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HEMOSTASIS & ANGIOGENESIS

Cancer progression requires consecutive transformation events through which tumor cells escape proliferative checkpoint controls and regulatory cues from the extracellular milieu. In this process, tumor cells also acquire the ability to shape the tumor microenvironment for their survival advantage. Virtually, all clinically relevant carcinomas have undergone the “angiogenic switch”, i.e., developed mechanisms to sustain an appropriate blood supply for further tumor expansion. In principle, tumor cells utilize the same programs of angiogenesis that restore organ function after ischemic, mechanical, or microbial injury. Whereas regenerative angiogenesis typically progresses to restore a functional, organ-specific hierarchical vascular bed, tumor vessels retain various degrees of immaturity and tortuous architecture. Thus, tumor vessels have inconsistent directions of flow, imperfect vessel wall architecture including abnormal pericyte recruitment, and—most importantly for the current review—increased permeability and extravasation of blood plasma components.

Vascular endothelial growth factor (VEGF) is the major hypoxia-induced and tumor-derived cytokine that is responsible for angiogenic progenitor cell recruitment, endothelial cell proliferation and survival as well as vascular hyperpermeability. Although VEGF-driven proliferation of tumor vessels is clearly beneficial for tumor expansion, the immature nature of the endothelial lining potentially exposes tumors to increased immune surveillance. However, tumor cells modulate immune responses by recruitment of immature dendritic cell and monocyte/macrophage populations that establish immunosuppressive cytokine networks. These antagonize antigen presentation and locally attenuate CD8-mediated, antigen-specific tumor killing. Moreover, immature myeloid populations and tumor-associated macrophages in the tumor microenvironment are increasingly appreciated as important facilitators of tumor metastatic niches and as local angiogenic regulators that support endothelial progenitors in neoangiogenesis. In addition to immune cells, the tumor environment is further shaped by reactive myofibroblasts that play important roles in the recruitment and retention of proangiogenic progenitors. As will be discussed in this review, both immune and mesenchymal cells are important participants in the interplay of the hemostatic system with angiogenesis.

Tumor and stromal cells express the initiator of the coagulation cascade, tissue factor (TF), which constitutes a strong procoagulant stimulus that activates coagulation factors extravasated from hyperpermeable tumor vessels. The deposition of fibrin is a well-characterized feature of the tumor stroma that enables functional cross talk of tumor and host cells. The hemostatic system not only shapes the tumor microenvironment but also organizes endothelial barriers by recruiting platelets at gaps in the hyperpermeable endothelium. This chapter will discuss pathways by which the hemostatic system regulates angiogenesis and contributes to endothelial homeostasis. These pathways show multiple synergies that may be relevant to sustain an immature angiogenic network with some degree of functionality. Conversely, regulatory mechanisms by which the hemostatic system controls angiogenesis are potential therapeutic modalities for antiangiogenic therapy.

COAGULATION ACTIVATION AND THE TUMOR MICROENVIRONMENT

Angiogenic regulators. The VEGF family of growth factors consists of several genes that undergo additional splicing to yield variants with distinct cell-surface and matrix-binding properties. VEGFA/vascular permeability factor is essential for developmental angiogenesis, signals through VEGF receptors 1 and 2, and thereby serves as the most important growth factor in the angiogenic switch induced by tumors. VEGF receptor 2 signaling achieves endothelial proliferation and survival. Targeting the VEGFA signaling pathways has proven to be of clinical antiangiogenic benefit, but other VEGF family members, such as placental growth factor (PlGF), VEGFB, VEGFC, and VEGFD, may represent additional targets for tumor therapy. The VEGFC/VEGF receptor 3 axis regulates lymphangionesis and expression of VEGFC in tumors establishes lymphatic routes of metastasis, for example, in breast cancer. VEGFA is frequently a component of regulatory networks by which other proangiogenic factors regulate tumor angiogenesis. Platelet-derived growth factor (PDGF) BB acts synergistically with VEGF to attract mural pericytes to stabilize neovessels. Pericyte recruitment is a highly dynamic process at endothelial sprouts and thus PDGF signaling is an accessory pathway that can be targeted to block angiogenesis. However, other growth factors or chemokines, such as basic fibroblast growth factor (bFGF) or interleukin (IL)-8, may compensate for loss of VEGF signaling and thereby escape currently applied antiangiogenic therapy.

