uPAR in the modulation of angiogenesis.

Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 105–122 (DOI: 10.1159/000353310)

Role of uPA/uPAR in the Modulation of Angiogenesis Nunzia Montuori a ⋅ Pia Ragno b

a

Department of Translational Medical Sciences, ‘Federico II’ University, Naples, and of Chemistry and Biology, University of Salerno, Salerno, Italy

b Department

Abstract

Blood vessels represent the highways connecting all districts of the body, thus allowing oxygen and nutrients to reach every cell in the organism. Dysregulation of blood vessel formation or functionality is the origin of a large number of diseases. New blood vessels can form de novo by endothelial progenitor cells (EPCs) (vasculogenesis) or by sprouting from pre-existing vessels (angiogenesis), even though it is now generally accepted that locally recruited EPCs can also contribute to angiogenesis [1–2]. During new vessel formation, endothelial cells (ECs) degrade their basement membrane, migrate into the interstitial matrix and proliferate. Thus, angiogenesis requires dynamic, temporal and spatial interactions among ECs and extracellular matrix (ECM), highly regulated by the subtle balance between pro- and antiangiogenic factors. Main angiogenic factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibro-

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Blood vessels connect all districts of the body and allow blood oxygen and nutrients to reach every cell in the organism. Dysregulation of blood vessel formation or functionality is the origin of a large number of diseases. During new vessel formation, endothelial cells degrade their basement membrane, migrate into the interstitial matrix and proliferate. Migrating endothelial cells need to be polarized, to focus at their leading edge the proteolytic machinery, which is essential for extracellular matrix degradation; thus, proteases and their receptors play a crucial role in angiogenesis. The urokinase-mediated plasminogen activation system is a complex system of serine proteases strongly involved in angiogenesis. The plasminogen activation system includes plasminogen/plasmin, activators, inhibitors and cell receptors. In the last decades, a large body of evidence has clearly indicated that the role of this system is not limited to extracellular matrix proteolysis but can contribute to all Copyright © 2014 S. Karger AG, Basel phases of the angiogenic process.

blast growth factor-2 (FGF-2), angiopoietin 1 (Ang-1), stromal cell-derived factor 1 (SDF-1/CXCL12), endothelin-1 (ET-1), monocyte chemoattractant protein-1 (MCP1) [2–3]. Angiostatic factors include endostatin, angiostatin, thrombospondin-1, and angiopoietin 2 [3]. Migrating ECs need to be polarized, to focus at their leading edge the proteolytic machinery, which is essential for matrix degradation; thus, proteases and their receptors play a crucial role in angiogenesis [4]. The urokinase (uPA)-mediated plasminogen activation (PA) system is a complex system of serine proteases strongly involved in angiogenesis. The PA system includes plasminogen/plasmin, activators, inhibitors and cell receptors [5]. In the last decades, a large body of evidence has clearly indicated that the role of this system is not limited to ECM proteolysis but can contribute to all the angiogenic steps.

The enzymes belonging to the PA system are serine proteases, because their active site consists of a catalytic triad (His603, Asp646, and Ser741) including the amino acid serine; this active site is located in the C-terminal domain. The N-terminal region contains one or more functional domains, such as finger domains, epidermal growth factor-like domains and triple-loop structures called kringle domains [6]. The main inhibitors of the PA system are grouped into the serpin (serine protease inhibitor) superfamily. Serpins have, in their C-terminal region, a reactive center loop containing a specific peptide bond, which is cleaved by their target enzyme, resulting in the release of a peptide from the inhibitor and formation of an inactive enzymeinhibitor complex [6]. The zymogen plasminogen is predominantly synthesized as a single chain in the liver and distributed throughout the human body. Plasminogen is converted to the two-chain active plasmin by cleavage of a single Arg561–Val562 peptide bond. Plasmin is composed of an N-terminal heavy chain containing five kringles and a C-terminal light chain containing the catalytic triad. The plasminogen/plasmin kringles contain lysine-binding sites that mediate their specific binding to fibrin, to membrane-binding sites and to the physiological inhibitor alpha2-antiplasmin [7]. Plasmin is a broad-spectrum protease. It binds fibrin and is a potent fibrinolytic agent; thus, a primary in vivo function of plasmin is to regulate fibrinolysis, i.e. the process wherein the fibrin clot, end product of the coagulation process, is degraded. Plasminogen/plasmin also binds to the cell surface, with low affinity but high capacity; cell-bound plasmin is protected from its natural inhibitors, since both receptors and inhibitors utilize plasminogen/plasmin kringle lysine-binding sites [7]. Plasmin, beside fibrin, can promote the degradation of various components of the ECM, directly, or by activating several metalloproteases (MMPs). The plasminogen/plasmin ability to bind low-affinity cellular receptors and to degrade ECM proteins has impli-

