Review TRENDS in Cell Biology Vol.13 No.1 January 2003 51

Extracellular matrix in vascular morphogenesis and disease: structure versus signalq

Benjamin S. Brooke, Satyajit K. Karnik and Dean Y. Li

Program in Human Molecular Biology and Genetics, Departments of Medicine and Oncological Science, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

The vascular system matures during embryonic elastic fibers and smooth-muscle cells, separated by development to form a stable, well-organized tubular interlamellar matrix containing , microfibrils, network. In vivo data have established that the extra- , and ground substance cellular matrix (ECM) is crucial in providing structural (Fig. 1). Finally, the adventitia extends beyond the support to the vascular system. In vitro studies are external elastic lamina and is composed mainly of defining the involvement of ECM–smooth-muscle cell and fibroblasts. Although these ECM molecules signaling in establishing and maintaining the mature have been identified within the vessel wall through tubular structure. However, correlating cell signaling ultrastructural analyses and biochemical studies, their with established structural functions for the ECM and precise interactions are relatively unknown. The com- determining the relative importance of these two roles plex relationship between vascular cells and the ECM in vivo is often difficult. Here, we examine human remainstobedetermined. genetics, murine gene targeting and cell biology to During the past two decades, human genetic studies, better understand the relationship between structural murine knockout models, biomechanical testing and other and signaling roles for the ECM in vascular morphogen- methods have established the importance of the ECM in esis and disease. maintaining the mature tubular structure of the vascular system. These studies indicate that ECM molecules such The assembly and maintenance of a mature tubular as fibrillins, collagens and provide crucial mech- network for blood circulation are crucial during embryo- anical support to the vessel wall during development and nic development. There are three major components of in the mature state. Specifically, these components this network: endothelial cells (ECs), vascular smooth- maintain the competence of the tubular network under muscle cells (VSMCs) and the hemodynamic pressure [4]. More recently, in vitro studies (ECM). ECs differentiate and form a primordial network of tubes through intussusception, growth and regres- sion. These EC tubes in turn provide signals that lead to the recruitment of VSMCs [1]. The third component consists of ECM molecules produced and organized Vascular smooth by VSMCs within the developing vessel wall. The ECM muscle cell can account for over 50% of the dry weight of the vasculature and is largely deposited towards the end of Collagen type I development [2]. and type III TheECMofthematurevesselwallisacomplex arrangement of fibrous proteins and associated glyco- Microfibril proteins embedded in a hydrated ground substance of and proteoglycans [3]. These ECM TRENDS in Cell Biology molecules and the vascular cells they associate with are responsible for organizing the vessel wall into three Fig. 1. Simplified schematic representation of mature vessel wall. The media of the discrete layers: the intima, the media and the adven- vessel wall is organized into lamellar units consisting of concentric layers of elastic titia. The intima is composed mostly of ground lamellae, vascular smooth-muscle cells (VSMCs) and interlamellar matrix. Elastic fibers are composed predominantly of elastin, whereas the interlamellar matrix substance separating ECs from the internal elastic includes type-I collagen, type-III collagen and microfibrils such as fibrillin. All of lamina (IEL). The media begins at the IEL and is these extracellular-matrix components provide structural organization to the organized into concentric lamellar units composed of vessel wall through interactions with VSMCs and associated extracellular-matrix elements such as glycoproteins, proteoglycans and glycosaminoglycans. Col- q lagens bind and signal VSMCs via specific matrix receptors. Elastic fibers are This article is the fourth review in our Tube Morphogenesis series linked to VSMCs through a microfibril scaffold composed of fibrillin and microfi- that commenced in the August 2002 issue of TCB. bril-associated glycoproteins. A direct interaction between elastin and VSMCs still Corresponding author: Dean Y. Li ([email protected]). remains to be elucidated. http://ticb.trends.com 0962-8924/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)00007-7 52 Review TRENDS in Cell Biology Vol.13 No.1 January 2003

