The Extracellular Matrix of Blood Vessels

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The Extracellular Matrix of Blood Vessels Johannes A. Eble* and Stephan Niland Center for Molecular Medicine, Dept. Vascular Matrix Biology, Excellence Cluster Cardio-Pulmonary System, Frankfurt University Hospital, 60590 Frankfurt, Germany Abstract: Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. They are able to autonomously regulate the blood flow, thus providing the tissues an optimal support of oxygen and nutrients and an efficient removal of waste products. In higher organisms, the blood vessel forms a closed circuit system, which additionally has the ability to seal itself in case of leakage as a result of injury. The blood vessel system does not only transport soluble substances, but also serves as “highway” system for leukocytes to patrol the body during the immunological surveillance and to reach the inflammation site quickly. In a complex interplay with the vascular wall, leukocytes are able to penetrate the blood vessel without any obvious leakage. Pathologically, tumor cells subvert the blood vessel system to disseminate from the primary tumor and colonize distant organs during metastasis. The extracellular matrix (ECM) of a blood vessel contributes substantially to the diverse functions of the blood vessel. First, the ECM constitutes the scaffold which keeps the histological structure of the vessel wall in shape but also bears the enormous and permanent mechanical forces levied on the vessel by the pulsatile blood flow in the arteries and by vasoconstriction, which regulates blood flow and pressure. The complex network of elastic fibers and tensile forces-bearing networks are well adapted to accomplish these mechanical tasks. Second, the ECM provides informational cues to the vascular cells, thus regulating their proliferation and differentiation. Third, ECM molecules can store, mask, present or sequester growth factors, thereby modulating their effects remarkably. Furthermore, several ECM molecules serve additional functions within the blood vessel. Their expression is altered in a spatial and temporal pattern during blood vessel formation and remodeling. In contrast to vasculogenesis during embryonic development, blood vessel shows a remarkably and life-long plasticity, which allows the formation and regeneration of new blood vessel even in adulthood. Both physiologically during wound healing and pathologically during tumor growth, the sprouting of new blood vessels during angiogenesis is an important process, in which the ECM takes a key role.

Key Words: Blood vessel, extracellular matrix, endothelial cells, vascular smooth muscle cells, angiogenesis. 1. INTRODUCTION Animals have adapted a circulatory system during evolution in order to ensure a constant supply of nutrients and oxygen to all organs and tissues as well as a permanent removal of waste metabolites. Vertebrates, the evolutionarily most advanced deuterostome animals, have a closed circulatory system and a heart that drives blood through closed blood vessels, whereas in insects, the highest developed protostomes, the body fluid, called hemolymph, is pumped by a dorsal heart through the tissue without closed vessels. Although enclosed in vessels, the blood is able to exchange nutrients, oxygen and waste products through the blood vessel wall with the surrounding tissue. Because of the pressure conditions, the body fluid is pressed through the vessel walls to form the lymph, which then is drained by lymph vessels back to the blood circulation. The different types of blood vessels have different histological and molecular structures to fulfill their various *Address correspondence to this author at the Center for Molecular Medicine, Dept. Vascular Matrix Biology, Excellence Cluster Cardio-Pulmonary System, Frankfurt University Hospital, Bldg. 9, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany; Tel: +49-69-6301 87651; Fax: +49-69-6301 87656; E-mail: [email protected] 1381-6128/09 $55.00+.00

physiological functions. Pumped out from the heart with high pressure, the blood exerts enormous pulsatile forces on the heart-proximal arteries, which withstand these forces by elastic deformation and use these elastic forces to drive the blood into the downstream vessels during the diastolic heart phase and to smooth out pressure fluctuations. Subsequent to these elastic arteries, muscular arteries transport the blood further to the different organs of the body. The muscular arteries and the smaller-lumen sized arterioles are able to decrease their lumen diameter due to the contraction of their muscular cuffs. This vasoconstriction leads to a higher flow resistance and hence a diminished blood flow, thus regulating the blood flow through the following small caliber blood vessels. The branching of the blood vessel reaches its climax in the capillaries. The capillary beds allow the exchange of metabolites with the surrounding tissue. Thereafter, the blood is drained into venules and veins, which bring the blood back to the heart. The venule is the place along blood vessels where leukocytes preferentially extravasate for their immunological surveillance patrol. Taken the heart as axis of symmetry, the body circuit is mirrored in the lung circuit, the capillary bed of which is required for oxygen loading and for removal of carbon dioxide from the blood.

© 2009 Bentham Science Publishers Ltd.

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2. THE HISTOLOGICAL STRUCTURE OF BLOOD VESSELS Blood vessels are generally composed of three concentric layers: tunica intima, tunica media and tunica adventitia [1]. These layers are histologically separated by two sheet-like structures of extracellular matrix proteins: the inner and outer limiting membranes (membrane limitans interna and externa), which segregate the tunica media from the tunica intima and the tunica adventitia, respectively (Fig. (1)). On the molecular level, these ECM structures do not segregate the histological layers of the blood vessel but rather connect them. In fact, too weak interactions of these layers may cause life-threatening aneurysms. The tunica intima contains a simple layer of squamous endothelial cells lining the inner surface of the vessel tube. The tunica media is usually the thickest of the three layers in arteries. It houses the mural cells, which are smooth muscle cells in larger blood vessels and pericytes in capillaries. The tunica adventitia, mostly the thickest layer in veins, links the blood vessel to the surrounding connective tissue. This generic architecture of a blood vessel is modified in the different types of blood vessels, arteries, veins, and capillaries, to fulfill their individual tasks. For example, the endothelial cell lining in capillaries is continuous in most tissues, whereas capillaries in exocrine and

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endocrine glands are often fenestrated. Moreover, sinusoidal enlarged capillaries of the liver, the spleen, and the bone marrow are even discontinuous allowing the direct contact of blood with subendothelial structures. These modifications, fenestration and holes, increase the exchange of hormones and metabolites, respectively, between the blood and the surrounding tissue. Another example are the elastic and muscular arteries. Whereas in elastic arteries, the tunica media is particularly rich in extracellular matrix molecules with elastic properties, such as elastin and fibrillin, the muscular arteries have numerous concentric layers of smooth muscle sheaths. This mirrors the fact that elastic arteries conduct the blood, whereas the muscular arteries distribute the blood to different organs. By contraction and release, the muscular `cuff´ of the latter ones mediates vasoconstriction and vasodilation, respectively, thus regulating the blood flow into the supplied organ. A similar sphincter function is found in those arterioles which regulate blood flow into the downstream capillary bed. Vasoconstriction not only directs the blood flow but also regulates blood pressure. Although the cellular aspects of blood vessel functions are very important, this review will focus on the extracellular matrix (ECM) which not only provides an inert scaffold or stage for the vascular cells but in fact essentially contributes to the physiological functions of the blood vessels.

