Growth factors, cytokines, and vascular injury

1990) . When subjected to appropriate stimuli, a subpopulation of medial SMCs develops the ability to migrate and regains the capacity to proliferate . These relatively dedifferentiated SMCs develop a fibroblastlike appearance and secrete extracellular matrix proteins such as elastin and collagen type I . This is accompanied by suppression of production of SMCspecific a-actin and myosin, and loss of contractile function (Gabbiani et al. 1984) . Proliferating SMCs are the predominant histologic feature of the neointimal proliferative response occurring after balloon angioplasty . It is important to define the signaling mechanisms that determine SMC activation, since this may provide us with better strategies to modify the biologic response to arterial injury. Here, we discuss the biologic consequence of three major injury-mediated processes : (a) loss of the endothelial layer, (b) trauma to the media, and (c) paracrine production of growth factors .

Growth Factors, Cytokines, and Vascular Injury James A . Fagin and James S . Forrester

Restenosis after angioplasty can be considered as the undesirable consequence of the vascular response to injury. The repair process, which manifests as smooth muscle cell hyperplasia and extracellular matrix deposition, is controlled by growth factors and cytokines from a variety ofsources . Vascular smooth muscle cells themselves synthesize mitogens, such as platelet-derived growth factor and insulinlike growth factor I, which synergize to promote medial cell proliferation . Additionally, growth factors such as fbroblast growth factor are probably released by trauma from damaged smooth muscle cells or from the matrix that surrounds them. The abundance and cellular distribution of cell membrane receptors for the various growth factors is also modulated after arterial injury, and probably plays a role in determining the characteristics and magnitude of the proliferative response . It is • Role of Deendothellalization becoming increasingly apparent that multiple redundant and often As an immediate consequence of balloon overlapping control mechanisms are involved in modulating restenosis . dilatation, the endothelial layer is reAs the sequence of signaling events is unraveled, and antagonists to the moved from the injured site. At least critical mediators are developed, it is likely that more effective treatment three major events result from deendoprotocols will become available to improve the long-term outcome of thelialization: platelet adhesion, loss of growth inhibitory factors made by endoangioplasty procedures. (Trends Cardiovasc Med 1992 ;2 :90-94)

Based on serial angiography studies, the rate ofrestenosis afterballoon angioplasty is now reported to be 40%50% (Nobuyoshi et al. 1988) . There is still no established therapy to prevent or ameliorate this complication. In this review, we discuss the pathogenesis of restenosis, with special emphasis on the role of growth factors in the vascular repair process that takes place after arterial injury . Most of the vessel dilatation achieved by balloon inflation is due to stretching of the normal arterial wall. The atheromatous plaque is largely nondistensible and may fissure or crack, but does not significantly contribute to luminal enlargement . The endothelial layer, in turn, James A. Fagin and James S . Forrester are at the Division of Cardiology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048, USA . 90

is stripped away from the balloon- treated surface, exposing the underlying media . These immediate effects of angioplasty on the vessel wall trigger a series of events destined to repair the injury . Restenosis can be considered as an undesirable consequence of this repair process (Forrester et al . 1990) . Vascular smooth muscle cells (SMCs) provide structure and elasticity to the vessel wall and are therefore primarily involved in the control of blood pressure and flow . They lie embedded in the medial layer of the vessel, where they are normally in a near-quiescent state, with a replication rate of -0 .02% . Each cell is surrounded by an extmeellular matrix composed of collagen type IV, laminin, entactin (nidogen), and heparan sulfate proteoglycans. This macromolecular network plays an important role in regulating homeostasis of the cells and in establishing a link with surrounding extracellular matrix proteins (Thyberg et al . 01992, Elsevier

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thelial cells, and absence of the normal permeability barrier to compounds present in plasma. The impact of endothelial loss on SMC proliferates includes the following : 1 . Platelets adhere to denuded area and release growth factors (for example, platelet-derived growth factor) 2 . Loss of growth-inhibitory products elaborated by endothelial cells (for instance, heparin) 3 . Absence of permeability barrier allows access of plasma-derived proteins with activity on SMC (for example, fibronectin)

Within minutes of endothelial injury, platelets adhere to the denuded surface and rapidly degranulate . A number of platelet factors stored in a granules are released locally into the exposed intimal layer . Prominent among them is plateletderived growth factor (PDGF), a glycoprotein dimer with powerful mitogenic and chemotactic activity on vascular SMCs . PDGF is a glycoprotein homo- or heterodimer made of two chains (A and B)

