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thrombosis in angiogenesis and tumour progression
Matrix Biology 19 Ž2000. 345᎐351
Effects of thrombinrthrombosis in angiogenesis and tumour progression Michael E. MaragoudakisU , Nikos E. Tsopanoglou, Paraskevi Andriopoulou, Michael-Emmanuel M. Maragoudakis Uni¨ ersity of Patras, Medical School, Department of Pharmacology, 261 10 Rio, Patras, Greece
Abstract Laboratory, histopathological, pharmacological and clinical evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. On the other hand, epidemiological studies provide evidence of an increased risk of cancer diagnosis following primary thromboembolism. Moreover, the metastatic ability of human breast cancer cells is correlated with the number of thrombin receptors of these cells, and thrombin treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats. We have proposed that these tumour-promoting effects of thrombin can be explained by the ability of thrombin to activate angiogenesis, an essential requirement for tumour progression. Many of the cellular events involved in the angiogenic cascade can be activated by thrombin. At the molecular level, brief exposure of endothelial cells to thrombin causes an upregulation of the receptors ŽKDR and Flt-1. of VEGF, the key angiogenic mediator. This results in a synergistic effect of thrombin and VEGF in the activation of angiogenesis. In addition, thrombin activates cancer cells to secrete VEGF, thus causing a mutual stimulation between EC and CA cells. Cancer cells exposed to thrombin secrete metalloproteinase 92 KD and overexpress the integrin av b3 , all of which are involved in tumour metastasis. 䊚 2000 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Thrombin; Angiogenesis; Tumour; Metastasis
1. Cancer and thrombosis The association between blood coagulation and metastatic dissemination was first recognised by Trousseau more than 128 years ago. This observation has subsequently been verified by many investigators who provided laboratory and clinical evidence sug-
Abbre¨ iations: CA, cancer cells; EC, endothelial cells; VEGF, vascular endothelial growth factor; TRAP, thrombin receptor agonist peptide; CAM, chick chorioallantoic membrane; FGF, fibroblast growth factor; RT-PCR, reverse transcriptase-polymerase chain reaction; PKC, protein kinase C; MAP, mitogen activated protein; t-PA, tissue-plasminogen activator; PAI, plasminogen activator inhibitor; NO, nitric oxide; PDGF, platelet derived growth factor; CPB, collagenous protein biosynthesis. U Corresponding author. Tel.: q30-30-61-997691r997638; fax: q30-30-61-994720. E-mail address: [email protected] ŽM.E. Maragoudakis..
gesting the systemic activation of the blood-clotting cascade in patients with cancer. Measurements of circulating fibrino-peptides have shown that patients with cancer often have inappropriately high intravascular coagulation and fibrinolysis ŽRickles and Edwards, 1983.. Many tumour cells elicit procoagulant activity by interaction with the blood’s thrombin-generating systems and also with platelets, leukocytes and endothelial cells ŽSloane et al., 1986.. Some tumour cells express the transmembrane tissue factor, which when exposed to circulating coagulation factor VII, activates factor X, leading to the generation of thrombin and the formation of fibrin. Other tumour-derived activators of factor X, such as mucin and cysteine protease, have been described. In all of these cases, local thrombin generation occurs, resulting in fibrin deposition, which is thought to be important for tumour growth and platelet activation. Recently
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Zacharsky et al. Ž1995. have shown the presence of thrombin in a variety of tumour cell types by immunostaining. Two recent large epidemiological studies ŽBaron et al., 1998; Sorensen et al., 1998. involving 86 000 ¨ patients with thromboembolism, 4200 of whom had cancer, suggest that the risk of deep vein thrombosis or pulmonary thromboembolism is 3.2 times higher in cancer patients. The risk of thrombosis is increasing with anti-neoplastic treatment. Cytotoxic therapy damages the endothelium and releases procoagulant substances and cytokines, which activate blood coagulation. As a result of these findings, anti-thrombotic therapy is considered a rational approach to prevent thromboembolism in cancer patients ŽKakkar and Williamson, 1998..
2. Thrombosis and tumour progression The aforementioned studies explain why hypercoagulability occurs in cancer patients, but does not answer the question of whether thrombosis can contribute directly to tumour promotion and metastasis. Indeed, in the epidemiological study by Sorensen et ¨ al. Ž1998. it was shown that 6᎐12 months after a primary thromboembolism, the risk of cancer diagnosis increases threefold. These clinical observations were in line with animal experiments, where the thrombin-treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats ŽNierodzik et al., 1992.. Until recently, the prometastatic effects of thrombin were attributed to the effects of thrombin on platelets, endothelial cells or fibrin formation. However, new evidence shows that the pretreatment of several tumour cells with thrombin increases their ability to adhere to platelets and extracellular matrix proteins ŽNierodzik et al., 1991.. Thrombin-pretreated B16 tumour cells showed an increase in pulmonary metastasis of up to 156-fold in mice compared to sham-treated cells. Based on these data, the anti-coagulant drug warfarin was used in animal tumour models with positive results. Following this experience with animal tumour models, controlled clinical trials of warfarin in several human cancer patients was initiated with encouraging results. Warfarin prolonged the length of survival of patients with a small cell carcinoma of the lung. Besides small cell lung carcinomas, other human cancer cells such as renal cell carcinomas and malignant melanomas are known to generate thrombin and are likely to be susceptible to anti-coagulant therapies. It is of importance in this respect that the metastatic ability of human breast cancer cells is related to the number of thrombin receptors on these cells ŽEvenRam et al., 1998.. This association between metastatic
ability and the number of thrombin receptors needs to be investigated in other types of tumours. The mechanism of action of warfarin in cancer therapy is not clear from these data. However, clinical studies support the concept that anti-coagulant drugs may have a therapeutic use for certain types of cancer as well as in preventing cancer-associated hypercoagulability, particularly during chemotherapy, when there is an increased risk.
