It is firmly established that angiogenesis is an absolute

Brief Review Role of the Matrix Metalloproteinase and Plasminogen Activator Plasmin Systems in Angiogenesis Michael S. Pepper Abstract Extracellular proteolysis is an absolute requirement for new blood vessel formation (angiogenesis). This review examines the role of the matrix metalloproteinase (MMP) and plasminogen activator (PA) plasmin systems during angiogenesis. Specifically, a role for gelatinases (MMP-2, MMP-9), membrane-type 1 MMP (MMP-14), the urokinase-type PA receptor, and PA inhibitor 1 has been clearly defined in a number of model systems. The MMP and PA-plasmin systems have also been implicated in experimental vascular tumor formation, and their role during this process will be examined. Antiproteolysis, particularly in the context of angiogenesis, has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. (Arterioscler Thromb Vasc Biol. 2001;21:1104-1117.) Key Words: extracellular matrix endothelium metalloproteinase plasminogen cancer It is firmly established that angiogenesis is an absolute requirement for the growth of normal and neoplastic tissues. 1 Many biological processes, including angiogenesis, depend on tightly controlled interactions between cells and the extracellular matrix (ECM). These interactions are mediated by (1) integral membrane proteins, including integrins, which provide a link between the ECM and the cytoskeleton, and (2) extracellular proteinases and their inhibitors, which mediate focal degradation of components of the ECM, some of which (eg, the fibrillar collagens) are highly resistant to broad-spectrum proteases. Most of the relevant extracellular proteolytic enzymes belong to one of two families: the serine proteases, in particular, the plasminogen activator (PA) plasmin system, and the matrix metalloproteinases (MMPs). 2 4 Like most other biological processes, angiogenesis is the result of subtle and often complex interactions between regulatory and effector molecules. To facilitate its analysis, it is useful to divide angiogenesis into a phase of activation (sprouting) and a phase of resolution. The phase of activation encompasses (1) increased vascular permeability and extravascular fibrin deposition; (2) vessel wall disassembly; (3) basement membrane degradation; (4) cell migration and ECM invasion; (5) endothelial cell proliferation; and (6) capillary lumen formation. The phase of resolution includes (1) inhibition of endothelial cell proliferation; (2) cessation of cell migration; (3) basement membrane reconstitution; (4) junctional complex maturation; and (5) vessel wall assembly, including recruitment and differentiation of smooth muscle cells and pericytes. Implicit in the definition of the resolution phase is the establishment of blood flow in the newly formed vessel. 1 Extracellular proteolysis has been implicated in many of these processes, including basement membrane degradation, cell migration/ecm invasion, and capillary lumen formation (the Table). 5 The classic descriptions by Clark and Clark in 1939 6 of new blood vessel formation in transparent chambers in the rabbit ear clearly demonstrate the production of fibrinolytic activity by growing capillary sprouts: As new blood vessels advanced... the fibrin outside them dissolved, leaving a clear space surrounding the new vessels which was filled at times with fluid but more commonly with a transparent gelatinous substance containing occasional blood cells, macrophages and fibroblasts. Although long, tapering, solid tips might be seen extending directly into the fibrin, which was still present beyond the advancing capillary network, the new capillaries, and even capillaries in which the circulation had not yet started, but which possessed a lumen and a rouleaux of erythrocytes, were all surrounded by a characteristic clear space from which fibrin had disappeared. A similar phenomenon of fibrin dissolution was described in studies of acute inflammation induced by subcutaneous implantation of a fibrin clot in the rat. 7 Capillary sprouts were associated with empty zones in the fibrin clot, which was indicative of fibrin degradation. Fibrinolytic activity decreased progressively in the later stages of the inflammatory process as the fibrin clot was replaced by connective tissue. These two articles were among the first to describe the presence of extracellular proteolytic activity in the endothelial pericellular environment during angiogenesis. They also highlighted the existence of a temporal sequence of matrix dissolution and assembly, in which fibrin provides a temporary matrix scaffold for migrating endothelial cells, which with time is replaced by a more mature collagenous matrix. Received April 6, 2001; revision accepted May 25, 2001. From the Department of Morphology, University Medical Center, Geneva, Switzerland. Correspondence to Dr M.S. Pepper, Département de Morphologie, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Genève 4, Switzerland. E-mail: michael.pepper@medecine.unige.ch 2001 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org 1104

Pepper Extracellular Proteolysis and Angiogenesis 1105 Extracellular Proteolysis and Angiogenesis Breaching of mechanical barriers Basement membrane degradation Cell migration and ECM invasion Lumen formation Regulation of cytokine activity Activation of latent TGF- Release of matrix-bound bfgf, VEGF Release of membrane-anchored TNF- Inactivation of MCP-3 (and generation of a chemokine antagonist) Generation of bioactive degradation products ECM components Plasminogen 3 angiostatin, kringle 5 (inhibitory) Collagen IV 3 arresten, canstatin, tumstatin (inhibitory) Collagen XV 3 restin (inhibitory) Collagen XVIII 3 endostatin (inhibitory) Thrombospondin (inhibitory) Hyaluronan (stimulatory) SPARC (stimulatory and inhibitory) Other molecules Prolactin (inhibitory) Platelet factor 4 (inhibitory) MMP-2 3 PEX (inhibitory) Antithrombin (inhibitory) Calreticulin 3 vasostatin (inhibitory) High-molecular-weight kininogen domain 5 3 kininostatin (inhibitory) One of the earliest events in the activation (sprouting) phase of angiogenesis is basement membrane degradation, and that this process may be mediated by matrix metalloproteinases (MMPs) was demonstrated almost 20 years ago. 8 These authors described the presence of type IV and V collagenolytic activity produced by bovine endothelial cells induced to migrate down a chemotactic gradient. No collagenolytic activity was observed in the absence of the gradient (even in the presence of the chemotactic agent), illustrating that enzyme activity was associated with the migration process. Considerable progress has since been made in understanding the mechanisms of angiogenesis. Thus, angiogenesis is controlled by the net balance between molecules that have positive and negative regulatory activity. 1 With the exception of angiogenesis that occurs in response to tissue injury or in female reproductive organs, endothelial cell turnover in the healthy, adult organism is very low. The maintenance of endothelial quiescence is thought to be due to the presence of endogenous negative regulators, because positive regulators are frequently detected in adult tissues in which there is apparently no angiogenesis. The converse is also true, namely, that positive and negative regulators often coexist in tissues in which endothelial cell turnover is increased. This has led to the notion of the angiogenic switch, in which endothelial activation status is determined by a balance between positive and negative regulators: in activated (angiogenic) endothelium, positive regulators predominate, whereas endothelial quiescence is achieved and maintained by the dominance of negative regulators. The switch involves either the induction of a positive regulator and/or the loss of a negative regulator. 9,10 Used initially in the context of tumor progression to describe the passage from the prevascular to the vascular phase, the notion of the switch can also be applied to developmental, physiological, and pathological angiogenesis. Positive regulators of angiogenesis include, but are not limited to, the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) families of cytokines and their tyrosine kinase receptors. 11 14 Interestingly, it has been observed that VEGF and basic FGF (bfgf, or FGF-2) induce a synergistic angiogenic response, both in vitro 15,16 and in vivo. 17,18 A number of endogenous negative regulators have been described, including inhibitors of extracellular proteinases, thrombospondins 1 and 2, and bioactive fragments of the ECM and other molecules (see below). 1,19 21 The notion of the balance applied to positive and negative regulators of angiogenesis can also be applied to extracellular proteolysis. The resulting notion of the proteolytic balance, 5,22 which highlights the importance of a precise protease-antiprotease equilibrium, has become one of the cornerstones on which our understanding of the molecular mechanisms of angiogenesis is based. Thus, although it has consistently been observed that endothelial cell migration and invasion are associated with increased proteolysis, protease inhibitors play an important albeit permissive role during angiogenesis by preserving matrix integrity. For example, in wound healing and inflammatory skin lesions, protease inhibitors have been localized to stromal cells surrounding vessels in regions of active vascular proliferation, where they may serve to prevent uncontrolled matrix destruction. 23 Excessive proteolysis is incompatible with normal angiogenesis, because it results either in destruction of the matrix scaffold that is required for invasion or in the formation of aberrant vascular structures. 24 In the absence of sufficient proteolysis, on the other hand, invasion and lumen formation are inhibited. 25 Another important notion is that of spatial restriction of extracellular proteolysis. The objective of spatial restriction is to amplify cell surface activity, while at the same time limiting proteolysis to the immediate pericellular environment. This objective is achieved through the local production of protease inhibitors as outlined above, as well as through the assembly of components of one or more protease systems on the cell surface, which brings the enzymes into close proximity to one another. The result is a highly efficient proteolytic cascade on the cell surface, which increases the efficiency of invasion without destroying the matrix scaffold required for invasion (for example, see Mazzieri et al 26 ). In addition to its requirement for matrix invasion and capillary morphogenesis, extracellular proteolysis has been implicated in the regulation of cytokine activity (the Table). Examples include activation of latent transforming growth factor- (TGF- ), 27 release of matrix-bound bfgf and VEGF, 27,28 release of membrane-anchored tumor necrosis factor- (TNF- ), 29 and inactivation of monocyte chemoattractant protein 3, with the resultant generation of a chemokine antagonist. 30 Finally, extracellular proteolysis has been implicated in the generation of proteolytic fragments of the ECM and other molecules, a number of which have been reported to have