Fig. (1). Histological structure of a blood vessel. This schematic drawing of the vessel section shows the three generic layers of blood vessels: tunica intima, media, and externa. The structure and thickness of each layer vary characteristically in different vessel types (arteries, capillaries, and veins). Shown here is a typical wall structure of an elastic (left side) and muscular (right side) artery. The elastic arteries are characterized by the abundant presence of elastic lamellae (shown in blue), which are arrayed as meshwork and fibrils in lower density in the muscular arteries. The vascular smooth muscle cells are symbolized by spindle-like cells, indicating their orientation within the tunica media. On the subendothelial BM (shown in yellow) the collagen IV meshwork and the cruciform laminin molecules are indicated. However, they are not drawn to scale. The endothelial cells are oriented along the vessel axis and the blood flow. Marker proteins preferentially found in a certain layer of the vessel wall are named on the left side. However, some of these “marker” proteins are not exclusively found in the indicated layer, such as versican, which occurs in high amounts in the tunica adventitia of muscular arteries, but is also detectable in almost any layer. Moreover, the presence and locations of some of these proteins vary during vasculogenesis and angiogenesis.

The Extracellular Matrix of Blood Vessels

The tunica adventitia consists of fibroelastic tissue and links the blood vessel to the surrounding connective tissue. In veins, the tunica adventitia is the thickest layer. Additionally, it is characterized by the vasa vasorum, the capillaries which provide the support of nutrients and oxygen to the vessel layer which is most distant from the blood-conducting lumen. 3. THE SUBENDOTHELIAL BASEMENT MEMBRANE, A CENTRAL PART WITHIN THE TUNICA INTIMA Components of the Subendothelial BM The tunica intima consists of the simple layer of endothelial cells which are anchored on a basement membrane, a thin sheet of subendothelial connective tissue underneath the basement membrane and the inner limiting membrane (membrana limitans interna). The subendothelial basement membrane (BM) not only serves as foundation to which the endothelial cells attach but it also is the tissue compartment border between the endothelium and the vascular connective tissue. The molecular architecture of BMs has recently been reviewed ([2-4] and the review by Yurchenco in this issue). The major components of all BMs are the network-forming collagens IV and XVIII, laminins, nidogens/entactins, and the proteoglycan perlecan. Additional components are found in BMs of certain organs and tissues. A characteristic constituent for the subendothelial BM is von Willebrand-factor (vWF). The collagen family has been described in detail by Zent and coworkers in this issue and by others [5-7]. 13 different collagen types of the collagen superfamily are present in the vascular wall [8, 9]. Among the members of the collagen family, collagen IV with its isoforms forms a special twodimensional network within the BM. Four collagen IV molecules align via their N-terminal 7S-domains to a spider-like tetramer [10]. The C-terminal ends of two spider arms interact in a head-to head fashion via its non-collagenous (NC)domains [11], resulting in a sheet structure with a chicken wire-like appearance. Covalent cross-linkage of the collagen IV molecules both via their 7S- and NC-domains contributes to the mechanical stability of the BM which is necessary to withstand the blood pressure with considerable peaks of pressure in the arterial system. This key role of collagen IV for the mechanical stability of vascular basement membranes is corroborated by the observation, that knockout mice for the two major collagen IV isoforms, 1 and 2, die because of dilated blood vessels with frequent ruptures of vascular BMs both in embryonic tissues and placenta during embryonic development at days E10.5-11.5, when the heart commences to pump and blood pressure rises [12]. Other collagen types, such as collagens type XV and XVIII are part of or in close association with the BM [4, 13]. They belong to the multiplexin subfamily of collagens and are characterized by a central triple-helical collagen domain, which is interrupted several times and flanked by non-collagenous regions [4]. Collagen XVIII is probably involved in anchoring the BM to the underlying connective tissue [13]. Whereas collagen IV-deficiency does not ablate the assembly of other ECM molecules into the BM, laminins are indispensably required for BM formation [14, 15]. Members

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of the laminin family form a collagen IV-independent network within the BM [2, 16]. All laminins consists of three chains, , , and (reviewed by [17]). In an -helical coiled-coil structure, the three chains are wound around each other. C-terminally adjacent to this rod-like domain, the globular (G) domain which is solely made of the chain, folds into five homologous LG-subdomains, each about 180200 amino acids in length [18]. The G-domain harbors the binding sites for several cell adhesion receptors, such as distinct integrins [19], dystroglycans [20], Lutheran blood group receptor [21] and membrane-bound heparan sulfate proteoglycans, such as syndecans [22]. The trimeric combination of five , four , and three chains leads to 15 different laminin isoforms. Whereas laminin-111 is expressed in the subendothelial BM during embryogenesis, the tissue distribution of other laminin isoforms shows a considerable variability in different organs and tissue [23]. The two isoforms, laminin-411 and laminin511 (formerly laminin-8 and -10, respectively), are the major laminins in vascular BMs [21]. Laminin-411 is expressed by endothelial cells and deposited in all subendothelial BMs throughout the entire vascular system during all stages of development. Therefore, it can be considered as the typical vascular laminin [24-26]. Spatially and temporarily more restricted, laminin-511 is especially found in the vascular BM of capillaries and venules, but only postnatally. The expression of both laminin isoforms is regulated by cytokines during inflammation, and in turn it regulates diapedesis of immune cells (see below). The two independent networks of collagen IV and laminins within the BM are connected to each other by nidogens [24, 25]. Nidogen-1 and -2 have been described with almost identical localization and functions. They are present in all BMs, albeit in different amounts. Interestingly, nidogen-2 also occurs outside of basement membranes, e.g. in the elastic tissue around larger vessels [26, 27]. Deficiency of either nidogen isoform alone did not show any phenotype, whereas mice lacking both nidogens die shortly after birth with intense signs of cyanosis, indicating lethal functional deficiencies in essential organs, such as heart and lung [28]. The BMs of these mice are mostly absent or highly disorganized. Bleedings around the heart capillaries indicate leaking blood vessels. However, no pericardial haemorrhages were observed. Another main component of BMs is perlecan, which is the major heparan sulfate proteoglycan (HSPG) in the vascular ECM [29]. In general, ECM components have modular architectures consisting of several domains each of which serves one or more functions (e.g. interaction with other ECM molecules, binding of growth factors). Thus, like Swiss army-knifes, they can act as multi-task molecules. Perlecan, in particular, represents this principle. In addition to its five distinct protein domains I to V, it contains four heparan sulfate chains, which adds further functions to this versatile molecule. Through its interactions with various other BM molecules, such as nidogen-1 and -2, collagen IV, and fibronectin, it seems to stabilize the BM [30]. Consistently, perlecan seems to prevent proteolytic degradation of laminin [31, 32]. Likely due to the homology of its domain II with the LDL (Low density lipoprotein) receptor, perlecan