and collagen [5,6]. The specific importance of fibrillin-1 microfibrils to vessel-wall structure was discovered by Arterial dilation, dissection and human genetic studies linking mutations in the gene rupture encoding fibrillin-1 (FBN1 ) to Marfan syndrome [7]. Cardiovascular abnormalities are the major source of ¥ Mutations in -1 ¥ Mutations in type I morbidity and mortality in Marfan syndrome, and involve collagen dilation of the aortic root with subsequent risk of aortic ¥ Mutations in type III dissection and rupture, and sudden death (Fig. 2). collagen Subsequently, the generation and analysis of two mouse strains with mutations in FBN1 confirmed the crucial role of fibrillin-1 in stabilizing elastic-fiber structure in the mature vessel [8–10]. Both of these mutant mouse strains died postnatally from vessel dissection and rupture (Fig. 2). Specifically, a deficiency of fibrillin-1 appeared to ¥ Mutations in destabilize the elastic-fiber architecture and to make the elastin vessel susceptible to injury from hemodynamic forces and Normal artery inflammation. Finally, human genetic studies and murine gene targeting have failed to reveal a role for fibrillin-2 in vascular morphogenesis or disease [7,11]. Therefore, the in Arterial occlusion TRENDS in Cell Biology vivo data support a specific role for fibrillin-1 in main- taining elastic-fiber structure and integrity in the vessel wall (Table 1). Fig. 2. Pathology of vascular disease resulting from extracellular-matrix gene mutations. There are two main types of pathology associated with vascular dis- The role of fibrillin-1 in cell signaling is less well ease – that in which the arterial lumen becomes occluded and that in which the characterized. Fibrillin-1 polypeptides with RGD vessel wall becomes weakened and subsequently dilates, dissects and ruptures. sequences are recognized by avb3 integrins in vitro, Human genetic studies and murine gene targeting have demonstrated that mutations in vascular extracellular-matrix components can result in pathological and this interaction has been shown to play a role in cell features of each type. Mutations in fibrillin-1, type-I collagen and type-III collagen attachment and adhesion [12] (Fig. 3a). The avb3 lead to arterial dilation, dissection and rupture. By contrast, mutations in elastin lead to arterial occlusion. integrin is known to regulate VSMC activity, including migration, proliferation, adhesion and survival [13,14]. However, avb3 integrins also recognize a wide range of have defined specific signaling interactions between the RGD-containing ECM components, including vitro- ECM and VSMCs of the vessel wall. Together, these nectin, fibronectin and collagen [14]. Mice lacking the experiments implicate the ECM as an integrated scaffold av integrin are embryonic lethal and have multiple that has both structural and signaling functions. However, defects in vasculogenesis, angiogenesis and organo- the relative importance of these two roles is not always genesis, including distended vessels of the perineural clear. Here, we highlight some of the issues to consider plexus [15]. The promiscuous binding of the avintegrin, when reconciling in vitro and in vivo data supporting however, precludes us from making any definite con- structural and signaling roles for ECM in vascular clusions from these in vivo data. morphogenesis and disease. We limit our examination to A model of fibrillin–VSMC signaling was proposed fibrillins, collagens and elastin. based on pathological data showing that VSMCs from fibrillin-1 mutant mice have cytoplasmic changes charac- Fibrillin teristic of synthetic, proliferative cells [10]. This model The fibrillins are a class of extracellular microfibrils that proposes that the loss of fibrillin–VSMC interactions leads associate with elastic fibers in the arterial wall. There are to phenotypic changes in VSMCs from a contractile two highly homologous isoforms: fibrillin-1, encoded by a quiescent state to a synthetic proliferative state. These gene on chromosome 15, and fibrillin-2, encoded by a gene cells subsequently release matrix metalloproteinases that on chromosome 5 [5,6]. Both fibrillin microfibrils are degrade the vessel wall. However, these data are based on known to interact with multiple ECM components within descriptive characterization of mutant mice and need to be the vessel wall, including elastin, vitronectin, fibronectin correlated with in vitro cell signaling experiments. At

Table 1. Vascular phenotypes resulting from extracellular-matrix (ECM) gene mutations in humans and mice

ECM fibrillar protein Human vascular disease Human vascular phenotype Mouse knockout phenotype Refs Fibrillin-1 Marfan syndrome Arterial dilation, dissection and rupture Die by 3 weeks from [6–10] ruptured blood vessels Type-I collagen Osteogenesis imperfecta Arterial dilation, dissection and rupture Die by embryonic day 14 [16,18,20,21] from ruptured blood vessels Type-III collagen Ehlers–Danlos syndrome type IV Arterial dilation, dissection and rupture Die around 6 months from [16,19,22] ruptured blood vessels Elastin Supravalvular aortic stenosis Arterial occlusion Die by neonatal day 4 [42–45] from arterial occlusion http://ticb.trends.com Review TRENDS in Cell Biology Vol.13 No.1 January 2003 53

In summary, both in vivo and in vitro evidence support (a) Fibrillin the conclusion that fibrillin-1 microfibrils primarily serve as structural molecules in the vessel wall. Their presence αV appears to stabilize the interaction between vascular cells and the integrated matrix scaffold. Although there is some ¥ Adhesion pathological evidence that fibrillin-1 might regulate the β 3 phenotypic modulation of VSMCs, there are no in vitro data to confirm these findings. Examining the activity of VSMCs from fibrillin-1 mutant mice in cell-culture αV experiments might help to answer these questions. ¥ Adhesion Specifically, it would be important to show that phenotypic β3 alterations in VSMCs from fibrillin mutant mice are not secondary to hemodynamic stress and that they could be Fibrillin reversed by the exogenous treatment of fibrillin-1. These Cell membrane Elastic fiber future experiments would help to establish a direct cell signaling role for fibrillin-1 in vascular morphogenesis and (b) Collagen disease.