1106 Arterioscler Thromb Vasc Biol. July 2001 angioregulatory activity, either positive or negative (the Table). 31 Much attention has been focused on angiostatin and endostatin, which are negative regulators derived from plasminogen and collagen XVIII, respectively. 32,33 Fragments of collagen IV, which are also negative regulators, include arresten, from the 1 chain, 34 canstatin, from the 2 chain, 35 and tumstatin, from the 3 chain. 36,37 Other ECM proteins from which fragments with angioregulatory activity have been derived include thrombospondin (inhibitory), hyaluronan (stimulatory), SPARC (secreted protein, acidic, rich in cysteine; stimulatory and inhibitory), and collagen XV/restin (inhibitory). 38 Non-ECM molecules from which fragments with angioinhibitory activity have been derived include prolactin, platelet factor 4, MMP-2 (PEX; see below), calreticulin (vasostatin), 39 and high-molecular-weight kininogen domain 5 (kininostatin). 40 Finally, a cleaved form of antithrombin has also been shown to have antiangiogenic activity. 41 It should be pointed out, however, that despite a large amount of information that has accumulated on their antiangiogenic activity, a degree of caution is still required when interpreting the effects of many of these molecules, including the mechanisms and precise molecular nature of their biological activity. Role of the MMP System in Angiogenesis MMPs are a family of zinc-dependent enzymes that can be divided into 2 structurally distinct groups, namely, secreted MMPs and membrane-type MMPs (MT-MMPs). Secreted MMPs include (but are not limited to) collagenases (interstitial collagenase, or MMP-1; neutrophil collagenase, or MMP-8; and rodent interstitial collagenase, or MMP-13), gelatinases (gelatinase A, or MMP-2; gelatinase B, or MMP- 9), stromelysins (stromelysin-1, or MMP-3; stromelysin-2, or MMP-10; stromelysin-3, or MMP-11), and other MMPs (matrilysin, or MMP-7; metalloelastase, or MMP-12). The catalytic activity of secreted MMPs is tightly regulated. They are secreted as inactive proenzymes (zymogens), with activation occurring in the extracellular compartment. In the case of some MMPs (eg, MMP-2), this occurs in association with the plasma membrane. 42,43 At least 5 genetically distinct MT-MMPs have been identified (MMP-14, -15, -16, -17, -21). With the exception of MMP-17 (which is glycosyl-phosphatidyl inositol (GPI)- anchored), these enzymes are single-pass type I membrane proteins with an extracellular N-terminus and a short, cytoplasmic C-terminal domain. MT-MMPs are activated intracellularly in the secretory pathway by furinlike enzymes. By virtue of its localization and activity, MT1-MMP is endowed with 2 characteristics important for cell migration: association with the plasma membrane, which focuses matrix digestion close to the cell surface, and the capacity to catalytically activate the precursor of another extracellular protease, namely MMP-2, which allows for amplification of the degradative process. MT1-MMP also has direct activity against a number of ECM proteins, including gelatin, fibronectin, vitronectin, fibrillar collagens, and aggrecan. 44,45 In addition to zymogen activation, MMP activity is subject to regulation by tissue inhibitors of metalloproteases (TIMPs), four of which have thus far been described. TIMPs form a 1:1 stoichiometric complex with all activated MMPs, with which they become covalently linked. TIMP-1 also binds pro MMP-9. TIMP-2 and -4 also bind pro MMP-2. TIMP-2 and -3 are effective inhibitors of MT-MMPs, whereas TIMP-3 is a good inhibitor of TNF- converting enzyme. TIMP-3 is deposited in the ECM and is notoriously difficult to extract from tissues. 46 The functional interaction between the two classes of MMPs has been the subject of intense research over the past few years. For example, MT1-MMP is a cellular receptor for and activator of pro MMP-2, with which it forms a trimolecular complex on the cell surface with TIMP-2. 44,45 The importance of MMP-2 and MT1-MMP during angiogenesis has been reviewed. 47 MMP-2, MT1-MMP, TIMP-2, and integrin v 3 have been colocalized in caveolae in the basolateral compartment of human endothelial cells. 48 In contrast, MMP-9 and TIMP-1 are uniformly distributed on the cell surface and are concentrated in the Golgi region. Interestingly, it has been demonstrated that TIMP-2 and -3 derived from perivascular cells (pericytes and smooth muscle cells) are capable of inhibiting the activation of MMP-2 by MT1-MMP in endothelial cells. 49 This interplay may be important during the resolution phase of angiogenesis and for the maintenance of endothelial quiescence. With regard to localization studies, basal levels of expression of MMPs and TIMPs are absent or weakly positive in endothelial cells of normal tissues. However, these molecules are upregulated in endothelial cells (and in some instances, in pericytes) in a variety of physiological and pathological settings. Examples in which MMPs and TIMPs have been detected in endothelial cells include embryogenesis (MMP-1, -2), wound healing (MMP-2), endometrial blood vessels during the menstrual cycle (MMP-2, -3, -9; TIMP-1, -2), placental microvessels (MMP-2, -9; TIMP-1), rheumatoid arthritis (MMP-9), and a variety of tumors, including breast (MMP-2), endometrial (MMP-2, -9), hepatocellular (MMP-2, TIMP-2), colorectal (MMP-7), pancreatic (MMP-7), gastric (MMP-7), renal (MMP-7), and lung (MMP-7) carcinomas, as well as various brain tumors (MMP-2, -9, -14). 50 61 Cultured endothelial cells have been shown to express MMP-1, -2, -3, -9, and -14 and TIMP-1 and -2. 62 Studies on the regulation of MMPs and TIMPs in endothelial cells by angiogenic cytokines have revealed a variable response that is in part cell-type dependent. Thus, VEGF increased MMP-2 and MT-1 MMP, had no effect on MMP-1, and decreased TIMP-1 and -2 in human dermal microvascular endothelial cells. 63,64 VEGF increased MMP-1, MMP-2, and TIMP-1 in human umbilical vein endothelial cells, and this change was accompanied by increased activation of MMP-2. 65,66 bfgf induced MMP-1 in human omentum derived 67 or dermal 68 microvascular endothelial cells. As a rule, the magnitude of the increase in MMP gene expression in response to angiogenic cytokines is significantly less than the increase seen with components of the PA-plasmin system (Figure 1A). Furthermore, expression of naturally occurring inhibitors of the MMP and PA-plasmin systems are not necessarily coordinately regulated: in response to TGF- 1, plasminogen activator inhibitor 1 (PAI-1) is increased whereas TIMP-1 is decreased (Figures 1B and 1C). In addition to regulation by angiogenic cytokines, endothelial cell MMP-2 and MT1- MMP expression is markedly increased when endothelial cells are cultured in suspension in a 3-dimensional collagen

Pepper Extracellular Proteolysis and Angiogenesis 1107 Figure 1. Regulation of protease and inhibitor gene expression in bovine microvascular endothelial (BME) cells. (A) Confluent monolayers of BME cells were exposed to bfgf at the indicated concentrations for 15 hours. Total cellular RNA (5 g/lane) was analyzed by Northern blotting. Replicate filters were probed with 32P-labeled bovine upa, bovine upar, human MMP-2, and human MT1-MMP crna probes. Filters were stained with methylene blue to reveal 28S and 18S ribosomal RNAs. (B) Confluent monolayers of BME cells were exposed to TGF- 1 at the indicated concentrations for 12 hours. Total cellular RNA (5 g/lane) was analyzed by Northern blotting. Replicate filters were probed with 32P-labeled bovine TIMP-1 and PAI-1 crna probes. Filters were stained with methylene blue to reveal 28S and 18S ribosomal RNAs. (C) Confluent monolayers of BME cells were exposed to TGF- 1 for the times indicated. Total cellular RNA (5 g/lane) was analyzed by Northern blotting. Replicate filters were probed with 32P-labeled bovine TIMP-1 and PAI-1 crna probes. Filters were stained with methylene blue to reveal 28S and 18S ribosomal RNAs. (PAI-1 Northern blots in Figures 1B and 1C are from Mandriotta and Pepper169 [J Cell Sci. 1997;110:2293-2302.] and are reproduced with permission from the Company of Biologists Ltd.) gel compared with monolayer (2-dimensional) cultures,69 which in turn results in a 15-fold increase in MMP-2 activity. The collagen-dependent increase in MT1-MMP is mediated by the transcription factor Egr-1,70 whereas the increase in MMP-2 activation appears to require integrin 2 1.71 Changes in Arg-Gly-Asp dependent integrin clustering and cell shape have also been shown to alter MMP-2 activation in endothelial cells.72 Finally, MMPs -2 and -9 have been found to colocalize with 1 integrin in focal contacts.73 That MMPs are required for angiogenesis has been firmly established.62,74 76 Support for this notion has come mainly from studies in which angiogenesis, or various components thereof, could be inhibited by naturally occurring or synthetic MMP inhibitors. Most studies have addressed the activation (sprouting) phase of angiogenesis. For example, increased expression of TIMPs -1 and -2 inhibited tumor-associated angiogenesis, which in the case of TIMP-2 was associated with a marked reduction in expression of tumor-derived VEGF.77,78 Only recently has the requirement for inhibition of MMP activity during vessel stabilization in the resolution phase of angiogenesis become apparent (induction of endothelial quiescence, increased adhesion, and basement membrane deposition).79 81 The presence of unchecked proteolysis during this phase was associated with regression of newly formed vessels. Although a large number of studies have demonstrated the antiangiogenic effect of synthetic MMP inhibitors,62 virtually all of these inhibitors lack specificity for a single MMP. For example, decreased vessel density and increased tumor cell apoptosis were observed in primary tumors and metastases in mice treated with KB-R7785, which inhibits MMPs -1, -3, and -9.82 Similar effects were observed with N-biphenylsulfonylphenylalanine hydroxamic acid, which potently inhibits MMPs -2, -9, and -14 but not MMPs -1, -3, or -7.83 MMPs have also been implicated in the generation of protein fragments that have angioinhibitory activity. These include, but are not limited to, angiostatin and endostatin. Thus, certain MMPs, including metalloelastase (MMP-12), gelatinase B (MMP-9), stromelysin-1 (MMP-3), and matrilysin (MMP-7), are capable of converting plasminogen into the