Several cell types contribute to the procoagulant character of the tumor microenvironment. Local expression of TF activates a crucial axis in the cross talk of the hemostatic system and angiogenic mechanisms in the tumor microenvironment (Fig. 1). Hypoxia- induced VEGF secreted from tumor cells triggers TF expression in angiogenic endothelial cells and monocytes. Inflammatory cells further enhance endothelial cell TF expression by providing tumor necrosis factor that synergizes with VEGF to induce TF. However, tumor endothelium and tumor-associated macrophages are not the only TF-positive cell types in tumor tissues. Myofibroblasts and tumor cells stain positive to variable degrees. Tumor stroma myofibroblasts upregulate TF in response to transforming growth factor (TGF)-β stimulation. In tumor cells, transformation, including ras mutations and loss of p53, is associated with TF upregulation, and hypoxia induces TF in glioblastoma cells after loss of the tumor suppressor PTEN, a key regulator of the phosphatidylinositol-3 kinase pathway. Thus, nonoverlapping pathways on host and tumor cells produce sustained TF expression in the tumor microenvironment.

Table 1.   Angiogenic Regulators

 

Sources

Main functions

Proangiogenic

VEGFA

Tumor cells, platelets

Endothelial growth factor, hyperpermeability

VEGFC

Platelets

Growth factor, lymphangiogenesis

IL-8

Tumor cells, EC

CXC chemokine, TAM recruitment

bFGF (FGF-2)

Tumor cells, platelets

Growth factor, synergy with VEGF

PDGF

EC, platelets

Growth factor, pericyte recruitment

Cyr61, CTGF

Tumor cells, platelets

Cys knot angiogenic growth factor

Antiangiogenic

Thrombospondin

Platelets

Matrix protein, CD36 ligand

PF4

Platelets

CXC chemokine, heparin neutralizing

TGF-β

Fibroblasts, platelets

Antiproliferative growth factor

Angiostatin

TME, platelets

Plasminogen fragment

Endostatin

TME, platelets

Collagen XVII fragment

 

Abbreviations: VEGF, vascular endothelial growth factor; IL-8, interleukin-8; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; CTGF, connective tissue growth factor; EC, endothelial cell; TME, tumor microenvironment; TAM, tumor-associated macrophages; TGF, transforming growth factor; PF4, platelet factor 4

Figure 1.   Multiple pathways converge to upregulate TF in the tumor microenvironment. VEGF produced by tumor cells not only triggers the angiogenic switch but also induces TF in ECs and monocytes that can mature into TAM. Transforming mutations in tumor cells and TGF-β activation of reactive myofibroblasts further contribute to local TF upregulation. Abbreviations: TF, tissue factor; VEGF, vascular endothelial growth factor; TAM, tumor-associated macrophages; TGF, transforming growth factor; ECs, endothelial cells.

Role of fibrin in the tumor stroma. Coagulation activation in the tumor stroma leads to fibrin deposition, a key feature of the transitional extracellular matrix in tumors. Matrix interactions are important for localizing growth factors in order to establish concentration gradients that guide sprouting angiogenesis. Existing matrix may serve as “vascular memory,” i.e., matrix guides the regeneration of vascular beads along existing basement structures after antiangiogenic therapy or vessel regression. Replacing an organ-specific, organized extracellular matrix by fibrin is a significant contributor to the immature and transitional character of the tumor microenvironment.

Fibrin stimulatesangiogenesis by several mechanisms. Fibrin and fibronectin, which readily associates from the plasma with fibrin, serve as ligands for several integrins on tumor cells and angiogenic endothelial cells, thus orchestrating the dynamic interplay between tumor and host. Fibrin cooperates with activated platelets in the recruitment and differentiation of endothelial cell progenitors. The importance of coagulation activation in bone marrow–derived progenitor recruitment is further underscored by studies in which coagulation inhibitors were overexpressed at sites of vascular injury.