α ¥ Migration 1, 2, Collagens ¥ Proliferation 10,11 The collagens are a family of ECM proteins that includes ¥ Adhesion at least 24 different types and ,38 distinct polypeptide ¥ Cytoskeletal rearrangement β1 chains [16]. Collagen types I and III are the most abundant in the vessel wall and are distributed throughout the media and adventitia [16,17].Human genetic studies and murine gene targeting revealed crucial roles for type-I and type-III collagens in vessel- ¥ Migration DDR 1, 2 wall structure. First, mutations in the genes that encode ¥ Proliferation type-I procollagen (COL1A1 and COL1A2 )resultin osteogenesis imperfecta [16,18]. The vascular compli- cations of osteogenesis imperfecta include aortic dilation, Cell membrane Collagen Elastic fiber dissection and rupture (Fig. 2). Second, mutations in the gene encoding type-III procollagen (COL3A1 )are (c) Elastin responsible for Ehlers–Danlos syndrome type IV [16,19]. Patients with this syndrome are particularly susceptible to dilation and rupture of arteries throughout the vasculature (Fig. 2). Third, murine gene targeting ¥ Migration confirmed the importance of type-I and type-III collagen ¥ Proliferation to the integrity of the vessel wall. Mice with mutations in ¥ Adhesion ? type-I and type-III collagen are both found to die ¥ Actin prematurely from ruptured blood vessels [20–22] polymerization (Fig. 2). Together, these in vivo findings indicate that the proper assembly and organization of type-I and type- III collagen fibers are necessary for normal mechanical and structural integrity of the vessel wall (Table 1). Many in vitro studies have established a role for Cell membrane Elastic fiber collagen matrix in cell signaling. Collagens are recog- nized by two major classes of receptors: (1) the 1 family TRENDS in Cell Biology b of integrins (a1b1, a2b1, a10b1anda11b1); and (2) members of the discoidin-domain receptor (DDR) family Fig. 3. Potential role of extracellular-matrix signaling in the vascular wall. (a) Fibril- lin-1 regulates vascular smooth-muscle cell (VSMC) adhesion via avb3 integrins in (DDR-1 and DDR-2) [23–32]. Both of these receptor vitro. There is currently no additional evidence for fibrillin-1 cell signaling. (b) Col- groups are produced by VSMCs and are thought to lagen regulates VSMC adhesion, migration, proliferation and cytoskeletal arrange- mediate cellular processes such as migration, adhesion ment through multiple receptor pathways, including the integrins a1b1, a2b1, a10b1 and a11b1 integrins, and the DDR-1 and DDR-2 receptors in vitro. Although and proliferation through specific downstream signal- defined signaling pathways have been studied for the a1b1 and a2b1 integrins, transduction pathways (Fig. 3b). Specifically, a1b1 and for the DDR receptors, the a10b1 and a11b1 integrins are less well character- integrin stimulates cell proliferation by activating ized. (c) Elastin regulates VSMC migration, proliferation and cytoskeletal arrange- ment in vitro. The molecular mechanism of this signaling pathway has not been ERKs in the mitogen-activated-protein-kinase pathway elucidated. [28]. By comparison, a2b1 inhibits cell proliferation by upregulating the cyclin-dependent-kinase (CDK) inhibi- present, the contribution of fibrillin-cell signaling to the tors p27 and p21, and suppressing cyclin-E–Cdk2 pathogenesis of vascular defects in humans and mice with activity [25,29].Thea2b1 signaling pathway is import- fibrillin-1 mutations has not been clearly established. ant in regulating VSMC adhesion, proliferation and http://ticb.trends.com 54 Review TRENDS in Cell Biology Vol.13 No.1 January 2003 differentiation on polymerized fibrillar collagen [30]. Elastin Moreover, it was recently shown that a lateral activation Elastin is the dominant ECM protein deposited in the of a2b1 with a G-protein subunit is needed for VSMC arterial wall and can compose up to 50% of its dry weight chemotaxis to collagen [30]. Although the a10b1and [2,40]. Unlike the fibrillins and collagens, there is only one a11b1 integrins have only recently been described, gene encoding elastin, located on chromosome 7. The recent in vitro data