1108 Arterioscler Thromb Vasc Biol. July 2001 angiogenesis-inhibiting angiostatin. 84 87 Interestingly, it has been reported that tissue levels of MMPs -7 and -9 are increased in 1 -integrin null mice and that these mice have increased circulating levels of angiostatin. 88 Tumor growth and vascularization were decreased in these mice, and this finding was attributed to the increase in circulating angiostatin levels. Endostatin is generated from the NC1 domain of collagen XVIII by several different proteinases (principally elastase and cathepsin L, but also several MMPs and other cathepsins). 89 91 Requirement for Gelatinases and MT1-MMP During Angiogenesis The importance of gelatinase activity in angiogenesis has been demonstrated in a rat Swarm chondrosarcoma model, in which the transition to the angiogenic phenotype was characterized by an increase in MMP-2 activity. 92 In this model, angiogenesis and tumor growth could be inhibited by antisense MMP-2 oligonucleotides. In the ongoing search for clinically relevant MMP inhibitors, some degree of selectivity for the gelatinases has been achieved. For example, specific gelatinase-inhibitory peptides have been identified from phage display libraries. 93 Cyclic peptides containing the sequence His-Trp-Gly-Phe, which are potent and selective inhibitors of MMP-2 and MMP-9, have been shown to inhibit tumor and endothelial cell migration in vitro and tumor growth and invasion in vivo. Moreover, peptide-displaying phage specifically targeted angiogenic blood vessels in vivo. Interestingly, endostatin has been reported to block MT1- MMP mediated activation and catalytic activity of MMP-2 in endothelial cells and to form a stable complex with pro MMP-2. 94 Similarly, the properdin-like type 1 repeats found in thrombospondins -1 and -2 (which, like endostatin, are endogenous inhibitors of angiogenesis) bind specifically to MMP-2 and inhibit its catalytic activity. 95 The C-terminal hemopexin-like domain of MMP-2 (termed PEX) has been shown to inhibit bfgf-induced angiogenesis in the chicken chorioallantoic membrane (CAM) and to disrupt tumor growth. 96 The antiangiogenic activity of PEX has been demonstrated with both a recombinant protein and a lentiviral delivery approach. 96,97 Physiologically active levels of PEX can purportedly be recovered from sites of active neovascularization in vivo. Not surprisingly, PEX prevents cell surface collagenolytic activity by blocking MMP-2 activation and enzyme activity. 96 It has also been reported that PEX prevents binding of MMP-2 to the integrin v 3. 96 An organic compound (TSR1359) selected for its ability to inhibit MMP-2 binding to v 3 has also been reported to have potent antiangiogenic activity in the CAM assay. 98 Additional evidence for a requirement for gelatinases during angiogenesis has come from genetic studies in mice. With regard to MMP knockouts, although mice deficient in MMP-2 99 and MMP-9 100 are viable, both have a clear angiogenic phenotype. Thus, in MMP-2 deficient mice, tumor-induced angiogenesis was markedly reduced in a dorsal air sac assay with B16-BL6 melanoma cells. 101 This finding indicates that host-derived MMP-2 plays an important role in angiogenesis and tumor progression, with an important contribution from stromal cells. In MMP-9 deficient mice, bone growth plate angiogenesis was reduced, probably owing to delayed release of a positive angiogenesis regulator by hypertrophic cartilage. 100 It has recently been reported that tumor angiogenesis is VEGF dependent in a transgenic mouse model of pancreatic beta-cell carcinogenesis (Rip1Tag2 mice). However, in these mice, there is no net increase in the level of expression of VEGF or its tyrosine kinase receptors. Instead, extracellular VEGF is mobilized from matrix stores, making it more available to VEGF receptors. This mobilization is dependent on MMP-9 activity (probably macrophage derived), which is upregulated during tumor progression, more specifically, during the switch from the prevascular to the vascular phase. 102 Tumor progression was inhibited in Rip1Tag2 MMP-9 null double-transgenic mice. In contrast, although MMP-2 activity was increased in parallel with MMP-9 in Rip1Tag2 mice, tumor progression was unaffected in Rip1Tag2 MMP-2 null double transgenics. With regard to the PAs (urokinase-type PA [upa] and tissue-type PA [tpa]), no reproducible changes in activity were noted during tumor progression, and tumor progression was unaffected in Rip1Tag2 upa null double-transgenic mice. Taken together, these findings reveal a specific requirement for MMP-9 in the angiogenic switch in this murine model of pancreatic beta-cell carcinogenesis. MT1-MMP deficient mice display severe skeletal defects that lead to death by 3 to 16 weeks of age. 103,104 Other tissues in these null mice develop relatively normally. The skeletal defects are due in part to decreased vascular invasion of calcified cartilage. In the corneal angiogenesis assay, MT1- MMP deficient mice also failed to respond to implanted FGF-2. Reduced activation of latent MMP-2 (but not MMP-9) was observed in various tissues of MT1-MMP null mice. MMP Requirement for Fibrinolysis During Angiogenesis Traditionally, physiological fibrinolysis has been ascribed to the PA-plasmin system, and studies in null mice have confirmed this notion. 105 Surprisingly, however, no overt phenotype related to aberrant angiogenesis has been observed in these mice, during either development or wound healing (see below). This finding raised the possibility that other protease systems might assume a fibrinolytic role during angiogenesis, ie, that in contrast to other cell types, endothelial cells invade fibrin matrixes in a plasmin-independent manner. 106 This hypothesis has recently been confirmed in an ex vivo model in which tissue fragments from null mice were embedded in a 3-dimensional fibrin matrix. 107 Thus, with the use of inhibitors of serine, cysteine, and aspartate proteinases, it was found that endothelial cell invasion and capillary morphogenesis progressed unabated. In contrast, these processes were completely inhibited by natural and synthetic MMP inhibitors. When a series of MMPs, including MMPs -1, -2, and -3 and a soluble form of MT1-MMP, were assessed for their fibrinolytic activity in a cell-free assay, all had the capacity to solubilize fibrin gels, and this activity could be completely blocked by MMP inhibitors. Quantitatively, the most potent fibrinolytic activity was ascribed to MT1-MMP. 107 In a recent study on antiglomerular basement membrane nephritis, MMP-9 was found to be required for

Pepper Extracellular Proteolysis and Angiogenesis 1109 fibrinolysis, and in its absence, fibrin accumulated in glomeruli.108 These unexpected and highly novel observations suggest that when endothelial cells invade a provisional fibrin ECM scaffold, this event occurs in a plasmin-independent manner and may require the activity of MMPs such as MT1-MMP and possibly MMP-9. These observations have potentially far-reaching mechanistic and therapeutic implications. Role of the PA-Plasmin System in Angiogenesis Plasmin is a broad-spectrum protease of tryptic specificity that either directly or indirectly, through the activation of certain pro-mmps, is presumed to hydrolyze many extracellular proteins, the most notable of which is fibrin. upa and tpa, the principal activators of plasminogen, are the products of separate genes that are regulated independently by a variety of factors, including hormones and cytokines. Like plasmin, upa and tpa are serine proteases of tryptic specificity, but in contrast to plasmin, they have a very restricted substrate specificity, namely, plasminogen. upa (or, more precisely, its inactive precursor pro-upa) is secreted as a soluble protein and binds with high affinity to a specific GPI anchored cell surface receptor (upa receptor, or upar) that is present on a variety of cells, including endothelial cells. Plasminogen and plasmin also associate with plasma membranes, and colocalization of upa and plasminogen on the cell surface increases the efficiency of plasminogen activation and subsequent plasmin-dependent proteolysis. The existence of multiple, specific, physiological inhibitors of both plasmin (ie, 2-antiplasmin) and PAs (ie, PAI-1 and PAI-2) provides additional points of regulation along this protease cascade. The specificity, activity, and localization of serine protease inhibitors can be regulated by the ECM and other glycoproteins.3,105 With regard to localization studies, the overall picture is that upa, upar, and PAI-1 are not expressed by quiescent endothelium. tpa, on the other hand, has been detected in the quiescent endothelium of a small percentage of arterioles, venules, and vasa vasorum of normal human tissues. In contrast, upa, upar, and PAI-1 are all expressed during angiogenesis in vivo in a variety of settings. upa and upar appear to be expressed by endothelial cells, and, depending on the situation, PAI-1 is expressed either by endothelial cells themselves or by nonendothelial (stromal and epithelial) cells, where it is thought to play a role in the preservation of matrix integrity.5,109 Little is known about tpa expression during angiogenesis. These in vivo observations are amply supported by results obtained in vitro. That cultured endothelial cells (which are closer to activated than quiescent endothelium) produce upa, upar, tpa, and PAI-1 has been known for some time, and their regulation by bfgf and VEGF has been described (Figure 2).2,5 It should be pointed out, however, that endothelial cells of human origin respond less well than do cells of bovine origin with regard to induction of PAs by bfgf and VEGF. The reason for the lack of response may be related to the chronic stimulation of these cells by the components of endothelial growth supplements (FGFs and probably VEGF) used to culture the cells. The response to VEGF also appears Figure 2. Regulation of upa, upar, tpa, and PAI-1 gene expression in bovine microvascular endothelial (BME) cells by VEGF. Confluent monolayers of BME cells were exposed to VEGF at the indicated concentrations for 4 or 15 hours. Total cellular RNA (5 g/lane) was analyzed by Northern blotting. Replicate filters were probed with 32P-labeled bovine upa, bovine upar, human tpa, and bovine PAI-1 crna probes. Filters were stained with methylene blue to reveal 28S and 18S ribosomal RNAs. to be dependent on the vascular bed from which the cells were isolated. Thus, VEGF induces upa and tpa in endothelial cells derived from the microvasculature but not in cells derived from the aorta (Figure 3).110,111 This distinction may have important implications for the regulation of angiogenesis, which traditionally has been considered to occur from preexisting vessels of the microvasculature. Hypoxia, a major stimulus for angiogenesis, has been reported to increase upar112 and PAI-1 (Figure 4)113 in endothelial cells. We have previously demonstrated that upa, upar, and PAI-1 are increased in endothelial cells in a 2-dimensional woundinduced migration assay and that both migration and protease/inhibitor induction are dependent on endogenous bfgf.114 116 In a coculture system of endothelial cells and fibroblasts, PAI-1 mrna and promoter activity were induced only in those fibroblasts apposed to sprouting but not resting endothelium.117 The authors reasoned that this may be of importance during sprouting, which constitutes the only period during which endothelial cells establish direct contact with fibroblasts. They suggested that the paracrine induction of PAI-1 provides a mechanism to negate excessive pericellular proteolysis. Angiogenesis results in the formation of new vessels in the blood vascular system. A comparable process, called lymphangiogenesis, is responsible for the formation of new