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Montuori · Ragno Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 105–122 (DOI: 10.1159/000353310)


Plasminogen Activation System

uPA/uPAR and Angiogenesis Marone G, Granata F (eds): Angiogenesis, Lymphangiogenesis and Clinical Implications. Chem Immunol Allergy. Basel, Karger, 2014, vol 99, pp 105–122 (DOI: 10.1159/000353310)

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cated plasmin in processes requiring degradation of protein barriers, in particular in cell migration [7]. Cell migration is a crucial event in various physiologic and pathologic processes such as embryogenesis, wound healing, angiogenesis, inflammation, tumor growth and dissemination [8]. Plasminogen can be activated by different proteases, but its specific physiologic activators are the tissue-type (tPA) and the urokinase-type (uPA) plasminogen activators. Both serine proteases are secreted as single chains and are activated by a single cleavage that yields two chains held together by a disulfide bond [6]. t-PA binds fibrin with high affinity by a finger and a kringle domain. The assembly of plasminogen and tPA at the surface of fibrin results in efficient fibrin degradation, thus tPA has a key role in fibrinolysis [6]. uPA is synthesized as pro-uPA, a single chain consisting of a growth factor-like domain (GFD, residues 1–49), a kringle domain (residues 50–131) and a serine protease domain (residues 159–411). Pro-uPA is almost inactive and may be activated by a cleavage at the Lys158-Ile159 residues, which yields a two-chain molecule, held together by a disulfide bond [9]. Cleavage may be catalyzed by various proteases including plasmin, cathepsin B and kallikrein, as shown in the urine from plasminogendeficient mice. A further proteolytic cleavage at Lys135-Lys136 releases the aminoterminal fragment (ATF, amino acids 1–135), which is able to bind the specific cellular receptor for uPA but is devoid of catalytic activity; the remaining molecule, consisting of the 136–158 fragment linked through a disulfide bridge to the carboxyterminal catalytic region, represents the low-molecular-weight form of uPA, which retains full ability to activate plasminogen but is unable to bind uPA receptors [5]. uPA does not bind fibrin as tPA, thus it seems less implicated in fibrinolysis; however, both uPA- and tPA-deficient mice show an increased thrombotic susceptibility, and only combined uPA/tPA-deficient mice show severe spontaneous thrombosis like plasmin-deficient mice, thus suggesting that these enzymes can substitute for each other in thrombolysis [10]. On the other hand, the discovery of a highly specific cellular receptor for uPA suggested that this plasminogen activator contributes mainly to the non-fibrinolytic activity of plasmin, i.e. the ECM degradation [11]. In fact, binding of plasminogen and its urokinase-type activator to the cell surface highly enhances plasminogen activation, strongly potentiating the invasive ability of the cell [12]. The activity of the plaminogen activation system is strictly regulated. The primary plasmin inhibitor is the alpha2-antiplasmin. Two specific inhibitors of the plasminogen activators (PAs) have been largely described. The type1 inhibitor (PAI1) is the primary inhibitor of both tPA and uPA, it reacts with two-chain but not single-chain pro-uPA [6]. The serpin is present at a low concentration in normal plasma, but at higher concentrations in many clinical conditions. PAI1 has been detected in two different activity states in vivo (active and latent), dependently on conformational changes in the reactive center loop of the inhibitor. In the active form, the reactive center is exposed on the surface of the molecule and is able to interact with its target proteases.

This form is synthesized and secreted by cells but is unstable in solution and spontaneously converts into the inactive (latent) form. The latent form can be converted into the active form by treatment with denaturants or negatively charged phospholipids [6]. All active PAI1 in plasma seems to circulate in complex with vitronectin (VN) and this interaction stabilizes the inhibitor. The high-affinity binding site for PAI1 in VN has been mapped in the somatomedin B (SMB) domain [13]. The type 2 inhibitor (PAI2) shows lower inhibitory efficiency, as compared to PAI1; its intracellular detection in nonglycosylated forms suggested a regulatory role for PAI2 in the activity of enzymes other than uPA [14]. uPA binds a specific high-affinity cell-anchored receptor, the urokinase-type plasminogen activator receptor (uPAR), which, in the last decades, has been largely characterized in its expression, structure and function [15].

