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Adrenomedullin and the microvasculature
Research Update
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
101
Research News
Adrenomedullin and the microvasculature Leonid L. Nikitenko, David M. Smith, Stephen Hague, Caroline R. Wilson, Roy Bicknell and Margaret C.P. Rees The peptide adrenomedullin shows a remarkable range of effects on the vasculature that include vasodilatation, regulation of vascular smooth muscle cell proliferation, inhibition of endothelial apoptosis and promotion of angiogenesis. It is becoming clear that either activation or disruption of adrenomedullin signalling might contribute to many pathologies including cardiovascular disease, pulmonary hypertension, atherosclerosis, renal failure, neoplastic growth, inflammatory disease and disorders of the reproductive tract. Recent advances in this area are reviewed.
The discovery of adrenomedullin [1] and its characterization as a multifunctional regulatory peptide [1,2] have opened up an exciting new area of vascular biology and significantly contributed to the field of endocrinology [3]. Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) both actively secrete adrenomedullin and express its receptors [4,5]. Adrenomedullin expression is increased in cardiovascular disease, septic shock and hypertension and it might be involved in compensatory mechanisms that induce vasodilatation to reduce the tissue damage found in these pathologies [2–4]. Transgenic and gene knockout models further emphasize that adrenomedullin is crucial to vascular morphogenesis and function [6,7]. Molecular characterization of the protein interactions that underlie the exhibited pharmacology of adrenomedullin receptors [8] has also provided insight into understanding the mechanisms of the diverse effects of this peptide on the vasculature. Vascular effects of adrenomedullin
Expression of adrenomedullin and its receptors by a variety of cell types, including ECs and VSMCs [2–4,9,10], defines the paracrine, autocrine and endocrine modes of adrenomedullinmediated vascular effects [1,7,9–16] (Fig. 1). The paracrine action of adrenomedullin has implications in atherogenesis and neoplasia [3,11] because it promotes crosstalk http://tips.trends.com
Adrenomedullin
Endothelial and vascular smooth muscle cells
Modulation of cell growth, movement and fate
Angiogenesis, vascular remodelling
Modulation of cellular shape and metabolism
Vasodilatation, regulation of blood coagulation and fibrinolysis, permeability
Vascular health and disease
TRENDS in Pharmacological Sciences
Fig. 1. Multiple effects of adrenomedullin define its significant role in the regulation of vascular development and function.
between ECs, VSMCs and fibroblasts, macrophages or tumour cells. Endothelial secretion of adrenomedullin is thought not only to coordinate vascular tone [15] but also to regulate fibroblast and VSMC proliferation [9,12]. Fibroblasts, macrophages and tumour cells [2–4] are a significant source of adrenomedullin that might affect the vascular microenvironment. Macrophagederived adrenomedullin might be involved in the inhibition of the growth and migration of VSMCs in atheromatous plaques [16], whereas adrenomedullin produced from tumour cells is thought to inhibit hypoxic death of these cells and also to promote angiogenesis [11]. Expression of functional adrenomedullin receptors by adrenomedullin-secreting vascular cells [4,5] identifies an autocrine
mode of peptide action, particularly in inflammatory [4] and hypoxic [17] conditions, in which the expression of adrenomedullin is upregulated in ECs. Autocrine effects of adrenomedullin could be essential for the development of the vasculature during embryogenesis [7], in reproductive organs [10] and during tumorigenesis [11]. Moreover, ECs, in addition to VSMCs and tumour cells, are thought to be a source of circulating adrenomedullin that potentially contributes to the endocrine mode of its action. Hypoxia [17] and cytokine production in septic shock or chronic heart failure, shear stress in hypertension, mechanical stress in heart failure [2,3], and hyperglycaemia in diabetes [18] induce adrenomedullin secretion by vascular cells. This might contribute to the increased plasma concentrations of the peptide in these conditions and hence constitute a protective action by which cardiovascular homeostasis is maintained. Molecular mechanisms of vascular responses to adrenomedullin
The mechanisms of adrenomedullinmediated vasodilatation have been well described [2,4]; however, how the peptide elicits other effects in vascular and extra-vascular tissues is less well defined. Vasodilatory effect