The β-chain sequence 15 to 42 binds heparin and vascular endothelial (VE)-cadherin and thereby regulates endothelial cell migration and tube formation. Fibrin particularly synergized with bFGF to promote angiogenesis. Fibrin recruits platelets through αIIbβ3  that bind to RGD sites in the α-chain (Aα 572–575 and potentially 95–98) or the chain sequence (γA400–411). The fibrinogen γ-chain sequence 390-396 mediates interaction with αMβ2  integrin and fibrin deposition and is an important contributor to leukocyte recruitment. Furthermore, fibrin not only binds VEGF and bFGF but also IL-1β, providing evidence for a synergistic role of fibrin to promote angiogenesis and to sustain local inflammation in the tumor environment.

Although fibrinogen deficiency only minimally perturbed tumor expansion in mice, regulated fibrin turnover by the fibrinolytic system impacts tumor development. Recent data have localized a cryptic sequence in fibrinogen that regulates angiogenesis through the induction of endothelial cell apoptosis. In part, such negative regulatory effects may have masked important contributions of fibrin to tumor growth and angiogenesis in fibrinogen-deficient mice. Fibrin promotes tissue plasminogen activator-dependent plasminogen activation and thereby supports matrix remodeling. The dynamic interplay of urokinase receptor–mediated pericellular proteolysis and matrix metalloproteases is another key link by which extracellular proteolysis regulates angiogenesis. Matrix proteolysis yields key angiogenic regulators that bind and influence the function of important integrins involved in angiogenesis. Macrophage-derived matrix metalloproteinases participate in the generation of plasminogen-derived angiostatin. Degradation of collagen XVIII yields a carboxyl-terminal, zinc-binding fragment, endostatin, and degradation of collagen IV yields a similar fragment, termed tumstatin. The hemostatic and fibrinolytic systems are thus upstream and part of mechanisms that generate key angiogenic regulators.

Coagulation activates FXIII, which cross-links fibrin between chains and to fibronectin. Phage display screening has recently identified tumor stroma–homing peptides that require both fibronectin and fibrin deposition for binding, demonstrating that fibrin–fibronectin complexes are an important component of tumor stroma. FXIII directly and indirectly, through α2-antiplasmin cross-linking, counteracts fibrin degradation and thus stabilizes the transitional matrix of the tumor microenvironment. Indeed, FXIII has proangiogenic effects and FXIII-deficient mice display reduced angiogenesis and wound healing. FXIII supports angiogenesis by multiple pathways, including changes in endothelial proangiogenic signaling by cross-linking of VEGF receptor 2 with integrin αvβ3. This results in enhanced endothelial proliferation and downregulation of thrombospondin that promotes endothelial apoptosis. FXIII stabilizes platelet–endothelial interactions and thus prolongs the proangiogenic effects of platelet-released growth factors. FXIII also facilitates monocyte/macrophage migration and may participate in the recruitment of inflammatory cells into the tumor microenvironment. The coagulation and fibrinolytic systems are thus key regulators of matrix organization in the tumor microenvironment.

PROTEASE-ACTIVATED RECEPTOR SIGNALING IN ANGIOGENESIS

TF as a regulator of the angiogenic switch in tumor cells. TF expression by tumor cells directly contributes to the angiogenic switch by suppressing antiangiogenic thrombospondin and upregulating proangiogenic VEGF. Although the mechanism has not been delineated completely, TF regulates the angiogenic switch through signaling of the cytoplasmic domain. The TF cytoplasmic domain regulates integrin activation and cell migration in part through the small GTPase rac and p38 kinase-dependent pathways (48). Regulation of cell migration by TF has also been documented for transendothelial migration of dendritic and endothelial cells. In tumor cells, TF regulates α3β1-depen- dent migration on laminin 5, a key integrin–matrix interaction for metastatic homing. The cross talk of the TF cytoplasmic domain with integrin signaling likely contributes to mechanisms by which TF regulates the angiogenic switch in tumor cells.