A cellular binding site with high specificity and affinity for uPA in human blood monocytes and in cells of the monocytic line U937 was identified in 1985 [11, 16]; the isolation of the purified protein and the sequencing of its cDNA were reported 5 years later [17–18]. The cDNA sequence suggested that the human uPAR consisted of 313 amino acid residues without any obvious transmembrane sequence. Accordingly, it was later shown that uPAR undergoes posttranslational modifications consisting in the removal of a 30-residue C-terminal signal sequence and the addition to Gly283 of a glycosylphosphatidylinositol (GPI) tail, anchoring the receptor to the cell membrane [19]. Mature uPAR is organized into three domains (DI-DII-DIII from the N-terminus) with homologous cysteine repeats characteristic of the Ly-6 protein superfamily; the domains are connected by short linker regions (fig. 1). uPAR is extensively N-glycosylated at residues in all three domains; the glycosylation pattern of uPAR differs among cell types. The function of these glycosylations is poorly understood, but they can enhance the affinity of uPAR for uPA, affect intracellular trafficking and protect from proteolysis [20–21]. Solved structures of uPAR-ligand complexes show that the three domains pack together into a concave structure with a large surface in the central cavity, containing residues from all three domains [22]. Alanine scanning mutagenesis of uPAR showed that the residues for uPA interaction are located within or at the rim of the central cavity formed by the assembly of all three domains, even half of the uPA/uPAR binding interface is located in DI. These data confirmed the previous observations showing the predominant role of DI in uPA binding but the requirement of all three uPAR domains for an efficient binding [23]. uPAR-bound uPA can form complexes with its inhibitor PAI1; the ternary complex can be internalized, via a member of the LDL receptor family, and degraded. Thereafter, uPAR can recycle back from the endocytotic compartment to the cell surface [24].

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Interestingly, uPAR also acts as a non-integrinic receptor for VN, a component particularly abundant in ECM associated with tumor tissues. uPAR binds the N-terminal SMB domain of VN, thus competing with PAI1 for the binding to VN [25]. The functional epitope on uPAR that is responsible for its interaction with VN has been identified by alanine scanning mutagenesis of uPAR; the five residues identified as ‘hot spots’ for VN binding are located in domain I and in the flexible linker region connecting uPAR domains I and II [26]. Since the VN- and uPA-binding sites are distinct, uPAR can simultaneously bind both ligands, allowing coordinated regulation of proteolysis and cell adhesion; indeed, uPA binding to uPAR enhances its binding to VN [5] (fig. 1). Cell-membrane uPAR can be cleaved in the DI–DII linker region by proteases such as uPA, plasmin and MMPs, thus generating a soluble DI fragment and cellmembrane truncated uPAR forms, which can expose or not the DI–DII linker region containing the sequence SRSRY (residues 88–92), according to the cleavage site [27– 28]. Both full-length and cleaved uPARs can be shed from the cell surface by phospholipases, generating full-length and cleaved uPAR soluble forms (fig. 1). Cleaved


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Fig. 1. Different forms of the uPA receptor. The cell-surface uPA receptor (uPAR) is organized into three domains (DI–DII–DIII from the N-terminus); the domains are connected by short linker regions. The uPA-binding site is located in the DI domain, but all the three domains are required for a highaffinity binding. uPAR also binds VN, an extracellular matrix component. Cell-surface uPAR can be cleaved in the DI–DII linker region by proteases, such as uPA, plasmin and MMPs, thus generating a cell-membrane truncated uPAR form, which can expose the SRSRY sequence (residues 88–92). Both full-length and cleaved uPARs can be shed from the cell surface by phospholipases, generating fulllength and cleaved uPAR soluble forms (suPARs). The cleaved suPAR, exposing the SRSRY sequence, is able to interact with the fMLF family of G-protein-coupled chemotaxis receptors (fMLF-Rs).

uPAR forms are unable to bind both uPA and VN, suggesting the cleavage of cellmembrane uPAR as a regulatory mechanism to abolish uPAR activities at the cell surface, i.e. focused proteolysis and binding to the ECM [5]. However, the cleaved form of soluble uPAR exposing the SRSRY sequence at the N-terminus exerts a novel activity, since it is a ligand of chemotaxis receptors for formylated peptides (fMLF) [29] (fig. 1). Like other GPI-anchored proteins, uPAR associates with cholesterol- and sphingolipid-rich membrane microdomains, termed ‘lipid rafts’ [30]. Lipid raft partitioning, which is promoted by uPA binding, increases the ability of uPAR to bind VN, suggesting a mechanism for the uPA-induced increase in uPAR binding to VN [31].