The vascular response to adrenomedullin is thought to be ~60 times more potent than such a response to proadrenomedullin N-terminal 20 peptide (PAMP) [19], which is generated from post-translational enzymatic processing of the same 185-amino-acid precursor. Two pathways might be involved in adrenomedullininduced vasodilatation: an endotheliumdependent mechanism mediated by nitric oxide (NO) and cGMP, or an endothelium-independent mechanism mediated by cAMP [15]. Upregulation of NO by adrenomedullin is mediated via the phosphatidylinositol 3-kinase (PI3K)–Akt (protein kinase B) pathway, known to be involved in the activation
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01983-0
102
Research Update
of endothelial NO synthase (eNOS). eNOS activity is modulated by the Ca2+–calmodulin pathway either through PKB phosphorylation or by direct binding of calmodulin, leading to conformational changes of the eNOS enzyme. The importance of NO in the vasodilatory response to adrenomedullin was also shown by inhibition of NOS in the adrenomedullin gene overexpression model [6], resulting in normalized blood pressure. Growth effects
Reports of the effects of adrenomedullin on EC and VSMC growth are conflicting and activities might depend on the microvascular bed [4,10], vascular cell types and experimental conditions [12] involved. Adrenomedullin might play dual roles in VSMC growth in vitro as a growth promoter or inhibitor in quiescent or proliferative cells, respectively [4,12]. In vivo the proliferative effects of injury on the vascular intima are enhanced in heterozygous knockouts but reduced in mice that overexpress adrenomedullin [6]. However, a decreased level of adrenomedullin expression might result in reduced VSMC development in large arteries [20]. The mitogenic activity of the peptide in quiescent rat VSMCs is mediated via a cAMP-dependent pathway [4] and/or via mitogen-activated protein kinase [12]. Inhibition of apoptosis
Adrenomedullin antagonizes endothelial apoptosis by upregulation of the transcription factor MAX, which is the heterodimeric partner of c-MYC, in both an autocrine and a paracrine manner [13]. Increased expression of MAX results in the formation of dimers with its alternative partners, MAD and MXI-1, and subsequent binding of the dimers to the E-box. This binding suppresses apoptosis by antagonizing the effect of the MYC–MAX transcription complex through competition for recognition sites. The anti-apoptotic effect of adrenomedullin is thought to be mediated via the NO pathway, but not via cGMP- or cAMP-dependent signal transduction [21]. Regulation of blood coagulation and fibrinolysis
Adrenomedullin has recently been postulated to play a role in haemostasis [14]. Thus, adrenomedullin inhibits angiotensin-II-induced expression of tissue factor and plasminogen activator inhibitor 1 http://tips.trends.com
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
(PAI-1) in vascular ECs via cAMP-mediated pathways and the activation of NOS. Enhanced expression of tissue factor and PAI-1 might promote thrombus formation and the development of ischaemia by initiating extrinsic blood coagulation and inhibiting fibrinolytic activity. Involvement in embryonic vascular development
Strong expression of adrenomedullin has been found in the developing murine vasculature [20]. Adrenomedullin knockouts have a lethal homozygous phenotype [7,20] with abnormal vascular development, further supporting the hypothesis that adrenomedullin is a vasculogenic factor. A poor vascularization of the yolk sac, very thin umbilical cord and few fully formed angiographically leaky placental vessels were found in homozygous embryos. However, the precise pathways of the involvement of adrenomedullin in embryonic vascular development also require further investigation. Adrenomedullin receptors
The effects of adrenomedullin on ECs and VSMCs are thought to be mediated via adrenomedullin receptors [2,4]. Despite the widespread demonstration of adrenomedullin-binding sites, it is only recently that the G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signalling system [8]. Expression of RAMPs dictates the cell-surface expression and the unique pharmacologies of CRLR. Adrenomedullin and its related peptide, calcitonin gene-related peptide (CGRP), bind with highest affinities to the heterodimers CRLR–RAMP2 and CRLR–RAMP1, respectively. RAMPs are now thought to induce conformational changes of CRLR to facilitate differential binding of the two peptides. Furthermore, other cellular components, such as CGRP receptor component protein (RCP), appear to be important for the intracellular signalling mediated via CRLR [22]. The predominant localization of CRLR in blood vessels [5] suggests that the microvasculature is one of the main adrenomedullin targets. RAMPs are expressed to a much higher degree than is CRLR and have wider tissue distribution [8]. Moreover, CRLR and RAMPs are