Because hypoxic tumor cells also frequently synthesize TFs proteases ligand factor VIIa, direct signaling of the TF-VIIa complex through cleavage of protease-activated receptors (PARs) is another pathway by which TF expressed by tumor cells regulates angiogenesis. The four members of the PAR or thrombin receptor family are activated by proteolytic cleavage of the extracellular domain, followed by insertion of the neo-amino- terminus into the binding pocket of the G protein–coupled receptor. The TF-VIIa complex activates PAR2, the only PAR that is not cleaved by thrombin. TF-VIIa signaling through PAR2 upregulates IL-8  and PAR2 signaling induces VEGF. Our recent studies have shown that TF can exist in two alternative conformations that are regulated by protein disulfide isomerase–mediated thiol/disulfide exchange. This regulatory switch can turn off TFs ability to trigger coagulation, while maintaining signaling of the TF-VIIa complex through PAR2. Tumor cell TF signaling may thereby regulate tumor angiogenesis prior to detectable signs for local coagulation activation in the tumor stroma.

PARs are targets for diverse proteases. In addition to the direct signaling of the TF- VIIa complex, TF-initiated coagulation generates Xa and thrombin, which are also relevant activators of PARs. Xa cleaves and activates PAR1 and PAR2. Thrombin cleaves PAR1, 3, and 4 and, in addition, can cross-activate PAR2, because the neoaminoterminus of PAR1 acts as a ligand for PAR2. Indeed, certain thrombin-dependent responses in tumor or endothelial cells require the simultaneous activation of PAR1 and PAR2. In the fibrinolytic system, plasmin regulates cell migration through PAR1 and PAR4, depending on whether the protease is bound through kringle domains to integrin α9β1 or αvβ3, respectively. PAR1 also cooperates with integrin αvβ6 in TGF-β activation during inflammation.

Matrixmetalloproteinase 1 is another potential activator of PAR1 in tumor biology. The list of proteases that activates PARs is steadily expanding and PAR2 is the target for diverse enzymes including bacterial proteases, the sperm protease acrosin, as well as mast cell tryptase and proteinase 3 of relevance for immune functions. Tumor cells also frequently show aberrant expression of proteases that activate PAR2, including trypsin expressed in gastrointestinal cancers, TMPRSS2, and matriptase. Additional PAR-activating proteases of relevance for angiogenesis are likely to be discovered in the emerging family of membrane-anchored serine proteases that can be expressed in endothelial and tumor cells.

Although tumor andendothelial cells have been most frequently studied as targets for proteases, PARs are known to be expressed by cells in the tumor stroma. Reactive myofibroblasts in breast cancer tissue, but not normal resident fibroblasts in normal breast tissue, prominently express PAR1 and PAR2. PARs are also found in inflammatory cells. PAR1 is the predominant receptor in monocytes, but PAR2 is upregulated after macrophage differentiation. PAR2 also plays a role in dendritic cell maturation and activation. Proteases may therefore regulate inflammation or influence immunological networks in the tumor environment through PAR signaling.

Overlapping and specific effects of PAR signaling in angiogenesis. A role for PARs in angiogenesis was indicated from mouse knockout studies. PAR1 deficiency produces partial embryonic lethality in mice due to vascular failure. In contrast, no apparent developmental defects in the vasculature result from deletion of PAR2. There are several mechanisms by which PAR1 signaling can influence endothelial function in angiogenesis, including regulation of TGF-β receptor internalization, attenuation of endothelial cell proliferation, and regulation of endothelial progenitor cell differentiation. Thrombin supports tumor or endothelial cell survival and proliferation, but TF and  PAR2 signaling can induce similar cellular effects. PAR1 and PAR2 signaling also overlap in the ability to cross-activate the epidermal growth factor receptor to promote proliferation. Proangiogenic growth factors are upregulated by either PAR1 or 2 signaling in tumor or stromal cells, including IL-8, VEGF, angiopoietin 2, and the cysteine knot proteins Cyr61 and connective tissue growth factor.