3. Proposed mechanism for tumour progression following thrombosis The final step in thrombosis is thrombin generation and fibrin formation. The thrombin in circulation is rapidly inactivated by anti-thrombin, but the thrombin trapped within the thrombi is protected and slowly released with the progression of fibrinolysis. We have reported that thrombin is a potent activator of angiogenesis ŽTsopanoglou et al., 1993. and proposed that this new action of thrombin may be the basis for the promotion of tumour progression following thrombosis. The present day dogma in oncology is that angiogenesis is an essential requirement for tumour growth and metastasis ŽFolkman, 1985.. Tumours do not grow beyond 1᎐2 mm3 in size unless they recruit their own blood supply for delivering nutrients and O2 . In addition, the malignant phenotype requires the ability of cancer cell for invasion and uncontrolled growth. Angiogenesis is activated in many other conditions besides cancer. Wound healing, diabetic retinopathy, atherosclerosis, the follicular phase of the menstrual cycle etc., are some examples. Considering the broad spectrum of physiological and pathophysiological processes that angiogenesis is involved in, we can strongly theorise the involvement of angiogenesis activation by thrombin is a more general phenomenon. We have studied the angiogenic action of thrombin initially in the model of the CAM system, in vivo ŽFig. 1.. It was shown that the effect was dose-dependent, specific and required the catalytic site of thrombin. Heparin or hirudin completely blocked the angiogenic action of thrombin. Also, the chemically inactivated thrombin at the active site ŽPPACK thrombin. was without effect and competed with thrombin for its angiogenic action. An analog of thrombin Ž ␥-thrombin., which lacks the anion binding exocite for binding fibrinogen and, therefore, cannot form fibrin, was also as active as thrombin in activating angiogenesis. This implied that the angiogenic action of thrombin was independent of fibrin formation. Most of the actions of thrombin were mediated by the activation of a thrombin receptor. This activation involved the proteolytic cleavage of the receptor by thrombin, thus generating a new NH2-terminus, which
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Fig. 1. Effect of thrombin on collagenous protein biosynthesis ŽCPB. in CAM in vivo. Results are expressed as mean cpmrmg protein " S.E.
acted as a tethered ligand binding to a new receptor on the cell membrane, coupled to various G-proteins ŽVu et al., 1991.. The repertoire of G-proteins coupled to the thrombin receptor and the cellular effectors activated determined the nature of the cellular responses generated by thrombin ŽDery et al., 1998.. A decatetrapeptide ŽTRAP., representing the NH2terminal of the activated thrombin receptor, mimicked many of the cellular effects of thrombin. TRAP is also a promoter of angiogenesis in the same fashion as thrombin. Moreover, FPR the antagonist peptide of TRAP, cancelled out the angiogenic effect of TRAP in the CAM ŽMaragoudakis et al., 1995.. This provided further support that the thrombogenic action of thrombin Žfibrin formation. was not essential for the angiogenic action. In addition to the CAM system, the angiogenic action of thrombin was established in the Matrigel system in vivo ŽHaralambopoulos et al., 1997..
4. Effects of thrombin on the angiogenic cascade Angiogenesis is a complex biological phenomenon in which many cell types and factors are involved. Briefly, it starts by the activation of the normally quiescent endothelial cells of the parent vessel Žusually a venule. by an angiogenic stimulus. This stimulus can be one of the many growth factors and endogenous substances identified thus far, which act as angiogenic factors, including thrombin ŽFolkman, 1985.. The activation of endothelial cells causes the secretion of metalloproteinases, which results in the local dissolution of basement membranes and the extracellular matrix, allowing the migration of the
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endothelial cell towards the angiogenic stimuli. This is followed by the proliferation of endothelial cells, alignment to form the lumen of the new vessel, anastomosis, synthesis of basement membrane of the new vessel, blood flow and the recruitment of pericytes and smooth muscle cells ŽFolkman and Shing, 1992.. This process is tightly regulated by endogenous promoters and inhibitors and is dormant under physiological conditions in an adult organism. However, it can be activated at a moment’s notice, like the blood coagulation cascade, probably by different mechanisms in each tissue that converge in the control of the activity of a single factor such as VEGF. VEGF is considered to be the specific endothelial cell mitogen and is expressed in all cases of activation of angiogenesis, both physiological and pathological. It has been proposed that under physiological conditions, angiogenesis is dormant, because of a balance in the activity of the many promoters and inhibitors of angiogenesis. It is believed that activation of angiogenesis is a result of an imbalance between angiogenic and anti-angiogenic factors ŽFolkman, 1985.. However, angiogenesis is a physiological process too important to be controlled only by a simple imbalance of redundant promoters and inhibitors. Strict controls must exist, probably unique to every tissue, and the immediate activation of angiogenesis at a short notice must be possible. This can only be accomplished by intricate interactions of the modulators of angiogenesis. We have established that thrombin modulates the activity of VEGF by up-regulating the expression of VEGF receptors in endothelial cells ŽTsopanoglou and Maragoudakis, 1999..
5. Activation of the angiogenic cascade by thrombin As mentioned above, angiogenesis proceeds in many distinct stages, which can be studied in several in vitro and in vivo systems. We have initiated a systematic approach to establish which of these steps are modulated by thrombin and the mechanisms involved. Brief exposure Žless than 15 min. of endothelial cells to thrombin causes a marked inhibition of their ability to adhere to type IV collagen and laminin ŽFig. 2.. The endothelial cell phenotype, which results after exposure to thrombin, can survive without attachment to the extracellular matrix. This effect is reversible, and the removal of thrombin and the re-incubation of cells in growth medium completely restores their ability to adhere to type IV collagen and laminin. These effects of thrombin are specific, since hirudin and heparin block this action, and require the active site of thrombin, since PPACK thrombin is without effect. It is also receptor-mediated since TRAP, the activated receptor agonist, has the same effect as throm-
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Fig. 2. Effect of thrombin on cell attachment. HUVECs in suspension were incubated for 15 min with serum-free M199 alone Žnonercontrol. or various concentrations of thrombin. Cells Ž40 000 cellsrwell. were plated on collagen IV or laminin and allowed to adhere for 60 min at 37⬚C. Adhered cells were counted with a computerised digital image analyser. Results were expressed as the mean of the percentage change of control " S.E., which is taken as 100%.
