6 ____________________________________________________________________________ Vascular Extracellular Matrix and Aortic Development Cassandra M. Kelleher, Sean E. McLean, and Robert P. Mecham Washington University School of Medicine Department of Cell Biology and Physiology St. Louis, Missouri 63110 I. Introduction II. Vessel Wall Formation and Structure III. The Vascular Extracellular Matrix IV. Collagens A. Genotype–Phenotype Correlations Resulting from Mutations in the Vascular Fibrillar Collagens V. The Elastic Fiber A. Elastin B. Fibrillin and Microfibrils VI. Fibulins A. Fibulin-1 B. Fibulin-2 C. Fibulins-3 and -4 D. Fibulin-5 VII. EMILIN/Multimerin Family VIII. Fibronectin IX. The Basement Membrane A. Laminins B. Entactin/Nidogen X. Proteoglycans A. Large Proteoglycans That Form Aggregates by Interaction with Hyaluronan B. Small Leucine Rich Proteoglycans XI. Matricellular Proteins A. Thrombospondins B. Tenascins C. SPARC (osteonectin) XII. Correlation of Matrix Gene Expression Profile with Cytoskeletal Markers XIII. Conclusions Acknowledgments References Current Topics in Developmental Biology, Vol. 62 Copyright 2004, Elsevier Inc. All rights reserved. 153 0070-2153/04 $35.00 154 Kelleher et al. I. Introduction With the emergence of a high-pressure, pulsatile circulatory system in verte- brates came a remarkable change in blood vessel structure and function. Blood vessels no longer acted as simple tubes for channeling blood or other body fluids from a low-pressure heart. In this closed circulatory system, large arteries became an important component of proper cardiac function by serving as elastic reservoirs, enabling the arterial tree to undergo large- volume changes with little change in pressure. Without elastic vessels, the tremendous surge of pressure as blood ejected from the heart would inhibit the heart from emptying, and the pressure in the vessels would fall so rapidly that the heart could not refill. Furthermore, distension of the elastic arterial wall by blood pushed from the heart is translated into kinetic energy when the arterial wall contracts, which helps move the blood down the vascular tree. The change that brought about this critical step in the evolution of higher organisms was the emergence of a vascular wall containing cells specialized in the production and organization of an extracellular matrix (ECM) uniquely designed to provide elastic recoil. In addition to providing the structural and mechanical properties required for vessel function, the ECM provides instructional signals that induce, define, and stabilize smooth muscle phenotypes. There are many examples of ECM molecules playing critical roles in the regulation of gene expression by interacting with specific matrix receptors on cells and by binding and storing growth factors that influence cellular function. This reciprocal instructive interaction between the cell and its ECM is important in directing the developmental transitions that occur in embryogenesis, postnatal devel- opment, and in response to injury. How vascular cells interpret these regulatory signals is a major area of research today. This review will discuss the ECM molecules made by vessel wall cells during vascular development, with the primary focus on the developing mouse aorta. Several excellent reviews have summarized our current under- standing of smooth muscle cell phenotypes based on expression of cytoskel- etal and other marker proteins (Glukhova and Koteliansky, 1995; Hungerford et al., 1996; Owens, 1995). There are also numerous ultrastruc- tural studies documenting the architecture of the developing vessel wall (Albert, 1972; Berry et al., 1972; Gerrity and CliV, 1975; Haust et al., 1965; Karrer, 1961; Paule, 1963; Pease and Paule, 1960; Thyberg et al., 1979), although most of these studies have been in animals other than mouse. The morphogenesis of the aortic wall in the rat, however, has been well investigated (Berry et al., 1972; CliV, 1967; Gerrity and CliV, 1975; Nakamura, 1988; Paule, 1963; Pease and Paule, 1960) and shows many similarities with mouse wall structure (Davis, 1993; Karrer, 1961). For the 6. Vascular Matrix and Aortic Development 155 interested reader, extensive information on the vascular smooth muscle cell and a still timely discussion of questions and issues driving research in vascular biology can be found in a monograph by Schwartz and Mecham (1995). II. Vessel Wall Formation and Structure While the role of endothelial cells in the formation of the vascular primordia is beginning to be well understood (Carmeliet, 2000; Drake et al., 1998; Rossant and Howard, 2002), surprisingly little is known about how vessels acquire their coat of smooth muscle cells that make up the vessel wall. Presumptive vascular smooth muscle cells (VSMCs) form