binds LDL and therefore may contribute to arteriosclerosis. The glycosaminoglycan (GAG) chains of perlecan interact with growth factors, such as fibroblast growth factor (FGF) 2, vascular endothelial growth factor (VEGF), and plateletderived growth factor (PDGF)-BB, thereby influencing their biological activities on cells [33]. As the GAG chains are added posttranslationally to the proteoglycan core protein, the heparan sulfate chains of perlecan molecules may vary in their lengths and sulfation patterns depending on their cellular sources. Thus, perlecan produced by endothelial cells may considerably differ from perlecan which is synthesized by vascular smooth muscle cells. Together with the different microenvironments of the vessel layers, this may explain the `perlecan paradox`, which describes the observation that perlecan induces proliferation of endothelial cells whereas it suppresses the growth of vascular smooth muscle cells [32]. In contrast, the C-terminal proteolytic fragment of perlecan is an anti-angiogenic factor inhibiting endothelial cell proliferation and migration [34]. Without completing the list of proposed functions of perlecan in vascular biology, its antithrombotic effect by binding anti-thrombin III is worth mentioning in this context [31, 32]. Although ablation of the perlecan gene does not compromise vasculogenesis severely, abnormalities in cardiac development are observed in the knockout mice [35, 36]. Perlecan deficiency causes disintegrity of the myocardial wall leading to hemorrhages. Additionally, an augmented proliferation of vascular smooth muscle cells occludes the heart-proximal arteries, which together with the transposition of the large arteries leads to a lethal crisis of most embryos at day E10.5 [35, 36]. Human disorders caused by a functional loss of perlecan have recently been identified as dyssegmental dysplasia of the Silverman-Handmaker type and as Schwart-Jampel syndrome type I, which become more manifest in skeletal dysplasias, similar to the mouse knock-out models [37, 38]. Expressed both by endothelial cells of the tunica intima and by vascular smooth muscle cells of the tunica media, SPARC (secreted protein acidic and rich in cysteine, also named BM40 or osteonectin) is a component of BMs. It is associated with collagen fibrils in the interstitial connective tissue [39]. In addition to its structural role of influencing the supramolecular aggregates of collagen fibrils, SPARC also modulates the biological effects of VEGF, PDGF, FGF-2 and other angiogenic factors. Under normal conditions, it is detected in the vascular layers only in low amounts. However, after vessel injury, SPARC is strongly upregulated during angiogenesis [40], similar to other matricellular proteins, such as thrombospondins [41] and osteopontin [42]. The von Willebrand factor (vWF) is a specific marker of the subendothelial BM, although its abundance varies in capillary beds of different organs, e.g. high expression in lung and brain vs. lower expression in liver and kidney. It is synthesized majorly by endothelial cells and marginally by megakaryocytes [43]. It is made of four different types of modules, termed A, B, C, and D, which line up in the molecule like beads on a string in the order D1, D2, D´, D3, A1, A2, A3, D4, B, C1, C2, CK [44, 45]. Each domain harbors distinct binding site for other ECM-molecules, such as collagen, or for cell adhesion molecules, such as the vWFreceptor GP Ib-IX-XI on the platelet surface. Additionally, vWF binds the blood clotting factor VIII.

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vWF molecules occur in at least two conformations: either rolled up in a compact conformation or stretched in an extended shape. The equilibrium between the two conformations depends on the flow rate and hence shear stress of the blood [46, 47]. The conformation of vWF rules its ability to assemble into supramolecular complexes and hence is highly relevant for its biological functions in hemostasis. Platelets preferentially attach to the extended conformation of vWF in higher aggregates. vWF is a large molecule of 2050 amino acids, which further associates into supramolecular complexes of up to 100 monomers. Endothelial cells constitutively secrete dimers and small aggregates of vWF, but can also store it in intracellular granules (Palade-Weibel bodies in endothelial cells, similar to the -granules in megakaryocytes), in which aggregation of high number-complexes occur. The latter ones are secreted by endothelial cells after various stimuli, such as bacterial or viral infections as well as under diabetic conditions [48]. As vWF is an ideal adhesion substrate for thrombocytes, an increased vWF level in blood is a risk factor for thrombosis, comparable to obesity, hypercholesteremia, smoking and hypertension [43, 48]. Lack of vWF leads to severe bleeding in von Willebrand disease, whereas increased levels of high number-aggregates of vWF caused by a lack of the specifically vWF-cleaving proteases ADAMTS13 result in enhanced propensity to thrombosis (thrombotic thrombocytopenia purpurea, TTP) [49]. vWF is not only stored in platelets and endothelial cells, but also forms an extended network underneath the endothelial cells within the subendothelial BM. After injury of the endothelial cell layer, this vWF carpet becomes accessible to platelets. They adhere to vWF via the GP Ib subunit of the GP Ib-IX-XI complex and via the integrin IIb3, which interact with the A1 domain and an arginine-glycine-aspartate peptide sequence (RGD in the one letter code of amino acids) within the C1 domain of vWF, respectively. In deeper wounds, also collagens become exposed and are decorated with vWF via the A3 domain. Thus, collagen is recognized by GP Ib-IX-XI. Alternatively, collagen also serves as direct ligand for collagen receptors, integrin 21 and GP VI, on thrombocytes [50]. Collagen interaction stimulates platelet activation and adhesion to collagen, fibrin, vWF and other ECM molecules. Consequently, the newly formed thrombus closes the leaking blood vessel and restores the integrity of the circulatory system, thus protecting the organism from hemorrhages and blood loss. Moreover, by secreting growth factors, platelets attract endothelial cells and fibroblasts, thus setting the stage for the subsequent steps of wound healing. As the subendothelial collagens and the vWF network are among the strongest stimuli for platelet activation, ECM molecules have to be covered by endothelial cells to avoid inappropriate thrombus formation and embolism. This is of particular interest in capillaries with discontinuous endothelia, such as the sinusoids of spleen and liver, where the perisinusoidal ECM within the red pulp and Disse´s space, respectively, is accessible to blood components through the gaps in the endothelial cell lining. The Subendothelial BM as Signaling Platform and as Cell-Impermeable Tissue Compartmentalization Border Endothelial cells are anchored to the BM of the tunica intima via different cell adhesion molecules, among which


integrins play an important role ([54-57], see also the review by Heino and Käpylä, this issue). Integrins are N-glycosylated proteins, consisting of two non-covalently associated, genetically non-related subunits, and . 18 and 8 subunits can combine to 24 different integrins. The integrins containing the 1 and 3 subunits are major receptors for ECM molecules [51]. The ectodomains of both subunits together form a globular head, which harbors the composite binding site for the ECM-ligand [52, 53]. Upon ligand binding, the ectodomain changes its conformation from a bent to an upright, activated position [54]. Thus, the conformational change is conveyed through the entire molecule, via the transmembrane domains towards the cytoplasmic tails, where cytoskeletal components and signaling molecules accept this information and convert it to an intracellular signal [51, 55]. Through the integrins, the BM does not only serve as a mechanical platform, to which endothelial cells are anchored, but it also provides microenvironmental cues for the cells, which trigger and sustain the differentiation state of the endothelial cells, such as their tight cell-cell contacts [56]. In addition, the BM as the intermediate environment of endothelial cells affects the metabolic activity of endothelial cells, such as production of leukocytes-adhesion molecules [57] and antithrombotic prostacyclins [58]. Syndecans, another group of cell adhesion molecules, are a family of four type I transmembrane proteoglycans in vertebrates that function with their GAG chains as coreceptors for many different ligands, including growth factors, cytokines, chemokines, and ECM molecules [59]. Syndecans 1, 2, and 4 are involved in angiogenesis, albeit with different effects. Syndecan-1 knockout mice show an increased angiogenesis in the cornea [60], whereas overexpression of syndecan-1 impairs angiogenesis [61]. Syndecan-2 knockdown leads to defects in angiogenesis [62], and ablation of syndecan-4 causes delayed wound healing with decreased angiogenesis in the granulation tissue [63]. The BM is impermeable to cells and constitutes the histological basis of tissue compartmentalization. The exceptions to this rule are physiologically leukocytes and pathologically tumor cells. Leukocytes transverse the subendothelial BM during their immunological surveillance patrol through tissues. They first adhere to the vessel wall, attach to the endothelial cells, and then sneak through or aside the endothelial cells to the BM, which they subsequently penetrate on their way to the underlying connective tissue [64]. This process (diapedesis) is strongly enhanced under the influence of selected cytokines during inflammation. The preferred extravasation sites of leukocytes are the venules, which are characterized by a special composition of the subendothelial BM [65]. Within these venular BM, collagen IV, laminin-511, and nidogen-2, but not perlecan, are less abundant compared to the subendothelial BM of other vessel sections. Organs with restricted accessibility for immune cells, such as the brain, Langerhans islets and seminiferous tubules of the testis, have even two basement membranes [66-68]. In the brain, the subendothelial BM surrounds the endothelial cells of the cerebral microvasculature [69]. A second sleeve of BM, the parenchymal BM, is formed by the astrocyte end feet. Whereas laminin-411 and -511 are abundant in the former one, the latter one contains preferentially laminin-111 and -211. Laminin-411 permits the extravasa-


