon 1A renin transcript is increased (103, 542). The recent findings on the differential regulation of preprorenin, i.e., the classical full-length renin, and exon 1A truncated renin in the heart might explain some of the previous controversies and discrepancies in the literature regarding renin expression in the heart, since investigators were unable to differentiate between the two isoforms before the identification of exon 1A renin (541).
Less controversial is the evidence for the presence of renin protein in the heart attributed to uptake from the circulation (134) either due to nonspecific uptake (diffusion) into the cellular interstitium (144, 315, 542) or through the actions of specific functional binding sites or receptor for prorenin and renin (87, 488, 490, 541). The M6P receptor has been shown to bind prorenin and renin cells on cardiomyocytes (601, 603). Hypertrophy of isolated neonatal cardiomyocytes in culture and increase in protein synthesis were only detected during coincubation of prorenin with angiotensinogen, while prorenin binding alone had no effect (603). The effects of prorenin plus angiotensinogen were comparable to those of 100 nM ANG II, although the ANG II levels in the medium during exposure of the cells to prorenin plus angiotensinogen were <1 nM. This suggests that cardiac ANG II generation by circulating renin occurs predominantly on the cell surface (315, 603). In addition, the newly identified prorenin and renin receptor that is capable of activating signal transduction via MAP and ERK kinases independently from ANG II generation shows high expression levels in the heart (488, 490). Taken together, these findings support the concept that the physiological role of the tissue RAS in the heart depends on the assembly of prorenin and renin binding receptors including M6P and ANG receptors (488, 603). This favorable microenvironment would allow maximal efficiency of local ANG II generation, i.e., immediate binding of ANG II to its receptors with minimal loss into the extracellular space (315, 488, 603).
A recent study could show that unglycosylated renin is rapidly taken up by cardiomyocytes by a mechanism that is independent from the M6P receptor and that transgenic rats with overexpression of the mouse Ren-2 gene (which have high prorenin levels in the plasma) exhibit strongly enhanced intracellular levels of unglycosylated renin in their hearts (542).
The existence of local ACE production in the heart is, in contrast to the renin, no matter of controversy. Cardiac ACE mRNA can be easily detected by a number of methods in rat (277, 362, 626) and human hearts (532). ACE activity is also readily detectable, for example, by autoradiography (796) or enzymatic assay (278, 362, 726). Immunohistochemistry has been used to localize the predominant source of ACE expression in cardiac blood vessels and the endocardium (190), whereas mRNA studies on cultured cardiac cells also found ACE expression in cardiomyocytes (536). These studies have been confirmed by expression studies showing that ACE is present in viable human cardiomyocytes after myocardial infarction (284). ACE2 expression has also been demonstrated in the heart in both animals (119, 713) and humans (67, 238).
Human heart chymase activates ANG I to ANG II but is not inhibited by ACE inhibitors and could act as an activator for alternative pathways of ANG II formation. Using whole heart homogenates, Urata et al. (729) described that up to 80% of ANG II forming activity in the heart was due to chymase, while only 11% was based on ACE activity, suggesting an increased importance of the chymase pathway in the human heart. When comparing studies in humans and animals, it is important to consider the important species differences in the pathways of intracardiac ANG II generation (30). In this regard, chymase predominates over ACE activity in human heart, accounting for considerably higher total ANG II formation in human heart compared with dog, rat, rabbit, and mouse hearts (30). The ultimate functional importance of a chymase-dependent pathway of ANG II formation in the heart remains questionable due to a number of factors: 1) clinically, ACE inhibitors are extremely efficient in the treatment of cardiac disease; 2) under experimental conditions, most of the ANG II generated by intact cardiac blood vessels can be blocked by ACE inhibitors; and 3) the expression of human heart chymase is highly compartmentalized and mostly restricted to mast cells (728). Nevertheless, ANG II-generating pathways in the heart that are independent from ACE might be particularly important in disease states such as cardiac hypertrophy (407) and heart failure (781).
4. Angiotensinogen, ANG I, and ANG II
The detection of angiotensinogen mRNA in the heart has been described for mouse (174), rat (268, 417), dog (382), and human (532). Although the cardiac mRNA levels of angiotensinogen are more readily detectable than those of renin, they are low compared with those found in liver, the major source of angiotensinogen production (174). Arguments against local mRNA synthesis of cardiac angiotensinogen stem from experiments in isolated perfused rat hearts where there was no endogenous angiotensinogen release detectable (144). These authors concluded that the major percentage of cardiac angiotensinogen is due to plasma uptake and presented evidence that the protein is rapidly taken up into the cardiac interstitium when added to the perfusate.