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linked by disulfide bonds . It has been postulated that this growth factor may be the major initiator of the local vascular response to injury (Ross 1986) . It is noteworthy that adhering platelets largely disappear within hours of injury, yet SMC migration and proliferation takes place over a time course of days or weeks (Clowes et al, 1986) . PDGF can bind to the glycosaminoglycan (GAG) heparin, which is present in the extracellular milieu of the vascular wall, and could conceivably serve as a reservoir for the growth factor long after platelets are no longer present in the injured vessel surface . Several lines of evidence indicate, however, that platelets are not an absolute requirement for vascular repair. Inhibition of platelet aggregation with antiplatelet drugs prior to endothelial denudation results in only partial decrease of neointimal proliferation in New Zealand rabbits (Faxon et al . 1984) and has almost no effect in rats (Clowes and Karnovsky 1977) . In clinical studies, the incidence of restenosis after coronary angioplasty is not affected by prior therapy with aspirin and dipyridamole (Schwartz et al . 1988) . These data support the concept that the restenotic process does not depend entirely on platelet function, and that other regulatory pathways must be involved in controlling the response to injury . After angioplasty, areas that remain denuded of endothelium for a longer period have greater SMC hyperplasia, whereas reendothelialization is often associated with cessation of SMC growth . Because of this, it has been postulated that endothelial cells may elaborate factors that inhibit SMC proliferation (Castellot et al . 1987) . Endothelial cellconditioned medium blocks vascular SMC replication in vitro, an effect largely abolished by prior digestion with a highly purified Flavobacterium heparinase, suggesting that the inhibitory effect is due to heparinlike products made by endothelial cells (Castellot et al . 1981) . Heparin and heparinlike species are effective antiproliferative agents for vascular SMCs . The action of heparin is probably not due to binding and inactivation of mitogens such as PDGF or fibroblast growth factors (FGFs), but rather to a specific receptor-mediated inhibition of cell division . Thus, SMC proliferation after injury may be facilitated through loss of the tonic growth inhibition norTCM Vol. 2, No . 3, 1992

mally exerted by heparinlike species made by endothelial cells . Deendothelialization is associated with removal of the normal permeability barrier of the vessel wall, thus allowing unrestricted access of factors present in plasma to the inner layers of the artery . In this manner, plasma-derived mitogens (for example, insulinlike growth factors [IGFs], epidermal growth factor, serotonin, and many others) may be directly involved in inducing migration and/or growth of medial SMC . The plasma protein fibronectin has also been shown to have major effects on the phenotypic properties of SMCs . Fibronectin promotes the transition of rat aortic smooth muscle cells from a contractile to a synthetic phenotype (Thyberg et al . 1990) . The dedifferentiating effects of fibronectin on SMCs are mediated through the cell attachment sequence of the protein (ArgGly-Asp-Ser, RGDS) . Interestingly, DNA replication and SMC proliferation do not take place in the presence of fibronectin unless serum or PDGF is also present. This suggests that plasma-derived fibronectin may modulate SMC phenotype, rendering the cells competent to divide in response to growth factors . • Arterial Wall Trauma

In general, injuries that cause little or no trauma to the media elicit only minor SMC hyperplasia (Fingerle et al . 1990) . Trauma might disrupt the basement membrane and/or the extracellular matrix network surrounding SMCs, therefore allowing free interaction with mitogens or other regulatory proteins . Additionally, SMC death may promote growth of neighboring cells through liberation of stored growth factors or cytokines . Vascular SMCs synthesize angiogenic factors such as FGFs, with powerful mitogenic activity for SMCs and endothelial cells (Klagsbrun and Edelman 1989) . There is solid evidence indicating that FGFs remain largely cell associated. As they lack a signal peptide sequence, they cannot be secreted in a similar fashion as other growth factors such as PDGF and IGF-I . For reasons that are unclear at present, FGFs are found not only associated with cells, but also stored in the basement membrane and extracellular matrix . After traumatic injury to the arterial media, FGFs could be released from damaged or dying SMCs. Furthermore, heparinases

and proteases from infiltrating macrophages have the potential to degrade the extracellular matrix and release FGFs stored in the pericellular space . The potential biologic relevance of these observations was recently demonstrated in vivo (Lindner and Reidy 1991) . Rats treated with a blocking antibody to basic FGF had decreased smooth muscle proliferation after balloon catheter injury, suggesting that this growth factor may be significant in the process of vessel repair and restenosis . • Autocrine or Paracrine Growth