expressed in different cell types within individual tissues [5]. This suggests that additional adrenomedullin receptor subtypes might exist [2,5] and there is much still to be learned. Further analysis of CRLR and its phenotypes and/or subtypes is required because some drugs that act on the GPCR can fail to differentiate between subtypes. Therefore, despite the fact that adrenomedullin is involved in pivotal processes in the vasculature, the precise signal transduction pathways remain to be defined. Adrenomedullin as a contributory peptide in microvascular dysfunction
Adrenomedullin appears to play a role in modulating blood pressure and vascular development, and hence organ function in cardiovascular, renal and pulmonary disease and in reproductive disorders [2–4,7]. Recent data on the structure and function of the peptide and its receptor system offer novel therapeutic strategies in the treatment of these pathologies. The investigation of the stability of the genomic structure for the genes encoding adrenomedullin and CRLR has recently attracted considerable interest. A genetically determined absence of adrenomedullin might be one cause of non-immune hydrops fetalis and haemorrhage, as a result of cardiovascular abnormalities and disturbance of lymphangiogenesis and angiogenesis [7,20]. Expansion of data on the polymorphisms of genes encoding adrenomedullin [23] and CRLR [24] and other potential components of the adrenomedullin receptor system (e.g. RAMPs) might further uncover mutations (single nucleotide or microsatellite polymorphisms) responsible for hypertension, where these genes might act in concert with others to determine an individual’s blood pressure [25]. Together with biochemical and molecular studies on ligand–receptor conformational changes, genomic approaches will further facilitate the detection of alterations in the amino acid sequences that lead to the changes in the structure and ligand-binding activity of the adrenomedullin receptor(s). The mechanism of adrenomedullin and CGRP receptor(s) blockade with or without desensitization [26] needs further investigation particularly with agents such as non-peptide antagonists [27], which are potentially beneficial for the suppression of adrenomedullinmediated proliferative effects in tumours.
Research Update
Gene transfer might represent an attractive treatment for pulmonary hypertension and salt-related cardiovascular and renal diseases [28]. Moreover, endocrine effects of adrenomedullin could be modulated by interaction with other proteins in the blood [29] or by proteolytic enzymes such as metalloproteases and aminopeptidases [2]. The complex of the peptide with adrenomedullin-binding protein 1 (namely factor H) is active in terms of receptor binding, and factor H actually enhances the effects of adrenomedullin [29]. Reciprocally, the peptide can modulate factor H activity, implicating adrenomedullin as a new regulatory component of the complement cascade. Furthermore, vasopeptidase inhibitors exhibit the therapeutic potential for the control of the endocrine activity of the peptide. Neprilysin (neutral endopeptidase) is known to be involved in the biosynthesis and/or inactivation of adrenomedullin and other peptides that play a major role in the regulation of cardiovascular and renal functions [19,30]. The selective inhibition of neprilysin might exert a beneficial effect by potentiation of the haemodynamic (vasodilatory and natriuretic) and antiproliferative effects of adrenomedullin in hypertension and heart failure [30]. In summary, the difference in the mechanisms of the adrenomedullinmediated disorders might determine the preference for one treatment over another to target the specific vascular dysfunction without causing adverse effects. Concluding remarks
Adrenomedullin is clearly a more significant player in vascular function than previously thought. Disruption of the genomic structure or signal transduction pathways mediated through adrenomedullin receptor(s) by genetic and environmental factors might contribute to common disorders that affect the vasculature. A heterogeneous range of drugs (including gene delivery and biochemical and molecular approaches) should be considered for selective targeting of the specific mechanisms involved in distinct adrenomedullinmediated pathology. Further studies are essential to elucidate the precise mechanisms of adrenomedullin action in individual organs before treatment of disorders that affect vascular development and function. http://tips.trends.com
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