ANG peptides have been detected in the heart (132, 416) at concentrations higher than those found in the plasma compartment. Although this could be seen as an indicator of cardiac synthesis, the issue of cardiac uptake and local storage of ANG II should be considered. Alternatively, renin and angiotensinogen taken up from the circulation could be interacting with local ACE to lead to intracardiac ANG II formation. To address this question, van Kats et al. (736) used infusions of radiolabeled ANG I and ANG II peptides in pigs and measured plasma and tissue levels of endogenous as well as the radiolabeled peptides. The results of this study indicated that >90% of cardiac ANG I is synthesized locally in the heart and that >75% of cardiac ANG II is synthesized locally, most of it using local ANG I generation as a basis. These findings clearly show the local synthesis of ANG peptides in the heart as a relevant mechanism and point out that the concept of a cardiac RAS is not dependent on the local synthesis of angiotensinogen and renin.
Both the AT1 and the AT2 receptors are expressed in the heart where they appear to be localized on cardiomyocytes (28, 55, 577, 590, 729). On cardiac fibroblasts, the receptor population appears to be dependent from the presence or absence of cardiac disease. Normal fibroblasts express AT1 only but can recruit the AT2 receptor under certain pathological conditions (118, 510, 635). The function of the two ANG receptors in the heart has been seen in perspective of a "Ying-Yang" principle, meaning that the AT1 is a stimulator of hypertrophy and proliferation of cardiac cells, whereas AT2 is mediating the opposite effects (773). Transgenic and knock-out studies in mice, however, have not supported this concept in the heart, since knockout of the AT2 receptor in mice has suggested that the receptor is also needed for the mediation of hypertrophic stimuli (705). Moreover, conflicting experimental data obtained more recently in animal studies using either selective AT2 antagonists or genetically modified mice have raised some concern regarding the beneficial role of AT2 stimulation in both the heart and vasculature in disease states (403).
A) INOTROPIC EFFECTS. Koch-Weser (351) first suggested that ANG II acts as an inotropic agent (351) but only at "supraphysiological" concentrations. The effect could at least in part be indirect by ANG II acting, for example, on the sympathetic nervous system (352). Nevertheless, direct effects by ANG II have been verified (148), which are thought to be mediated by intracellular calcium influx and changes of the plateau phase of the cardiac action potential. In vitro studies in human preparations carried out physiological conditions in right atrial and right and left ventricular myocardial preparations of patients with a variety of cardiac diseases suggested that ANG II exerts positive inotropic effects only in atrial preparations (286). However, transgenic overexpression of the human AT1 receptor on cardiac myocytes in a transgenic rat model has supported the early findings since transgenic AT1 upregulation led to an enhanced intracellular calcium response after ANG II stimulation (280).
B) HYPERTROPHIC EFFECTS.
ANG II mediates myocyte hypertrophy due to activation of the AT1 receptor as an adaptive response to increased myocardial stress. While hypertrophy of cardiomyocytes acts initially as a compensatory mechanism to preserve cardiac function, it becomes a major risk factor for congestive heart failure and sudden cardiac death and overall mortality (172). In vitro studies have demonstrated this effect in cultured cardiomyocytes (738), and it has been suggested that this effect is secondary to the release of other growth factors such as endothelin-1 and transforming growth factor (TGF)-
(239). Left ventricular hypertrophy due to enhanced ANG II production in the heart has also been described in transgenic rat models with overexpression of RAS components such as mouse renin (549), the human AT1 receptor (279, 280), human ACE (555, 711), and double transgenic rats expressing human renin and human angiotensinogen (470). In some of these models it has been clearly shown that the ANG II effects occur independently from its effects on blood pressure, suggesting a functional role of the local cardiac RAS in mediating these changes. Hypertrophic changes induced by ANG II are mediated by several distinct intracellular pathways such as the activation of tyrosine kinase and RhoA cascades which include activation of MAP kinase and JAK/STAT pathways (142, 435, 613, 725). An important maladaptation in left ventricular hypertrophy relates to diastolic dysfunction that results from functional changes such as impaired diastolic calcium handling and/or structural changes such as cardiac fibrosis (101, 586, 629). Studies in rats with experimental left ventricular hypertrophy indicated that locally generated ANG II may disturb relaxation, i.e., diastolic function, of the heart (629). This notion was supported by