1110 Arterioscler Thromb Vasc Biol. July 2001 Figure 3. Differential regulation of PAs in bovine microvascular and aortic endothelial (BME and BAE, respectively) cells by bfgf and VEGF. (A) PA plaque assay. BME and BAE cells were seeded at 1 and 5 10 4 cells, respectively, per 35-mm tissueculture dish in serum-containing medium. Twenty-four hours later, cells were treated with bfgf (10 ng/ml) or VEGF (100 ng/ml). Twelve to 15 hours later, cells were overlaid with a mixture containing agar, plasminogen, and casein. After incubation for various times at 37 C, dishes were photographed under dark-field illumination; black spots around individual cells are indicative of plasmin-mediated casein lysis. This experiment was performed in duplicate; representative dishes are shown. Both VEGF and bfgf increased plaque number and size in BME cells. In contrast, in BAE cells, although bfgf dramatically increased PA activity (resulting in complete lysis of the overlay), VEGF was without effect. (B) Zymographic and reversezymographic analysis of BME and BAE cells exposed to VEGF. Confluent monolayers were exposed to VEGF (20 ng/ml) for 15 hours. VEGF was from Genentech (V G ) or Peprotech (V P ). Cell extracts (CE) and culture supernatants (Sup) were analyzed by zymography (upper panels) and reverse zymography (lower panels). VEGF induced upa and tpa in BME but not in BAE cells. vessels in the lymphatic system. Lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on angiogenesis. This is due in part to the lack of identification of lymphangiogenic factors as well as suitable markers with which to distinguish blood from lymphatic vascular endothelium. This scenario is changing rapidly after discovery of the first lymphangiogenic factors (VEGF-C and -D) as well as a limited number of lymphatic endotheliumspecific markers. 118 In adult tissues, new lymphatic capillaries grow by sprouting as extensions from preexisting lymphatics, much in the same way as new blood capillaries arise by sprouting from the preexisting microvasculature during angiogenesis. Although very little is known about the mechanisms of lymphangiogenesis, it has been demonstrated that VEGF, VEGF-C, and bfgf are capable of stimulating upa, upar, tpa, and PAI-1 in bovine large-vessel lymphatic endothelial cells. 119 122 The relevance of these in vitro observations to lymphangiogenesis in vivo remains to be determined. The generation of mice deficient in components of the PA-plasmin system 109 provided an opportunity to assess the role of individual components of this system during angiogenesis in vivo. Previous studies, both in vivo and in vitro, had pointed to a crucial role for the PA-plasmin system during angiogenesis. Unexpectedly, however, it was found that embryonic and postnatal development was unperturbed in these mice, at least from the perspective of an angiogenesis-associated phenotype. 5 A seminal study on wound healing in plasminogen-deficient mice clearly defined a role for the PA-plasmin system during keratinocyte migration in vivo. 123 However, although keratinocyte migration was severely impaired, there were no overt qualitative differences in the granulation tissue (inflammatory cell infiltration, fibroblast migration, and neovascularization) between wildtype and plasminogen -/- mice, suggesting that in this setting, angiogenesis is not PA-plasmin dependent. In addition, expression of 6 MMPs, including MMPs -1, -3, and -9 by keratinocytes and MMPs -2 and -11 and MT1-MMP by cells in granulation tissue, was unaffected in plasminogen -/- mice. These findings suggest that although keratinocyte migration is plasmin dependent, implementation of the genetic program that controls keratinocyte migration is plasmin independent. Although developmental and wound healing associated angiogenesis appears to be unaffected in upa-, upar-, tpa-, PAI-1, and plasminogen-null mice, several in vivo studies have demonstrated a requirement for this system during angiogenesis in other settings. For example, tumor-associated angiogenesis (vascular density) was inhibited in an athymic mouse model utilizing a human prostate carcinoma cell line, PC-3, that was stably transfected with human PAI-1. 124 Although this study pointed to a role for PAs in tumor Figure 4. Induction of PAI-1 by hypoxia in bovine microvascular and aortic endothelial (BME and BAE, respectively) cells. Confluent monolayers were exposed to hypoxia/anoxia (H) for 15 hours. N indicates normoxia. (A) Total cellular RNA (5 g/lane) was analyzed by Northern blotting. Filters were probed with a 32 P-labeled bovine PAI-1 crna probe. Filters were stained with methylene blue to reveal 28S and 18S ribosomal RNAs. (B) Cell extracts were analyzed in duplicate by reverse zymography.

Pepper Extracellular Proteolysis and Angiogenesis 1111 angiogenesis, it provided no mechanistic information, including the cell type specificity of the effect. More precise information has been provided in a murine model of myocardial infarction, in which postinfarction myocardial revascularization (angiogenesis) was severely impaired in upa / mice. 125 Because this phenotype could not be rescued by exogenously added VEGF, and because endothelial and smooth muscle cells expressed upa during revascularization, it was concluded that the defect in angiogenesis was due to impaired endothelial cell invasion. Interestingly, when assayed in the rabbit cornea or on the chicken CAM, upa itself had a proangiogenic effect. 126 128 In the CAM, this event could be ascribed to the release of endogenous bfgf. 128 upa-upar Interactions Are Required for Angiogenesis The importance of the interaction between upa and upar during angiogenesis has been demonstrated in a number of in vivo systems. Thus, bfgf-induced angiogenesis in subcutaneously injected Matrigel was inhibited by a fusion protein consisting of the receptor-binding amino-terminal fragment of upa and the Fc portion of human IgG, which functions as a high-affinity upar antagonist. 129 Similarly, in syngeneic and xenograft murine tumor models, adenovirally delivered amino-terminal fragment specifically inhibited tumor angiogenesis. 130 In a homologous immunocompetent rat model of tumor cells transfected with a mutant murine upa that retains receptor binding but not proteolytic activity, microvessel density was markedly reduced. 131 Tumor angiogenesis in several murine models was also inhibited by an octamer peptide derived from the non receptor-binding region of upa. 132,133 These studies defined a novel epitope in upa involved in the upa-upar interaction. PAI-1 Is Required for Angiogenesis Localization studies have demonstrated that PAI-1 is strongly expressed in endothelial cells in a number of tumor types. 5 Studies in PAI-1 null mice have revealed an absolute requirement for PAI-1 in tumor-induced angiogenesis. In one model, tumor and stromal cells are separated by the interposition of a type I collagen gel, which allows independent assessment of exogenously added tumor cell as well as of host stromal cell invasion. 134,135 The absence of PAI-1 (but not of upa, upar, or tpa) from the host markedly impaired tumor cell invasion and host-derived neovascularization. Reversal of this phenotype was achieved by adenovirally delivered human PAI-1 to PAI-1 null mice. In wild-type mice, PAI-1 expression in the stroma could be localized to endothelial and nonendothelial cells. Interestingly, although upa was localized to newly formed blood vessels in wildtype mice, upa was replaced by tpa in mice deficient in upa, demonstrating the capacity for compensation between the PAs. The requirement for host PAI-1 (but not upa) during tumor angiogenesis has also been demonstrated in a fibrosarcoma model in gene inactivation mice. 136 bfgf-induced corneal angiogenesis was similarly impeded in PAI-1 but not in upa-deficient mice. Two potential mechanisms may explain why PAI-1 is essential for angiogenesis. First, by protecting the ECM against excessive degradation, PAI-1 may serve to maintain the matrix scaffold required for endothelial cell migration and tube formation. 22 Second, a complex series of interactions have been described between PAI-1, upar, integrins, and the ECM component vitronectin. 137 139 Alterations in PAI-1 expression and activity would therefore be expected to alter the adhesive, migratory, and growth properties of endothelial cells, which in turn would regulate angiogenesis. To address this question directly, adenovirus was used to deliver two mutant forms of PAI-1 to PAI-1 deficient mice in the collagen gel interposition model described above. 135 The PAI-1 mutants either (1) bound vitronectin normally but failed to inhibit upa or tpa or (2) inhibited PAs normally but had negligible binding to vitronectin. Angiogenesis was restored by the PAI-1 mutant that retained PA inhibitory activity but not by the mutant that bound vitronectin but failed to inhibit the PAs, demonstrating that the requirement for PAI-1 in this model is due to inhibition of excessive proteolysis rather than inhibition of cellular adhesion to vitronectin. In a separate study, exogenously added PAI-1 ( at therapeutic concentrations ) was shown to inhibit bfgfinduced angiogenesis in the CAM. 140 With use of a variety of PAI-1 mutants, this activity could be ascribed both to PAI-1 s antiprotease activity as well as its vitronectin-binding capacity. Does tpa Play a Role in Angiogenesis? Little is known about the role of tpa in angiogenesis. In vivo, tpa antigen is localized to the endothelium of the microvasculature, 141 which, as morphological studies have revealed, is the region of the vascular bed from which new blood vessels are formed during angiogenesis. The presence of tpa in angiogenic endothelium has yet to be described. In vitro, tpa has been implicated in capillary-like tube formation on matrices constituted of type I collagen, Matrigel, and the explanted amnion. 142 146 The requirement for tpa in endothelial cell migration in vitro has been demonstrated in some studies 147 but not in others. 148 Interestingly, the combination of bfgf and VEGF induced tpa mrna and enzyme activity in a synergistic manner in cultured endothelial cells. 111 In this study, immunofluorescence analysis revealed that cellassociated tpa was colocalized with von Willebrand factor, a marker for Weibel-Palade bodies, which are regulated, endothelial cell specific storage granules. A combination of bfgf and VEGF increased the number of tpa-positive cells as well as the number of tpa-positive granules per cell. Angiostatin, an endogenous inhibitor of angiogenesis, has been shown to inhibit tpa-catalyzed plasminogen activation in endothelial cells. 149 At present, one can only speculate on the function of tpa in Weibel-Palade bodies in the context of angiogenesis. An attractive role would be to protect newly forming blood vessels from thrombotic occlusion. Indeed, stagnant blood in the forming dead end of new blood vessels would be particularly prone to coagulation, first because of the potential exposure of blood to the ECM (and in particular, to collagen) in the hyperpermeable and rapidly remodeling migrating sprout and second, because activated blood coagulation would be less easily washed away. To overcome this problem, locally generated thrombin would result in the acute release of tpa from Weibel-Palade bodies, 150,151 which in turn would aid in the removal of fibrin deposits.