References 1 Kitamura, K. et al. (1993) Adrenomedullin: a novel hypotensive peptide isolated from human phaeochromocytoma. Biochem. Biophys. Res. Commun. 192, 553–560 2 Hinson, J.P. et al. (2000) Adrenomedullin, a multifunctional regulatory peptide. Endocr. Rev. 21, 138–167 3 Takahashi, K. (2001) Adrenomedullin from a phaeochromocytoma to the eye: implications of the adrenomedullin research for endocrinology in the 21st century. Tohoku J. Exp. Med. 193, 79–114 4 Minamino, N. et al. (2000) Adrenomedullin: a new peptidergic regulator of the vascular function. Clin. Hemorheol. Microcirc. 23, 95–102 5 Nikitenko, L.L. et al. (2001) Differential and cell-specific expression of calcitonin receptor-like receptor and receptor activity modifying proteins in the human uterus. Mol. Hum. Reprod. 7, 655–664 6 Shindo, T. et al. (2000) Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 101, 2309–2316 7 Shindo, T. et al. (2001) Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circulation 104, 1964–1971 8 Sexton, P.M. et al. (2001) Receptor activity modifying proteins. Cell. Signal. 13, 73–83 9 Upton, P.D. et al. (2001) Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations. Am. J. Respir. Cell Mol. Biol. 24, 170–178 10 Nikitenko, L.L. et al. (2000) Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium. Mol. Hum. Reprod. 6, 811–819 11 Zhao, Y. et al. (1998) PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non-estrogenic mechanism in the human endometrium. Oncogene 16, 409–415 12 Iwasaki, H. et al. (1998) Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 139, 3432–3441 13 Shichiri, M. et al. (1999) Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol. Endocrinol. 13, 1353–1363 14 Sugano, T. et al. (2001) Adrenomedullin inhibits angiotensin II-induced expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells. Arterioscler. Thromb. Vasc. Biol. 21, 1078–1083 15 Nishimatsu, H. et al. (2001) Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta. Circ. Res. 89, 63–70 16 Nakayama, M. et al. (1999) Adrenomedullin in monocytes and macrophages: possible involvement of macrophage-derived adrenomedullin in atherogenesis. Clin. Sci. 97, 247–251 17 Ogita, T. et al. (2001) Hypoxic induction of adrenomedullin in cultured human umbilical vein endothelial cells. J. Hypertens. 19, 603–608 18 Hayashi, M. et al. (1999) Hyperglycemia increases vascular adrenomedullin expression. Biochem. Biophys. Res. Comm. 258, 453–456 19 Wilkinson, I.B. et al. (2001) Adrenomedullin (ADM) in the human forearm vascular bed: effect of neutral endopeptidase inhibition and
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comparison with proadrenomedullin NH2-terminal 20 peptide (PAMP). Br. J. Clin. Pharmacol. 52, 159–164 Caron, K.M. et al. (2001) Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene. Proc. Natl. Acad. Sci. U. S. A. 98, 615–619 Sata, M. et al. (2000) Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism. Hypertension 36, 83–88 Evans, B.N. et al. (2000) CGRP-RCP, a novel protein required for signal transduction at calcitonin generelated peptide and adrenomedullin receptors. J. Biol. Chem. 275, 31438–31443 Ishimitsu, T. et al. (2001) Microsatellite DNA polymorphism of human adrenomedullin gene in normotensive subjects and patients with essential hypertension. Hypertension 38, 9–12 Nakazawa, I. et al. (2001) Human calcitonin receptor-like receptor for adrenomedullin: genomic structure, eight single-nucleotide polymorphisms, and haplotype analysis. J. Hum. Genet. 46, 132–136 Rubin, E.M. and Tall, A. (2000) Perspectives for vascular genomics. Nature 407, 265–269 Iwasaki, H. et al. (1998) Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells. Eur. J. Pharmacol. 352, 131–134 Doods, H. et al. (2000) Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist. Br. J. Pharmacol. 129, 420–423 Zhang, J.J. et al. (2000) Human adrenomedullin gene delivery protects against cardiac hypertrophy, fibrosis and renal damage in hypertensive dahl salt-sensitive rats. Hum. Gene Ther. 11, 1817–1827 Pio, R. et al. (2001) Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J. Biol. Chem. 276, 12292–12300 Bralet, J. and Schwartz, J-C. (2001) Vasopeptidase inhibitors: an emerging class of cardiovascular drugs. Trends Pharmacol. Sci. 22, 106–109
Leonid L. Nikitenko* Nuffield Dept of Obstetrics and Gynaecology, and Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine Stephen Hague Caroline R. Wilson Margaret C.P. Rees§ Nuffield Dept of Obstetrics and Gynaecology Roy Bicknell§ Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK OX3 9DU. David M. Smith AstraZeneca, CVGI, Alderley Park, Macclesfield, Cheshire, UK SK10 4TG. *To whom correspondence should be addressed; e-mail: [email protected] §Authors share senior authorship.