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tion of leukocytes. In contrast, Laminin-511 restricts diapedesis of leukocytes, but is downregulated during inflammation [21, 66]. As a hallmark of malignancy, tumor cells extravasate and colonize tissues which are distant from their primary node. Adhesion of tumor cells to the endothelial cells and the subsequent penetration are key steps of the metastatic cascade [70, 71]. Different cell-adhesion molecules, integrins and matrix metalloproteinases (MMPs) are involved in this process [72-74]. Blocking cell adhesion molecules, such as integrins, may delay or even inhibit tumor cell infiltration [75]. Considering the pivotal role for cardiovascular biology and blood vessel integrity, the subendothelial BM is one of the key targets of snake venoms, which aim to hamper or kill the snake’s prey or predator [76]. Snake venoms contain proteases, which degrade the ECM molecules of blood vessels [77]. By destroying the BM, they result in aneurysm formation and hemorrhages. This effect is further acerbated by venom components, which block the interaction of platelets with ECM molecules, thus preventing hemostasis and leading to life-threatening bleedings, such as hemorrhagic stroke [78]. 4. THE ECM OF THE TUNICA MEDIA AND ITS (PATHO)PHYSIOLOGICAL ROLE Matrix Components Among the different vascular layers, the tunica media shows the most prominent variability in ECM structure, depending on the type of blood vessels. Heart-proximal elastic arteries contain an enormous amount of elastic fibers endowing them with their characteristic elastic properties. In the heart-distal arteries, the muscle cell layers are more pronounced. In capillaries, the tunica media is missing with the exception of a thin sheath of scattered pericytes. This minimizes the diffusion distance between blood and tissue. Thus, the exchange of nutrients, oxygen, and waste products is maximized. Endowing arteries with their elasticity and resilience to the pulsatile mechanic stretching, elastin contributes up to 50 % of the vessel’s dry weight [79]. Supramolecularly assembled into lamellae and fibers in elastic and muscular arteries, respectively, the elastic network makes up concentric layers within the tunica media, between which the clusters of vascular smooth muscle cells are sandwiched (Fig. (1)). In a cross-section of a vessel, the smooth muscle cells align in a fish bone-like array. Upon contraction, they reduce the distances between the elastic lamellae, thus decreasing the caliber of the vessel. Contracted smooth muscle cells typically show cork screw-shaped nuclei. Elastin occurs as a single isoform, which is genetically encoded on human chromosome 7 [79, 80]. Secreted as soluble tropoelastin by vascular smooth muscle cells, it is extracellularly processed to elastin by proteases. Several elastin molecules aggregate in a phase transition-like process called coacervation. The supramolecular elastin aggregates are further stabilized by cross-linking lysyl oxidases (LOX, LOXL) [79, 81]. Thus, an insoluble elastin network is formed.


The elastin molecule consists of several domains. Although crystallographic structural data are still missing, elastin is remarkably rich in sheets and -turns. The latter ones are likely to contain the hydrophobic repeat peptides VPGVG, VPGG, and APGVGV. The hydrophobic domains are usually flanked by lysine-rich regions, through which several elastin molecules are cross-linked. Different models exist to explain the remarkable elastic properties of the elastin molecules, which under mechanical forces can be reversibly extended to up to 220 % of the unstretched length. After the strain is relieved, the molecule elastically returns to its initial length [79]. This property explains the vessel resilience to the pulsatile pressure load on the arteries. Furthermore, like a spring, it stores a portion of the mechanically energy by allowing the artery to elastically widen under the systolically expelled blood, and releases this energy by driving the blood downstream during diastole under restoring its original caliber [82]. In addition to its structural role, elastin also regulates proliferation of vascular smooth muscle cells. Elastin and even peptides thereof inhibit growth of smooth muscle cells much stronger than BM proteins and collagen [83]. Although the mechanism for this effect is not yet clear and the cellular receptors for elastin has not been fully characterized [83, 84], elastin knock-out mice and patients suffering from loss-offunction mutations of the elastin gene show an overshooting proliferation of vascular smooth muscle cells, which eventually leads to vessel occlusion [83, 85, 86]. The human phenotype is called supravalvular aortic stenosis (SVAS) [87]. The proliferation of vascular smooth muscle cells and vessel occlusion caused by elastin deficiency resembles arteriosclerosis, yet without any sign of inflammation [88]. Both its mechanical properties and its ability to regulate proliferation make elastin an indispensable component of tissue-engineered vascular substitutes, which are used in the surgical replacement of occluded blood vessels, e.g. coronary arteries after heart attack [82]. The concentric elastin lamellae are connected with each other via microfibrils, which radially reach from one elastin lamella to another. Furthermore, these bridging molecules, among them fibrillin-1 and fibulin-5, play a role in the formation of these elastin sheets [81] and are candidates for mediating the interaction of the elastin network with its cellular receptors [83]. Thus, the vascular muscle cells are anchored within the microfibril network between the elastic lamellae. Among the microfibril components, fibrillin-1 belongs to a protein family consisting of multi-modular proteins with high molecular mass and self-aggregating ability [89, 90]. Fibrillin-1 is the major isoform of the three family members and is expressed in the adult organism. It can be reversibly extended by mechanical forces, albeit to a lesser extent and with a stiffness, which is two orders of magnitude higher than the one from elastin [81]. Marfan syndrome is caused by mutations within the fibrillin-1 gene [91] and becomes manifest in hyperflexibility of skin and joints as well as in the dilatation of blood vessels followed by acute aortic dissection, which is a frequent death cause of Marfan patients. In addition to its structural role, fibrillin-containing microfibrils also take part in transforming growth factor (TGF) signaling. The latent TGF binding protein (LTBP)

