Extracellular matrix gene expression in the developing mouse aorta Sean E. McLean,1 Brigham H. Mecham,2 Cassandra M. Kelleher,1 Thomas J. Mariani2 and Robert P. Mecham1 1 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 2 Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts Contents 1. Introduction ................................ 81 2. Expression profiling of the developing mouse aorta . ......... 83 3. The vascular ECM . ............................ 84 3.1. Collagens . ............................ 86 3.2. Elastic fiber proteins . ........................ 86 3.3. Fibronectin . ............................ 87 3.4. Basement membrane components.................. 87 3.5. Proteoglycans ............................ 88 4. The functional context of SMC diVerentiation .............. 89 4.1. Group A: Genes upregulated in embryogenesis . ......... 89 4.2. Group B: Genes with constant expression except for variation at P7toP14............................... 99 4.3. Group C: Genes with low expression in the embryo but increasing expression in postnatal stages . ................... 100 4.4. Group D: Genes that decrease at birth . .............. 112 5. Conclusions................................. 118 Advances in Developmental Biology Copyright # 2005 Elsevier B.V. Volume 15 ISSN 1574-3349 All rights reserved. DOI: 10.1016/S1574-3349(05)15003-0 82 S. E. McLean, B. H. Mecham, C. M. Kelleher, T. J. Mariani and R. P. Mecham 1. Introduction Embryonic development requires the establishment of a functional circula- tory system early in embryogenesis. The general outline of the forming vascular network is established in the absence of blood flow by endothelial cells through angiogenic or vasculogenic processes. With the initiation of blood flow, recruit- ment and diVerentiation of cells that make up the vascular wall begins in a process that is highly sensitive to local hemodynamic forces (Langille, 1996). 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 (SMCs) between sheets of elastin (the elastic laminae); and the tunica adventitia, made up of myofi- broblasts that produce mainly collagen fibers. Within the medial layer, the 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 (Wolinsky and Glagov, 1967; Berry et al., 1972; Gerrity and CliV, 1975). As the vessel wall matures, the SMCs go through multiple overlapping phenotypic transitions, character- ized broadly by cellular proliferation, matrix production, and the assembly of an appropriate contractile apparatus within the cell cytoplasm. Defining the functional characteristics of developing vascular wall cells is diYcult because of the transient nature of many marker proteins that are character- istic of the SMC phenotype. Expression of most known SMC diVerentia- tion markers in other cell types either during development or in response to injury is also a problem with cell-type determination. Further complicating our understanding of the vascular SMC is the cellular heterogeneity (Frid et al., 1994; Gittenberger-de Groot et al., 1999) and phenotypic plasticity (Schwartz and Mecham, 1995) observed during embryogenesis and vessel maturation. In medium and large vessels, a major function of the SMC is to synthesize and organize the unique extracellular matrix (ECM) responsible for the mechanical properties of the wall. Unlike cells in the small muscular and resistance vessels, the SMCs of the elastic conducting vessels contribute little to the static mechanical properties of the wall. Hence, their ability to pro- duce ECM can be considered to be their ‘‘diVerentiated’’ phenotype. Because the formation of a functional ECM must occur in an organized sequence, the ‘‘matrix phenotype’’ is changing throughout the entire period of vessel wall development. In addition to providing the structural and mechanical properties required for vessel function, the ECM provides instructional signals that induce, Extracellular matrix gene expression in the developing mouse aorta 83 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 develop- ment, and in response to injury. How vascular cells interpret these regulatory signals is a major area of research today. This chapter will discuss the vascular SMC phenotype and ECM mole- cules made by vessel wall cells during vascular development, with the pri- mary focus on the developing mouse aorta. Several excellent reviews have summarized our current understanding of SMC phenotypes based on expression of cytoskeletal and other marker proteins (Glukhova and Koteliansky, 1995; Owens, 1995; Hungerford et al., 1996; Owens et al., 2004). There are also numerous ultrastructural studies documenting the architecture of the developing vessel wall (Pease and Paule, 1960; Karrer, 1961; Paule, 1963; Haust et al., 1965; Albert, 1972; Gerrity and CliV, 1975; Thyberg et al., 1979). 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). 2. Expression profiling of the developing mouse aorta To better understand the functional properties of vessel wall cells during diVerentiation and maturation, gene expression profiling of developing mouse aorta was done using oligonucleotide microarray technology (MU74Av2 chip from AVymetrix, Santa Clara, CA). The entire aorta was removed starting at the most distal aspect of the aortic arch up to, but not including, the common iliac vessels. The time points used in the study were embryonic day 12 (E12), E14, E16, E18, postnatal day 0 (P0) (representing the first 24 hours of life), P4, P7, P10, P14, P21, P30, P60, 5.5 months of life, and 6 months of life. Ten micrograms of total ribonucleic acid (RNA) from each sample of pooled aortae were used to create the target. Twenty-five micrograms of each complementary RNA were hybridized to the MU74Av2 chip (AVymetrix) and four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) were included in each hybridization reaction to verify consistent hybridization eYciency.The image intensities of each scanned chip were analyzed by the GeneChip Analysis Suite software (AVymetrix) and were scaled to an average of 1,500 U. Probe sets were removed from the analysis if the diVerence between their maximum and minimum raw average diVerence values over the time series was <300. The normality of the chips themselves was determined by reassessing the CEL files produced by the 84 S. E. McLean, B. H. Mecham, C. M. Kelleher, T. J. Mariani and R. P. Mecham Microarray Suite software (AVymetrix) with DNA-Chip Analyzer (dChip) software package as described by Li and Wong (2001). 3. The vascular ECM The array data identify several major patterns for matrix gene expression. The first and most prevalent begins around day 14, shortly after mesenchy- mal cells recruited to the vessel wall organize into layers that closely approx- imate the number that will be found in the mature tissue. This expression pattern consists of a major increase in matrix protein expression at embry- onic day 14 followed by a steady rise through the first 7 to 14 days after birth. This is followed by a decrease in expression over 2 to 3 months to low levels that persist in the adult (Fig. 1). Most of the structural matrix proteins follow this pattern. The second most prevalent pattern was one of consistent expression throughout the time series and was typical of base- ment membrane components, fibronectin, most integrins, and some matrix metalloproteinases. The third pattern consists of high expression levels in the embryonic/fetal period followed by decreased expression postnatally. The final and least populated pattern was low expression throughout develop- ment with an increase in the adult period. Our expression data are in agreement with the appearance of structural matrix proteins in the vessel wall as assessed by ultrastructural studies (Nakamura, 1988). The electron micrographs in Fig. 2 compare the vessel wall of the developing mouse aorta at E12, E14, and E18. At day 12, there are few discernable collagen or elastin fibers in the extracellular space. By Fig. 1. Typical expression pattern for structural matrix proteins in developing mouse aorta. Expression of structural matrix proteins begins around E14, shortly after mesenchymal cells recruited to the vessel wall organize into layers that closely approximate the number that will be found in the mature tissue. This expression pattern consists of a major