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
101
Research News
Adrenomedullin and the microvasculature Leonid L. Nikitenko, David M. Smith, Stephen Hague, Caroline R. Wilson, Roy Bicknell and Margaret C.P. Rees The peptide adrenomedullin shows a remarkable range of effects on the vasculature that include vasodilatation, regulation of vascular smooth muscle cell proliferation, inhibition of endothelial apoptosis and promotion of angiogenesis. It is becoming clear that either activation or disruption of adrenomedullin signalling might contribute to many pathologies including cardiovascular disease, pulmonary hypertension, atherosclerosis, renal failure, neoplastic growth, inflammatory disease and disorders of the reproductive tract. Recent advances in this area are reviewed.
The discovery of adrenomedullin [1] and its characterization as a multifunctional regulatory peptide [1,2] have opened up an exciting new area of vascular biology and significantly contributed to the field of endocrinology [3]. Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) both actively secrete adrenomedullin and express its receptors [4,5]. Adrenomedullin expression is increased in cardiovascular disease, septic shock and hypertension and it might be involved in compensatory mechanisms that induce vasodilatation to reduce the tissue damage found in these pathologies [2–4]. Transgenic and gene knockout models further emphasize that adrenomedullin is crucial to vascular morphogenesis and function [6,7]. Molecular characterization of the protein interactions that underlie the exhibited pharmacology of adrenomedullin receptors [8] has also provided insight into understanding the mechanisms of the diverse effects of this peptide on the vasculature. Vascular effects of adrenomedullin
Expression of adrenomedullin and its receptors by a variety of cell types, including ECs and VSMCs [2–4,9,10], defines the paracrine, autocrine and endocrine modes of adrenomedullinmediated vascular effects [1,7,9–16] (Fig. 1). The paracrine action of adrenomedullin has implications in atherogenesis and neoplasia [3,11] because it promotes crosstalk http://tips.trends.com
Adrenomedullin
Endothelial and vascular smooth muscle cells
Modulation of cell growth, movement and fate
Angiogenesis, vascular remodelling
Modulation of cellular shape and metabolism
Vasodilatation, regulation of blood coagulation and fibrinolysis, permeability
Vascular health and disease
TRENDS in Pharmacological Sciences
Fig. 1. Multiple effects of adrenomedullin define its significant role in the regulation of vascular development and function.
between ECs, VSMCs and fibroblasts, macrophages or tumour cells. Endothelial secretion of adrenomedullin is thought not only to coordinate vascular tone [15] but also to regulate fibroblast and VSMC proliferation [9,12]. Fibroblasts, macrophages and tumour cells [2–4] are a significant source of adrenomedullin that might affect the vascular microenvironment. Macrophagederived adrenomedullin might be involved in the inhibition of the growth and migration of VSMCs in atheromatous plaques [16], whereas adrenomedullin produced from tumour cells is thought to inhibit hypoxic death of these cells and also to promote angiogenesis [11]. Expression of functional adrenomedullin receptors by adrenomedullin-secreting vascular cells [4,5] identifies an autocrine
mode of peptide action, particularly in inflammatory [4] and hypoxic [17] conditions, in which the expression of adrenomedullin is upregulated in ECs. Autocrine effects of adrenomedullin could be essential for the development of the vasculature during embryogenesis [7], in reproductive organs [10] and during tumorigenesis [11]. Moreover, ECs, in addition to VSMCs and tumour cells, are thought to be a source of circulating adrenomedullin that potentially contributes to the endocrine mode of its action. Hypoxia [17] and cytokine production in septic shock or chronic heart failure, shear stress in hypertension, mechanical stress in heart failure [2,3], and hyperglycaemia in diabetes [18] induce adrenomedullin secretion by vascular cells. This might contribute to the increased plasma concentrations of the peptide in these conditions and hence constitute a protective action by which cardiovascular homeostasis is maintained. Molecular mechanisms of vascular responses to adrenomedullin
The mechanisms of adrenomedullinmediated vasodilatation have been well described [2,4]; however, how the peptide elicits other effects in vascular and extra-vascular tissues is less well defined. Vasodilatory effect