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interacts firmly with fibrillin-1. After its proteolytic release, TGF is a potent regulator of cell survival, proliferation, differentiation and ECM protein synthesis [92]. Another component of the microfibrils is fibulin-5. Five fibulins have been described and several splice variants increase their diversity [93]. Whereas fibulin-1 shows a widespread tissue distribution, including the BMs of blood vessels as well as the heart valves and septa, fibulin-3 and -4 are mainly detected in the vessel walls of capillaries and larger blood vessels [94]. Fibulin-3, -4 and particularly fibulin-5 is found along the vascular elastic fibers. The physiological importance of fibulin-5 and -4 in vascular physiology becomes evident in the respective knock-out mice. They both show tortuous aortas with a loss of compliance, with aneurysms, ruptures and hemorrhages, as well as severe emphysema and loose skin [95, 96]. Whereas the fibulin-5-deficient mice survive to adulthood, the mice lacking fibulin-4 die perinatally. The fibulin-1 knockout mice also die perinatally with hemorrhages in skin, muscles and perimuscular tissue which is caused by rupture of the endothelial lining of capillaries but not of larger blood vessels [97]. Patients with mutant fibulin-1 also bear a high risk of aneurysms as a vascular phenotype of the Ehlers-Danlos syndrome IV [83]. Among the various microfibril components [81], both fibulin-5 and fibrillin-1 are necessary for the formation of the elastic network in blood vessels. In a model suggested by Hirai and coworkers (2007) [98], fibulin-5 associates with the fibrillin-1 containing microfibrils to form the platform, to which secreted tropoelastin binds and undergoes coacervation. Lysyl oxidases (LOX and LOXL) also join and covalently cross-link these aggregates, thus forming the elastic fibers or lamellae. At the interface between the amorphous elastin phase and cells or microfibrils, yet another family of ECM proteins, the EMILINs, were identified [99]. Their name has been derived from their location: elastin microfibril interface located protein. All three known isoforms of EMILINs are homotrimers and consist of several domains, such as a collagenous domain, coiled-coil domain and a C1q domain, which the EMILINs share with the complement factor C1q and the vascular collagen VIII. Along with the elastic microfibrils, collagen fibrils are found between the elastic lamellae. In contrast to elastin and the microfibrils, the collagen fibers are not elastically deformable, but bear the tensile forces of the vessel and set the limits of its elastic expansibility. As components of these collagen fibers, type I and III collagens are the major collagens within this portion of the tunica media. Although the phenotype of highly fragile bones (brittle bone disease, osteogenesis imperfecta) is more known, patients with a deficiency of functional collagen I also suffer from vascular ruptures. Similarly, collagen I-deficient mice, one of the first knockout experiments of an ECM protein, are embryonic lethal [100]. Between days E12 and E13, they die due to ruptures of their blood vessels, which do not withstand the pulsatile pressure caused by the beating heart. The innermost and outermost elastic lamellae are called inner and outer limiting membrane, respectively (membrana limitans interna and externa) (Fig. (1)). They represent the


borderline between the tunica media towards the tunica interna and adventitia, respectively. How the subendothelial BM is anchored to the underlying interstitial connective tissue and the inner limiting membrane is still an open question. Type XVIII collagen interacts C-terminally with the collagen IV network and N-terminally with fibrillin-containing microfibrils [13] and thus may serve as good candidate for anchoring the subendothelial BM with the elastic fiber network. Type XVI collagen may also contribute to the connection between the elastic fiber network and the collagen matrix. Expressed by VSMC, it is found in close vicinity of microfibrils and additionally associated with the fibrillar collagens (type I, III) within the arterial tunica media [101, 102]. Furthermore, collagen XVI contains a binding site for 11 integrin, the major collagen receptor on VSMCs [103, 104]. Whereas fibril-forming collagens (I, III) are majorly found within the tunica media, collagen VI and VIII, which do not form D-staggered fibrils [6], are detected in the interstitial connective tissue between the subendothelial BM and the inner limiting membrane. Collagen VI molecules aggregate into beaded filaments, which are usually found close to fibrillin-containing microfibrils [105, 106]. Despite the obvious phenotypes of collagen VI mutations in Bethlem myopathy and Ullrich congenital muscle dystrophy [107], no vascular defects, such as arterial dissection, vessel rupture or aneurysms have been described in these patients. Collagen VIII belongs to the subclass of collagens, which form hexagonal networks [6]. Although its function is not fully understood yet, it may play a role in arteriosclerosis [9]. The Mural Cells of the Tunica Media The extracellular matrix of the tunica media is mainly produced by vascular smooth muscle cells (VSMCs). Embedded in the network of microfibrils and fibril-forming collagen-I and –III, every smooth muscle cell in the tunica media is encapsulated by a BM [106], which contains typical BM proteins, such as laminins and collagen-IV, yet it appears incomplete in electron micrographs [108]. Both in vitro and in situ, VSMCs show biological plasticity and can appear in two different phenotypes, generally named contractile (differentiated) and secretory (less differentiated, migratory) phenotype. Which phenotype is adopted by a VSMC, depends on the surrounding ECM, on growth factors and cytokines, as well as certain (patho)physiological situations, such as vessel repair and arteriosclerosis [104, 108]. Most VSMCs of the tunica media show a contractile (differentiated) phenotype with a low number of secretory granules and a high content of -smooth muscle actin [108]. The latter is the molecular basis for their ability to contract and exert mechanical forces onto their pericellular matrix. These forces are transduced by cell-adhesion molecules, especially the collagen-binding integrin 11 as well as the lamininbinding integrin 71 and dystroglycan [104]. Whereas the integrins are located in adherens junctions which are aligned in prolate ribs along the longitudinal cell axis, the dystroglycan complexes are found in caveolar domains in the plasmalemma between these ribs [104]. In contrast to this contractile phenotype, the other phenotype of VSMC is characterized by its ability to proliferate, to migrate and to produce and secrete ECM proteins. This capability is mirrored by a


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high number of secretory vesicles and a comparatively low amount of -smooth muscle actin within these cells [108]. In the secretory phenotype, another subset of integrins, such as the fibronectin receptor 51, the integrins 91 and 41 are predominantly expressed. In parallel, the fibronectin splice variants V (IIICS) and EIIIA, which harbor binding sites for the integrins 51, 41, and 91, are abundant around these cells [108], and they are typical for wound healing [109]. The subcellular arrangement of the integrins in longitudinal ribs on VSMCs is lost in the secretory phenotype. In addition, the secretory VSMC are characterized by their less polarized rhomboid cell morphology [104]. The two phenotypes of VSMCs can interconvert under certain physiological or pathophysiological conditions. After vasculogenesis is completed, the VSMCs in the tunica media are differentiated and contractile, thus they are able to regulate the vessel caliber. Upon vessel repair after injury or as a step in arteriosclerosis [88], the contractile VSMCs dedifferentiate to the secretory phenotype under the influence of inflammatory cytokines and growth factors, such as PDGFBB and TGF, and in the presence of fibronectin and other ECM molecules which are typical for regenerating tissue. Interestingly, elastin and its fragments are among the most effective inhibitors of this dedifferentiation process. Thus, less-differentiated VSMCs migrate into the wound area of an injured vessel and form a neointima [88]. Then they synthesize and secrete vessel-characteristic ECM proteins, and the pericellular BM reappears. Under physiological conditions, VSMCs consequently resume their differentiated and contractile phenotype. Under dysregulated conditions, such as in arteriosclerosis, the less differentiated, proliferative phenotype persists. This results in an overshooting thickening of the tunica media due to an increase in cell number and an augmented production of ECM [8], which may occlude the vessel lumen. Furthermore, caused by an increased production of matrix-degrading proteinases, especially MMPs, the arteriosclerotic plaque may rupture leading to thrombosis and embolism [8, 88]. The internal lining of capillaries with endothelial cells is covered by an outer sheet of pericytes, which adjoin the endothelial cell sheet and the subendothelial BM of the capillary in a scattered manner. They express -smooth muscle actin and are individually encapsulated by a BM, similar to the smooth muscle cells of the tunica media. Thus, pericytes can be considered as the residual homologue of a tunica media along capillaries. The mural cells stabilize the blood vessel not only mechanically but they also support the endothelial cell lining. Direct cell-cell-interactions via neural cell adhesion molecule (N-CAM) as well as paracrine cross-talks via PDGF-BB and angiopoietins between pericytes and endothelial cells mediate and regulate this support, inducing and maintaining the physiological properties of pericytes and the tight lining of endothelial cells [110-112]. 5. THE MATRIX OF THE TUNICA ADVENTITIA The tunica adventitia consists of fibroelastic connective tissue. Its ECM is characterized by matrix molecules, such as versican, which however are not exclusively found in the outer vessel layer. Vascular versican comes in three different splice variants, V0, V1, and V3, differing in the length of their