bin ŽTsopanoglou and Maragoudakis, 1998.. These results suggest that thrombin enables endothelial cells to detach from their anchorage site on the basement membrane of the parent vessel, to survive without attachment to the extracellular matrix, and to migrate to distant sites. The exposure of endothelial cells to thrombin causes an increase in the activated form of progelatinase A Ž68 kDa. in zymograms of the culture medium. Activation of the 72-kDa metalloproteinase, which degrades collagen type IV, is probably related to the local degradation of basement membrane collagen, thus allowing for the movement of the activated endothelial cells. In addition, the dissolution of the extracellular matrix liberates sequestered growth factors such as b-FGF, which further promote the angiogenic cascade. It has been shown that thrombin and TRAP act as endothelial cell mitogens and promote cell growth. Like all of the aforementioned effects of thrombin, this action of thrombin on endothelial cells is specific and requires the active catalytic site of thrombin ŽTsopanoglou and Maragoudakis, 1999.. In combination with VEGF, the endothelial cell specific mitogen, we obtained a synergistic effect in the rate of DNA synthesis and cell proliferation ŽFig. 3.. Although the exposure to thrombin needs to be brief Ž15 min., the time required for the synergistic effect to become manifested is 8᎐12 h after thrombin-treatment. At
earlier times Ž0.5, 1.5 and 4 h. this potentiating effect of thrombin on VEGF-induced DNA synthesis and endothelial cell proliferation is not evident. The specificity of this effect is established by the fact that hirudin cancels out this action of thrombin. PPACK thrombin is without effect, and TRAP has the same effect as thrombin, indicating that this is also a thrombin receptor-mediated event. We concluded from these findings that thrombin effectively enhances the mitogenic potency of VEGF on endothelial cells ŽTsopanoglou and Maragoudakis, 1999.. This potentiating effect of thrombin on VEGFinduced endothelial cell proliferation can be explained by the upregulation of VEGF receptors. This was shown by a sensitive quantitative RT-PCR technique. The mRNA for both receptors of VEGF ŽKDR and flt-1. was upregulated 8᎐12 h after exposure to thrombin. Thrombin stimulates KDR and flt-1 mRNA expression dose-dependently, with a maximum obtained at 1.5 IUrml. The over-expression of mRNA for KDR and flt-1 is a receptor-mediated event and can be obtained using TRAP, the agonist of the activated thrombin receptor. We have established that the increase in KDR mRNA was accompanied by an increase in the functional KDR protein. The endothe-
Fig. 3. Temporal effect of thrombin on VEGF-induced DNA synthesis in human endothelial cells. HUVECs were preincubated with M199r1%BSA alone or with thrombin Ž1.5 IUrml. for the indicated times, and were subsequently incubated either with M199r4%FBS alone or with VEGF Ž5 ngrml. for 18 h. All cells were pulsed with w3 Hx-thymidine for an additional 6 h. Control: cells were preincubated with M199r1% BSA and the with M199r4%FCS. Thr: cells were preincubated with thrombin and then with M199r4%FCS. VEGF: cells were preincubated with M199r1%BSA and then with VEGF. ThrrVEGF: cells were preincubated with thrombin and then with VEGF. Results are expressed as mean of percent over the control " S.E. of six wells, which is taken as 0%. Similar results were obtained in three separate experiments.
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lial cells, after exposure to thrombin, were lysed and, after immunoprecipitation, the band corresponding to KDR Ž190 kDa. was increased by 200% over that of control cells. The upregulated KDR receptor was functional, as shown by the phosphorylation of KDR protein, after treatment of the endothelial cells with VEGF ŽTsopanoglou and Maragoudakis, 1999.. In order to exclude the possibility that the upregulation of the VEGF receptors by thrombin was due to stabilisation of mRNA by decreasing its rate of degradation, we measured KDR mRNA in the presence of actinomycin D. Inhibition of the transcription rate by actinomycin D did not affect the rate of decay of KDR mRNA. Both the control and thrombin-treated endothelial cells had a half-life of KDR mRNA of approximately 2.8 h. Nuclei isolated from control and thrombin-treated endothelial cells were incubated in vitro to determine the rate of transcription of KDR. The thrombin-treated nuclei showed an 80% increase in transcription rate after 1 h. The transduction mechanisms involved in the overexpression of VEGF receptors were investigated using agents that modulate key cellular transduction mechanisms known to be involved in the cellular actions of thrombin, namely the PKC and MAP kinase-dependent pathways. Using PMA as a PKC activator, and calphostin C as a selective PKC inhibitor, we demonstrated that PMAlike thrombin upregulates KDR mRNA, while calphostin C completely abolishes the upregulation of KDR by thrombin and PMA. Similarly, using a specific inhibitor of MAP kinase ŽPD98059., we showed that the upregulation of KDR and flt-1 mRNA was mediated by the activation of the MAP kinase pathway. In contrast, the activation of adenyl cyclase by forskolin did not effect the levels of KDR and flt-1 mRNA induced by thrombin. We concluded from these experiments that thrombin upregulates the VEGF receptors via the activation of PKC and MAP kinase pathways downstream after the activation of the thrombin receptor, coupled to G-proteins. The final result is the amplification of the role of VEGF in angiogenesis.
6. Effects of thrombin that may lead to activation of angiogenesis In addition to the effects of thrombin in blood coagulation Ži.e. amplification: FVª FVa, FVIII ª FVIIIa; clot formation: fibrinogen ª fibrin, FXIII ª FXIIIa; platelet aggregation: secretion and granule release ., thrombin has many other actions on various cell types, which may be related to promotion of angiogenesis ŽKanthou et al., 1998..
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On endothelial cells thrombin is known to have many effects: 䢇
䢇
䢇
䢇
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Activation leading to CAM expression and leukocyte adhesion; secretion of PGI2 , t-PA, NO, PDGF, PAI-1, and endothelin; anticoagulant by thrombomodulinrprotein C activation; pro-coagulant by tissue factor and microparticle shedding; and re-traction leading to an increase in vascular permeability
In leukocytes, thrombin stimulates proliferation, chemotaxis and cytokine production. These actions of thrombin on endothelial, inflammatory cells and platelets probably contribute to tumour and inflammatory angiogenesis ŽTapparelli et al., 1993..
7. Thrombin effects on the metastatic phenotype of cancer cells
As mentioned already, the association between malignancies and coagulopathies has long been established. A common clotting abnormality in cancer patients is an elevated amount of fibrinogenrfibrin degradation products, indicating thrombin generation. This fibrin-rich product, together with the extracellular matrix, may provide the optimal tumour microenvironment. This may also provide a storage site for thrombin within the fibrin clot, from which thrombin is released to activate the thrombin receptors. Such receptors are overexpressed in metastatic cancer cells ŽEven-Ram et al., 1998.. Preliminary evidence from our laboratory has shown that PC3 human prostate carcinoma cells, when treated with thrombin or thrombin receptor agonists ŽTRAP. increases the secretion of the 92-kDa metalloproteinase. In addition, there is an increase in mRNA expression for VEGF and the secretion of the VEGF protein in the medium. Furthermore, thrombin treatment causes an overexpression of the av b3 integrin Žunpublished observations.. These results suggest that thrombin treatment promotes the metastatic and angiogenic phenotype of cancer cells. The overexpression of VEGF in cancer cells, in connection with the upregulation of VEGF receptors in endothelial cells after thrombin treatment, probably results in the mutual stimulation of endothelial and cancer cells and the activation of the angiogenic cascade.