The primary role of uPAR, at its discovery, was considered the focusing of proteolytic activities on the cell surface [11, 16]. This function is crucial to promote localized ECM degradation, which allows cell migration throughout the surrounding matrix. Subsequently, several reports showed that uPAR was also able to activate intracellular signaling and to promote cell adhesion, migration, differentiation, proliferation and survival, independently of proteolytic activities [15]. In fact, uPAR-dependent signaling can be activated by uPA or its aminoterminal fragment, devoid of proteolytic activity, by VN, and by uPAR overexpression itself [32]. uPAR stimulation activates a number of signaling molecules such as Src family kinases, Rho family GTPases, ERK1/2 Ser/Thr kinases; among Rho GTPases, uPAR system has been reported to act through Rac, Cdc42, RhoA, and, very recently, RhoB [32–33]. However, because of its lack of transmembrane and cytosolic regions, uPAR requires signaling partners able to transduce signals inside the cell. uPAR capability to associate to ‘lipid rafts’, which are important platforms for signal transduction, might increase protein-protein interactions between uPAR, its signaling partners and intracellular effectors, allowing downstream signaling. Indeed, the solved structure of uPAR suggested that since uPA engages the uPAR central cavity, the external receptor surface is accessible for other protein interactions [22]. Many signaling partners have been proposed for uPAR; the most important transmembrane receptors associated with uPAR belong to integrin families, even if a direct and physical uPAR interaction with integrins has been questioned [32] (fig. 2). Fluorescence resonance energy transfer analysis, immunolocalization and co-immunoprecipitation have identified uPAR in complex with several integrin families, such as beta1, beta2, beta3 and beta5 [32]. uPAR co-capping with the beta2 integrin Mac-1 was firstly demonstrated in resting neutrophils [34]; subsequently, the presence of uPAR and beta2 integrins was identified in large receptor complexes that included signaling molecules. In vivo, the beta2 integrin-dependent recruitment of leukocytes to inflamed peritoneum and of

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neutrophils to the lung in response to Pseudomonas aeruginosa infection is significantly reduced in uPAR-deficient mice [35–36]. uPAR-beta1 integrin association mediates cell adhesion, migration, invasion of ECM, proliferation, epithelial-mesenchymal transition (EMT), and, in addition, increases the expression of uPA and metalloproteases, suggesting that uPAR signaling through beta1 integrins can contribute to cell migration also by increasing pericellular proteolysis [37–38]. alphav integrins have also been strongly implicated in uPAR signaling [37]. Both uPAR and alphav integrins bind VN, uPAR recognizing the SMB domain and alphav integrins the Arg-Gly-Asp sequence. The uPAR-alphav integrin interaction has an important role in signaling for cell migration [32]. uPAR-binding sites have been identified on alpha and beta1 integrin chains, and, on the other hand, putative integrin-binding sites on uPAR have been identified in the uPAR domains II and III [5]. However, uPAR binding to integrins requires the full-length receptor, as in the case of uPAR binding to uPA and VN [39]. uPAR-beta1


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Fig. 2. uPAR activity at the cell surface. uPAR, upon binding to uPA, activates signaling pathways through lateral interactions with integrins, thus regulating various cell functions. uPAR also focuses uPA proteolytic activity at the cell surface and, at the same time, mediates cell adhesion to the ECM by binding VN. uPAR-bound uPA can be inactivated by specific inhibitors (PAIs) or can transform plasminogen (Plg) in the broad spectrum serine protease plasmin (Plm) that, in turn, degrades ECM components directly or through the activation of latent metalloproteases (pro-MMPs).

and uPAR-alphav integrin interactions have been often associated with the activation of focal adhesion kinase/src signaling and increased activities of the small GTP-binding protein Rac1 or RhoB and/or extracellular regulated kinases 1–2 [32–33]. Functional interactions of cell-surface uPAR with the chemotaxis receptors for fMLF (fMLF-Rs) have been also reported [15, 38] and uPAR has been proposed as an endogenous ligand for fMLF-Rs [40]. GPI-uPAR can also cross-talk with growth factor receptors, such as epidermal growth factor receptor (EGFR) and PDGFR-beta. Indeed, uPA binding to uPAR can initiate a cell-signaling pathway that is mediated by EGFR, and, conversely, uPARdependent cell-signaling may prime cells to proliferate in response to EGF [5]. Recently, the cross-talk between uPAR and CXCR4, the receptor for the stromal-derived factor 1 (SDF1) chemokine has been reported [41]; uPAR expression regulates CXCR4 activity on specific extracellular matrix components by a mechanism involving fMLF receptors (fMLF-Rs) and alphav integrins [41].