protein cores and the number of attached chondroitin sulfate GAG chains. Due to their capability to swell in aqueous environment, versican, like other proteoglycans, such as biglycan and decorin, contributes to the compressibility of the vessel wall. Lipoproteins also associate with the chondroitin sulfate side chains of versican. Under pathological conditions, this interaction contributes to arteriosclerosis [113]. During inflammation and arteriosclerosis, expression of versican is highly upregulated in endothelial cells, VSMCs and fibroblasts of the tunica adventitia. In healthy muscular arteries, versican is most prominent in the tunica adventitia, but in elastic arteries and veins it is found in all vessel wall layers. The potential of versican to interact with fibrillin-1 [114], with fibulins-1 and -2 [115, 116] and with other vascular ECM molecules makes versican a well apt component of the matrix scaffold of the tunica adventitia, which connects the blood vessel with the surrounding connective tissue [117]. 6. VASCULAR ECM IN ANGIOGENESIS Angiogenesis is the formation of new capillaries from already existing vessels. It is mostly driven by tissue hypoxia and occurs physiologically, e.g. during endometrial growth as part of the menstrual cycle, or in wound healing, graft reception and after ischemic lesions. Pathologically, however, it can also be induced by a tumor node. Angiogenesis has to be distinguished from vasculogenesis and arteriogenesis. The former term is used for the de novo formation of vessels during embryonic development by condensation of mesenchymal cells into capillary plexus that differentiate into arteries, veins, and capillaries [112]. The latter term denotes the remodeling of newly formed or preexisting blood vessels into larger arteries and collateral vessels. In angiogenesis, an existing capillary meshwork is remodeled by sprouting, intussusceptive microvascular growth and fusion into a mature and functional vascular bed [118, 119]. Angiogenesis also includes penetration by sprouting of vessels into as yet avascular regions of the tissue and the recruitment of mural cells. Thereto correct interactions among endothelial cells, pericytes, and surrounding cells, as well as cell-matrix interactions with the surrounding ECM, in particular the BM, are crucial (Fig. (2)). The last few years have seen great achievements in deciphering the molecular basis of both vasculogenesis and angiogenesis. Especially, signaling mechanisms, such as the pathways of vascular endothelial growth factor (VEGF) and Notch, have been unraveled (reviewed in several recent reviews, such as [116, 121-125]. In this review, we attempt to describe the role of vascular ECM molecules in the angiogenic process, although it must be stated that ECM-molecules have an intense crosstalk with growth factors, by storing, releasing, masking, or degrading them. The Angiogenic Cascade For successful angiogenesis multiple consecutive steps are necessary, which all require interaction between cells and ECM. The angiogenic cascade comprises the following steps: (i) degradation of BM with MMPs (soluble and cellanchored MMPs), (ii) outgrowth of a tip cell and proliferation of stalk cells influenced by cell-matrix contacts, (iii)

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tube formation with a lumen sealed by tight junctions at cellcell contacts, (iv) synthesis of BM proteins and assembly of a new basement membrane, and finally (v) pruning of excess endothelial tubes that are not further supported, and simultaneous stabilization of the remaining tubes by pericytes and maturation into capillaries. The Basement Membrane is Degraded by MMPs To advance into avascular tissue regions, the basement membrane ensheathing an existing capillary has to be opened as the first step of angiogenesis. Furthermore, the surrounding ECM needs to be partially degraded in order to facilitate the infiltration of cells. Therefore, at the onset of angiogenesis, endothelial cells acquire a proteolytic phenotype [120, 121]. Of major importance in this connection are MMPs [74, 122]. Ghajar et al. (2008) reviewed recent findings how MMPs control capillary morphogenesis [123]. In contrast to MT-MMPs, soluble MMPs seem to play rather a minor part in angiogenesis, although MMP-1 and MMP-9 are induced during tube formation, but this may vary in different vascular beds [124]. MMP-9, which can be inhibited by TSP-1, can act both angiogenic, by releasing VEGF, and anti-angiogenic, by generating tumstatin [125, 126]. MMP activity is also necessary for the angiogenic activity of VEGF and bFGF in collagen I implant-induced angiogenesis in vivo, and MMP-2 association is necessary for V3 integrinmediated angiogenesis, where it seems to facilitate cell migration through the extracellular matrix [127, 128]. As a consequence of basement membrane degradation, endothelial cells lose their contacts with laminin and get into contact with interstitial collagen, which activates signaling cascades that lead to reorganization of the cytoskeleton, to changes of their cell shapes and finally to sprouting morphogenesis [124, 129]. Furthermore, they become motile and align into chords [130]. A precisely regulated temporal and spatial degradation of the ECM is important for undisturbed angiogenesis. Cell-Matrix Contact Influences the Outgrowth of Tip Cells and Proliferation of Stalk Cells Macromolecular cues as well as diffusible gradients are responsible for directing the outgrowing endothelial cells into the region to be vascularized. Utilizing the same molecules in some cases, endothelial sprouts are guided similarly to growing nerve fibers (reviewed by [112]). Upon stimulation by VEGF-A, an endothelial cell differentiates into a socalled tip cell, and upon opening of the basement membrane, it leads the proliferating stalk cells that form a new vessel sprout along a gradient of VEGF-A [131, 132]. DLL4Notch1 signaling controls VEGF-A-induced differentiation of endothelial cells into tip cells, thus regulating vessel sprouting and branching in an appropriate pattern. It also determines the adequate ratio between tip and stalk cells required for a correct sprouting and branching pattern within the angiogenic sprout [133]. Sprout extension can occur either by migrating endothelial cells that follow the tip cell or by EC proliferation along the sprout longitudinal axis [125, 137, 140]. The tip cell of an endothelial sprout expresses MT1-MMP activity, with which it opens its way into the surrounding matrix [134]. Later in angiogenesis, when the stalk cells get in contact with pericytes, the endothelial cells