1112 Arterioscler Thromb Vasc Biol. July 2001 It is of interest to note that secretion of MMP-1 and MMP-2, but not of TIMP-2, can occur through a posttranscriptional mechanism in endothelial cells. 152 MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, providing a mechanism for their rapid mobilization in the early phases of angiogenesis. Whether these enzymes, like tpa, are localized to Weibel- Palade bodies (regulated, endothelial cell specific storage granules) remains to be determined. The intracellular accumulation of MMP-9 in secretory vesicles in response to phorbol esters has been reported. 71 Requirement for the MMP and PA-Plasmin Systems in Polyomavirus Middle T Oncogene Induced Vascular Tumor Formation Endothelial cells are the primary target of the transforming ability of the polyomavirus middle T (PymT) oncogene. Tumors induced by PymT (called endotheliomas) have been used as a model system for endothelial cell tumors. Although significant morphological differences exist between endotheliomas and human vascular tumors (including hemangiomas), the PymT system does provide a good functional model for hemangiomas because (1) the tumors are endotheliumspecific and organ-nonspecific; (2) there appears to be rapid, uncontrolled endothelial cell proliferation; and (3) mice develop many signs of the Kasabach-Merritt syndrome (ie, thrombocytopenia, anemia, and associated splenomegaly), which may be a life-threatening complication of hemangiomas in humans. It has also been suggested that endotheliomas may serve as a model system for vascular malformations. This is due to the cavernous nature of the lesions, which is a characteristic feature of cavernous angiomas in humans. Finally, because recruited host cells are an important component of these tumors, endotheliomas have also been used as a model system for Kaposi s sarcoma. 153 PymT-transformed endothelial cells (End. cells), which can be isolated with relative ease, have the potential to grow indefinitely, and they retain many important features of differentiated endothelium. Endotheliomas can be induced not only by PymT itself but also by subcutaneous or intraperitoneal injection of End. cells. The following is a working hypothesis for endothelioma formation after subcutaneous injection. 153 End. cells invade and integrate into host blood vessels, leading to their rupture and subsequent hemorrhage. This event in turn is followed by rapid recruitment of host endothelial and nonendothelial cells. Recruited endothelial cells form new capillary blood vessels, which feed the growing cystic tumor. Recruitment is dependent on the continuous presence of End. cells and mainly involves host cell migration with low levels of proliferation. The signal for host endothelial cell recruitment is not known, but it probably originates from the injected End. cells as well as from the intense inflammatory reaction observed at the periphery of the hemorrhagic cysts. It is likely that in addition to host cell recruitment, proliferating End. cells themselves contribute significantly to tumor expansion during the later phases of tumor growth. Taraboletti et al 154 and Vergani et al 155 have partially characterized an End. cell derived motility factor Figure 5. In vitro morphogenetic behavior of normal mouse brain endothelial cells (BECs) and PymT-expressing endothelioma cells grown within 3-dimensional fibrin gels. (a) Phasecontrast view of a network of branching and anastomosing cords formed by primary mouse BECs; fine slitlike lumina are indicated by the small arrows. (b) Spherical cyst formed by PymT-expressing endothelial cells. In this picture, the focus is approximately on the equatorial plane of the cyst. The endothelial cells lining the floor and roof of the cavity appear blurred in the center of the cyst. Bar 100 m. (Reprinted from Cell, vol 62, Montesano et al 24 : Increased proteolytic activity is responsible for the aberrant morphogenetic behaviour of endothelial cells expressing middle T oncogene, pp 435 445, copyright 1997, with permission from Elsevier Science.) that may be responsible for recruitment of host endothelial cells. An in vitro model has been developed to study the autonomous, morphogenetic behavior of End. cells. 24 This consists of growing End. cells in suspension in 3-dimensional fibrin gels. In these fibrin gels, End. cells proliferate rapidly, and after 10 to 14 days form large cystic structures lined by a monolayer of endothelial cells (Figure 5). These cystlike structures are strikingly reminiscent of the cavernous endotheliomas that develop in vivo. Under the same experimental conditions, nontransformed endothelial cells from a variety of

Pepper Extracellular Proteolysis and Angiogenesis 1113 species and organs form elongated, branching, tubelike structures (Figure 5); in no instance are cystlike structures seen. The fibrin gel model, in which normal vascular morphogenesis is disrupted, ie, formation of endothelium-lined cystlike structures rather than tubelike structures, provides a powerful in vitro system for investigating the mechanisms of proteasedependent tumor formation. With regard to MMPs and endothelioma formation in vivo, it has been demonstrated that End. cell induced endothelioma growth can be inhibited by the broad-spectrum hydroxamic acid based peptide derivative Batimastat (also known as BB94). 156 Daily local injection of the inhibitor at the site of End. cell injection increased tumor doubling time in a dose-dependent manner. Inhibition of tumor growth lasted as long as Batimastat was given, and when treatment was stopped, the tumors resumed a pattern of growth similar to that seen in controls. Histologically, treated tumors consisted of solid, cellular aggregates that circumscribed small- to medium-size blood-filled spaces. Subcutaneous hemorrhage was rarely observed in treated animals. In a subsequent study, these authors demonstrated that tumor growth could be efficiently inhibited by overexpressing TIMP-2 in End. cells. 155 When compared with the cavernous tumors induced by wild-type cells, tumors induced by TIMP-2 overproducers had small-to-medium blood-filled spaces and multiple apoptotic foci. With regard to the PA-plasmin system, End. cells display increased PA activity when compared with nontransformed endothelial cells, which can be accounted for by an increase in upa and, in some cell lines, by a concomitant decrease in PAI-1. 24,157 159 The mechanism for this increase is not known, although it has been shown that PymT increases upa gene activation without influencing upa mrna stability. 158 Taken together with the observation that transformed endothelial cells form cystlike structures when embedded in fibrin gels (see above), these findings demonstrate that excessive proteolysis is associated with aberrant vascular morphogenesis. Reduction of this activity by inhibiting plasmin restores normal morphogenetic properties to these cells, ie, the formation of capillary-like tubes. These findings clearly implicate increased PA-plasmin mediated proteolysis in aberrant vascular morphogenesis in vitro. 24 Based on these in vitro observations, it was hypothesized that the PA-plasmin system would also be required for endothelioma formation in vivo. This hypothesis was tested directly in upa-, tpa-, PAI-1, and plasminogen-deficient mice. Essentially, tumor formation after viral infection/transduction was unaffected in the different genotypes, suggesting that the primary transformation process is PA-plasmin independent. However, compared to tumors induced by injection of wild-type End. cells, tumor incidence and latency were increased in upa / and double-transgenic upa:tpa / mice but not in tpa / or PAI-1 / mice. Tumor incidence and latency were also increased when upa / and upa:tpa / End. cells were injected into wild-type mice. 160 These genetic studies demonstrated that components of the PA-plasmin system, in particular upa, are critical for tumor growth in vivo. With respect to the in vitro morphogenetic behavior of End. cells derived from protease-knockout mice, although PA activity was necessary, it did not appear to be sufficient for cyst formation. 160 The plasminogen-dependent and -independent proteolytic activity of wild-type and mutant End. cells has been determined. 161 Thus, plasminogen activation with wild-type and tpa / cells was comparable but was reduced 4-fold with upa / cells and increased 3-fold with PAI-1 / cells. Lysis of an 125 I-labeled matrix in the presence of plasminogen was comparable in wild-type, tpa /, and PAI-1 / cells but was significantly reduced in upa / cells. Lysis of a human umbilical vein endothelial cell derived, [ 3 H]proline-labeled ECM in the presence of plasminogen with wild-type, tpa /, and PAI-1 / cells was comparable but was virtually abolished in upa / cells. These findings provide a mechanism for the reduction in tumor growth seen in the absence of upa (see above). Finally, using the in vitro fibrin gel model, we have infrequently observed the formation of cystlike structures by End. cells deficient in both upa and tpa (Pepper et al, unpublished observations, 2000). Clonal populations of these cells have been obtained and in which cyst formation can be completely inhibited by synthetic MMP inhibitors (Pepper et al, unpublished observations, 2000). This result implicates another protease system(s) (possibly MMPs) in the fibrinolysis required for cyst formation and is in accord with the emerging notion of MMPmediated fibrinolysis in endothelial cells. Conclusions and Perspectives Increased understanding of the mechanisms of angiogenesis has opened up novel and exciting therapeutic avenues, and considerable benefit can now be derived in the clinical setting from manipulating angiogenesis, either positively or negatively. 1,162 With respect to MMPs, potential targets for inhibiting angiogenesis include the gelatinases and MT-MMPs. The additional role of MT1-MMP in fibrinolysis during angiogenesis makes this a particularly attractive target. Several novel synthetic inhibitors are currently being investigated in clinical trials. 163,164 However, despite the abundance of promising preclinical data, the clinical application of MMP inhibitors has to date met with limited success, and a number of phase III trials have been discontinued. 165 Because MMPs have been implicated in the generation of molecules with antiangiogenic activity (eg, endostatin), MMP inhibition may result in the stimulation rather than inhibition of angiogenesis. Furthermore, one hydroxamate-based MMP inhibitor has been reported to promote the formation of liver metastases in a murine breast cancer xenotransplantation model, 166 and systemic administration of human TIMP-4 stimulates the growth of human breast cancer cells in a murine xenotransplantation model. 167 At present, therefore, a degree of uncertainty surrounds the use of MMP inhibitors in cancer, and it appears that under certain circumstances, MMP inhibition may even be detrimental to cancer patients. The studies on the PA-plasmin system cited in this review provide ample evidence for the requirement for this system during angiogenesis in vivo. In particular, a role for upar and PAI-1 has been clearly defined, with upa playing a more ancillary role. The counterintuitive possibility that inhibition of PAI-1 might serve to inhibit angiogenesis is one that merits further investigation. To date, little is known about the role of tpa in angiogenesis. The tumor vasculature has become a key target in the war against cancer. 168 Increased understanding of the complex