from the sur- rounding mesenchyme and/or cardiac neural crest in response to soluble factors secreted by endothelial cells. The angiopoietin/Tie receptor pathway (Dumont et al., 1995; Sato et al., 1993) is clearly a major player in early stages of this process, but questions remain about what other factors guide smooth muscle diVerentiation through the various stages of vessel wall formation. Complicating our understanding of the VSMC is the cellular heterogeneity (Frid et al., 1994; Gittenberger-de Groot et al., 1999) and phenotypic plasticity (Schwartz and Mecham, 1995) observed during em- bryogenesis and vessel maturation. As the vessel wall matures, the SMCs go through multiple overlapping phenotypic transitions, characterized broadly by cellular proliferation, matrix production, and the assembly of an appropriate contractile apparatus within the cell cytoplasm. In medium and large vessels, the major function of the SMC is to synthesize and organize the unique extracellular matrix responsible for the mechanical properties of the wall. Unlike cells in the small muscular and resistance vessels, the smooth muscle cells of the elastic conducting vessels contribute little to the static mechanical properties of the wall. Hence, their ability to produce ECM can be considered to be their ‘‘diVerentiated’’ phenotype. Because the formation of a functional extracellular matrix must occur in an organized sequence, the ‘‘matrix phenotype’’ is changing throughout the entire period of vessel wall development. As pointed out by Little and colleagues (Drake et al., 1998; Hungerford et al., 1996), the expression pattern of ECM proteins may be a better indicator of VSMC diVerentiation status than the presence or absence of intracellular markers. The general histological form of the large blood vessels includes three compartments: the tunica intima, consisting of a single layer of endothelial cells that sit directly on the internal elastic lamina (IEL); the tunica media, consisting of concentric layers of smooth muscle cells between sheets of elastin (the elastic laminae); and the tunica adventitia, made up of myofibro- blasts that produce mainly collagen fibers. Within the medial layer, the 156 Kelleher et al. collagen and elastin fibers are arranged to form a ‘‘two phase’’ system, in which circumferentially aligned collagen fibers of high tensile strength and elastic modulus bear most of the stressing force at and above physiologic blood pressure. Elastin, which is distensible and has a low tensile strength, functions primarily as an elastic reservoir and distributes stress evenly throughout the wall and onto collagen fibers (Berry et al., 1972; Gerrity and CliV, 1975; Wolinsky and Glagov, 1967). The number of lamellar units (generally defined as the elastic lamella and adjacent smooth muscle cells) in a vascular segment is related linearly to tensional forces within the wall (Clark and Glagov, 1985; Leung et al., 1977; Wolinsky and Glagov, 1967), with the greatest number of elastic layers occurring in the larger, more proximal vessels that experience the highest wall stress. A role for hemodynamics in vessel wall development (Folkow, 1983; Langille, 1996) and in modulating elastin production (Faury et al., 2003; Keeley and Alatawi, 1991; Keeley and Johnson, 1986) has been suggested from numerous studies of vascular remodeling in response to altered pres- sure and flow. In the developing chick coronary artery, for example, SMC recruitment from undiVerentiated mesenchyme does not occur until the connection to the aorta is made and actual blood flow through these vessels has begun (BergwerV et al., 1996). When the vessel wall is forming, SMC diVerentiation, lamellar number, and elastin content coordinately increase with the gradual rise in blood pressure until the proper number of lamellar units are organized (Nakamura, 1988; Roach, 1983). The relatively constant tension per lamellar unit and their uniformity of composition, regardless of species, indicate that the proportion of collagen, elastin, and SMCs in the media is optimal for the stresses to which the aorta is subjected (Wolinsky and Glagov, 1967). III. The Vascular Extracellular Matrix In addition to the structural matrix proteins (collagen, elastin, proteogly- cans, etc.), vascular cells must produce matrix macromolecules that are important for cell movement, polarization, and anchorage. These molecules, which include adhesive glycoproteins such as fibronectin, basement mem- brane components, and the matricellular proteins that modulate cell–matrix interactions, provide important informational signals to cells that can influ- ence gene expression and cellular function. To identify the types of matrix proteins produced by SMCs and to compare their expression pattern with