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Fig. (2). The equilibrium of angiogenesis. Angiogenesis is ruled by a delicate balance of factors, which belong to the groups of ECM-molecules, their respective cell adhesion receptors, and the matrix degrading metalloproteinases (MMPs), as well as growth factors and the cellular environment of endothelial cells. Fibrin and fibronectin as ECM proteins of the provisional ECM in wound healing and fibrillar collagens, typical of interstitial ECM, together with their respective integrin receptors, induce angiogenesis. The collagen-binding integrins, 11 and 21, and the RGD-dependent integrins, like the angiogenesis-related vitronectin receptor, V3 integrin, signal the information about the pericellular ECM into the endothelial cells, which then together with signals from the angiogenic growth factor, vascular endothelial growth factor (VEGF), triggers the angiogenic cascade. Certain MMPs are expressed by endothelial cells during angiogenic sprouting. On the other hand, the basement membrane and its proteins (laminins and the network-forming collagen IV) via their cellular receptors bring endothelial cells into the quiescent state, thus bringing angiogenic sprouting to an end, or keep endothelial cells in this quiescent state. Pericytes and certain growth factors sustain this quiescent state, thus stabilizing the vessel, which thereby matures. The named factors affect each other and must be seen as an interactive cross-talk network, which all together determine endothelial cells for angiogenic sprouting or for quiescence.

down-regulate MT1-MMP again [134]. The interaction of endothelial cells with pericytes also induces expression of TIMP-2 in endothelial cells and TIMP-3 in pericytes in order to switch off the proteolytic phenotype of the endothelial cells [135]. Tube Formation Initiated by the matrix-integrin-cytoskeletal signaling axis, an endothelial cell cord is transformed into a tube by the formation of vacuoles within the endothelial cells, by subsequent intra- and intercellular fusion of these vacuoles, and by strengthening of cell-cell contacts [136-138]. In an integrin-dependent process, pinocytotic vesicles are formed that coalesce into vacuoles, which then fuse to large intracellular compartments [130, 143, 146]. Subsequently, these compartments fuse by exocytotic events, thus forming a continuous lumen [124, 136, 139]. Interestingly, activity of MTMMPs on the endothelial cell surface is necessary for intracellular vacuole and lumen formation [140]. The new tubes

need to be carefully sealed for proper vessel function; the specific mechanisms of endothelial tight and adherens junctions are reviewed by Wallez and Huber (2008) [56]. A New Basement Membrane is Constructed by Synthesis and Assembly of BM Proteins In order to provide structural and organizational stability and to change the EC phenotype to a quiescent one, the endothelial tube is ensheathed by a new basement membrane (BM) (Fig. (2)). The BM is produced by endothelial cells in cooperation with surrounding cells [124]. It is built up of laminins as a scaffold and essential BM components, such as collagen IV, perlecan, nidogens, and collagen XVIII, while it simultaneously prevents contact to collagen I [124, 141, 142]. Maturation of New Capillaries An immature capillary plexus is excessively branched and characterized by a surplus of tubes. At this stage of an-


giogenesis, the vascular tree shows high plasticity by undergoing regression of unneeded vessels, called pruning, and by expansion of others to optimize the blood supply. As there are no pericytes around the newly formed vessel yet, pressure-regulating vasoreactivity is reduced. The pulsating blood flow dilates the vessel, which renders its wall hyperpermeable and leads to increased interstitial and reduced transcapillary pressure and blood flow [143]. Flow speed and shear stress are sensed by endothelial cells and the architecture of the vascular network is improved by pruning and removal of excess vessels to optimize perfusion [144]. In addition, vascular pruning is also proposed to represent an adjustment to surplus oxygen [145]. Moreover, the surrounding ECM also exerts an effect on growth and orientation of angiogenic microvessels, as growing microvascular structures align along the direction of mechanical strain and the traction which is generated by endothelial cells [146]. Some soluble MMPs appear to be produced especially for tube regression rather than for tube formation, e.g. MMP-1 and MMP-10 [140]. Recruitment of pericytes and association with mural cells is essential for maturation of endothelial cell tubes into blood vessels. VEGF not only increases vascular permeability while angiopoietin-1 (Ang-1) reduces it, but these growth factors also control vascular maturation by causing the recruitment of perivascular mural cells to the endothelial plexus [143, 145, 147-149]. These cells express low levels of -smooth muscle actin. Coming from the arterioles, they proliferate and migrate longitudinally along the previously formed vessels [145]. They are recruited by endothelial cells that secrete platelet-derived growth factor BB (PDGF-BB) and recruit pericytes by activating their respective receptor PDGFR. PDGF-BB is retained close to the secreting endothelial cells by a C-terminal retention motif that contains basic amino acids. With this retention motif it interacts with the heparan sulfate proteoglycans in the ECM around the endothelial cells, thus stabilizing a guidance cue for pericytes [150]. The degree of sulfation of heparan sulfate proteoglycans in the ECM around the endothelial cells governs the diffusibility of PDGF-BB and so controls immigration and settlement of the pericytes [151, 152]. PDGF-BB signaling through PDGFR is enhanced by MT-MMP-1 [153]. Pericytes are located at the ablumenal face of endothelia. Embedded in a shared basement membrane they represent the interphase between endothelium and surrounding tissue, where these cells and the matrix molecules synthesized by them contribute significantly to the stability and permeability of capillaries. Evidence that pericytes also can be considered as gatekeepers in hematogenous tumor cell metastasis is reviewed by Gerhardt and Semb (2008) [110]. Not by sheathing, but as a result of pericyte-endothelial interaction, mature vessels lose their dependence on VEGF-A, and thus the plasticity window is closed [145, 154]. Pericytes and vascular smooth muscle cells express Angiopoietin-1 (Ang-1) on their surface, whereas endothelial cells express the corresponding Tie-2 receptor. Binding of Ang-1 to Tie-2 causes tight endothelial cell-pericyte interaction and vessel stabilization, but it can be antagonized by Ang-2 expression [162, 163]. An optimal number of pericytes is necessary for adequate angiogenesis, because on the one hand too many pericytes stabilize the newly created vessels and thus inhibit further sprout-

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ing, and on the other hand too few pericytes render the vessels unstable to support adequate blood flow [127, 162, 164]. Regulation of Angiogenesis: Soluble Growth Factors and Insoluble ECM Proteins Angiogenesis is regulated by many local and circulating factors, such as VEGF, PDGF, angiopoietins, and basic fibroblast growth factor (bFGF) (Fig. (2)). Vascular endothelial growth factor A (VEGF-A) is the most important molecule for the control of blood vessel morphogenesis. It belongs to a family of potent angiogenic regulators, together with VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). Endothelial precursor cells (EPCs, angioblasts) use it as a chemotactic cue in vasculogenesis during embryonic development, and it is involved in nearly all steps of the angiogenic cascade under physiological and pathological conditions. The cellular response to VEGFs is influenced by different parameters, such as the expression of different VEGF family members [155], different splice variants [156], or binding to different receptors [157]. VEGF interacts with the tyrosine kinase receptors of the VEGF receptor (VEGFR) family. Binding of VEGF-A to VEGFR1 and VEGFR2 is of central importance in regulating angiogenesis, and the ratio of different VEGF-A isoforms modulates vessel growth and patterning. On the other hand, VEGF-C and VEGF-D stimulate lymphangiogenesis by binding to VEGFR3 [158, 159]. While initiation of angiogenesis is dependent on VEGF, platelet-derived growth factor (PDGF) is required for maintenance of angiogenesis and to prevent dissociation of pericytes from endothelial cells. By its positively charged C-terminal retention motif, VEGF is immobilized on heparan sulfate polysaccharide chains in the pericellular ECM of endothelial cells [150, 160] and can be released by the cleavage of the proteoglycan by MMP-9 [161]. Thus, the extracellular matrix acts on the one hand as a biochemical regulator of the endothelial phenotype, and on the other hand it regulates angiogenesis by serving as a structural framework to support sprout and neovessel structure and function [160]. Important signals for initiation as well as termination of cellular processes that are involved in vessel formation come from macromolecules of the extracellular matrix [129]. The most abundant proteins in the ECM are collagens. Fibrillar collagens, such as collagen I, which are substantial constituents of the interstitial connective tissue, may be considered as the main extracellular matrix components that drives angiogenesis [124, 128]. In contrast, laminins as typical components of BMs, keep endothelial cells in a quiescent state [124, 162]. In addition to fibrillar collagens of the interstitial ECM, fibronectin as a prominent component of the provisional ECM in wound beds acts also pro-angiogenic (Fig. (2)) [126, 163, 164]. A certain splice variant of fibronectin is transiently expressed around developing and sprouting vessels [109, 165]. During vessel maturation fibronectin expression ceases [165]. Although their regulative roles in angiogenesis have not been fully established, members of the tenascin and thrombospondin family show a spatially and temporary expression