subsequent studies in transgenic rats with activated tissue RAS showing significant impairment of diastolic relaxation (585, 586). Moreover, diastolic function could be restored by treatment with a non-blood pressure-lowering dose of an AT1 receptor antagonist (586). Functional analysis of left ventricular dysfunction in this setting indicated that the impairment of diastolic dysfunction was attributable to impaired diastolic sarcoplasmic reticulum calcium pump (SERCA2) activity (586). A similar effect could also be induced in the same rat model by treatment with a selective endothelin A receptor antagonist, demonstrating the activation of the endothelin system in rats with activated tissue RAS and its functional consequence in the heart (585). Chronically, activation of the cardiac RAS may not only lead to cardiac hypertrophy and diastolic function but also to progressive systolic dysfunction, cardiac enlargement, and heart failure. The independent role of cardiac RAS activation for these consequences has been recently demonstrated in transgenic TG1306/1R mice that develop ANG II-mediated cardiac hypertrophy in absence of elevated blood pressure (168). A long-term follow up study in these mice demonstrated that transgenic animals develop dilated cardiomyopathy with aging and exhibit a significant increase in mortality compared with wild-type mice. Cardiac hypertrophy in transgenic mice is also associated with SERCA2 activity and reduced Ca2+ transport. Moreover, systolic function was also impaired as evidenced by impaired contractility in isolated cardiomyocytes (168). Equivocal results have been obtained with regard to the role of the AT2 receptor in cardiac hypertrophy (403). While the AT2 receptor has been initially associated with antihypertrophic effects (403), some studies in AT2-deficient mice indicated that AT2 has a significant effect on cardiac hypertrophy induced by aortic banding (635) or ANG II infusion (297).
C) MECHANICAL STRETCH. Several lines of evidence indicate that ANG II pathways can be defined as a "rapid response system" for mechanical stretch in the heart, which may be involved in the mediation of cardiac hypertrophy (100). Stretch can induce ANG II release into the media of cultured cardiomyocytes in vitro (587) and in vivo (391), and virtually the expression of all gene transcripts of the RAS can be stimulated by stretch (432). Overload of the left ventricle, which represents a situation associated with chronic stretch of cardiomyocytes, results in a similar activation of the cardiac RAS (29, 626). The intracellular pathways activated by the induction of the cardiac RAS and local ANG II production in cultured neonatal cardiomyocytes are blocked by AT1 receptor antagonism (357). The signaling is mediated by p53 as well as by the JAK/STAT pathway (391). Whereas initially it was thought that these effects are mediated entirely through the AT1 receptor, recent AT2 knock-out studies in mice have revealed that the AT2 receptor is also involved in mediating these changes (635).
D) REMODELING. Proliferative stimuli by cardiac ANG II are probably most relevant for the fibroblast portion of cardiac cell population as has been shown by Schelling and Ganten (606, 608). Similar mechanisms have been described during cardiac remodeling where fibroblast proliferation has been shown as a cellular indicator of pathological changes. Local ACE formation appears to play an important role in this process, since previous studies in a rat heart failure model induced by experimental myocardial infarction (which goes along with increased fibrosis) have demonstrated an activation of cardiac ACE activity and mRNA, whereas plasma ACE activity was not changed (277).
In rat hearts, Sun and Weber (687) have shown that at weeks 1 and 4 after myocardial infarction myofibroblasts were the predominant cell expressing high-density ANG II receptors at this site, while fibroblasts, macrophages, and vessels demonstrated low-density ANG II receptor binding. After myocardial infarction in rats treated with the AT1 antagonist losartan, a significant reduction in collagen volume fraction at remote sites of the infarction was found compared with untreated animals (139).
Since either AT1 blockade or ACE inhibition is not associated with any normalization of elevated collagen mRNA after rat myocardial infarction, Dixon et al. (165) suggested that the reduction of cardiac fibrosis mediated by ACE inhibition and losartan treatment may reside at the posttranslational level in cardiac collagen metabolism. The RAS has apparently multiple targets involved in cardiac remodeling. Tan et al. (702) measured the cardiotoxic effects of ANG II in rats. The authors have shown that pathophysiological levels of endogenous as well as nonhypertensive low doses of exogenous ANG II produced multifocal antimyosin labeling of cardiac myocytes and myocytolysis, increased DNA synthesis rate and fibroblast proliferation. Both myocyte injury and fibroblast proliferation were prevented with captopril (702).