suggest that they mediate col- protein product of the elastin gene is synthesized by lagen-dependent adhesion and chemotaxis [31,32]. VSMCs and secreted as a monomer, tropoelastin. After Finally, the DDR family is a class of tyrosine-kinase post-translational modification, tropoelastin is cross-linked receptors activated by collagen that have recently been by lysyl oxidase and organized into elastin polymers that characterized [23,27]. DDR-1–collagen interactions form concentric rings of elastic lamellae around the have been suggested to regulate VSMC activity, includ- arterial lumen [2,40]. Each elastic lamella alternates ing the induction of migration, the activation of pro- with a ring of smooth-muscle and forms the structural unit liferation and the stimulation of matrix metalloprotease of the vessel wall [41]. The deposition of elastin is limited to production [23,33]. Together, these in vitro data indicate the medial layer of the vessel wall, extending from the IEL that collagen regulates VSMC activity through specific to the external elastic lamina. This biomechanical support integrin and DDR signaling pathways. provides the compliance that arteries need to absorb and To study the role of collagen receptors in vascular-cell transmit hemodynamic force [4]. signaling, knockout mouse strains have been generated for Human genetic studies demonstrated that loss-of- the a1, a2andb1 integrin subunits, as well as for the function mutations in one allele of the elastin gene are DDR receptors that are known to interact with collagens responsible for supravalvular aortic stenosis (SVAS), an [28,34–37]. Based on the phenotypes observed in mice obstructive vascular disorder that causes hemodynami- lacking type-I and type-III collagen, vascular defects cally significant narrowing of large arteries [42–44] would be expected in these mutants. However, the a12/2 (Fig. 2). Subsequently, mice lacking elastin (Eln 2/2 ) and a22/2 mice do not exhibit any significant vascular were generated and found to die from vascular occlusion phenotype [34,35] and there are no murine knockouts for in early postnatal life [45]. Vessel obstruction in Eln 2/2 the a10 and a11 integrins. Moreover, b12/2 mice were mice occurred because of the excessive subendothelial found to be embryonic lethal at embryonic day 5.5, proliferation and accumulation of VSMCs in the absence of precluding an analysis for their role in vascular develop- an inflammatory response (Fig. 2). Moreover, this obstruc- ment [36]. Finally, the characterization of mice lacking tive pathology was found throughout the arterial tree in DDR-1 or DDR-2 failed to reveal any vascular defects Eln 2/2 mice and occurred in the presence of a normal [23,38,39]. However, it is interesting that consistent with endothelium. These in vivo results suggest that elastin in vitro studies, DDR-12/2 mice had decreased VSMC matrix is a crucial regulator of smooth-muscle-cell activity proliferation and neointimal formation following vascular in the vessel wall (Table 1). injury [33]. Many in vitro studies suggest a crucial role for elastin in In summary, many in vitro data suggest that collagen VSMC signaling [45–49]. Organ-culture experiments has an important role in VSMC signaling through defined using aortae from Eln 2/2 mice revealed that the over- receptors and signal-transduction pathways (Fig. 3b). proliferation of VSMCs in these vessels occurs in the However, in vivo models do not clearly explain or predict absence of hemodynamic forces [45]. VSMCs grown on this biological function. The phenotypes of knockout mice elastin-coated substrates proliferate less than cells grown for collagen receptors do not recapitulate the vascular on uncoated substrates [46]. The amount of elastin phenotype observed in humans and mice with mutated deposited by smooth-muscle cells in patients with SVAS type-I and type-III collagen. There are several possible is inversely related to their rate of cell proliferation [47]. explanations for this apparent discordance. There might Moreover, this increased rate of cell proliferation of SVAS be extensive cross-reactivity between collagen’s integrin