Although PAR1 and 2 signaling show redundancy in the induction of proangiogenic mediators in tumor cells, it remains an important question which proteases are generated in sufficient concentrations to activate PARs in the tumor microenvironment. Protease coreceptors may further be expressed in a tumor-type specific manner and thus direct or amplify PAR signaling. Thrombin stimulates angiogenesis in certain angiogenesis models in vivo and PAR1 antagonists block these angiogenic responses. However, thrombin after binding to endothelial expressed thrombomodulin activates protein C. In turn, activated protein C (aPC) in complex with endothelial cell protein C receptor (EPCR) cleaves PAR1 of endothelial cells and PAR2 potentially on other cell types. The amount of local thrombin generation in combination with availability of the key receptors of the protein C pathway may determine whether thrombin activates PAR1 through direct cleavage or indirectly through the protein C pathway.

Importantly, PAR1 activation by thrombin and aPC/EPCR can produce opposing effects in endothelial cells exposed to inflammatory mediators. Direct thrombin signaling may produce apoptosis through upregulation of thrombospondin, whereas aPC/ EPCR has profound endothelial protective, antiapoptotic effects. Indeed, aPC has proangiogenic properties in vivo. However, in certain tumor and angiogenesis models, coagulation inhibitors that target the upstream TF signaling complex have considerably higher potency compared to inhibitors to downstream coagulation proteases, which reduce thrombin and aPC generation. The complex contributions of PARs to tumor progression may result from nonredundant roles of PAR signaling on tumor versus host or stromal cells. It will be necessary to combine specific inhibitors, genetically engineered mice, and PAR-deficient tumor cell lines to clarify the proangiogenic effects of PARs and coagulation signaling complexes on host and tumor cells.

Role of direct TF signaling in angiogenesis. Evidence for a role of TF signaling in host cells came from the characterization of TF cytoplasmic domain–deleted mice that show deregulated angiogenesis. The complete knockout of TF had documented that the TF pathway maintains vasculature integrity in early embryonic development. Because PAR1 deficiency in endothelial cells showed a similar developmental phenotype, TF is likely upstream of vascular protective PAR1 signaling. In contrast, TF cytoplasmic domain–deleted mice have no developmental lethality. In postnatal mice, TF is expressed in the endothelium during inflammation and tumor progression. In mice that lack the TF cytoplasmic domain, we found significantly enhanced growth of TF-positive, syngeneic tumors. Because the tumor-expressed TF drives local thrombin generation, accelerated tumor development in mice that carried the TF cytoplasmatic domain deletion provided clear evidence for nonredundant and independent function of TF on host cells during tumor angiogenesis.

Angiogenesis in TF cytoplasmic domain–deleted mice was characterized by the in vitro aortic ring endothelial cell sprouting assay. These experiments showed that TF-VIIa drives PAR2-dependent angiogenesis specifically in the presence of PDGF BB. PAR2 deletion per se had little effect on angiogenesis. One possible explanation for normal angiogenesis of PAR2-deficient mice is a balanced signaling cross talk with the TF cytoplasmic domain. PAR2 signaling, but not PAR1 signaling, leads to TF cytoplasmic domain phosphorylation. Indeed, phosphorylation of the TF cytoplasmic domain was specifically observed in abnormal, proliferative neovasculature of the eye, whereas TF in normal vessels was not phosphorylated. Conceivably, dephosphorylation of the TF cytoplasmic domain may limit PAR2-dependent neovascularization, and hyperphosphorylation may lead to uncontrolled angiogenesis similar to that observed in TF cytoplasmic domain–deleted mice. Suppression of integrin α3β1-dependent migration is reversed by TF cytoplasmic domain phosphorylation and integrin α3β1 has been shown to mediate antiangiogenic effects of tissue inhibitors of metalloproteinase 2. In ongoing studies, we have validated in a relevant hypoxia-driven model that the proangiogenic phenotype of TF cytoplasmic domain–deleted mice is dependent on PAR2 and growth factor signaling pathways in vivo.