Factors As mentioned above, a number of powerful mitogens are released by platelets upon adhesion to the damaged vessel wall (that is, PDGF, epidermal growth factor [EGF], and transforming growth factors [TGFs]) . However, the process of restenosis is not adequately prevented by blocking platelet function. Because of this, it has been postulated that growth factors or cytokines elaborated within the vessel wall may be involved in the induction or perpetuation of SMC migration and proliferation (Figure 1) . Balloon injury induces medial SMCs to produce PDGF AA, an isoform of PDGF that has strong mitogenic activity, but may not be as effective as PDGF AB or PDGF BB in promoting migration (Siegbahn et al. 1990) . Differences in potency between the PDGF isoforms may in part be explained by variations in the type of PDGF receptor expressed by the cells . Expression of PDGF AA peaks a few hours after injury and falls to near basal levels within 24 h (Majesky et al . 1990) . Additionally, regenerating endothelial cells and macrophages secrete PDGF BB, although the production of this PDGF isoform is not significantly modulated during experimental vascular injury . These data are compatible with the following role for PDGF in the first hours after balloon angioplasty : platelets release PDGF AB and BB, which serve as an initial stimulus to both SMC migration and proliferation ; upon activation, SMCs produce PDGF AA, which further induces cellular growth in a paracrine fashion but does not contribute to cell chemotaxis . A recent observation compatible with this hypothesis was made in rats rendered thrombocytopenic with antiplatelet antibodies . In these animals,

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Rp B Li

0 1 3 7

Figure 1 . Regulation of alternative 3' insulinlike growth factor I (IGF-I) messenger RNA (mRNA) transcripts in the rat aorta after balloon denudation . A solution hybridizationRNase protection assay with an IGF-I riboprobe complementary to IGF-Ib mRNA is shown. Lanes : Rp, labeled riboprobe (not treated with RNase) ; B, hybridization with 40 itg transfer RNA served as a negative control ; Li, hybridization with 40 gg total RNA from normal rat liver served as a positive control ; and 0-7, 40 .tg total RNA from aortas pooled from four rats before and 1, 3, and 7 days after balloon denudation, respectively. Arrows indicate protected fragments of 168 (IGF-Ib) and 116 bases (IGF-1a). The intermediate-sized band is an aberrant digestion product of IGF-Ia . From Cercek et al . (1990) .

SMC proliferation after balloon injury was unimpaired, whereas neointimal plaque did not form, demonstrating a platelet-dependent effect on SMC migration (Fingerle et al . 1989) . Cellular proliferation not only depends on the presence of growth factors, but on the ability of the cells to respond to them uniquely and specifically. This characteristic is contingent on the presence of plasma membrane receptors . Increasing evidence indicates that ex92

pression of receptors may also be regulated as part of the arterial response to injury. PDGF-p receptor is not expressed or expressed at very low levels in normal vessels . After balloon injury, PDGF-l3 receptor mRNA is induced in neointimal cells (Majesky et al . 1990), suggesting that modulation of PDGF responsiveness may play a role in the repair process . As discussed above, PDGF stimulates cell growth through interaction with specific plasma membrane receptors and activation of an intracellular signal cascade that ultimately leads to DNA synthesis and cell division . However, PDGF requires the presence of additional growth factors to maximally stimulate cell replication . PDGF has been termed a "competence facto' in that it enables cells to move from the quiescent (G o) to the G l phase of the cell cycle . The target cells require a second class of growth factors, termed "progression factors," in order for DNA synthesis to take place . One of the best-characterized progression factors forPDGF-mediated mesenchymal cell growth is IGF-I (Stiles et al . 1979) . IGF-I, also termed somatomedin C, mediates the growth-promoting effects of pituitary growth hormone and is abundantly present in plasma . IGF-I markedly synergizes with PDGF to induce SMC growth in vitro . Furthermore, there is evidence that smooth muscle cells can make IGF-I, and that this production is, in turn, stimulated by PDGF (Clemmons 1985) . Thus, PDGF can induce the production of its own progression factor. It is likely that this process is also occurring in vivo . IGF-I gene expression is markedly increased in rat aorta after balloon angioplasty, with maximal production 7 days after injury (Figure 1, Cercek et al . 1990) . Further evidence for local bioactivity of IGF-I is the fact that the expression of the IGF-I receptor decreases after injury in a reciprocal fashion (lowest levels at 7 days), suggesting that the receptor is downregulated in vivo by ligand binding . Besides functioning as a progression factor for PDGF-induced growth, local overproduction of IGF-I in the vascular media may be serving other important reparative functions . It is noteworthy that IGF-I is an effective stimulus for elastin biosynthesis (Foster et al . 1987) and thus may be involved in regenerating the damaged medial elastin layer after balloon trauma . The significance of the synergism be®1992, Elsevier