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8. Conclusions The molecular basis of the association between malignancies and coagulopathies, although it has been known for long, it remains poorly understood. Recently, Even-Ram et al. Ž1998. have shown that the thrombin receptor is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Thrombin receptor expression has been reported in solid tumours of rat and mouse origin ŽWojtukiewicz et al., 1995.. This may be a general phenomenon for all malignancies. These receptors can be activated by thrombin or with a urokinase-type plasminogen activator, both of which are expressed at high levels in invasive tumour cells ŽPyke et al., 1991; Zacharsky et al., 1995.. We have shown in our studies that the activation of these receptors promotes angiogenesis in model systems in vivo ŽTsopanoglou et al., 1993; Haralambopoulos et al., 1997.. Many of the cellular events involved in the complex cascade of angiogenesis have been shown to be activated by thrombin and thrombin-receptor agonist peptides. The demonstration that the activation of thrombin receptors upregulates the expression of the VEGF receptors is of special importance. VEGF is considered to be the specific endothelial cell mitogen and the key angiogenic factor. The upregulation of VEGF receptors in endothelial cells sensitises these cells to the action of VEGF for the activation of angiogenesis. This in connection with the overexpression of VEGF by tumour cells after thrombin receptor activation has resulted in the mutual activation of tumour and endothelial cells for new blood vessel formation, a process essential for tumour growth and metastasis. These findings suggest a potential role of thrombin inhibitors in tumour therapy. Indeed sulfo-hirudin treatment showed a successful and significant reduction of experimental metastasis in a murine melanoma model ŽEsumi et al., 1991.. Zacharsky et al. Ž1995. have reviewed evidence that thrombin may be a growth factor for certain types of malignancy. In addition, warfarin or heparin treatment has been shown to increase the survival of patients with small cell lung carcinomas ŽLebeau et al., 1994.. The molecular and cellular events described in this paper have provided a basis and a rationale for the anti-thrombin therapy of cancer. These events can be activated by peptides that mimic the activated thrombin receptor and, conversely, are inhibited by receptor antagonists. This opens the possibility of developing agents that interfere with the angiogenic action of thrombin without interfering with the blood coagulation cascade. Such agents would be easier to use than heparin and other anti-coagulants in the clinic, and would be more specific for the desired result. Several
low-molecular weight thrombin inhibitors are under development for coagulopathies ŽTapparelli et al., 1993.. It remains to be seen if they find therapeutic use as anti-angiogenic agents in cancer and other angiogenic diseases such as retinopathy, chronic inflammation, etc.
Acknowledgements This work was supported in part by grants from the Greek Ministry of Research and Technology ŽPENED 1999 ED 26., ŽEPET II, 97, EKBANZ-1.2-115. and a NATO collaborative grant ŽCRG 940677.. We thank Ms Anna Marmara for secretarial help. References Baron, J.A., Gridley, G., Weiderpass, E., Nyren, O., Linet, M., 1998. Venous thromboembolism and cancer. Lancet 351, 1077᎐1080. Dery, O., Correra, C.U., Steinhoff, M., Bunnett, N.W., 1998. Proteinase-activated receptors: novel mechanisms of signalling by serine proteases. Am. J. Physiol. 274, C1429᎐C1452. Esumi, N., Fan, D., Fidler, I.J., 1991. Inhibition of murine melanoma experiments metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor. Cancer Res. 51, 4549᎐4556. Even-Ram, S., Usiely, B., Cohen, P. et al., 1998. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat. Med. 4, 909᎐914. Folkman, J., 1985. Tumour angiogenesis. Adv. Cancer Res. 43, 172᎐203. Folkman, J., Shing, Y., 1992. Angiogenesis: mini-review. J. Biol. Chem. 267, 10931᎐10934. Haralambopoulos, G.C., Grant, D.S., Kleinman, H.K., Maragoudakis, M.E., 1997. Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Am. J. Physiol. 273, C239᎐C242. Kakkar, A.K., Williamson, R.C.N., 1998. Thrombosis anti-thrombotic therapy. Cancer Heamostaseologie ŽSuppl.., S46᎐S52. Kanthou, C., Kakkar, V.V., Benzakour, O., 1998. Cellular and molecular effects of thrombin in the vascular system. In: Maragoudakis, M.E. ŽEd.., Angiogenesis Models, Modulators and Clinical Applications, vol. 298. Plenum Press, pp. 263᎐282. Lebeau, B., Chastany, C., Brechot, J.M., Capron, F., Dautzenberg, B., Delaisements, C., Mornet, M., Brun, J., Hurdebourcq, J.P., Lemarie, E., 1994. Subcutaneous heparin treatment increases survival in small cell lung cancer. ‘Petites Cellules’ Group Cancer, 74Ž1., 38᎐45. Maragoudakis, M.E., Tsopanoglou, N.E., Sakkoula, E., Pipili-Synetos, E., 1995. On the mechanism of promotion of angiogenesis by thrombin. FASEB J. 9, A587. Nierodzik, M.L., Plotkin, A., Kajumo, F., Karpakin, S., 1991. Thrombin stimulates tumour᎐platelet adhesion in vitro and metastasis in vivo. J. Clin. Invest. 87, 229᎐236. Nierodzik, M.L., Kajumo, F., Karpatikin, S., 1992. Effect of thrombin treatment of tumour cells on adhesion of tumour cells to platelets in vitro and tumour metastasis in vivo. Cancer Res. 52, 3267᎐3272. Pyke, C. et al., 1991. Messenger RNA for two type IV collagenases is located in stromatic cells in human colon cancer. Am. J. Pathol. 138, 1059᎐1067.
M.E. Maragoudakis et al. r Matrix Biology 19 (2000) 345᎐351 Rickles, F.R., Edwards, R.L., 1983. Activation of blood coagulation in cancer: Trousseau’s syndrome revisited. Blood 62 Ž1., 14᎐31. Sloane, B.F., Rozhin, J., Jonhson, K., Taylor, H., Crissman, J.D., Honn, K.V., 1986. Cathepsin B: association with plasma membrane in metastatic tumours. Proc. Natl. Acad. Sci. ŽUSA. 83, 2483᎐2487. Sorensen, H.T., Mellem, K.L., Steffensen, F.H., Olsen, J.H., Nielsen, ¨ G.L., 1998. The risk of diagnosis of cancer after primary deep venous thrombosis or pulmonary embolism. N. Engl. J. Med. 338, 1169᎐1173. Tapparelli, C., Metternich, R., Ehrhardt, C., Cook, N.S., 1993. Synthetic low molecular weight thrombin inhibitor: molecular design and pharmacological profile. Trends Pharmac. Sci. 14, 366᎐376. Tsopanoglou, N., Maragoudakis, M.E., 1998. On the mechanism of thrombin-induced angiogenesis: Inhibition of attachment of endothelial cells on basement membrane components. Angiogenesis J. 2, 192᎐200.