THE HEMOSTATIC SYSTEM AS REGULATOR OF ENDOTHELIAL HOMEOSTASIS (FIG. 2)

The hemostatic system participates in the dynamics ofthe tumor microenvironment by regulating angiogenic growth factor expression, cell proliferation, and matrix remodeling. These pathways may directly influence tumor cell proliferation, invasion, and metastasis. Examples for proliferative effects span from coagulation protease signaling through PARs to tumor cell stimulation by platelet-derived bioactive lipids, i.e., lysophosphatidic acid. Equally important for the mechanism of angiogenesis is the maintenance of endothelial functions by hemostatic mechanisms. The hemostatic system participates in the regulatory control of endothelial cell barrier integrity, apoptosis, and integration of signals that orchestrate the transit of cells and transmission of information across the endothelium.

Synergistic effects of hemostatic pathways on endothelial cell barrier function. Although VEGF results in upregulation of TF in endothelial cells, blockade of the VEGF pathway paradoxically increases thrombosis risk in combination with certain chemotherapies, emphasizing the persistent procoagulant character of the tumor microenvironment. The clinical use of inhibitors that target the VEGF receptor–signaling pathway in tumors further demonstrated the crucial role of elevated VEGF levels in maintaining the immature character of the tumor vasculature. Indeed, the remodeling and pruning of tortuous tumor vessels after VEGF blockade improves perfusion and delivery of cytostatic drugs in cancer therapy. There are several pathways by which the hemostatic system counteracts VEGF-dependent hyperpermeability and maintains tumor perfusion through enhanced endothelial barrier function and prevention of bleeding.

Although thrombin can acutely increase endothelial permeability through PAR1 signaling, the aPC/EPCR signaling pathway, by activating PAR1, can significantly increase endothelial cell barrier function through sphingosine-1 phosphate (S1P) production. S1P is a potent bioactive lipid that activates predominantly S1P receptor 1 on endothelial cells. Platelets also store and release S1P upon activation. Local synthesis of S1P is probably responsible for the tonic maintenance of barrier integrity, whereas acute release from platelets may acutely “seal off endothelial cell barriers under increased stress.

Platelets stimulate angiogenesis by secretion of angiogenic growth factors VEGF, bFGF, and PDGF. The release of S1P may counteract VEGF-induced permeability increase and thereby contribute to the mechanisms by which platelets provide hemostatic protection during angiogenesis. Platelets also secrete platelet factor 4 (PF4), a CXC chemokine that interacts with IL-8 and thus regulates angiogenesis. PF4 plays roles in platelet thrombus formation and PF4 neutralizes heparin and thus attenuates antithrombin-dependent coagulation inhibition. PF4 also enhances thrombomodulin-dependent protein C activation and may thereby be integrated into pathways by which local platelet deposition initiates acute and sustained barrier protection of angiogenic endothelium.

                   

Figure 2.   Regulation of endothelial homeostasis by hemostatic mechanisms. The key targets for proteases and S1P signaling are the regulation of endothelial cell barrier protection. Endothelial cells release angiogenic mediators from WPB and recruit inflammatory cells by P-selectin exposure and platelets through vWF release. Coagulation activation leads to turnover of inhibitors that act as proapoptotic signals for endothelial cells. Abbreviations: S1P, sphingosine-1 phosphate; WPB, Weibel–Palade bodies; PAR, protease- activated receptor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; SDF-1, stroma-derived factor-1; PF4, platelet factor 4; PAI, plasminogen activator inhibitor.

Hemostatic factors that induce endothelial cell apoptosis. Angiostatin has led the way in the identification of angiogenic regulators that are derived from the hemostatic system. This plasminogen-derived fragment is a ligand for integrins expressed by endothelial cells and induces endothelial apoptosis. Integrin αVβ3, which is expressed by angiogenic endothelia cells binds prothrombin in dependence of integrin activation, lead- ing to prothrombin conversion. This may contribute to local accumulation of pro- thrombin kringle domains with antiangiogenic activities. Thrombin initiates fibrin deposition and subsequent proteolysis of fibrin exposes cryptic, proapoptotic epitopes that suppress angiogenesis. Kringle and fibrin fragments may exert antiangiogenic activities by concerted actions on integrin, VE-cadherin, and angiogenic growth factor pathways.