The mechanisms leading to ANG II-induced fibrosis are thought to be at least partially mediated through growth factor pathways induced by AT1 receptor activation (548, 686). In this context, TGF- has been implicated as a candidate. Studies on transgenic rats expressing the mouse Ren-2 gene have shown that inhibition of TGF- synthesis by the specific growth factor inhibitor tranilast did not affect blood pressure but resulted in a significant alleviation of interstitial cardiac fibrosis seen in this model, which was also associated with longer survival of treated transgenic animals (548).
Another mediator, osteopontin, which is involved in the vascular smooth muscle cell remodeling process, is increased at mRNA and protein levels after addition of ANG II to rat cardiac fibroblasts (24, 506). This effect is blocked by the AT1 receptor blocker losartan. This suggests that osteopontin is a potentially important mediator of ANG II regulation of cardiac fibroblast behavior in the cardiac remodeling process. Indeed, osteopontin mRNA is elevated in transgenic rats, the mouse Ren-2 gene already in the prehypertensive phase, which suggests that it contributes directly to the contractile dysfunction seen in this model and is blood pressure independent (584).
Yet another mediator in the inhibition of cardiac fibroblast proliferation appears to be the recently described alternative substrate of ACE, AC-SDKP, a hematopoetic stem cell regulator, is hydrolyzed by the NH2-terminal active site of ACE. It has been demonstrated that the administration of ACE inhibitors stimulates AC-SDKP plasma levels over fivefold (25), which could be a plasma marker for efficient ACE inhibition. Recently, it has also been demonstrated that AC-SDKP inhibits also fibroblast proliferation in a dose-dependent manner (554), suggesting that the alternative substrate could be the mediator of antiproliferative effects on fibroblasts in cardiac remodeling seen after ACE inhibition. This fascinating hypothesis, however, awaits final confirmation.
In addition to the structural abnormalities related to activation of the cardiac RAS, increased activity of the system has also been linked to changes in the electrical physiology that lead to arrhythmias both in the ventricle and atria (146, 266). Indeed, in human patients undergoing heart surgery, patients with a history of paroxysmal or persistent atrial fibrillation showed increased interstitial fibrosis and threefold higher ACE tissue levels compared with patients in sinus rhythm (233), while the densities for the AT1 receptor was decreased and increased for AT2 receptors (231). Most importantly, evidence obtained from recent pharmacological intervention studies has pointed to a new concept in which inhibition of the RAS by ACE inhibitors or AT1 antagonists may induce specific benefits in patients with atrial fibrillation (232, 265, 649).
E) APOPTOSIS. Whereas earlier studies carried out in PC-12 cells (a rat pheochromocytoma cell line) have suggested that programmed cell death is mediated by the AT2 receptor (797), it is generally accepted that apoptosis of cardiac myocytes is mediated via the AT1 receptor (99). The process is thought to be involved in cardiac remodeling, for example, after myocardial infarction (19), hypertensive cardiomyopathy (163), and diabetic cardiomyopathy (206). It can be effectively blocked by AT1 antagonists (160), which suggests that the beneficial effects of RAS blockade in heart failure could be due in part to this intracardiac mechanism.
The vascular wall is the effector organ for the hormonal or plasma RAS where AT1 receptors localized on vascular smooth muscle cells mediate vasoconstriction. The concept of a vascular RAS was generated when it became evident that ANG II can differentially affect growth properties of vascular cells and that RAS components can be formed intracellularly in the vasculature.
Whereas some studies have not found renin activity in blood vessels (210), others have found renin mRNA expression in conductance and resistance vessels of human (532) and rat (594). Nevertheless, renin mRNA has been difficult to detect due to the small sample size which very often required pooling of samples and the use of more sensitive assays such as RNase protection assays as well as RT-PCR. This has led to a similar controversy as that concerning renin in the heart, and it has been proposed that local renin synthesis is negligible if at all present in the vasculature (751). Although this may be true under physiological circumstances, it is possible that local renin production may be turned on in disease states. In this context, vascular renin induction has been demonstrated in the rat neointima model (309).