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Tsopanoglou, N., Maragoudakis, M.E., 1999. On the mechanism of thrombin-induced angiogenesis: potentiation of VEGF activity on endothelial cells by upregulation of its receptors. J. Biol. Chem. 274, 23969᎐23976. Tsopanoglou, N.E., Pipili-Synetos, E., Maragoudakis, M.E., 1993. Thrombin promotes angiogenesis by a mechanism independent of fibrin formation. Am. J. Physiol. 264, C1302᎐C1307. Vu, T.K.H., Hung, D.T., Wheaton, V.I., Coughlin, S.R., 1991. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 69, 1057᎐1068. Wojtukiewicz, M.Z. et al., 1995. Solid tumour cells express functional ‘tethered ligand’ thrombin receptor. Cancer Res. 55, 698᎐707. Zacharsky, L.R., Memoli, V.A., Morain, W.D., Schlaeppi, J.M., Rousseau, S.M., 1995. Cellular localization of enzymatically active thrombin in intact human tissues by hirudin binding. Thromb. Haemost. 73, 793᎐797.
Effects of thrombinrthrombosis in angiogenesis and tumour progression Michael E. MaragoudakisU , Nikos E. Tsopanoglou, Paraskevi Andriopoulou, Michael-Emmanuel M. Maragoudakis Uni¨ ersity of Patras, Medical School, Department of Pharmacology, 261 10 Rio, Patras, Greece
Abstract Laboratory, histopathological, pharmacological and clinical evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. On the other hand, epidemiological studies provide evidence of an increased risk of cancer diagnosis following primary thromboembolism. Moreover, the metastatic ability of human breast cancer cells is correlated with the number of thrombin receptors of these cells, and thrombin treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats. We have proposed that these tumour-promoting effects of thrombin can be explained by the ability of thrombin to activate angiogenesis, an essential requirement for tumour progression. Many of the cellular events involved in the angiogenic cascade can be activated by thrombin. At the molecular level, brief exposure of endothelial cells to thrombin causes an upregulation of the receptors ŽKDR and Flt-1. of VEGF, the key angiogenic mediator. This results in a synergistic effect of thrombin and VEGF in the activation of angiogenesis. In addition, thrombin activates cancer cells to secrete VEGF, thus causing a mutual stimulation between EC and CA cells. Cancer cells exposed to thrombin secrete metalloproteinase 92 KD and overexpress the integrin av b3 , all of which are involved in tumour metastasis. 䊚 2000 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Thrombin; Angiogenesis; Tumour; Metastasis
1. Cancer and thrombosis The association between blood coagulation and metastatic dissemination was first recognised by Trousseau more than 128 years ago. This observation has subsequently been verified by many investigators who provided laboratory and clinical evidence sug-
Abbre¨ iations: CA, cancer cells; EC, endothelial cells; VEGF, vascular endothelial growth factor; TRAP, thrombin receptor agonist peptide; CAM, chick chorioallantoic membrane; FGF, fibroblast growth factor; RT-PCR, reverse transcriptase-polymerase chain reaction; PKC, protein kinase C; MAP, mitogen activated protein; t-PA, tissue-plasminogen activator; PAI, plasminogen activator inhibitor; NO, nitric oxide; PDGF, platelet derived growth factor; CPB, collagenous protein biosynthesis. U Corresponding author. Tel.: q30-30-61-997691r997638; fax: q30-30-61-994720. E-mail address: [email protected] ŽM.E. Maragoudakis..
gesting the systemic activation of the blood-clotting cascade in patients with cancer. Measurements of circulating fibrino-peptides have shown that patients with cancer often have inappropriately high intravascular coagulation and fibrinolysis ŽRickles and Edwards, 1983.. Many tumour cells elicit procoagulant activity by interaction with the blood’s thrombin-generating systems and also with platelets, leukocytes and endothelial cells ŽSloane et al., 1986.. Some tumour cells express the transmembrane tissue factor, which when exposed to circulating coagulation factor VII, activates factor X, leading to the generation of thrombin and the formation of fibrin. Other tumour-derived activators of factor X, such as mucin and cysteine protease, have been described. In all of these cases, local thrombin generation occurs, resulting in fibrin deposition, which is thought to be important for tumour growth and platelet activation. Recently
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Zacharsky et al. Ž1995. have shown the presence of thrombin in a variety of tumour cell types by immunostaining. Two recent large epidemiological studies ŽBaron et al., 1998; Sorensen et al., 1998. involving 86 000 ¨ patients with thromboembolism, 4200 of whom had cancer, suggest that the risk of deep vein thrombosis or pulmonary thromboembolism is 3.2 times higher in cancer patients. The risk of thrombosis is increasing with anti-neoplastic treatment. Cytotoxic therapy damages the endothelium and releases procoagulant substances and cytokines, which activate blood coagulation. As a result of these findings, anti-thrombotic therapy is considered a rational approach to prevent thromboembolism in cancer patients ŽKakkar and Williamson, 1998..
2. Thrombosis and tumour progression The aforementioned studies explain why hypercoagulability occurs in cancer patients, but does not answer the question of whether thrombosis can contribute directly to tumour promotion and metastasis. Indeed, in the epidemiological study by Sorensen et ¨ al. Ž1998. it was shown that 6᎐12 months after a primary thromboembolism, the risk of cancer diagnosis increases threefold. These clinical observations were in line with animal experiments, where the thrombin-treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats ŽNierodzik et al., 1992.. Until recently, the prometastatic effects of thrombin were attributed to the effects of thrombin on platelets, endothelial cells or fibrin formation. However, new evidence shows that the pretreatment of several tumour cells with thrombin increases their ability to adhere to platelets and extracellular matrix proteins ŽNierodzik et al., 1991.. Thrombin-pretreated B16 tumour cells showed an increase in pulmonary metastasis of up to 156-fold in mice compared to sham-treated cells. Based on these data, the anti-coagulant drug warfarin was used in animal tumour models with positive results. Following this experience with animal tumour models, controlled clinical trials of warfarin in several human cancer patients was initiated with encouraging results. Warfarin prolonged the length of survival of patients with a small cell carcinoma of the lung. Besides small cell lung carcinomas, other human cancer cells such as renal cell carcinomas and malignant melanomas are known to generate thrombin and are likely to be susceptible to anti-coagulant therapies. It is of importance in this respect that the metastatic ability of human breast cancer cells is related to the number of thrombin receptors on these cells ŽEvenRam et al., 1998.. This association between metastatic
ability and the number of thrombin receptors needs to be investigated in other types of tumours. The mechanism of action of warfarin in cancer therapy is not clear from these data. However, clinical studies support the concept that anti-coagulant drugs may have a therapeutic use for certain types of cancer as well as in preventing cancer-associated hypercoagulability, particularly during chemotherapy, when there is an increased risk.