Potentially, the uPA/uPAR system may mediate all phases of the angiogenic process. In fact, following pro-angiogenic signals, ECs degrade their basement membrane, migrate into the interstitial matrix and proliferate [1–3]. The uPA/uPAR axis can control bioavailability of angiogenic factors, matrix remodeling, and activation of MMPs, which, in turn, can contribute to the angiogenic process. Further, upon binding to its cell surface receptor, uPA can activate signaling pathways regulating cell survival, proliferation and migration [32]. The pro-angiogenic role of uPA was initially proposed in models of corneal vascularization about 30 years ago [42]. Some years later, Montesano et al. showed that FGFs can induce capillary endothelial cells to form characteristic tubules similar to blood capillaries in a three-dimensional collagen matrix and, concomitantly, it stimulated endothelial cells to produce uPA [43]. A direct role for uPA in tumor angiogenesis was hypothesized in the same year [44]. To evaluate the role of PAs in physiological angiogenesis, Bacharach et al. [45] investigated the in vivo patterns of expression of uPA during neovascularization of the ovarian follicles, corpus luteum, and the maternal decidua. Using in situ hybridization, they detected uPA mRNA in the ovary along the route of capillary extension, originating at the existing ovarian vasculature, extending toward growing follicles, and terminating at the newly formed capillary sheaths surrounding each growing follicle. Following ovulation, uPA mRNA was expressed in capillary sprouts within the developing corpus luteum. During the process of decidual neovascularization, uPA expression was detected in endothelial cell cords traversing the maternal decidua in the direction of the newly implanted embryo. uPA mRNA was not detected in endothelial cells upon completion of neovascularization, clearly suggesting that uPA ex-

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pression was a part of the angiogenic response. During the in vitro angiogenesis assay with cultured aortic explants, uPA was expressed in capillary sprouts but not in underlying endothelial cell sheets, suggesting that the expression of uPA depends on the histological context of the endothelial cell [45]. In fact, many proangiogenic factors, including cytokines such as bFGF and VEGF, or hypoxia, can upregulate uPA/uPAR expression [46]. Studies in mice with targeted gene inactivation revealed that vascular injury-induced neointima formation was reduced in mice lacking uPA-mediated plasmin proteolysis. Arterial neointima formation was induced in mice with inactivated genes of the PA system, using an externally applied electric current to deplete a defined vessel fragment of viable cells. Comparative morphometric analysis revealed that deficiency of uPA or plasminogen greatly reduced and delayed neointima formation and neointimal accumulation of smooth muscle cells. However, the finding that genetically altered mice developed normally without overt vascular anomalies suggested the possibility of compensation by other proteases/protease receptors in physiologic angiogenesis [47]. Indeed, uPA directly activates VEGF and pro-HGF and, via generation of plasmin, leads to the release of other angiogenic growth factors from ECM, including bFGF, IGF and TGF-beta. In turn, these factors upregulate uPA and/or uPAR expression, increasing the proteolytic activity at the cell-surface [46]. VEGF can also contribute to pro-uPA activation; Prager et al. identified a mechanism, involving PI3-kinase and MMP2 activation, by which VEGF165, interacting with its receptor VEGFR-2, rapidly induces activation of uPAR-bound pro-uPA in endothelial cells, which contributes to a VEGF-dependent local fibrinolytic activity [48]. uPAR-bound uPA can then form complexes with its inhibitor PAI1, which can be internalized via a member of the LDL receptor family and degraded. Thereafter, uPAR itself can recycle back from the endocytotic compartment to the cell surface, not by random distribution, but focused on newly formed focal adhesions at the leading edge of migrating ECs [49]. An original study on endothelial microparticles (EMPs) from tumor necrosis factor-alpha-stimulated endothelial cells showed that they serve as a surface for the generation of plasmin, mediated by uPA bound to its receptor at the EMP surface. Low amounts of EMPs increase tube formation from endothelial progenitor cells, whereas higher concentrations inhibit it. Prevention of these effects by inhibitors of either uPA or plasmin further underscores the key role of uPA-induced plasmin generation in angiogenesis [50]. Since the role of uPA-uPAR axis was originally related to their capability to regulate localized ECM degradation and first studies in mice with targeted uPA/plasmin gene inactivation confirmed this role [47], several reports aimed to evaluate the importance of uPA-mediated proteolysis and the effects of inhibition of uPA activity or of uPA/uPAR interaction in angiogenesis models. Several of these studies were carried out in in vivo or in vitro tumor models, characterized by abnormal vessel growth. A protein resulting from the binding domain of mouse uPA (GFD 1–48) fused to the