The vascular response to adrenomedullin is thought to be ~60 times more potent than such a response to proadrenomedullin N-terminal 20 peptide (PAMP) [19], which is generated from post-translational enzymatic processing of the same 185-amino-acid precursor. Two pathways might be involved in adrenomedullininduced vasodilatation: an endotheliumdependent mechanism mediated by nitric oxide (NO) and cGMP, or an endothelium-independent mechanism mediated by cAMP [15]. Upregulation of NO by adrenomedullin is mediated via the phosphatidylinositol 3-kinase (PI3K)–Akt (protein kinase B) pathway, known to be involved in the activation
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01983-0
102
Research Update
of endothelial NO synthase (eNOS). eNOS activity is modulated by the Ca2+–calmodulin pathway either through PKB phosphorylation or by direct binding of calmodulin, leading to conformational changes of the eNOS enzyme. The importance of NO in the vasodilatory response to adrenomedullin was also shown by inhibition of NOS in the adrenomedullin gene overexpression model [6], resulting in normalized blood pressure. Growth effects
Reports of the effects of adrenomedullin on EC and VSMC growth are conflicting and activities might depend on the microvascular bed [4,10], vascular cell types and experimental conditions [12] involved. Adrenomedullin might play dual roles in VSMC growth in vitro as a growth promoter or inhibitor in quiescent or proliferative cells, respectively [4,12]. In vivo the proliferative effects of injury on the vascular intima are enhanced in heterozygous knockouts but reduced in mice that overexpress adrenomedullin [6]. However, a decreased level of adrenomedullin expression might result in reduced VSMC development in large arteries [20]. The mitogenic activity of the peptide in quiescent rat VSMCs is mediated via a cAMP-dependent pathway [4] and/or via mitogen-activated protein kinase [12]. Inhibition of apoptosis
Adrenomedullin antagonizes endothelial apoptosis by upregulation of the transcription factor MAX, which is the heterodimeric partner of c-MYC, in both an autocrine and a paracrine manner [13]. Increased expression of MAX results in the formation of dimers with its alternative partners, MAD and MXI-1, and subsequent binding of the dimers to the E-box. This binding suppresses apoptosis by antagonizing the effect of the MYC–MAX transcription complex through competition for recognition sites. The anti-apoptotic effect of adrenomedullin is thought to be mediated via the NO pathway, but not via cGMP- or cAMP-dependent signal transduction [21]. Regulation of blood coagulation and fibrinolysis
Adrenomedullin has recently been postulated to play a role in haemostasis [14]. Thus, adrenomedullin inhibits angiotensin-II-induced expression of tissue factor and plasminogen activator inhibitor 1 http://tips.trends.com
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
(PAI-1) in vascular ECs via cAMP-mediated pathways and the activation of NOS. Enhanced expression of tissue factor and PAI-1 might promote thrombus formation and the development of ischaemia by initiating extrinsic blood coagulation and inhibiting fibrinolytic activity. Involvement in embryonic vascular development
Strong expression of adrenomedullin has been found in the developing murine vasculature [20]. Adrenomedullin knockouts have a lethal homozygous phenotype [7,20] with abnormal vascular development, further supporting the hypothesis that adrenomedullin is a vasculogenic factor. A poor vascularization of the yolk sac, very thin umbilical cord and few fully formed angiographically leaky placental vessels were found in homozygous embryos. However, the precise pathways of the involvement of adrenomedullin in embryonic vascular development also require further investigation. Adrenomedullin receptors
The effects of adrenomedullin on ECs and VSMCs are thought to be mediated via adrenomedullin receptors [2,4]. Despite the widespread demonstration of adrenomedullin-binding sites, it is only recently that the G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signalling system [8]. Expression of RAMPs dictates the cell-surface expression and the unique pharmacologies of CRLR. Adrenomedullin and its related peptide, calcitonin gene-related peptide (CGRP), bind with highest affinities to the heterodimers CRLR–RAMP2 and CRLR–RAMP1, respectively. RAMPs are now thought to induce conformational changes of CRLR to facilitate differential binding of the two peptides. Furthermore, other cellular components, such as CGRP receptor component protein (RCP), appear to be important for the intracellular signalling mediated via CRLR [22]. The predominant localization of CRLR in blood vessels [5] suggests that the microvasculature is one of the main adrenomedullin targets. RAMPs are expressed to a much higher degree than is CRLR and have wider tissue distribution [8]. Moreover, CRLR and RAMPs are