in sprouting vessels. Tenascin-C, -R, -X, and -W form a family of large hexameric ECM glycoproteins that can interact with several other ECM proteins and cell surface receptors, such as integrins [166]. Tenascin-C, classified as an adhesion-modulating protein, is an important regulator during embryogenesis, wound healing and cancer progression [167]. It serves as microenvironmental cue that mediates postnatal neovascularization by modulating the activity of endothelial cells and bone marrow-derived endothelial progenitor cells [168]. Although tenascin-C is not expressed in normal adult myocardium, it is involved during cardiac development in several important steps, e.g. in the initial differentiation of cardiomyocytes, and in coronary vasculogenesis and angiogenesis [169]. The thrombospondin family comprises five multifunctional large glycoproteins that can be subdivided into two groups with respect to their quaternary structure; thrombospondins-1 and -2 form trimers, whereas thrombospondin3, -4, and -5 are pentamers [170]. Thrombospondin-1 and -2 can shape the structure of the ECM in several ways: they can bind directly to collagen and fibronectin, and in addition they can modulate proteases, such as MMPs and plasmin. By activating TGF, trombospondin-1 can also modulate the synthesis of other ECM proteins. Although expressed around sprouting tumor vessels, thrombospondin-1 and -2 are potent inhibitors of angiogenesis, as they inhibit migration and induce apoptosis of endothelial cells [44, 179, 180]. Some of these anti-angiogenic effects can be mimicked by peptides derived from the thrombospondin sequence [171, 172]. However, in some experimental models thrombospondin-1 can act pro-angiogenic by attracting and stimulating smooth muscle and inflammatory cells that release pro-angiogenic factors ([171] and references therein). Matrikines: Angiogenesis-Related Fragments of ECMMolecules Beyond their structural roles, some ECM proteins even are functional in form of their proteolytic fragments which are soluble and have cytokine-like activities. These peptides generated by partial degradation of ECM macromolecules that influence cell activities are named matrikines. Some of them have recently raised hopes in antiangiogenic tumor therapy (for non-matrix- and matrix-derived angiogenesis inhibitors see also the reviews of [122, 173]). For example, the C-terminal fragment of collagen XVIII, named endostatin, strongly inhibits angiogenesis [174]. It disrupts the cytoskeleton of endothelial cells via a heparan sulfate-dependent 51 integrin interaction [175], however the molecular mechanism of its effect is not fully understood [176]. Another homologous angiostatic molecule, called restin, is cleaved from the 1 chain of collagen XV [177]. While perlecan is typically described as angiogenic, a Cterminal fragment from domain V, called endorepellin, is strongly anti-angiogenic [29, 178]. Endorepellin interacts with 21 integrin which is the functional receptor for its angiostatic activity [34, 179]. From collagen IV the angiogenesis inhibitors arresten, canstatin, tumstatin, and the unnamed NC1 domain of the 6(IV) chain can be released [180, 181]. Arresten corresponds to the NC1 domain cleaved off from the collagen IV 1 chain, and it inhibits endothelial


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cell migration and proliferation as well as tumor growth and can induce apoptosis via integrin 11 [182]. Canstatin comprises the NC1 domain of the 2 chain [183]. It inhibits endothelial cell proliferation, migration, and tube formation via the integrins v3 and v5 [184]. The C-terminal NC1 domain from the collagen IV 3 chain can also be cleaved off efficiently by MMP-9 and is then called tumstatin [125, 185]. It suppresses tumor-driven angiogenesis presumably via integrin V3 [125, 186]. Also vastatin, the recombinantly produced collagen VIII(1) NC1 domain, inhibits endothelial cells [187]. A C-terminal fragment of the fibronectin III1C region, called anastellin [188] can induce fibronectin polymerization in the absence of cells, which again has an inhibitory effect on angiogenesis [189-191] by interfering with ERK signaling in endothelial cells [192]. Although not derived from an ECM protein but from an ECM-degrading protease, angiostatin, a 38kDa catalytically inactive fragment of the protease plasminogen is also a potent angiogenesis inhibitor [193]. In contrast to the anti-angiogenic matrikines, elastinderived fragments, called elastokines, are highly proangiogenic [194-196]. All biologically active elastin fragments show the consensus sequence XGXXPG that promotes a turn conformation [197, 198]. In a human microvascular endothelial cell line, -Elastin, which is a mixture of hydrolytically generated elastin fragments as well as the peptide (VGVAPG)3 induce MT1-MMP production via activation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) [199, 200]. 7. APPLICATIONS AND OUTLOOK Excessive or deficient regulation of angiogenesis underlies many pathological situations. Thus it is important to exactly monitor angiogenesis and find ways to take modulating action within a pharmacological treatment. Advances in noninvasive imaging of angiogenesis by magnetic resonance imaging (MRI), computer tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) have been made recently by developing radiotracers that target integrins, ECM molecules, VEGF and its receptors, MMPs and activated endothelial cells [143, 201]. These novel technologies along with molecular and pharmacological studies on pro- and antiangiogenic factors will provide new insights in the complex process of vessel sprouting and its pharmacological treatment in tumor angiogenesis. ACKNOWLEDGEMENT: We thank the Deutsche Forschungsgemeinschaft for its financial support (grant: Eb 177/5-1). We wish to apologize to authors of important work not cited here for reasons of space limitation. ABBREVIATIONS BM

= Basement membrane

ECM

= Extracellular matrix

EMILIN = Elastin microfibril interface located protein


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FGF

= Fibroblast growth factor

[22]

GAG

= Glycosaminoglycan

[23]

LDL

= Low density lipoprotein

LTBP

= Latent TGF binding protein

MMP

= Matrix metalloproteinase

PDGF

= Platelet derived growth factor

SPARC

= Secreted protein acidic and rich in cysteine

TGF

= Transforming growth factor-beta

VEGF

= Vascular endothelial growth factor

VSMC

= Vascular smooth muscle cells

vWF

= Von Willebrand factor

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[25]

[26]

[27]

[28]

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