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tween PDGF and IGF-I is highlighted by recent studies on another in vivo model of wound healing (Lynch et al . 1989) . When applied topically to partialthickness porcine skin wounds, the combination of EDGE and IGF-I was by far the most effective stimulator of healing (compared to nine individual factors and eight other combinations) . This synergism was apparent in the promotion of dermal cell proliferation as well as hydroxyproline content of the 7-day-old wounds . IGFs circulate in plasma complexed with a number of high-affinity binding proteins . Vascular SMCs also elaborate IGF-binding proteins, which may be released into the vessel wall to target and/or modulate the bioactivity of IGF-I (Giannella-Veto et al., unpublished) . Their precise role in vascular injury is currently under study. • Extracellular Matrix Production

In the clinical setting, restenosis occurs 3-6 months after intervention, well beyond the time course of SMC migration and proliferation. The bulk of the increase in plaque size is due to deposition of extracellular matrix proteins by synthetic vascular smooth muscle cells . Histologic evaluation of human restenotic lesions reveals that the predominant matrix components are collagen and GAGs . GAGs appear to be the most abundant component of the early restenotic lesions, followed by collagen deposition after 6 months . The two major GAGs synthesized by vascular SMCs are dermatan sulfate and chondroitin sulfate . Transforming growth factor (3 (TGF(3) is a homodimeric polypeptide ubiquitously expressed by mammalian cells, which is released by cells localized at the site of tissue repair, such as platelets, activated macrophages, lymphocytes, and possibly vascular SMCs (Sporn et al, 1987) . TGF-(3 is a powerful stimulus to the production of GAG and may be a major factor regulating the early matrix deposition phase of vascular repair (Chen et al . 1987) . In fibroblasts, TGF-(3 can also activate the expression of other matrix proteins, such as collagen and fibronectin (Ignotz and Massague 1986) . In contrast, the effects of TGF-(3 on SMC production of collagen subtypes and fibronectin are at best modest Wait and Chan 1989) .

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INJURY

I 0

I 7

From :

certain antiplatelet drugs to affect myointimal thickening following arterial endothelial injury in the rat. Lab Invest 36 :452-464 . 14 days

Clowes AW, Reidy MA, Clowes MM : 1986 . Kinetics of cellular proliferation after arterial injury. III . Endothelial and smooth muscle cell growth in chronically denuded vessels . Lab Invest 54:295-303 .

Plasma

Growth factors : IGF, serotonin, others Fibronectin Angiotensinogen

Platelets

PDGF AB/BB TGF-beta EGF

Faxon DP, Sanborn JA, Haudenschild CC, Ryan TJ: 1984 . Effect of antiplatelet therapy on restenosis after experimental angioplasty. Am J Cardiol 53 :72C-76C . Fingerle J, Johnson R, Clowes AW, Majesky MW, Reidy MA : 1989 . Role of platelets in smooth muscle proliferation and migration after vascular injury in rat carotid artery . Proc Natl Acad Sci USA 86 :8412-8416 .

SMC/ECM

r- -

FGF

SMC

OW-

PDGF-AA

SMC

IGF-1

Figure 2. Contribution of blood and vessel components to vascular repair during the first 2 weeks after balloon angioplasty. SMC, smooth muscle cells; and ECM, extracellular matrix .

Fingerle J, Tim Au YP, Clowes AWL Reidy MA: 1990. Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury . Arteriosclerosis 10 :1082-1087 . Forester JS, Fishbein M, Helfant R, Fagin JA : 1990 . A paradigm for restenosis based on cell biology: clues for the development of new prevention therapies . J Am Coll Cardiol 17 :758-769 .