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Fc fragment of IgG was reported to inhibit bFGF and VEGF-stimulated branching morphogenesis in an endothelial cell tube formation assay on a fibrin matrix in vitro and to inhibit angiogenesis and primary tumor growth in syngeneic mice [51]. A noncompetitive antagonist (A6) of the uPA-uPAR interaction derived from a nonreceptor-binding region of uPA (amino acids 136–143) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time [52]. Delivery of antisense sequences of uPAR and uPA in a single adenoviral vector inhibited glioma cell-induced angiogenesis. Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting uPA and its receptor was shown to retard angiogenesis and to inhibit intracranial tumor growth in nude mice [53]. A fusion protein, comprising the ATF of urokinase and VAS, the antiangiogenic functional domain of vasostatin, exhibited an improved inhibitory efficacy against endothelial cell proliferation and capillary vessel formation in a 3D angiogenesis model [54]. Abnormal vessel growth promotes also ocular disorders. An adenoviral vector carrying a uPA antagonist, the murine ATF coupled to human serum albumin (ATFHSA), reduced retinal neovascularization in retinopathy induced by exposing old mice to high levels of oxygen [55]. Besides regulating pericellular proteolytic activity, uPAR is also able to transduce signals inside the cells [32–33]. In fact, several reports showed that the uPA/uPAR axis mediates intracellular signal transduction also in endothelial cells. Bovine aortic endothelial cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF; however, migration is stimulated via a signaling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis [56]. bFGF and VEGF-dependent endothelial cell migration in vitro was inhibited by the uPA antagonist ATF-HSA. ATFHSA did not affect cell proliferation but was more potent than plasmin inhibitors, suggesting that it exerted its effects not solely by inhibiting ECM remodeling. In fact, analysis of the cell shape change during migration revealed that its effect was related to a decrease in cell deformability [57]. According to these observations, uPAR occupancy on endothelial cells is able to activate signaling pathways. First reports showed that uPA induced neovascular growth in the avascular rabbit cornea and promoted growth, chemotaxis and matrix invasion of cultured endothelial cells by PKC activation, and that uPA, upon binding uPAR, induced phosphorylation of focal adhesion proteins and activation of mitogen-activated phosphokinases in endothelial cells [58–59]. uPA also protects endothelial cells from apoptosis by transcriptional upregulation and partially by stabilization of the mRNA coding for the inhibitor of apoptosis proteins via NF-kappaB activation [60]. The uPA-uPAR axis can also interfere with other signaling pathways: the suppression of uPA and uPAR inhibits angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/forkhead (FKHR) pathway [61].


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uPA-uPAR role in angiogenesis has been examined also in pathologies in which, contrarily to tumors or ocular diseases, angiogenesis is defective, in particular in systemic sclerosis (SSc), showing in vitro and in vivo evidence for unexpected roles of uPAR in angiogenesis [62]. The events underlying the SSc disease appear to involve endothelium, fibroblasts and immunological mediators, resulting in an accumulation of ECM constituents, which replace the normal tissue architecture. Vascular involvement is an early event during SSc, since vascular morphologic changes, such as endothelial cell injury and apoptosis, may be apparent before the onset of fibrosis. The progressive losses of capillaries on one hand and the vascular remodeling of arteriolar vessels on the other, result in insufficient blood flow, causing severe and chronic hypoxia. Hypoxia is a major stimulus of angiogenesis [63]; however, despite severe hypoxia and consequent elevated levels of VEGF in skin and serum of SSc patients throughout different disease stages, the adaptive angiogenic response is largely defective [62]. Expression of the PA system components has been investigated in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI1 secretion increased in fibroblasts from limited SSc lesions compared to normal fibroblasts, suggesting a role for uPA and PAI1 in SSc, likely related to the activation of latent forms of cytokines and to the accumulation of ECM components [64]. Microvascular endothelial cells (MVECs) from SSc patients showed poor spontaneous and uPAdependent invasion, proliferation, and capillary morphogenesis. They expressed higher levels of uPAR, as compared with MVECs from healthy skin; however, in SSc MVECs, uPAR was cleaved between domains I and II by MMP-12, which is overexpressed in SSc MVECs. Full-length uPAR showed a connection with the actin cytoskeleton in ECs, mediated by beta2 integrin that was lost following uPAR cleavage occurring in SSc MVECs. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of small GTPases Rac1 and Cdc42, thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility [65]. Interestingly, a genetic variation located in the promoter region of the uPAR gene has been found to be associated with the vascular complications of systemic sclerosis [66]. A proangiogenic role has been reported also for the cleaved form of soluble uPAR (DII–DIII suPAR). DII–DIII suPAR exposing the SRSRY sequence promotes the formation of cord-like structures in vitro. This effect is independent of uPA proteolytic activity and involves the fMLF receptors, whose DII–DIII suPAR is a ligand, in endothelial cells. DII–DIII suPAR leads to a remarkable degree of sprouting in human saphenous vein rings and promotes a marked response in angioreactors implanted into the dorsal flank of nude mice [67]. An efficient angiogenic program relies on the subtle balance between pro- and anti-angiogenic factors [3]. PA system components contribute to angiogenesis also by providing angiostatic molecules. Angiostatin (plasminogen kringles 1–4) is produced through the proteolytic cleavage of plasminogen; several enzymes including macrophage elastase and uPA have been implicated in this process [68]. A second fragment