3. Proposed mechanism for tumour progression following thrombosis The final step in thrombosis is thrombin generation and fibrin formation. The thrombin in circulation is rapidly inactivated by anti-thrombin, but the thrombin trapped within the thrombi is protected and slowly released with the progression of fibrinolysis. We have reported that thrombin is a potent activator of angiogenesis ŽTsopanoglou et al., 1993. and proposed that this new action of thrombin may be the basis for the promotion of tumour progression following thrombosis. The present day dogma in oncology is that angiogenesis is an essential requirement for tumour growth and metastasis ŽFolkman, 1985.. Tumours do not grow beyond 1᎐2 mm3 in size unless they recruit their own blood supply for delivering nutrients and O2 . In addition, the malignant phenotype requires the ability of cancer cell for invasion and uncontrolled growth. Angiogenesis is activated in many other conditions besides cancer. Wound healing, diabetic retinopathy, atherosclerosis, the follicular phase of the menstrual cycle etc., are some examples. Considering the broad spectrum of physiological and pathophysiological processes that angiogenesis is involved in, we can strongly theorise the involvement of angiogenesis activation by thrombin is a more general phenomenon. We have studied the angiogenic action of thrombin initially in the model of the CAM system, in vivo ŽFig. 1.. It was shown that the effect was dose-dependent, specific and required the catalytic site of thrombin. Heparin or hirudin completely blocked the angiogenic action of thrombin. Also, the chemically inactivated thrombin at the active site ŽPPACK thrombin. was without effect and competed with thrombin for its angiogenic action. An analog of thrombin Ž ␥-thrombin., which lacks the anion binding exocite for binding fibrinogen and, therefore, cannot form fibrin, was also as active as thrombin in activating angiogenesis. This implied that the angiogenic action of thrombin was independent of fibrin formation. Most of the actions of thrombin were mediated by the activation of a thrombin receptor. This activation involved the proteolytic cleavage of the receptor by thrombin, thus generating a new NH2-terminus, which
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Fig. 1. Effect of thrombin on collagenous protein biosynthesis ŽCPB. in CAM in vivo. Results are expressed as mean cpmrmg protein " S.E.
acted as a tethered ligand binding to a new receptor on the cell membrane, coupled to various G-proteins ŽVu et al., 1991.. The repertoire of G-proteins coupled to the thrombin receptor and the cellular effectors activated determined the nature of the cellular responses generated by thrombin ŽDery et al., 1998.. A decatetrapeptide ŽTRAP., representing the NH2terminal of the activated thrombin receptor, mimicked many of the cellular effects of thrombin. TRAP is also a promoter of angiogenesis in the same fashion as thrombin. Moreover, FPR the antagonist peptide of TRAP, cancelled out the angiogenic effect of TRAP in the CAM ŽMaragoudakis et al., 1995.. This provided further support that the thrombogenic action of thrombin Žfibrin formation. was not essential for the angiogenic action. In addition to the CAM system, the angiogenic action of thrombin was established in the Matrigel system in vivo ŽHaralambopoulos et al., 1997..
4. Effects of thrombin on the angiogenic cascade Angiogenesis is a complex biological phenomenon in which many cell types and factors are involved. Briefly, it starts by the activation of the normally quiescent endothelial cells of the parent vessel Žusually a venule. by an angiogenic stimulus. This stimulus can be one of the many growth factors and endogenous substances identified thus far, which act as angiogenic factors, including thrombin ŽFolkman, 1985.. The activation of endothelial cells causes the secretion of metalloproteinases, which results in the local dissolution of basement membranes and the extracellular matrix, allowing the migration of the
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endothelial cell towards the angiogenic stimuli. This is followed by the proliferation of endothelial cells, alignment to form the lumen of the new vessel, anastomosis, synthesis of basement membrane of the new vessel, blood flow and the recruitment of pericytes and smooth muscle cells ŽFolkman and Shing, 1992.. This process is tightly regulated by endogenous promoters and inhibitors and is dormant under physiological conditions in an adult organism. However, it can be activated at a moment’s notice, like the blood coagulation cascade, probably by different mechanisms in each tissue that converge in the control of the activity of a single factor such as VEGF. VEGF is considered to be the specific endothelial cell mitogen and is expressed in all cases of activation of angiogenesis, both physiological and pathological. It has been proposed that under physiological conditions, angiogenesis is dormant, because of a balance in the activity of the many promoters and inhibitors of angiogenesis. It is believed that activation of angiogenesis is a result of an imbalance between angiogenic and anti-angiogenic factors ŽFolkman, 1985.. However, angiogenesis is a physiological process too important to be controlled only by a simple imbalance of redundant promoters and inhibitors. Strict controls must exist, probably unique to every tissue, and the immediate activation of angiogenesis at a short notice must be possible. This can only be accomplished by intricate interactions of the modulators of angiogenesis. We have established that thrombin modulates the activity of VEGF by up-regulating the expression of VEGF receptors in endothelial cells ŽTsopanoglou and Maragoudakis, 1999..
5. Activation of the angiogenic cascade by thrombin As mentioned above, angiogenesis proceeds in many distinct stages, which can be studied in several in vitro and in vivo systems. We have initiated a systematic approach to establish which of these steps are modulated by thrombin and the mechanisms involved. Brief exposure Žless than 15 min. of endothelial cells to thrombin causes a marked inhibition of their ability to adhere to type IV collagen and laminin ŽFig. 2.. The endothelial cell phenotype, which results after exposure to thrombin, can survive without attachment to the extracellular matrix. This effect is reversible, and the removal of thrombin and the re-incubation of cells in growth medium completely restores their ability to adhere to type IV collagen and laminin. These effects of thrombin are specific, since hirudin and heparin block this action, and require the active site of thrombin, since PPACK thrombin is without effect. It is also receptor-mediated since TRAP, the activated receptor agonist, has the same effect as throm-
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Fig. 2. Effect of thrombin on cell attachment. HUVECs in suspension were incubated for 15 min with serum-free M199 alone Žnonercontrol. or various concentrations of thrombin. Cells Ž40 000 cellsrwell. were plated on collagen IV or laminin and allowed to adhere for 60 min at 37⬚C. Adhered cells were counted with a computerised digital image analyser. Results were expressed as the mean of the percentage change of control " S.E., which is taken as 100%.