Cell surfaceproteoglycans are critical for angiogenic growth factor binding. Heparinase as well as heparin neutralization by, e.g., PF4, interfere with growth factor binding and attenuate VEGF- and bFGF-induced angiogenesis. Antiangiogenic antithrombin is a cleaved and latent form of this serine protease inhibitor (serpin) that blocks thrombin and factor Xa. The potent antiangiogenic activity of the latent serpin conformation is due to preferential binding to proteoglycans involved in angiogenic growth factor binding, in comparison, to native antithrombin that interacts more tightly with anticoagulant heparins (126). A cleaved form of plasminogen activator inhibitor 1 also induces endothelia apoptosis, indicating a common theme of how protease action on serpins can produce feedback inhibition of angiogenesis.

TF pathway inhibitor (TFPI) is another coagulation inhibitor with antiangiogenic activity. TFPI has three Kunitz-type protease inhibitory domains and the third domain in conjunction with the basic carboxyl-terminus contributes to heparin binding. TFPI is the major inhibitor of the TF initiation complex and controls both TF-dependent initiation of coagulation and direct cell signaling. TFPI is tightly bound to endothelial cells by a glycosylphosphatidylinositol anchor attached either directly to an alternative spliced form of TFPI (130) or indirectly available through TPFI receptors. TFPI also interacts with versican and the very-low-density lipoprotein receptor (VLDLR). VLDLR is expressed on endothelial cells and the interaction was mapped to a sequence in the very carboxyl-terminus of TFPI (134). Interaction of this sequence with VLDLR triggers endothelial apoptosis and may thus regulate angiogenesis independent of the anti- coagulant activity of TFPI.

The turnover of coagulation and fibrinolytic factors and their inhibitors thus either directly through endothelial receptor interaction or indirectly by angiogenic growth factor displacement induce endothelial cell apoptosis. This may contribute to pruning and partial maturation of the tumor vasculature. The resulting improved perfusion may benefit tumor growth and survival. Conversely, these mechanisms provide opportunities for improved antiangiogenic therapy in cancer.

The endothelium as a gatekeeper for inflammatory and stem cell recruitment. The endothelium is actively involved in recruiting and directing the transit of blood-derived inflammatory cells and precursors into the extravascular space. In addition to other agonists, coagulation protease–mediated PAR activation plays a key role in triggering the release of Weibel–Palade bodies, storage compartments specifically found in endothelial cells. PAR1 and PAR2 are involved in Weibel–Palade body release, but the intermediate signaling pathways appear to differ with PAR1 predominantly triggering calcium fluxes, whereas cAMP pathways play predominant roles in PAR2-mediated release. Weibel–Palade body release leads to P-selectin exposure that mediates leukocyte rolling and thus initiates the transendothelial migration and recruitment of tumor-associated macrophages. Weibel–Palade bodies also store angiogenic regulators IL-8 and angiopoietin 2, as well as eotaxin-3. Furthermore, IL-8 synthesis and PAR2 expression in the endothelium are induced by inflammatory mediators. Resident macrophages in the tumor micro- environment may thereby maintain continuing influx of inflammatory cells in conjunction with protease-mediated activation of the endothelium.

Weibel–Palade body release triggers the local exposure of ultralarge von Willebrand Factor multimers that potently recruit platelets. The crucial role for platelets as hemostatic effectors in angiogenesis has been documented. However, platelets participate in multiple facets of the angiogenic process by locally releasing angiogenic mediators after activation (Table 1). Platelet activation also induces the release of microvesicles that are emerging as significant vehicles to transmit proangiogenic signals to the host.