In addition to local synthesis, renin uptake via unspecified binding sites on endothelial cells or specific prorenin/renin receptors (87, 315, 488, 490) has been suggested as a relevant mechanism. The M6P receptor was shown to bind renin and prorenin on human endothelial cells (5), and studies in endothelial cells indicated that uptake of prorenin by M6P may represent a clearance mechanism for prorenin (733). This could in fact provide a protective mechanism during activation of the RAS either systemically or at the tissue level. The potential importance of this mechanisms was shown by generation of a transgenic rat model that directed high prorenin expression and release into the plasma from the liver (740). This led to dramatic increases in plasma prorenin that were up to 400-fold. Interestingly, these animals did not have higher blood pressures than controls, yet developed severe vascular lesions, suggesting that prorenin is taken up from the circulation and stimulating the intracellular RAS leading to pathological trophic effects (740). Moreover, in humans, increases in circulating prorenin levels have been associated with disease states such as diabetic microvascular complications (143, 423, 481). It thus appears of interest for further studies to evaluate the functional role of prorenin clearance and elimination in the endothelium by M6P or other mechanisms. These effects have to be balanced against mechanisms such as binding by the prorenin/renin receptor (490) or nonproteolytic activation of prorenin (296, 689) that would lead to the activation of the RAS (87, 315, 488, 490).
In the vascular wall, ACE is readily detectable, where it is localized predominantly on the surface of endothelial cells (190, 811). There are controversial data regarding the distribution of ACE expression in different layers of vascular wall. Wilson et al. (776) found a predominant labeling in the endothelium and adventitia. This finding was confirmed by Rogerson et al. (578) in human, dog, rabbit, and sheep arteries. Arnal et al. (22) have shown high ACE mRNA and protein expression as well as immunoreactivity in the media of rat aorta where the expression level is almost as high as in the endothelium, while expression is low in the adventitia. Several reports have indicated that vascular smooth muscle cells, which do not appear to express ACE, can do so in certain pathophysiological situations such as neointima formation (197). Low amounts of ACE have also been detected in the adventitia of blood vessels (578).
ACE2 mRNA expression is ubiquitously found in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied by Hamming et al. (251). An important role for ACE and ACE2 in the pulmonary vasculature and epithelial cells during injury and/or SARS virus infection has been recently supported (301, 364, 491). However, further studies are required to delineate the cell types responsible for RAS component expression in the lung and to identify the physiological role of the pulmonary RAS (436). This will also provide a better understanding of the activated RAS, and particularly the potential protective effect of ACE2 in lung disease (436, 491).
3. Angiotensinogen, ANG I, and ANG II
The substrate of the RAS cascade has been detected in blood vessels at the mRNA level (74, 268). In situ hybridization studies showed that it is abundantly expressed in periadventitial fat cells (73, 84). This led to the suggestion that angiotensinogen is secreted by these cells and diffuses through the vascular wall where it gets in contact with vascular renin.
Both AT1 and AT2 receptors were identified in the vasculature (54, 475). Initially the function of these receptors was investigated on cultured primary vascular cells. Vascular smooth muscle cells in culture express only the AT1 receptor, whereas cultured endothelial cells expressed both AT1 and AT2 (681). ANG II stimulated growth of vascular smooth muscle cells, while the peptide inhibited the growth of quiescent coronary endothelial cells in response to stimulation by basic fibroblast growth factor (681). In the presence of an AT2 receptor antagonist, this effect was abolished, suggesting a growth-inhibiting role of AT2 (681). Later, this interesting concept was also reproduced in an in vivo experiment of AT2 gene transfer in balloon-injured rat carotid arteries, which led to a reduction of neointima formation, an effect which was neutralized by an AT2 antagonist (461). The distribution of the AT2 receptor in the vascular wall is nevertheless a matter of debate (39). Overall, functional studies in isolated arteries from both animals and humans accumulated a large body of evidence that the endothelium is the most important side for AT2 receptor expression (38, 39, 46, 234, 254, 622). In addition, since AT2 may form heterodimers with AT1 receptors (1), this would require a colocalization of both receptors and thus points to the additional expression of AT2 expression on vascular smooth muscle cells in the vascular wall (39).
A) VASCULAR TONE AND ENDOTHELIAL FUNCTION. The tissue RAS contributes to the maintenance of cardiovascular homeostasis by the dual impact on vessel function mediated through the opposing effects of its two receptors. In in vivo studies, i.e., in the whole body situation, it is not possible to clearly </