Recently, angiotensin-converting enzyme (ACE) inhibitors were found to

be an "amplifying factor" for SMC growth, a final common pathway for the

inhibit myointimal proliferation after balloon arterial injury in rats (Powell et al .

full-blown mitogenic effects of PDGF and FGF to take place . As the sequence of

1989) . A preliminary retrospective study suggests that ACE inhibitors may also

signaling events becomes clear (Figure 2) and specific antagonists to the critical

decrease restenosis in human subjects (Gottlieb et al . 1990) . Angiotensin II is not

mediators are developed, it is likely that better treatment protocols will become

a significant mitogen for smooth muscle

available to modify the overexuberant

cells, although it does significantly stimulate protein synthesis (Berk et al . 1989) .

biologic response to arterial angioplasty .

Angiotensin II markedly stimulates GAG synthesis, a fact that could explain in part

References

Gabbiani G, Kocher 0, Bloom WS, et al .: 1984 . Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media . J Clin Invest 73 :148-152 .

Berk BC, Vekshtein V Gordon HM, et al . : 1989. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells . Hypertension 13 :305-314 .

Gottlieb NB, Gottlieb RS, Morgamoth J, et al. : 1990 . Prevention of restenosis after PTCA by angiotensin converting enzyme inhibitors [abst] . JAm Coll Cardiol 17 :181A.

Castellot JJ, Addonizio ML, Rosenberg RD, Karnovsky MJ: 1981 . Cultured endothelial cells produce a heparin-like inhibitor of cell growth . J Cell Biol 90:372-379 .

Ignotz RA, Massague J : 1986 . Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 261 :4337-4345 .

the beneficial effects on restenosis of blocking angiotensin II formation (Bailey et al ., unpublished) . Much remains to be known, however, about the complex control mechanisms for matrix deposition after vascular injury .

Conclusions We have discussed the role of growth factors and cytokines in the vascular response to injury and restenosis . It is becoming increasingly apparent that the healing process is subject to multiple redundant and often overlapping control mechanisms . For instance, at least two powerful mitogens, PDGF and FGF, are likely to play a role in inducing smooth muscle proliferation . PDGF derives from a variety of sources (for example, platelets, SMCs, and macrophages), and simply preventing release from one of these (that is, platelets) provides only a marginal decrease in myointimal proliferation and clinical restenosis . IGF-I may

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Castellot JJ, Wright TC, Kamovsky MJ: 1987. Regulation of vascular smooth muscle cell growth by heparin and heparan sulphates . Semin Thromb Hemost 13 :489-503 . Cercek B, Fishbein MC, Forrester JS, Helfant RE, Fagin JA: 1990 . Induction of insulinlike growth factor I messenger RNA in rat aorta after balloon denudation . Circ Res 66 :1755-1760 . Chen JK, Hoshi H, McKeehan W : 1987. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells . Proc Nail Acad SO USA 84 :5287-5291 . Clemmons DR : 1985. Variables controlling the secretion of somatomedin-like peptide by culture porcine smooth muscle cells. Circ Res 56 :418-426 . Clowes AW, Karnovsky MJ: 1977 . Failure of

Foster J, Rich CB, Florin J : 1987 . Insulin-like growth factor I, somatomedin C, induces the synthesis of tropoelastin in aortic tissue . Coll Relat Res 7 :161-169 .

Klagsbrun M, Edelman ER : 1989. Biological and biochemical properties of fibroblast growth factors : implications for the pathogenesis of atherosclerosis . Arteriosclerosis 9 :269-278 . Liau G, Chan LM : 1989. Regulation of extracellular matrix RNA levels in cultured small muscle cells : relationship to cellular quiescence . J Biol Chem 264 :10,31510,320, Lindner V, Reidy MA: 1991 . Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor . Proc Natl Acad Sci USA 88 :3739-3743 . Lynch SE, Colvin RB, Antoniades HN : 1989. Growth factors in wound healing : single and synergistic effects on partial thickness por-

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thickness porcine skin wounds . J Clin Invest 84 :640-646 .

ous transluminal coronary angioplasty . N Engl J Med 318:1719-1724 .

Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM : 1990 . PDGF ligand and receptor gene expression during repair of arterial injury . J Cell Blot 111 :2149-2158 .

Siegbahn A, Hammacher A, Westermark B, Heldin C-H: 1990. Differential effects of the various isoforms of platelet-derived growth factor on chemotaxis of fibroblasts, monocytes, and granulocytes . J Clin Invest 85 :916-920.