of plasminogen, kringle 5, generated through the activity of macrophage elastase, has been demonstrated to inhibit endothelial cell growth and migration more potently than angiostatin [46, 69]. Recently, uPAR involvement in the antiangiogenic activity of endostatin has been reported [70]. Endostatin, the C-terminal fragment of collagen XVIII, is a potent anti-angiogenic factor that significantly modulates the gene expression pattern in endothelial cells [3]. Upon cell surface binding, endostatin can function not only extracellularly but also translocating to the nucleus within minutes [70]. Nucleolin and integrin α5β1, two widely accepted endostatin receptors, mediate this clathrin-dependent uptake process. Either mutagenesis study, fluorescence resonance energy transfer analysis, or fluorescence cell imaging demonstrates that nucleolin and integrin α5β1 interact with uPAR simultaneously upon endostatin stimulation. Blockade of uPAR decreases not only the interaction between nucleolin and integrin α5β1, but also the uptake process, suggesting that the nucleolin/uPAR/integrin α5β1 complex facilitates the internalization of endostatin [70].

Vasculogenesis, the formation of new vessels by circulating EPCs independently from pre-existing vessels, was regarded to be restricted to embryogenesis. However, the discovery of EPCs in adult bone marrow and peripheral blood has challenged this theory [1–2, 62]. Bone marrow-derived CD34+ progenitor cells can generate mature ECs, express EC markers and can be incorporated into new capillary vessels at sites of ischemia. In fact, EPCs are mobilized from their bone marrow niches into the circulation in response to stress- and/or damage-related signals, migrate through the bloodstream and home to the sites of vascular injury, where they contribute to the formation of neovessels as well as to the repair of damaged vessels, collaborating with preexisting mature ECs [1–2, 62]. The SDF-1 chemokine has a major role in the recruitment and retention of progenitor cells expressing the SDF1 receptor CXCR4 [71]. SDF-1, expressed by ECs at the site of injury, probably has an important role in triggering cell arrest and emigration into the neoangiogenic niches [1, 62]. Interestingly, a functional connection between the SDF1/CXCR4 axis and uPAR has been shown. In fact, evidence of regulation of the CXCR4 activity, mediated by a cross-talk between uPAR and fMLF-Rs, has been recently reported [41]. The uPA/uPAR expression and function has been analyzed in EPCs outgrown from human umbilical cord blood. Cells derived from EPC, presenting typical features of late outgrowth endothelial cells, displayed higher levels of uPA and uPAR as compared to mature endothelial cells, represented by human umbilical vein endothelial cells. Inhibiting uPA proteolytic activity or uPA-uPAR binding significantly reduced EPDC proliferation, migration and capillary-like tube formation. Moreover,

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uPA-uPAR in Endothelial Progenitor Cells

tumor necrosis factor-alpha and VEGF, known to be locally secreted in ischemic areas, further increased the proteolytic potential of EPDC by upregulating uPA and uPAR expression [72]. An interesting role for uPAR in EPCs has been recently proposed in a study on the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of EPCs matched with a high proliferative rate [73]. VEGF treatment of ECFCs upregulated both caveolin-1 and full-length uPAR expression and their colocalization in lipid rafts. Inhibition of uPAR expression caused redistribution of caveolin-1 similarly to the effect of known caveolae-disrupting agents, suggesting that uPAR is an inducer of caveolae organization. A monoclonal anti-uPAR antibody was able to reduce matrigel invasion and capillary morphogenesis from ECFCs treated with VEGF, thus demonstrating that the proangiogenic role of VEGF involves uPAR [73]. On the other hand, uPAR silencing abolished caveolae formation and specifically impaired ECFC invasion and capillary morphogenesis, similarly to caveolin-1 silencing. Overexpression of MMP12, which cleaves full-length uPAR, inhibited ECFC-dependent vascularization in vitro and in vivo, in a matrigel sponge assay in mice [73].

In the study on the expression of plasminogen activators during neovascularization of ovarian follicles, corpus luteum and maternal decidua [45], the authors found that during corpus luteum development and decidual neovascularization, as well as in aortic explants, PAI1 expression was preferentially activated in cells in the vicinity of uPA-expressing capillary-like structures. These findings suggested a functional interplay between uPA- and PAI1-expressing cells, supporting the idea that natural inhibitors of plasminogen activators protect neovascularized tissues from excessive proteolysis during angiogenesis. The studies in mice with targeted gene inactivation of PA system components initially showed that the deficiency of PAI1 significantly accelerated neointima formation and neointimal accumulation of smooth muscle cells [47]. However, in another model of angiogenesis, deficient PAI1 expression in host mice was shown to prevent tumor vascularization of transplanted malignant keratinocytes [74]. When this PAI1 deficiency was circumvented by intravenous injection of an adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrated that host-produced PAI1 is essential for cancer cell invasion and angiogenesis [74]. Further, both tumoral and choroidal vascularization are impaired in PAI1–/– mice and can be restored in vivo by intravenous injection of a recombinant adenovirus-expressing PAI1 [75]. These observations partially explained the paradoxical clinical data that high levels of PAI1 in the primary tumor tissue of patients with various types of solid cancer correlate with disease recurrence and reduced survival.