expressed in different cell types within individual tissues [5]. This suggests that additional adrenomedullin receptor subtypes might exist [2,5] and there is much still to be learned. Further analysis of CRLR and its phenotypes and/or subtypes is required because some drugs that act on the GPCR can fail to differentiate between subtypes. Therefore, despite the fact that adrenomedullin is involved in pivotal processes in the vasculature, the precise signal transduction pathways remain to be defined. Adrenomedullin as a contributory peptide in microvascular dysfunction
Adrenomedullin appears to play a role in modulating blood pressure and vascular development, and hence organ function in cardiovascular, renal and pulmonary disease and in reproductive disorders [2–4,7]. Recent data on the structure and function of the peptide and its receptor system offer novel therapeutic strategies in the treatment of these pathologies. The investigation of the stability of the genomic structure for the genes encoding adrenomedullin and CRLR has recently attracted considerable interest. A genetically determined absence of adrenomedullin might be one cause of non-immune hydrops fetalis and haemorrhage, as a result of cardiovascular abnormalities and disturbance of lymphangiogenesis and angiogenesis [7,20]. Expansion of data on the polymorphisms of genes encoding adrenomedullin [23] and CRLR [24] and other potential components of the adrenomedullin receptor system (e.g. RAMPs) might further uncover mutations (single nucleotide or microsatellite polymorphisms) responsible for hypertension, where these genes might act in concert with others to determine an individual’s blood pressure [25]. Together with biochemical and molecular studies on ligand–receptor conformational changes, genomic approaches will further facilitate the detection of alterations in the amino acid sequences that lead to the changes in the structure and ligand-binding activity of the adrenomedullin receptor(s). The mechanism of adrenomedullin and CGRP receptor(s) blockade with or without desensitization [26] needs further investigation particularly with agents such as non-peptide antagonists [27], which are potentially beneficial for the suppression of adrenomedullinmediated proliferative effects in tumours.
Research Update
Gene transfer might represent an attractive treatment for pulmonary hypertension and salt-related cardiovascular and renal diseases [28]. Moreover, endocrine effects of adrenomedullin could be modulated by interaction with other proteins in the blood [29] or by proteolytic enzymes such as metalloproteases and aminopeptidases [2]. The complex of the peptide with adrenomedullin-binding protein 1 (namely factor H) is active in terms of receptor binding, and factor H actually enhances the effects of adrenomedullin [29]. Reciprocally, the peptide can modulate factor H activity, implicating adrenomedullin as a new regulatory component of the complement cascade. Furthermore, vasopeptidase inhibitors exhibit the therapeutic potential for the control of the endocrine activity of the peptide. Neprilysin (neutral endopeptidase) is known to be involved in the biosynthesis and/or inactivation of adrenomedullin and other peptides that play a major role in the regulation of cardiovascular and renal functions [19,30]. The selective inhibition of neprilysin might exert a beneficial effect by potentiation of the haemodynamic (vasodilatory and natriuretic) and antiproliferative effects of adrenomedullin in hypertension and heart failure [30]. In summary, the difference in the mechanisms of the adrenomedullinmediated disorders might determine the preference for one treatment over another to target the specific vascular dysfunction without causing adverse effects. Concluding remarks
Adrenomedullin is clearly a more significant player in vascular function than previously thought. Disruption of the genomic structure or signal transduction pathways mediated through adrenomedullin receptor(s) by genetic and environmental factors might contribute to common disorders that affect the vasculature. A heterogeneous range of drugs (including gene delivery and biochemical and molecular approaches) should be considered for selective targeting of the specific mechanisms involved in distinct adrenomedullinmediated pathology. Further studies are essential to elucidate the precise mechanisms of adrenomedullin action in individual organs before treatment of disorders that affect vascular development and function. http://tips.trends.com
TRENDS in Pharmacological Sciences Vol.23 No.3 March 2002
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Leonid L. Nikitenko* Nuffield Dept of Obstetrics and Gynaecology, and Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine Stephen Hague Caroline R. Wilson Margaret C.P. Rees§ Nuffield Dept of Obstetrics and Gynaecology Roy Bicknell§ Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK OX3 9DU. David M. Smith AstraZeneca, CVGI, Alderley Park, Macclesfield, Cheshire, UK SK10 4TG. *To whom correspondence should be addressed; e-mail: [email protected] §Authors share senior authorship.