VSMCs can be reversed by the addition of elastin to the and non-integrin signaling receptors. Alternatively, culture medium. Several groups have demonstrated the redundancies between a-integrin subunits or DDRs chemotactic effect of elastin matrix and elastin peptides for might compensate for individual deficiencies in vivo.A VSMCs [48,49]. Growing VSMCs on elastin in cell-culture potential solution for this problem might be to develop preserves the quiescent, contractile phenotype, as shown compound knockouts such as mice lacking a1 and a2 by the presence of contractile myofilaments [50]. Addition- integrin, and mice lacking DDR-1 and DDR-2. In addition, ally, adding elastin to Eln 2/2 VSMCs activates a specific our understanding of the b1 integrin is incomplete given cellular and biochemical pathway involving heterotri- its early lethality. A tissue-specific knockout of the b1 meric G-protein signaling, RhoA and actin polymerization integrin would allow us to investigate its role in the [51]. Finally, the delivery of elastin-covered stents reduces vasculature. Furthermore, the involvement of a10b1 and neointimal thickness in a porcine injury model of coronary a11b1 integrins in vascular morphogenesis and disease restenosis [51]. Together, these data suggest that elastin remains unclear given their recent discovery. Future signaling regulates VSMC migration, induces a contractile murine gene targeting and in vitro studies will hopefully phenotype and inhibits proliferation. clarify their role in VSMC biology. At present, the Despite this evidence supporting a role for elastin contribution of collagen signaling to the dramatic pheno- in regulating VSMCs, the precise molecular signaling types observed in human and mice with collagen gene pathway for this activity remains to be elucidated mutations remain unclear. (Fig. 3c). Several groups have attempted to identify an http://ticb.trends.com Review TRENDS in Cell Biology Vol.13 No.1 January 2003 55 elastin-signaling receptor by purifying proteins that bind studies clearly demonstrate the importance of collagen– to elastin. Three different proteins were described, includ- integrin signaling in modulating VSMC proliferation, ing a 59-kDa integral membrane protein, a 120-kDa migration and differentiation. Yet human genetic and integral membrane protein, and a 67-kDa extracellular gene-targeting studies emphasize the contribution of protein [52–57]. Work by Hinek and others indicates that collagens to the structural integrity of the vessel wall the 67-kDa protein is an alternatively spliced form of and not its signaling role. Conversely, there is both in vivo b-galactosidase called EBP. They propose that EBP com- and in vitro evidence for a role of elastin in regulating plexes with other proteins to transduce elastin signaling smooth-muscle-cell activity via a signaling pathway. What via pertussis-toxin-sensitive G-proteins. Prior to this is missing is an understanding of the molecular mechan- report, heterotrimeric G proteins were primarily activated ism of elastin signaling. by members of the 7-transmembrane GPCR family. To assign specific roles to ECM signaling in vascular Interestingly, these investigators suggest that elastin morphogenesis and disease, it will be necessary to activates proliferation of VSMCs [58]. These findings are correlate human genetics and gene-targeting and cell- 2/2 in contrast to prior work with Eln mice, primary culture studies. For this to occur, three lines of evidence cultures of rodent and murine VSMCs, and VSMCs should be consistent. First, gene mutations in specific derived from SVAS patients [45,47,50,51]. More work is ECM proteins should show a distinct vascular pheno- needed to correlate mechanistic findings with in vitro and type. Second, in vitro cell-culture systems should in vivo functional data. An alternative signal pathway for establish defined cell signaling pathways that explain elastin signaling was recently suggested by the identifi- the cellular activity in vivo.Third,mutationsinECM cation of fibulin-5, a matrix protein that binds to elastin receptors and their downstream effectors defined by