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PAI1 and Angiogenesis

To further determine the role of PAI1 in angiogenesis, Devy et al. used an aortic ring assay with vessels from knockout mice. Lack of PAI1 completely abolished angiogenesis; the addition of recombinant PAI1 led to a bell-shaped angiogenic response showing that the pro- or anti-angiogenic effect of PAI1 is dose dependent, thereby providing an explanation for the apparently contradictory results previously reported [76]. On this basis, protease inhibitors seem to play an important permissive role during angiogenesis by limiting extracellular proteolysis to the immediate pericellular environment and thereby preventing excessive or inappropriate matrix degradation. The colocalization of uPA and PAI1 on migrating cells emphasizes the importance of the concomitant production of both proteases and inhibitors for cell migration. These studies have led to the concept of a ‘proteolytic balance’ in which critical protease-inhibitor equilibrium is necessary for cell migration and differentiation.

Conclusions

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Endothelial cells, in order to form new vessels during angiogenesis, need to migrate, invade the ECM, survive, proliferate, and to re-establish new contacts with cells and the surrounding matrix; endothelial progenitor cells can be recruited in damage sites to contribute to angiogenesis. The uPA-mediated PA system can support all these activities, since plasmin formation allows bioavailability of various proangiogenic factors and promote ECM degradation, the uPA-uPAR axis can transduce signals regulating cell survival, proliferation and migration, PAI1 balances the proteolytic activity to avoid tissue damage, and angiostatic factors are provided by the system itself for a correct process. Further, uPAR seems required also for novel and unexpected roles, for instance as ruler of the activity of integrins and/or of receptors for angiogenic factors, or, as shown in more recent work, as an inducer of caveolae organization. Abnormal blood vessel formation or functionality is the origin of a large number of diseases. Insufficient vessel growth and/or vessel regression contribute to various disorders, for instance SSc; conversely, uncontrolled vessel growth promotes tumorigenesis and ocular disorders such as age-related macular degeneration. The uPAuPAR axis can be an important target for pro- or anti-angiogenic therapy, aiming to restore adequate vessel densities. Many efforts have been done in that direction, in particular in the field of tumor vascular biology, but they still have to be translated in clinical applications. Shedding light on pathologic angiogenesis mechanisms and on the constantly emerging new roles of this complicated system will allow the identification of new therapeutic agents which could contribute to rebalance vascularization in human diseases.

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Prof. Pia Ragno Department of Chemistry and Biology, University of Salerno Via Ponte don Melillo IT–84084 Fisciano, Salerno (Italy) E-Mail pragno @ unisa.it

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Roles of eosinophils in the modulation of angiogenesis.

Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo.

Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

SPRY1 promotes the degradation of uPAR and inhibits uPAR-mediated cell adhesion and proliferation.

Angiogenesis versus arteriogenesis: neuropilin 1 modulation of VEGF signaling.

The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

Modulation of thioacetamide-induced hepatic inflammations, angiogenesis and fibrosis by andrographolide in mice.

Novel paracrine modulation of Notch-DLL4 signaling by fibulin-3 promotes angiogenesis in high-grade gliomas.

Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM).

First-in-human uPAR PET: Imaging of Cancer Aggressiveness.

Marine compound catunaregin inhibits angiogenesis through the modulation of phosphorylation of akt and eNOS in vivo and in vitro.

uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder--Possible Clinical Implications.

Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells.

Modulation of Host Angiogenesis as a Microbial Survival Strategy and Therapeutic Target.

GPR126 protein regulates developmental and pathological angiogenesis through modulation of VEGFR2 receptor signaling.

Modulation of Salmonella Tumor-Colonization and Intratumoral Anti-angiogenesis by Triptolide and Its Mechanism.

Rho kinase pathway.

Correction: Modulation of Host Angiogenesis as a Microbial Survival Strategy and Therapeutic Target.

uPAR directed-imaging of head-and-neck cancer.

Angiogenesis and angiogenesis inhibitors in paediatric diseases.

Expression of uPAR in human trophoblast and its role in trophoblast invasion.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Angiogenesis in the atherosclerotic plaque.

Poststroke angiogenesis, con: dark side of angiogenesis.