bin ŽTsopanoglou and Maragoudakis, 1998.. These results suggest that thrombin enables endothelial cells to detach from their anchorage site on the basement membrane of the parent vessel, to survive without attachment to the extracellular matrix, and to migrate to distant sites. The exposure of endothelial cells to thrombin causes an increase in the activated form of progelatinase A Ž68 kDa. in zymograms of the culture medium. Activation of the 72-kDa metalloproteinase, which degrades collagen type IV, is probably related to the local degradation of basement membrane collagen, thus allowing for the movement of the activated endothelial cells. In addition, the dissolution of the extracellular matrix liberates sequestered growth factors such as b-FGF, which further promote the angiogenic cascade. It has been shown that thrombin and TRAP act as endothelial cell mitogens and promote cell growth. Like all of the aforementioned effects of thrombin, this action of thrombin on endothelial cells is specific and requires the active catalytic site of thrombin ŽTsopanoglou and Maragoudakis, 1999.. In combination with VEGF, the endothelial cell specific mitogen, we obtained a synergistic effect in the rate of DNA synthesis and cell proliferation ŽFig. 3.. Although the exposure to thrombin needs to be brief Ž15 min., the time required for the synergistic effect to become manifested is 8᎐12 h after thrombin-treatment. At
earlier times Ž0.5, 1.5 and 4 h. this potentiating effect of thrombin on VEGF-induced DNA synthesis and endothelial cell proliferation is not evident. The specificity of this effect is established by the fact that hirudin cancels out this action of thrombin. PPACK thrombin is without effect, and TRAP has the same effect as thrombin, indicating that this is also a thrombin receptor-mediated event. We concluded from these findings that thrombin effectively enhances the mitogenic potency of VEGF on endothelial cells ŽTsopanoglou and Maragoudakis, 1999.. This potentiating effect of thrombin on VEGFinduced endothelial cell proliferation can be explained by the upregulation of VEGF receptors. This was shown by a sensitive quantitative RT-PCR technique. The mRNA for both receptors of VEGF ŽKDR and flt-1. was upregulated 8᎐12 h after exposure to thrombin. Thrombin stimulates KDR and flt-1 mRNA expression dose-dependently, with a maximum obtained at 1.5 IUrml. The over-expression of mRNA for KDR and flt-1 is a receptor-mediated event and can be obtained using TRAP, the agonist of the activated thrombin receptor. We have established that the increase in KDR mRNA was accompanied by an increase in the functional KDR protein. The endothe-
Fig. 3. Temporal effect of thrombin on VEGF-induced DNA synthesis in human endothelial cells. HUVECs were preincubated with M199r1%BSA alone or with thrombin Ž1.5 IUrml. for the indicated times, and were subsequently incubated either with M199r4%FBS alone or with VEGF Ž5 ngrml. for 18 h. All cells were pulsed with w3 Hx-thymidine for an additional 6 h. Control: cells were preincubated with M199r1% BSA and the with M199r4%FCS. Thr: cells were preincubated with thrombin and then with M199r4%FCS. VEGF: cells were preincubated with M199r1%BSA and then with VEGF. ThrrVEGF: cells were preincubated with thrombin and then with VEGF. Results are expressed as mean of percent over the control " S.E. of six wells, which is taken as 0%. Similar results were obtained in three separate experiments.
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lial cells, after exposure to thrombin, were lysed and, after immunoprecipitation, the band corresponding to KDR Ž190 kDa. was increased by 200% over that of control cells. The upregulated KDR receptor was functional, as shown by the phosphorylation of KDR protein, after treatment of the endothelial cells with VEGF ŽTsopanoglou and Maragoudakis, 1999.. In order to exclude the possibility that the upregulation of the VEGF receptors by thrombin was due to stabilisation of mRNA by decreasing its rate of degradation, we measured KDR mRNA in the presence of actinomycin D. Inhibition of the transcription rate by actinomycin D did not affect the rate of decay of KDR mRNA. Both the control and thrombin-treated endothelial cells had a half-life of KDR mRNA of approximately 2.8 h. Nuclei isolated from control and thrombin-treated endothelial cells were incubated in vitro to determine the rate of transcription of KDR. The thrombin-treated nuclei showed an 80% increase in transcription rate after 1 h. The transduction mechanisms involved in the overexpression of VEGF receptors were investigated using agents that modulate key cellular transduction mechanisms known to be involved in the cellular actions of thrombin, namely the PKC and MAP kinase-dependent pathways. Using PMA as a PKC activator, and calphostin C as a selective PKC inhibitor, we demonstrated that PMAlike thrombin upregulates KDR mRNA, while calphostin C completely abolishes the upregulation of KDR by thrombin and PMA. Similarly, using a specific inhibitor of MAP kinase ŽPD98059., we showed that the upregulation of KDR and flt-1 mRNA was mediated by the activation of the MAP kinase pathway. In contrast, the activation of adenyl cyclase by forskolin did not effect the levels of KDR and flt-1 mRNA induced by thrombin. We concluded from these experiments that thrombin upregulates the VEGF receptors via the activation of PKC and MAP kinase pathways downstream after the activation of the thrombin receptor, coupled to G-proteins. The final result is the amplification of the role of VEGF in angiogenesis.
6. Effects of thrombin that may lead to activation of angiogenesis In addition to the effects of thrombin in blood coagulation Ži.e. amplification: FVª FVa, FVIII ª FVIIIa; clot formation: fibrinogen ª fibrin, FXIII ª FXIIIa; platelet aggregation: secretion and granule release ., thrombin has many other actions on various cell types, which may be related to promotion of angiogenesis ŽKanthou et al., 1998..
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On endothelial cells thrombin is known to have many effects: 䢇
䢇
䢇
䢇
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Activation leading to CAM expression and leukocyte adhesion; secretion of PGI2 , t-PA, NO, PDGF, PAI-1, and endothelin; anticoagulant by thrombomodulinrprotein C activation; pro-coagulant by tissue factor and microparticle shedding; and re-traction leading to an increase in vascular permeability
In leukocytes, thrombin stimulates proliferation, chemotaxis and cytokine production. These actions of thrombin on endothelial, inflammatory cells and platelets probably contribute to tumour and inflammatory angiogenesis ŽTapparelli et al., 1993..