Platelet-derived microparticles carry proangiogenic mediators VEGF, PDGF, and bFGF, and by dispersion into the circulation, microparticles serve as delivery vehicles for cargo to different areas of the tumor vasculature. Microparticles can alter the procoagulant properties of the endothelium, induce endothelial activation, and thus contribute to the recruitment of inflammatory cells. Release of microparticles from endothelial cells is conversely regulated by proteases and microparticles carry TF or EPCR as relevant protease receptors to modulate intravascular coagulation activation and control. In addition, microparticles derived from tumor cells can serve overlapping functions with platelet-derived microparticles and by releasing tumor cell TF may contribute to the prothrombotic state of tumor patients.

A particularly important function of platelets in orchestrating proangiogenic progenitor recruitment is emerging. Fibrin and platelets provide a matrix for homing and differentiation of endothelial progenitor populations that are incorporated into newly formed vessels. Another relevant population of proangiogenic progenitors are integrin CD11b positive, immature myeloid cell populations that play important supportive roles in angiogenesis and revascularization. Platelets are intimately linked to the homing and retention of these progenitor populations in neoangiogenic vessels. In addition, evidence is emerging that hematopoietic and endothelial progenitors express coagulation receptors, such as EPCR and PARs. The biology of proangiogenic progenitors cells may therefore be controlled and influenced directly by proteases of the coagulation cascade.

CONCLUSIONS

The hemostatic systems play crucial roles in maintaining the specific character of the tumor microenvironment and support angiogenesis by multiple mechanisms. Anticoagulant intervention has shown partial benefit to prolong survival in cancer patients and it is reasonable to assume that part of the therapeutic effects relates to interference with angiogenic mechanisms. However, the complexity by which the hemostatic system participates in angiogenesis suggests a number of potential targets that have not been explored for therapeutic intervention. Targeting the TF-VIIa complex rather than thrombin in cancer will provide broader suppression of coagulation proteases and more importantly begin to intervene in the direct signaling pathways of TF. Antibody-based strategies to exosites, active site directed inhibitors of VIIa, or agents that suppress the expression of TF on tumor or host cells are strategies to be considered.

The hemostatic system makes contributions to and regulates angiogenesis distinct from and synergistic with the major proangiogenic growth factor pathways. Exploiting the prothrombotic character of the tumor microenvironment as a platform to induce thrombosis remains a counterintuitive, but potentially feasible strategy to starve tumors of their blood supply. Additional studies are required to better define the overlap of proangiogenic pathways in order to identify true synergies that can be exploited for combination antiangiogenic therapy. Direct targeting of PARs and interference with platelet-induced angiogenic mechanism are potential avenues of interest. The unexpected association of thrombosis with antiangiogenic therapy has highlighted the close interdependence of angiogenesis and the hemostatic system. Continuing research in the cross talk of these pathways will be of critical importance for new advances as well as a safety consideration in antiangiogenic therapy. Angiogenicregulators are synthesized locally by tumor or stroma cells, including tumor-associated macrophages, or released from α-granules of activated platelets. However, these growth factors, chemokines, and cytokines have distinct molecular targets, a reflection of the complex cellular interactions that sustain tumor angiogenesis (Table 1). Several of the proangiogenic stimuli converge functionally in the recruitment of endothelial and hematopoietic progenitor population and tumor-associated macrophages. VEGFA gradients attract VEGF receptor 2 positive endothelial progenitors as well as VEGF receptor 1 positive hematopoietic, myeloid, and macrophage progenitors. The VEGF family member PlGF only activates VEGF receptor 1 and may thus play a more prominent role in the recruitment of certain progenitor populations. Motility and directed migration of progenitor populations is regulated by additional pathways. Stroma-derived factor-1 (SDF-1) is a CXC cytokine that is synthesized by stromal fibroblasts and endothelial cells. SDF-1 signaling through CXCR4 retains progenitor population in the tumor stroma. In addition, CXCR4 is upregulated in breast cancer cells and thereby reactive fibroblasts are involved in multiple cellular cross talks. The persistent activated state of myofibroblasts and the immaturity of myeloid populations determine by multiple pathways the overall character of pathological angiogenesis and of the tumor microenvironments as a “wound that does not heal”.

 

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