1114 Arterioscler Thromb Vasc Biol. July 2001 roles of the MMP and PA-plasmin systems in the regulation of angiogenesis will provide a rational basis for the development of antiangiogenesis-based therapeutic strategies for cancer and other angiogenesis-associated diseases. Acknowledgments Work in the author s laboratory was supported principally by the Swiss National Science Foundation. The author would like to thank Roberto Montesano and Egbert Kuithof for critically reading this manuscript. References 1. Pepper MS. Manipulating angiogenesis: from basic science to the bedside. Arterioscler Thromb Vasc Biol. 1997;17:605 619. 2. Mignatti P, Rifkin DB. Plasminogen activators and matrix metalloproteinases in angiogenesis. Enzyme Protein. 1996;49:117 137. 3. Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci. 2000;57:25 40. 4. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 2000;14:2123 2133. 5. Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein. 1996;49:138 162. 6. Clark ER, Clark EL. Microscopic observations on the growth of blood capillaries in the living mammal. Am J Anat. 1939;64:251 301. 7. Kwaan HC, Astrup T. Fibrinolytic activity of reparative connective tissue. J Pathol Bacteriol. 1964;87:409 414. 8. Kalebic T, Garbisa S, Glaser B, Liotta LA. Basement membrane collagen: degradation by migrating endothelial cells. Science. 1983;221: 281 283. 9. Bouck N. Tumor angiogenesis: the role of oncogenes and tumor suppressor genes. Cancer Cells. 1990;2:179 185. 10. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353 364. 11. Dvorak HF, Nagy JA, Feng D, Brown LF, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol. 1999;237:97 132. 12. Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol. 1999;237:1 30. 13. Dow JK, devere White RW. Fibroblast growth factor 2: its structure and property, paracrine function, tumor angiogenesis, and prostate-related mitogenic and oncogenic functions. Urology. 2000;55:800 806. 14. Gerwins P, Skoldenberg E, Claesson-Welsh L. Function of fibroblast growth factors and vascular endothelial growth factors and their receptors in angiogenesis. Crit Rev Oncol Hematol. 2000;34:185 194. 15. Pepper MS, Ferrara N, Orci L, Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun. 1992;189:824 831. 16. Goto F, Goto K, Weindel K, Folkman J. Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Invest. 1993;69:508 517. 17. Asahara T, Bauters C, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation. 1995;92(suppl II):II-365 II-371. 18. Hu DE, Fan T-PD. Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A. Br J Pharmacol. 1995; 114:262 268. 19. Cao Y. Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica. 1999;84:643 650. 20. Cirri L, Donnini S, Morbidelli L, Chiarugi P, Ziche M, Ledda F. Endostatin, a promising drug for antiangiogenic therapy. Int J Biol Markers. 1999;14:263 267. 21. Keshet E, Ben-Sasson SA. Anticancer drug targets: approaching angiogenesis. J Clin Invest. 1999;104:1497 1501. 22. Pepper MS, Montesano R. Proteolytic balance and capillary morphogenesis. Cell Differ Dev. 1990;32:319 327. 23. Saarialho-Kere UK, Pentland AP, Birkedal-Hansen H, Parks WC, Welgus HG. Distinct populations of basal keratinocytes express stromelysin-1 and stromelysin-2 in chronic wounds. J Clin Invest. 1994; 94:79 88. 24. Montesano R, Pepper MS, Möhle-Steinlein U, Risau W, Wagner EF, Orci L. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing middle T oncogene. Cell. 1990;62:435 445. 25. Pepper MS, Belin D, Montesano R, Orci L, Vassalli JD. Transforming growth factor- 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J Cell Biol. 1990;111:743 755. 26. Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, Mignatti P. Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J. 1997;16:2319 2332. 27. Flaumenhaft R, Rifkin DB. The extracellular regulation of growth factor action. Mol Biol Cell. 1992;3:1057 1065. 28. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267:26031 26037. 29. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor- from cells. Nature. 1997;385:729 733. 30. McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science. 2000;289:1202 1206. 31. Sage EH. Pieces of eight: bioactive fragments of extracellular proteins as regulators of angiogenesis. Trends Cell Biol. 1997;7:182 186. 32. O Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994;79:315 328. 33. O Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J. Endostatin, an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997;88:277 285. 34. Colorado PC, Torre A, Kamphaus G, Maeshima Y, Hopfer H, Takahashi K, Volk R, Zamborsky ED, Herman S, Sarkar PK, Ericksen MB, Dhanabal M, Simons M, Post M, Kufe DW, Weichselbaum RR, Sukhatme VP, Kalluri R. Anti-angiogenic cues from vascular basement membrane collagen. Cancer Res. 2000;60:2520 2526. 35. Kamphaus GD, Colorado PC, Panka DJ, Hopfer H, Ramchandran R, Torre A, Maeshima Y, Mier JW, Sukhatme VP, Kalluri R. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J Biol Chem. 2000;275:1209 1215. 36. Maeshima Y, Colorado PC, Kalluri R. Two RGD-independent v 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties. J Biol Chem. 2000;275:23745 23750. 37. Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, Hopfer H, Xiao Y, Stillman IE, Kalluri R. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J Biol Chem. 2000;275:21340 21348. 38. Ramchandran R, Dhanabal M, Volk R, Waterman MJF, Segal M, Lu H, Knebelmann B, Sukhatme VP. Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin. Biochem Biophys Res Commun. 1999;255:735 739. 39. Pike SE, Yao L, Jones KD, Cherney B, Appella E, Sakaguchi K, Nakhasi H, Teruya-Feldstein J, Wirth P, Gupta G, Tosato G. Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth. J Exp Med. 1998;188:2349 2356. 40. Colman RW, Jameson BA, Lin Y, Johnson D, Mousa SA. Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis. Blood. 2000;95:543 550. 41. O Reilly MS, Pirie-Shepherd S, Lane WS, Folkman J. Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science. 1999;285:1926 1928. 42. Woessner JF. The matrix metalloproteinase family. In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases. San Diego, Calif: Academic Press; 1998:1 14. 43. Johansson N, Ahonen M, Kähäri VM. Matrix metalloproteinases in tumor invasion. Cell Mol Life Sci. 2000;57:5 15. 44. Seiki M. Membrane-type metalloproteinases. APMIS. 1999;107: 137 143. 45. Murphy G, Stanton H, Cowell S, Butler G, Knäuper V, Atkinson S, Gavrilovic J. Mechanisms for pro matrix metalloproteinase activation. APMIS. 1999;107:38 44.

Pepper Extracellular Proteolysis and Angiogenesis 1115 46. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000; 1477:267 283. 47. Haas TL, Madri JA. Extracellular matrix-driven matrix metalloproteinase production in endothelial cells. Trends Cardiovasc Med. 1999; 9:70 77. 48. Puyraimond A, Fridman R, Lemesle M, Arbeille B, Menashi S. MMP-2 colocalizes with caveolae on the surface of endothelial cells. Exp Cell Res. 2001;262:28 36. 49. Lafleur MA, Forsyth PA, Atkinson SJ, Murphy G, Edwards DR. Perivascular cells regulate endothelial membrane type-1 matrix metalloproteinase activity. Biochem Biophys Res Commun. 2001;282: 463 473. 50. Salo T, Makela M, Kylmaniemi M, Autio-Harmainen H, Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing. Lab Invest. 1994;70:176 182. 51. Soini Y, Hurskainen T, Hoyhtya M, Oikarinen A, Autio-Harmainen H. 72 KD and 92 KD type IV collagenase, type IV collagen, and laminin mrnas in breast cancer: a study by in situ hybridization. J Histochem Cytochem. 1994;42:945 951. 52. Karelina TV, Goldberg GI, Eisen AZ. Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors. J Invest Dermatol. 1995;105:411 417. 53. Ahrens D, Kock AE, Pope RM, Stein-Picarella M, Niedbala MJ. Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis. Arthritis Rheum. 1996;39:1576 1587. 54. Musso O, Theret N, Campion JP, Turlin B, Milani S, Grappone C, Clement B. In situ detection of matrix metalloproteinase-2 (MMP2) and the metalloproteinase inhibitor TIMP2 transcripts in human primary hepatocellular carcinoma and in liver metastasis. J Hepatol. 1997;26: 593 605. 55. Nagashima Y, Hasegawa S, Koshikawa N, Taki A, Ichikawa Y, Kitamura H, Misugi K, Kihira Y, Matuo Y, Yasumitsu H, Miyazaki K. Expression of matrilysin in vascular endothelial cells adjacent to matrilysin-producing tumors. Int J Cancer. 1997;72:441 445. 56. Rooprai HK, Van Meter T, Rucklidge GJ, Hudson L, Everall IP, Pilkington GJ. Comparative analysis of matrix metalloproteinases by immunocytochemistry, immunohistochemistry and zymography in human primary brain tumours. Int J Oncol. 1998;13:1153 1157. 57. Vagnoni KE, Zheng J, Magness RR. Matrix metalloproteinases-2 and -9, and tissue inhibitor of metalloproteinases-1 of the sheep placenta during the last third of gestation. Placenta. 1998;19:447 455. 58. Iurlaro M, Loverro G, Vacca A, Cormio G, Ribatti D, Minischetti M, Ria R, Bruno M, Selvaggi L. Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with upgrading and myometrial invasion in endometrial carcinoma. Eur J Clin Invest. 1999;29: 793 801. 59. Forsyth PA, Wong H, Laing TD, Rewcastle NB, Morris DG, Muzik H, Leco KJ, Johnston RN, Brasher PM, Sutherland G, Edwards DR. Gelatinase-A (MMP-2), gelatinase-b (MMP-9), and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br J Cancer. 1999;79:1828 1835. 60. Freitas S, Meduri G, Le Nestour E, Bausero P, Perrot-Applanat M. Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biol Reprod. 1999;61:1070 1082. 61. Ribatti D, Nico B, Vacca A, Iurlaro M, Roncali L. Temporal expression of the matrix metalloproteinase MMP-2 correlates with fibronectin immunoreactivity during the development of the vascular system in the chick embryo chorioallantoic membrane. J Anat. 1999;195:39 44. 62. Moses MA. The regulation of neovascularization by matrix metalloproteinases and their inhibitors. Stem Cells. 1997;15:180 189. 63. Chan VT, Zhang DN, Nagaravapu U, Hultquist K, Romero LI, Herron GS. Membrane-type matrix metalloproteinases in human dermal microvascular endothelial cells: expression and morphogenetic correlation. J Invest Dermatol. 1998;111:1153 1159. 64. Lamoreaux WJ, Fitzgerald ME, Reiner A, Hasty KA, Charles ST. Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro. Microvasc Res. 1998;55:29 42. 65. Unemori EN, Ferrara N, Bauer EA, Amento E. Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells. J Cell Physiol. 1992;153:5575 5562. 66. Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, Jesty J. Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer. 1998;75:780 786. 67. Okamura K, Sato Y, Matsuda T, Hamanaka R, Ono M, Kohno K, Kuwano M. Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factortreated human microvascular endothelial cells. J Biol Chem. 1991;266: 19162 19165. 68. Cornelius LA, Nehring LC, Roby JD, Parks WC, Welgus HG. Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration. J Invest Dermatol. 1995;105:170 176. 69. Haas TL, Davis SJ, Madri JA. Three-dimensional type I collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells. J Biol Chem. 1998;273: 3604 3610. 70. Haas TL, Stitelman D, Davis SJ, Apte SS, Madri JA. Egr-1 mediates extracellular matrix-driven transcription of membrane type I matrix metalloproteinase in endothelium. J Biol Chem. 1999;274:22679 22685. 71. Nguyen M, Arkell J, Jackson CJ. Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP. Int J Biochem Cell Biol. 2000;32:621 631. 72. Yan L, Moses MA, Huang S, Ingber DE. Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells. J Cell Sci. 2000;113:3979 3987. 73. Partridge CA, Phillips PG, Niedbala MJ, Jeffrey JJ. Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts. Am J Physiol. 1997;272:L813 L822. 74. Sang QX. Complex role of matrix metalloproteinases in angiogenesis. Cell Res. 1998;8:171 177. 75. Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest. 1999;103: 1237 1241. 76. Raza SL, Cornelius LA. Matrix metalloproteinases: pro- and antiangiogenic activities. J Invest Dermatol Symp Proc. 2000;5:47 54. 77. Guedez L, McMarlin AJ, Kingma DW, Bennett TA, Stetler-Stevenson M, Stetler-Stevenson WG. Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt s lymphoma via divergent effects on tumor growth and angiogenesis. Am J Pathol. 2001;158:1207 1215. 78. Hajitou A, Sounni NE, Devy L, Grignet-Debrus C, Lewalle JM, Li H, Deroanne CF, Lu H, Colige A, Nusgens BV, Frankenne F, Maron A, Yeh P, Perricaudet M, Chang Y, Soria C, Calberg-Bacq CM, Foidart JM, Noel A. Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis. Cancer Res. 2001;61:3450 3457. 79. Kräling BM, Wiederschain DG, Boehm T, Rehn M, Mulliken JB, Moses MA. The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro. J Cell Sci. 1999;112: 1599 1609. 80. Zhu WH, Guo X, Villaschi S, Nicosia RF. Regulation of vascular growth and regression by matrix metalloproteinases in the rat aorta model of angiogenesis. Lab Invest. 2000;80:545 555. 81. Davis GE, Pintar Allen KA, Salazar R, Maxwell SA. Matrix metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices. J Cell Sci. 2001;114:917 930. 82. Lozonschi L, Sunamura M, Kobari M, Egawa S, Ding L, Matsuno S. Controlling tumor angiogenesis and metastasis of C26 murine colon adenocarcinoma by a new matrix metalloproteinase inhibitor, KB-R7785, in two tumor models. Cancer Res. 1999;59:1252 1258. 83. Maekawa R, Maki H, Yoshida H, Hojo K, Tanaka H, Wada T, Uchida N, Takeda Y, Kasai H, Okamoto H, Tsuzuki H, Kambayashi Y, Watanabe F, Kawada K, Toda K, Ohtani M, Sugita K, Yoshioka T. Correlation of antiangiogenic and antitumor efficacy of N-biphenylsulfonylphenylalanine hydroxamic acid (BPHA), an orallyactive, selective matrix metalloproteinase inhibitor. Cancer Res. 1999; 59:1231 1235. 84. Dong Z, Kumar R, Yang X, Fidler IJ. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell. 1997;88:801 810. 85. Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem. 1997;272:28823 28825. 86. Cornelius LA, Nehring LC, Harding E, Bolanowski M, Welgus HG, Kobayashi DK, Pierce RA, Shapiro SD. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol. 1998; 161:6845 6852.