7. Thrombin effects on the metastatic phenotype of cancer cells
As mentioned already, the association between malignancies and coagulopathies has long been established. A common clotting abnormality in cancer patients is an elevated amount of fibrinogenrfibrin degradation products, indicating thrombin generation. This fibrin-rich product, together with the extracellular matrix, may provide the optimal tumour microenvironment. This may also provide a storage site for thrombin within the fibrin clot, from which thrombin is released to activate the thrombin receptors. Such receptors are overexpressed in metastatic cancer cells ŽEven-Ram et al., 1998.. Preliminary evidence from our laboratory has shown that PC3 human prostate carcinoma cells, when treated with thrombin or thrombin receptor agonists ŽTRAP. increases the secretion of the 92-kDa metalloproteinase. In addition, there is an increase in mRNA expression for VEGF and the secretion of the VEGF protein in the medium. Furthermore, thrombin treatment causes an overexpression of the av b3 integrin Žunpublished observations.. These results suggest that thrombin treatment promotes the metastatic and angiogenic phenotype of cancer cells. The overexpression of VEGF in cancer cells, in connection with the upregulation of VEGF receptors in endothelial cells after thrombin treatment, probably results in the mutual stimulation of endothelial and cancer cells and the activation of the angiogenic cascade.
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8. Conclusions The molecular basis of the association between malignancies and coagulopathies, although it has been known for long, it remains poorly understood. Recently, Even-Ram et al. Ž1998. have shown that the thrombin receptor is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Thrombin receptor expression has been reported in solid tumours of rat and mouse origin ŽWojtukiewicz et al., 1995.. This may be a general phenomenon for all malignancies. These receptors can be activated by thrombin or with a urokinase-type plasminogen activator, both of which are expressed at high levels in invasive tumour cells ŽPyke et al., 1991; Zacharsky et al., 1995.. We have shown in our studies that the activation of these receptors promotes angiogenesis in model systems in vivo ŽTsopanoglou et al., 1993; Haralambopoulos et al., 1997.. Many of the cellular events involved in the complex cascade of angiogenesis have been shown to be activated by thrombin and thrombin-receptor agonist peptides. The demonstration that the activation of thrombin receptors upregulates the expression of the VEGF receptors is of special importance. VEGF is considered to be the specific endothelial cell mitogen and the key angiogenic factor. The upregulation of VEGF receptors in endothelial cells sensitises these cells to the action of VEGF for the activation of angiogenesis. This in connection with the overexpression of VEGF by tumour cells after thrombin receptor activation has resulted in the mutual activation of tumour and endothelial cells for new blood vessel formation, a process essential for tumour growth and metastasis. These findings suggest a potential role of thrombin inhibitors in tumour therapy. Indeed sulfo-hirudin treatment showed a successful and significant reduction of experimental metastasis in a murine melanoma model ŽEsumi et al., 1991.. Zacharsky et al. Ž1995. have reviewed evidence that thrombin may be a growth factor for certain types of malignancy. In addition, warfarin or heparin treatment has been shown to increase the survival of patients with small cell lung carcinomas ŽLebeau et al., 1994.. The molecular and cellular events described in this paper have provided a basis and a rationale for the anti-thrombin therapy of cancer. These events can be activated by peptides that mimic the activated thrombin receptor and, conversely, are inhibited by receptor antagonists. This opens the possibility of developing agents that interfere with the angiogenic action of thrombin without interfering with the blood coagulation cascade. Such agents would be easier to use than heparin and other anti-coagulants in the clinic, and would be more specific for the desired result. Several
low-molecular weight thrombin inhibitors are under development for coagulopathies ŽTapparelli et al., 1993.. It remains to be seen if they find therapeutic use as anti-angiogenic agents in cancer and other angiogenic diseases such as retinopathy, chronic inflammation, etc.
Acknowledgements This work was supported in part by grants from the Greek Ministry of Research and Technology ŽPENED 1999 ED 26., ŽEPET II, 97, EKBANZ-1.2-115. and a NATO collaborative grant ŽCRG 940677.. We thank Ms Anna Marmara for secretarial help. References Baron, J.A., Gridley, G., Weiderpass, E., Nyren, O., Linet, M., 1998. Venous thromboembolism and cancer. Lancet 351, 1077᎐1080. Dery, O., Correra, C.U., Steinhoff, M., Bunnett, N.W., 1998. Proteinase-activated receptors: novel mechanisms of signalling by serine proteases. Am. J. Physiol. 274, C1429᎐C1452. Esumi, N., Fan, D., Fidler, I.J., 1991. Inhibition of murine melanoma experiments metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor. Cancer Res. 51, 4549᎐4556. Even-Ram, S., Usiely, B., Cohen, P. et al., 1998. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat. Med. 4, 909᎐914. Folkman, J., 1985. Tumour angiogenesis. Adv. Cancer Res. 43, 172᎐203. Folkman, J., Shing, Y., 1992. Angiogenesis: mini-review. J. Biol. Chem. 267, 10931᎐10934. Haralambopoulos, G.C., Grant, D.S., Kleinman, H.K., Maragoudakis, M.E., 1997. Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Am. J. Physiol. 273, C239᎐C242. Kakkar, A.K., Williamson, R.C.N., 1998. Thrombosis anti-thrombotic therapy. Cancer Heamostaseologie ŽSuppl.., S46᎐S52. Kanthou, C., Kakkar, V.V., Benzakour, O., 1998. Cellular and molecular effects of thrombin in the vascular system. In: Maragoudakis, M.E. ŽEd.., Angiogenesis Models, Modulators and Clinical Applications, vol. 298. Plenum Press, pp. 263᎐282. Lebeau, B., Chastany, C., Brechot, J.M., Capron, F., Dautzenberg, B., Delaisements, C., Mornet, M., Brun, J., Hurdebourcq, J.P., Lemarie, E., 1994. Subcutaneous heparin treatment increases survival in small cell lung cancer. ‘Petites Cellules’ Group Cancer, 74Ž1., 38᎐45. Maragoudakis, M.E., Tsopanoglou, N.E., Sakkoula, E., Pipili-Synetos, E., 1995. On the mechanism of promotion of angiogenesis by thrombin. FASEB J. 9, A587. Nierodzik, M.L., Plotkin, A., Kajumo, F., Karpakin, S., 1991. Thrombin stimulates tumour᎐platelet adhesion in vitro and metastasis in vivo. J. Clin. Invest. 87, 229᎐236. Nierodzik, M.L., Kajumo, F., Karpatikin, S., 1992. Effect of thrombin treatment of tumour cells on adhesion of tumour cells to platelets in vitro and tumour metastasis in vivo. Cancer Res. 52, 3267᎐3272. Pyke, C. et al., 1991. Messenger RNA for two type IV collagenases is located in stromatic cells in human colon cancer. Am. J. Pathol. 138, 1059᎐1067.
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