and is recognized by avb3, avb5, and a9b1 integrins these in vitro studies should recapitulate the in vivo [59–61]. It is possible that elastin signals VSMCs through phenotypes caused by targeted mutation of the ECM a fibulin-5-mediated integrin pathway. However, mice ligand in question. Although correlating the in vivo lacking fibulin-5 are characterized by tortuous blood phenotype of matrix mutants with receptor mutants is 2/2 vessels, not the occlusive arterial disease seen in Eln not straightforward, it might be supported by the mice [59,60] (Fig. 2). Thus, it is unclear whether fibulin-5 generation of compound murine knockouts and vascu- has a role in mediating elastin signaling in the vessel wall. lature-specific mutants. Because of the complex inter- Both in vivo and in vitro data consistently suggest, actions and multiple components involved in an but do not prove, that elastin signaling is crucial for the integrated matrix scaffold, it is important to establish maintenance of tubular structure. Elastin is a major all three lines of evidence to confirm a specific role for component of the integrated scaffold and associates with matrix signaling in vascular morphogenesis. other ECM elements and growth factors. Thus, disrupt- Common human vascular diseases such as athero- ing elastin in vivo can affect vascular smooth-muscle sclerosis, restenosis and aortic aneurysms are a major biology both directly and indirectly. To establish a direct source of morbidity and mortality in developed nations. signaling role for elastin in the vessel wall, an elastin In this article, we have discussed the genetic defects receptor and downstream signaling cascade must be in specific ECM components that lead to familial identified that recapitulate the in vitro and in vivo data. vascular diseases, including SVAS, osteogenesis imper- If elastin signaling is required for arterial morpho- fecta,Ehlers–DanlossyndrometypeIV,andMarfan genesis, we predict that targeted disruptions in this syndrome. Although these vascular diseases are rare, signaling mechanism will produce an occlusive arterial the modest polymorphisms in the ECM genes and phenotype similar to Eln 2/2 mice (Fig. 2). The combin- receptors that cause these disorders might interact ation of in vitro and in vivo studiesemphasizesthe with known risk factors to contribute to the pathogenesis importance of elucidating the details of elastin signaling of the common vascular diseases. Preliminary reports as pharmacologic approaches for the treatment of suggest that restoring crucial matrix elements or vascular diseases develop. activating their signaling pathways in an injured arterial wall might be potential therapeutic strategies Concluding remarks for these vascular disorders. Fully developing these This article highlights some of the important findings that strategies requires a better understanding of the precise have emerged from an analysis of the ECM through combined human genetics, murine gene-targeting experi- structural and signaling roles of the ECM in vascular ments and cell-culture studies. Current evidence supports development and disease. the concept of an integrated scaffolding composed of elastic fibers, microfibrils and collagens that maintains vascular References 1 Risau, W. (1995) Vasculogenesis. Annu. Rev. Cell Dev. Biol. 11, 73 – 91 homeostasis through a combination of mechanical support 2 Parks, W.C. (1993) The extracellular matrix. Adv. Mol. Cell. Biol. 6, and biological signaling. However, the complexity of this 133–182 matrix scaffold makes it difficult to distinguish between 3 Raines, E.W. (2000) The extracellular matrix can regulate vascular cell the effects of structure and signaling. In the case of migration, proliferation, and survival: relationships to vascular fibrillin-1, existing evidence indicates that it serves disease. Int. J. Exp. Pathol. 81, 173–182 4 Faury, G. (2001) Function–structure relationship of elastic arteries in primarily a structural role in the vasculature. For evolution: from microfibrils to elastin and elastic fibres. Pathol. Biol. collagen, however, there is discordance between the in (Paris) 49, 310–325 vitro and in vivo data that needs to be reconciled. In vitro 5 Kielty, C.M. et al. (2002) Fibrillin: from microfibril assembly to http://ticb.trends.com 56 Review TRENDS in Cell Biology Vol.13 No.1 January 2003