1116 Arterioscler Thromb Vasc Biol. July 2001 87. Lijnen HR, Ugwu F, Bini A, Collen D. Generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3). Biochemistry. 1998;37:4699 4702. 88. Pozzi A, Moberg PE, Miles LA, Wagner S, Soloway P, Gardner HA. Elevated matrix metalloproteinase and angiostatin levels in integrin 1 knockout mice cause reduced tumor vascularization. Proc Natl Acad Sci USA. 2000;97:2202 2207. 89. Wen W, Moses MA, Wiederschain D, Arbiser JL, Folkman J. The generation of endostatin is mediated by elastase. Cancer Res. 1999;59: 6052 6056. 90. Felbor U, Dreier L, Bryant RAR, Ploegh HL, Olsen BR, Mothes W. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J. 2000;19:1187 1194. 91. Ferreras M, Felbor U, Lenhard T, Olsen BR, Delaissé JM. Generation and degradation of human endostatin proteins by various proteinases. FEBS Lett. 2000;486:247 251. 92. Fang J, Shing Y, Wiederschain D, Yan L, Butterfield C, Jackson G, Harper J, Tamvakopoulos G, Moses MA. Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model. Proc Natl Acad Sci U S A. 2000;97:3884 3889. 93. Koivunen E, Arap W, Valtanen H, Rainisalo A, Medina OP, Heikkila P, Kantor C, Gahmberg CG, Salo T, Konttinen YT, Sorsa T, Ruoslahti E, Pasqualini R. Tumor targeting with a selective gelatinase inhibitor. Nat Biotechnol. 1999;17:768 774. 94. Kim YM, Jang JW, Lee OH, Yeon J, Choi EY, Kim KW, Lee ST, Kwon YG. Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase 2. Cancer Res. 2000;60:5410 5413. 95. Bein K, Simons M. Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. J Biol Chem. 2000;275:32167 32173. 96. Brooks PC, Silletti S, von Schalscha TL, Friedlander M, Cheresh DA. Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Cell. 1998;92:391 400. 97. Pfeifer A, Kessler T, Silletti S, Cheresh DA, Verma IM. Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2. Proc Natl Acad Sci U S A. 2000;97: 12227 12232. 98. Silletti S, Kessler T, Goldberg J, Boger DL, Cheresh DA. Disruption of matrix metalloproteinase 2 binding to integrin v 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo. Proc Natl Acad Sci U S A. 2001;98:119 124. 99. Itoh T, Ikeda T, Gomi H, Nakao S, Suzuki T, Itohara S. Unaltered secretion of -amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J Biol Chem. 1997;272:22389 22392. 100. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 1998;93:411 422. 101. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res. 1998;58:1048 1051. 102. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol. 2000;2:737 744. 103. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, Birkedal- Hansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell. 1999;99:81 92. 104. Zhou Z, Apte SS, Soininen R, Cao R, Baaklini GY, Rauser RW, Wang J, Cao Y, Tryggvason K. Impaired enchondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci U S A. 2000;97:4052 4057. 105. Collen D. The plasminogen (fibrinolytic) system. Thromb Hemost. 1999;82:259 270. 106. Montesano R, Pepper MS, Vassalli JD, Orci L. Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors. J Cell Physiol. 1987;132:509 516. 107. Hiraoka N, Allen E, Apel IJ, Gyetko MR, Weiss SJ. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell. 1998;95:365 377. 108. Lelongt B, Bengatta S, Delauche M, Lund LR, Werb Z, Ronco PM. Matrix metalloproteinase 9 protects mice from anti-glomerular basement membrane nephritis through its fibrinolytic activity. J Exp Med. 2001; 193:793 802. 109. Carmeliet P, Collen D. Transgenic mouse models in angiogenesis and cardiovascular disease. J Pathol. 2000;190:387 405. 110. Cavallaro U, Tenan M, Castelli V, Perilli A, Maggiano N, Van Meir E, Montesano R, Soria MR, Pepper MS. The response of bovine endothelial cells to FGF-2 and VEGF is dependent on their site of origin: relevance to the regulation of angiogenesis. J Cell Biochem. In press. 111. Pepper MS, Rosnoblet C, Di Sanza C, Kruithof EKO. Synergistic induction of tissue-type plasminogen activator (tpa) by vascular endothelial growth factor and basic fibroblast growth factor and localization of tpa to Weibel-Palade bodies in bovine microvascular endothelial cells. Thromb Hemost. In press. 112. Kroon ME, Koolwijk P, van Der Vecht B, van Hinsbergh VWM. Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix. Blood. 2000;96:2775 2783. 113. Uchiyama T, Kurabayashi M, Ohyama Y, Utsugi T, Akuzawa N, Sato M, Tomono S, Kawazu S, Nagai R. Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 2000;20:1155 1161. 114. Pepper MS, Vassalli J-D, Montesano R, Orci L. Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells. J Cell Biol. 1987;105:2535 2541. 115. Pepper MS, Sappino A-P, Montesano R, Orci L, Vassalli J-D. Plasminogen activator inhibitor-1 is induced in migrating endothelial cells. J Cell Physiol. 1992;153:129 139. 116. Pepper MS, Sappino A-P, Stocklin R, Montesano R, Orci L, Vassalli J-D. Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Biol. 1993;122:673 684. 117. Bacharach E, Itin A, Keshet E. Apposition-dependent induction of plasminogen activator inhibitor type-1 expression: a mechanism for balancing pericellular proteolysis during angiogenesis. Blood. 1998;92: 939 945. 118. Pepper MS. Lymphangiogenesis and tumor metastasis: myth or reality. Clin Cancer Res. 2001;7:462 468. 119. Pepper MS, Wasi S, Ferrara N, Orci L, Montesano R. In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells. Exp Cell Res. 1994;210:298 305. 120. Pepper MS, Mandriota SJ, Jeltsch M, Kumar V, Alitalo K. Vascular endothelial growth factor (VEGF)-C synergises with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro, and alters endothelial cell proteolytic properties. J Cell Physiol. 1998;177: 439 452. 121. Liu N-F, He Q-L. The regulatory effects of cytokines on lymphatic angiogenesis. Lymphology. 1997;30:3 12. 122. Leak LV, Saunders M, Day AA, Jones M. Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium. Microvasc Res. 2000;60:201 211. 123. Rømer J, Bugge TH, Pyke C, Lund LR, Flick MJ, Degan LJ, Danø K. Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med. 1996;2:287 292. 124. Soff GA, Sanderowitz J, Gately S, Verrusio E, Weiss I, Brem S, Kwaan HC. Expression of plasminogen activator inhibitor type 1 by human prostate carcinoma cells inhibits primary tumor growth, tumorassociated angiogenesis, and metastasis to lung and liver in an athymic mouse model. J Clin Invest. 1995;96:2593 2600. 125. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999;5:1135 1142. 126. Berman M, Winthrop S, AusprunkD, Rose J, Langer R, Gage J. Plasminogen activator (urokinase) causes vascularization of the cornea. Invest Ophthalmol Vis Sci. 1982;22:191 199. 127. Goldfarb RH, Ziche M, Murano G, Liotta LA. Plasminogen activators (urokinase) mediate neovascularization: possible role in tumor angiogenesis. Semin Thromb Hemost. 1986;12:337 338. 128. Ribatti D, Leali D, Vacca A, Giuliani R, Gualandris A, Roncali L, Nolli ML, Presta M. In vivo angiogenic activity of urokinase: role of endogenous fibroblast growth factor-2. J Cell Sci. 1999;112:4213 4221. 129. Min HY, Doyle LV, Vitt CR, Zandonella L, Stratton-Thomas JR, Shuman MA, Rosenberg S. Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res. 1996;56:2428 2433.