Nobuyoshi M, Kimura T, Noksaka H, et al . : 1988 . Percutaneous transluminal coronary angioplasty : serial angiography follow-up of 229 patients. J Am Coll Cardiol 12 :616623 . Powell JS, Clozel JP, Muller RKM, et al . : 1989 . Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury . Science 245 :186-188 . Ross R: 1986 . The pathogenesis of atherosclerosis: an update. N Engl J Med 314 :488500 . Schwartz L, Bourassa MG, Lesperance J, et al. : 1988 . Aspirin and dipyridamol in the prevention of restenosis after percutane-

Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B : 1987 . Some recent advances in the chemistry and biology of transforming growth factor-beta . J Cell Biol 105 :1039-1045. Stiles CD, Capone GT, Scher CDE, Antoniades HN, Van Wyk JJ, Pledger WJ : 1979 . Dual control of cell growth of somatomedins and platelet-derived growth factor. Proc Nat! Acad Set USA 76 :1279-1282 . Thyberg J, Hedin U, Sjolund M, et al . : 1990. Regulation of differentiated properties and proliferation of arterial smooth muscle TCM cells . Arteriosclerosis 10 :966-990 .

The Tissue Renin-Angiotensin Systems in Cardiovascular Disease Martin Paul, Jurgen Bachmann, and Detlev Ganten The renin-angiotensin system, in its classic definition, is known as an endocrine system that exerts its actions through the effector peptide, angiotensin II, in various organs to act as a vasoconstrictor and a regulator of salt and volume homeostasis. The availability of more sensitive methods to study the biochemistry and pharmacology as well as the molecular biology of the RAS has expanded our knowledge of the system and provided new perspectives of autocrine and paracrine functions of the RAS in cardiovascular regulation . One of the more exciting of these recently described actions is the possible involvement of the RAS in the adaptive processes related to cardiovascular hypertrophy and angiogenesis. (Trends Cardiovasc Med 1992 ;2 :94-99)

many pieces of evidence that the components of the RAS are present and functional in a great number of tissues . Cellular and molecular studies demonstrated that in many organs, such as in the brain or in the vasculature, not all of the components of the RAS are synthesized in the same cells . Would this speak against a functional role of the tissue systems? The answer is no, since it is conceivable that various components of the RAS interact extracellularly to generate ANG II, which is then transported through the extracellular space to reach its receptor. Such a concept has been proposed in the brain (Fuze et al . 1988) and appears also to be a valid model for other tissues in which a functional RAS has been described. In the vasculature, renin is localized in the smooth muscle cell layer (Higashimori et al . 1991), whereas angiotensinogen is synthesized primarily in the adventitia (Cassis et al. 1988) but also in smooth muscle cells (Naftilan et al . 1991), whereas angiotensin-converting enzyme (ACE) can be found on the endothelial cell membrane. Despite these different cellular localizations, ANG II can be formed in the vasculature and can act both as an endocrine substance (after its release into the vasculature) or as an autocrine/paracrine substance when feeding back either to neighboring cells (paracrine action) or onto the secretory cell itself (autocrine action) . Plasma and tissue RAS are, therefore, not competing entities, but are interacting to maintain cardiovascular tone as well as water and electrolyte balance . Based on these observations, it has been proposed that plasma RAS is responsible for an acute regulatory mechanism, whereas the tissue systems act in a longer time frame .

• Recent studies on the role of the reninangiotensin system (RAS) have provided new insights into its function . Many of its effects fit the endocrine concept of the

Martin Paul and Jurgen Bachmann are at the German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, W-6900 Heidelberg, Germany . Dedev Ganten is at the MaxDelbrttck Center for Molecular Medicine, 0-1115 Berlin Buch, Germany .

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system. But there are other actions of the peptide, for example, its effects on the central nervous system (CNS), that cannot be explained by the classic definition alone . Angiotensin II (ANG II) is effective as an autocrine or paracrine factor and it has been hypothesized that it can even function as an intracrine substance (Dzau 1987) . Although the latter has not been proven, ANG II probably can act at both the intercellular and intracellular levels (Figure 1) . In addition, there are

The Components of the ReninAngiotensin System

The first step of angiotensin biosynthesis is the cleavage of the decapeptide ANG I from the substrate angiotensinogen by renin. ANG I is then activated by ACE to ANG II, the effector peptide of the RAS, which acts on specific receptors and is further cleaved to shorter peptides by endopeptidases (Figure 2) . Some of these fragments appear to be degradation products, whereas others may have distinct and important actions on cardiovascular homeostasis. Since the genes or cDNAs

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