biomechanical function. Phil. Trans. R. Soc. Lond. B Biol. Sci. 357, domain integrins – what do they do? Prog. Histochem. Cytochem. 37, 207–217 1–54 6 Handford, P.A. et al. (2000) Fibrillin: from domain structure to 33 Hou, G. et al. (2001) The discoidin domain receptor tyrosine kinase supramolecular assembly. Matrix Biol. 19, 457–470 DDR1 in arterial wound repair. J. Clin. Invest. 107, 727–735 7 Robinson, P.N. and Godfrey, M. (2000) The molecular genetics of 34 Bouvard, D. et al. (2001) Functional consequences of integrin gene Marfan syndrome and related microfibrillopathies. J. Med. Genet. 37, mutations in mice. Circ. Res. 89, 211–223 9–25 35 Chen, J. et al. (2002) The a2 integrin subunit deficient mouse. Am. 8 Pereira, L. et al. (1997) Targeting of the gene encoding fibrillin-1 J. Pathol. 161, 337–344 recapitulates the vascular aspect of Marfan syndrome. Nat. Genet. 17, 36 Fassler, R. and Meyer, M. (1995) Consequences of lack of b1 integrin 218–222 gene expression in mice. Genet. Dev. 9, 1896–1908 9 Pereira, L. et al. (1999) Pathologenetic sequence for aneurysm revealed 37 Rupp, P.A. and Little, C.D. (2001) Integrins in vascular development. in mice underexpressing fibrillin-1. Proc. Natl Acad. Sci. USA 96, Circ. Res. 89, 566–578 3819–3823 38 Labrador, J.P. et al. (2001) The collagen receptor DDR2 regulates 10 Bunton, T.E. et al. (2001) Phenotypic alteration of vascular smooth proliferation and its elimination leads to dwarfism. EMBO Rep. 2, muscle cells precedes elastolysis in a mouse model of Marfan 446–452 syndrome. Circ. Res. 88, 37–43 39 Vogel, W.F. et al. (2001) Discoidin domain receptor1 tyrosine kinase has 11 Arteaga-Solis, E. (2001) Regulation of limb patterning by extracellular an essential role in mammary gland development. Mol. Cell. Biol. 21, microfibrils. J. Cell Biol. 154, 275–281 2906–2917 12 Pfaff, M. et al. (1996) Cell adhesion and integrin binding top 40 Rosenbloom, J. et al. (1993) Extracellular matrix 4: the elastic fiber. recombinant human fibrillin-1. FEBS Lett. 384, 247–250 FASEB J. 7, 1208–1218 13 Hynes, R.O. (2002) Integrins. Bidirectional, allosteric signaling 41 Wolinski, H. and Glasgov, S.A. (1967) Lamellar unit of aortic medial machines. Cell 110, 673–684 structure and function. Circ. Res. 20, 99–111 14 Clyman, R.I. et al. (1992) Beta 1 and beta 3 integrins have different 42 Stamm, C. et al. (2001) Congenital supravalvar aortic stenosis: a roles in the adhesion and migration of vascular smooth muscle cells on simple lesion? Eur. J. Cardiothorac. Surg. 19, 195–202 extracellular matrix. Exp. Cell Res. 200, 272–284 43 Curran, M.E. et al. (1993) The elastin gene is disrupted by a 15 Bader, B.L. et al. (1998) Extensive vasculogenesis, angiogenesis, and translocation associated with supravalvular aortic stenosis. Cell 73, organogenesis precede lethality in mice lacking all av integrins. Cell 159–168 95, 507–519 44 Li, D.Y. et al. (1997) Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis. Hum. Mol. Genet. 6, 16 Myllyharju, J. and Kivirikko, K.I. (2001) Collagens and collagen- 1021–1028 related diseases. Ann. Med. 33, 7–21 45 Li, D.Y. et al. (1998) Elastin is an essential determinant of arterial 17 Fleischmajer, R. et al. (1983) Collagen fibril formation during morphogenesis. Nature 393, 276–280 embryogenesis. Proc. Natl Acad. Sci. USA 80, 3354–3358 46 Ito, S. et al. (1998) Effect of coacervated a-elastin on proliferation of 18 Kuivaniemi, H. et al. (1997) Mutations in fibrillar collagens (types I, II, vascular smooth muscle cells and endothelial cells. Angiology 49, III, and XI), fibril-associated collagen (type IX), and network-forming 289–297 collagen (type X) cause a spectrum of diseases of , , and 47 Urban, Z. et al. (2002) Connections between elastin haploinsufficiency blood vessels. Hum. Mutat. 9, 300–315 and cell proliferation in patients with supravalvular aortic stenosis 19 Pepin, M. et al. (2000) Clinical and genetic features of Ehlers– and Williams–Beuren syndrome. Am. J. Hum. Genet. 