Pepper Extracellular Proteolysis and Angiogenesis 1117 130. Li H, Lu H, Griscelli F, Opolon P, Sun LQ, Ragot T, Legrand Y, Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediated delivery of a upa/upar antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Ther. 1998;5: 1105 1113. 131. Evans CP, Elfman F, Parangi S, Conn M, Cunha G, Shuman MA. Inhibition of prostate cancer neovascularization and growth by urokinase-plasminogen activator receptor blockade. Cancer Res. 1997; 57:3594 3599. 132. Guo Y, Higazi AA, Arakelian A, Sachais BS, Cines D, Goldfarb RH, Jones TR, Kwaan H, Mazar AP, Rabbani SA. A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (upa) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 2000;14:1400 1410. 133. Mishima K, Mazar AP, Gown A, Skelly M, Ji XD, Wang XD, Jones TR, Cavenee WK, Huang HJ. A peptide derived from the non-receptorbinding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin. Proc Natl Acad Sci U S A. 2000;97:8484 8489. 134. Bajou K, Noel A, Gerard RD, Masson V, Brunner N, Holst-Hansen C, Skobe M, Fusenig NE, Carmeliet P, Collen D, Foidart JM. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med. 1998;4:923 928. 135. Bajou K, Masson V, Gerard RD, Schmitt PM, Albert V, Praus M, Lund LR, Frandsen TL, Brunner N, Dano K, Fusenig NE, Weidle U, Carmeliet G, Loskutoff D, Collen D, Carmeliet P, Foidart JM, Noël A. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin: implications for antiangiogenic strategies. J Cell Biol. 2001;152:777 784. 136. Gutierrez LS, Schulman A, Brito-Robinson T, Noria F, Ploplis VA, Castellino FJ. Tumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1. Cancer Res. 2000;60:5839 5847. 137. Loskutoff DJ, Curriden SA, Hu G, Deng G. Regulation of cell adhesion by PAI-1. APMIS. 1999;107:54 61. 138. Ossowski L, Aguirre-Ghiso JA. Urokinase receptor and integrin partnership: coordination of signalling for cell adhesion, migration and growth. Curr Opin Cell Biol. 2000;12:613 620. 139. Preissner KT, Kanse SM, May AE. Urokinase receptor: a molecular organizer in cellular communication. Curr Opin Cell Biol. 2000;12: 621 628. 140. Stefansson S, Petitclerc E, Wong MK, McMahon GA, Brooks PC, Lawrence DA. Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1. J Biol Chem. 2001;276:8135 8141. 141. Levin EG, del Zoppo GJ. Localization of tissue plasminogen activator in the endothelium of a limited number of vessels. Am J Pathol. 1994;144: 855 861. 142. Mignatti P, Tsuboi R, Robbins E, Rifkin DB. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J Cell Biol. 1989;108:671 682. 143. Yasunaga C, Nakashima Y, Sueishi K. A role of fibrinolytic activity in angiogenesis: quantitative assay using in vitro model. Lab Invest. 1989; 61:698 704. 144. Sato Y, Okamura K, Morimoto A, Hamanaka R, Hamaguchi K, Shimada T, Ono M, Kohno K, Sakata T, Kuwano M. Indispensable role of tissue-type plasminogen activator in growth factor-dependent tube formation of human microvascular endothelial cells in vitro. Exp Cell Res. 1993;204:223 229. 145. Ito K-I, Ryuto M, Ushiro S, Ono M, Sugenoya A, Kuraoka A, Shibata Y, Kuwano M. Expression of tissue-type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on Matrigel. J Cell Physiol. 1995;162: 213 224. 146. Schnaper HW, Barnathan ES, Mazar A, Maheshwari S, Ellis S, Cortez SL, Baricos WH, Kleinman HK. Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways. J Cell Physiol. 1995;165:107 118. 147. Mawatari M, Okamura K, Matsuda T, Hamanaka R, Mizoguchi H, Higashio K, Kohno K, Kuwano M. Tumor necrosis factor and epidermal growth factor modulate migration of human microvascular endothelial cells and production of tissue-type plasminogen activator and its inhibitor. Exp Cell Res. 1991;192:574 580. 148. Zablocki DK, Rade JJ, Alevriadou BR. Adenovirus-mediated expression of tissue plasminogen activator does not alter endothelial cell proliferation and invasion. In Vitro Cell Dev Biol Anim. 2000;36:625 628. 149. Stack MS, Gately S, Bafeti LM, Enghild JJ, Soff GA. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrixenhanced plasminogen activation. Biochem J. 1999;340:77 84. 150. Datta YH, Youssoufian H, Marks PW, Ewenstein BM. Targeting of a heterologous protein to a regulated secretion pathway in cultured endothelial cells. Blood. 1999;94:2696 2703. 151. Rosnoblet C, Vischer UM, Gerard RD, Irminger JC, Halban PA, Kruithof EKO. Storage of tissue-type plasminogen activator in Weibel- Palade bodies of human endothelial cells. Arterioscler Thromb Vasc Biol. 1999;19:1796 1803. 152. Taraboletti G, Sonzogni L, Vergani V, Hosseini G, Ceruti R, Ghilardi C, Bastone A, Toschi E, Borsotti P, Scanziani E, Giavazzi R, Pepper MS, Stetler-Stevenson WG, Bani MR. Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells. Exp Cell Res. 2000;258:384 394. 153. Pepper MS, Tacchini-Cottier F, Sabapathy K, Montesano R, Wagner EF. Endothelial cells transformed by polyomavirus middle T oncogene: a model for hemangiomas and other vascular tumours. In: Bicknell R, Lewis CE, Ferrara N, eds. Tumour Angiogenesis. Oxford, England: Oxford University Press; 1997:309 331. 154. Taraboletti G, Belotti D, Dejana E, Mantovani A, Giavazzi R. Endothelial cell migration and invasiveness are induced by a soluble factor produced by murine endothelioma cells transformed by polyoma virus middle T oncogene. Cancer Res. 1993;53:3812 3816. 155. Vergani V, Garofalo A, Bani MR, Borsotti P, Parker MP, Drudis T, Mazzarol G, Viale G, Giavazzi R, Stetler-Stevenson WG, Taraboletti G. Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas. Int J Cancer. 2001;91:241 247. 156. Taraboletti G, Garofalo A, Belotti D, Drudis T, Borsotti P, Scanziani E, Brown PD, Giavazzi R. Inhibition of angiogenesis and murine hemangioma growth by Batimastat, a synthetic inhibitor of matrix metalloproteinases. J Natl Cancer Inst. 1995;87:293 298. 157. Dubois-Stringfellow N, Jonczyk J, Bautch VL. Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells. Blood. 1994;83: 3206 3217. 158. Besser D, Urich M, Sakaue M, Messerschmitt A, Ballmer-Hofer K, Nagamine Y. Urokinase-type plasminogen activator gene regulation by polyomavirus middle-t antigen. Oncogene. 1995;11:2383 2391. 159. Primo L, Roca C, Ferrandi C, Lanfrancone L, Bussolino F. Human endothelial cells expressing polyoma middle T induce tumors. Oncogene. 2000;19:3632 3641. 160. Sabapathy KT, Pepper MS, Kiefer F, Möhle-Steinlein U, Tacchini- Cottier F, Fetka I, Breier G, Carmeliet P, Risau W, Montesano R, Wagner EF. Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system. J Cell Biol. 1997;137: 953 963. 161. Lijnen HR, Wagner EF, Collen D. Plasminogen-dependent and -independent proteolytic activity of murine endothelioma cells with targeted inactivation of fibrinolytic genes. Thromb Hemost. 1997;77: 362 367. 162. Griffioen AW, Molema G. Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev. 2000;52:237 268. 163. Brown PD. Clinical studies with matrix metalloproteinase inhibitors. APMIS. 1999;107:174 180. 164. Hidalgo M, Eckhardt SG. Development of matrix metalloproteinase inhibitors in cancer therapy. J Natl Cancer Inst. 2001;93:178 193. 165. Zucker S, Cao J, Chen WT. Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment. Oncogene. 2000;19: 6642 6650. 166. Kruger A, Soeltl R, Sopov I, Kopitz C, Arlt M, Magdolen V, Harbeck N, Gansbacher B, Schmitt M. Hydroxamate-type matrix metalloproteinase inhibitor batimastat promotes liver metastasis. Cancer Res. 2001; 61:1272 1275. 167. Jiang Y, Wang M, Celiker MY, Liu YE, Sang QXA, Goldberg ID, Shi YE. Stimulation of mammary tumorigenesis by systemic tissue inhibitor of matrix metalloproteinase 4 gene delivery. Cancer Res. 2001;61: 2365 2370. 168. Eatock MM, Schätzlein A, Kaye SB. Tumour vasculature as a target for anticancer therapy. Cancer Treatment Rev. 2000;26:191 204. 169. Mandriota SJ, Pepper MS. Vascular endothelial growth factor-induced in vitro angiogenesis and plasminogen activator expression are dependent on endogenous basic fibroblast growth factor. J Cell Sci. 1997;110:2293 2302.

Role of the Matrix Metalloproteinase and Plasminogen Activator Plasmin Systems in Angiogenesis Michael S. Pepper Arterioscler Thromb Vasc Biol. 2001;21:1104-1117 doi: 10.1161/hq0701.093685 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright 2001 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/21/7/1104 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/