71, 30–44 Danlos syndrome type IV, the vascular type. New Engl. J. Med. 48 Ooyama, T. et al. (1987) Substratum-bound elastin peptide inhibits 342, 673–680 aortic smooth muscle cell migration in vitro. Arteriosclerosis 7, 20 Lohler, J. et al. (1984) Embyronic lethal mutation in mouse collagen I 593–598 gene causes rupture of blood vessels and is associated with 49 Hinek, A. (1994) Nature and the multiple functions of the 67-kD erythropoietic and mesenchymal cell death. Cell 38, 597–607 elastin/laminin binding protein. Cell Adhes. Commun. 2, 185–193 21 Chipman, S.D. et al. (1993) Defective proa2(I) collagen synthesis in a 50 Yamamoto, M. et al. (1992) Identification of the phenotypic modulation recessive mutation in mice: a model of human osteogenesis imperfecta. of rabbit arterial smooth muscle cells in primary culture by flow Proc. Natl Acad. Sci. USA 90, 1701–1705 cytometry. Exp. Cell Res. 198, 43–51 22 Liu, X. et al. (1997) Type III collagen is crucial for collagen I 51 Karnik, S.K. et al. (2002) A critical role for elastin signaling in vascular fibrillogenesis and for normal cardiovascular development. Proc. morphogenesis and disease. Development in press Natl Acad. Sci. USA 94, 1852–1856 52 Wrenn, D.S. et al. (1988) Kinetics of receptor-mediated binding of 23 Hou, G. et al. (2002) Tyrosine kinase activity of discoidin domain tropoelastin to fibroblasts. J. Biol. Chem. 263, 2280–2284 receptor 1 is necessary for smooth muscle cell migration and matrix 53 Blood, C.H. et al. (1988) Identification of a tumor cell receptor for metalloproteinase expression. Circ. Res. 90, 1147–1149 VGVAPG, an elastin-derived chemotactic peptide. J. Cell Biol. 107, 24 Yamamoto, M. et al. (1993) Type I collagen promotes modulation of 1987–1993 cultured rabbit arterial smooth muscle cells from a contractile to a 54 Hornebeck, W. et al. (1986) Inducible adhesion of mesenchymal synthetic phenotype. Exp. Cell Res. 204, 121–129 cells to elastic fibers: elastonectin. Proc. Natl Acad. Sci. USA 83, 25 Koyama, H. et al. (1996) Fibrillar collagen inhibits arterial smooth 5517–5520 muscle proliferation through the regulation of CDK2 inhibitors. Cell 55 Hinek, A. et al. (1988) The elastin receptor: a galactoside-binding 87, 1069–1078 protein. Science 239, 1539–1541 26 Giancotti, F.G. (2000) Complexity and specificity of integrin signalling. 56 Mecham, R.P. and Hinek, A. (1996) Non-integrin laminin receptors. Nat. Cell Biol. 2, E13–E14 The Laminins (Ekblom, P., Timpl, R. eds), pp. 159–183, Harwood 27 Vogel, W. et al. (1997) The discoidin domain receptor tyrosine kinases Academic Publishers are activated by collagen. Mol. Cell 1, 13–23 57 Privitera, S. et al. (1998) The 67-kDa enzymatically inactive 28 Pozzi, A. et al. (1998) Integrin a1b1 mediates a unique collagen- alternatively spliced variant of b-galactosidase is identical to the dependent proliferation pathway in vivo. J. Cell Biol. 142, 587–594 elastin/laminin-binding protein. J. Biol. Chem. 273, 6319–6326 29 Henriet, P. et al. (2000) Contact with fibrillar collagen inhibits 58 Mochizuki, S. et al. (2002) Signaling pathways transduced through melanoma cell proliferation by upregulating p27KIP1. Proc. Natl elastin receptor facilitate proliferation of arterial smooth muscle cells. Acad. Sci. USA 97, 10026–10031 J. Biol. Chem. 277, 44854–44863 30 Brown, E.J. and Frazier, W.A. (2001) Integrin-associated protein 59 Yanagisawa, H. et al. (2002) Fibulin-5 is an elastin-binding protein (CD47) and its ligand. Trends Cell Biol. 11, 130–135 essential for elastic fibre development in vivo. Nature 415, 168–171 31 Tiger, C.-F. et al. (2001) a11b1 integrin is a receptor for interstitial 60 Nakamura, T. et al. (2002) Fibulin-5/DANCE is essential for collagens involved in cell migration and collagen reorganization on elastogenesis in vivo. Nature 415, 171–175 mesenchymal non-muscle cells. Dev. Biol. 237, 116–129 61 Midwood, K.S. and Schwarzbauer, J.E. (2002) Elastic fibers: building 32 Gullberg, D.E. and Lundgren-Akerland, E. (2002) Collagen-binding I bridges between cells and their matrix. Curr. Biol. 12, R279–R281 http://ticb.trends.com