NON-HUMAN TRANSGENIC ANIMALS DEFICIENT IN GAS6 FUNCTION AND THEIR USE

Abstract: It has been shown that Gas6 deficient endothelial cells are resistant to activation by endotoxin and TNF- alpha <i>in vitro</i> and <i>in vivo</i> and that leukocytes did not or much less extravasate in Gas6 deficient mice. The present invention also relates to the manufacture of a medicament for the treatment of endothelial dysfunction. The present invention further relates to the use of an inhibitor of the Gas6 function and/or an inhibitor of a Gas6 receptor for the manufacture of said medicament.

Description: Non-human transqenic animals deficient in Gas6 function and their use. The present invention relates to the manufacture of a medicament for the treatment of endothelial dysfunction. The present invention further relates to the use of an inhibitor of the Gas6 function and/or an inhibitor of a Gas6 receptor for the manufacture of said medicament. The endothelium is uniquely positioned at the interface between the blood and the vessel. As such, it performs multiple functions: it is involved in the regulation of coagulation, leukocyte adhesion in inflammation, vessel tone, and vascular smooth muscle cell growth, and also acts as a barrier to transvascular flux of liquids and solutes (Griendling & Alexander, 1996). Far from being a passive participant in these events, it is a dynamic tissue, secreting and modifying vasoactive substances, influencing the behaviour of other cell types, and regulating extracellular matrix production and composition. One of the most important events in the reaction to all forms of injury is adhesion of leukocytes to activated endothelium, a prelude to their emigration into tissues (Cotran & Mayadas-Norton, 1998). Not surprisingly, therefore, when endothelial cells become dysfunctional or deregulated, they may contribute significantly to the development of numerous disorders with major morbidity and mortality, some of which are listed hereunder. Cardiovascular disorders including atherothrombosis, stroke, arterial (re)-stenosis, ischemic heart disease, myocardial infarction, diabetic macro- and microangiopathy, allograft arteriosclerosis and the like constitute the leading cause of morbidity and mortality in Westernised societies. A common feature of all these disorders is activation of the endothelium, which induces interaction with leukocytes (Ross, 1999). For example, leukocytes adhere to and infiltrate underneath the endothelium during lipid-induced atherosclerosis (Ross, 1999; Libby et al., 1997; Vadas et al., 1997; Boyle et al., 1997). During immune-mediated transplant arteriosclerosis or cuff-induced restenosis, leukocytes adhere to the endothelium, subsequently infiltrate in the intima and media, where they activate medial smooth muscle cells to emigrate in the intima, thereby leading to obstructive intimal thickening (Libby et al., 1992). During ischemia/reperfusion, leukocytes have been involved in the production of toxic substances, leading to tissue destruction (Murohara et al., 1995; Dinerman & Mehta, 1990; DeGraba, 1998; Hallenbeck, 1997; Cavanagh et al., 1998). Excessive formation of new blood vessels (neovascularisation) promotes progression of cancers, induces diabetic retinopathy leading to blindness, and contributes to chronic inflammatory disorders (rheumatoid arthritis, non-healing ulcerations) (Folkman, 1995). In order for endothelial cells to form new blood vessels, they first need to become activated by angiogenic molecules (Carmeliet & Collen, 1998). Leukocytes and tumor cells, adhering to the endothelium, play an essential role in the induction of pathological neovascularization by production of angiogenic factors (Schaper & Ito, 1996). The morbidity and mortality associated with abnormal angiogenesis is significant. Conversely, insufficient angiogenesis has been related to impaired responsiveness of the endothelium, such as in the elderly or diabetic patients. Infection and resulting sepsis continue to be important causes of morbidity and mortality, in particular in surgical patients. Emerging evidence implicates the endothelium as a central effector in the inflammatory response (Ahmed & Christou, 1996; McGill et al., 1998; Pajkrt & van Deventer, 1996). Through the expression of surface proteins and secretion of soluble mediators, the endothelium controls vascular tone and permeability, regulates coagulation and thrombosis, and directs the passage of leukocytes into areas of inflammation. Derangements in these normal functions may contribute significantly to a maladaptive inflammatory response leading to systemic inflammation, multiple organ failure, and "diffuse intravascular coagulation", a life-threatening complication. Metastasis involves adhesion of tumor cells to the endothelium with subsequent extravasation and colonisation in distant tissues. Adhesion of tumor cells is mediated by similar adhesion molecules as used by leukocytes. Endothelial cells in tumors are activated, express more and distinct adhesion molecules, and are often dysfunctional. Collectively, a better understanding of the molecular basis leading to activation of endothelial cells is mandated in order to design more rationally future treatments for these life-threatening and debilitating disorders. Considering its enormous surface area (equivalent in adults to an entire soccer field) and accessibility, it is not surprising that this "organ" plays an important role in the above-mentioned disorders. The identification of candidates involved in this process may constitute attractive targets for development of drugs for treatment of such disorders. Previous studies using inhibitors or in gene-inactivated mice have amply demonstrated the importance of endothelial activation and endothelial-leukocyte interaction in cardiovascular disorders (Frenette & Wagner, 1997). Growth arrested gene 6 (Gas6) was originally identified as a gene, whose expression in fibroblasts increased by serum starvation and contact inhibition, and was therefore implicated in reversible growth arrest (Schneider et al., 1988). Gas6 has significant sequence similarity with anticoagulant protein S but lacks anticoagulant activity due to absence of two thrombin cleavage sites. Both molecules are composed of a Gla domain, four EGF-like repeats and a carboxyterminal tandem globular (G)-domains with homology to steroid hormone- binding globulin that are present in molecules involved in cell growth and differentiation (Nakano et al., 1997a). Gas6 binds members of the Axl family of receptor tyrosine kinases including Axl (also called Ark, Ufo), Sky (also called Rse, Tyro3, Dtk, Etk, Bit) and Mer (also called Eyk, Nyk) (Crosier and Crosier, 1997; Nagata et al., 1996; Stitt et al., 1995). The extracellular domain of these receptors posses Ig-like motifs and fibronectin type III modules, found in cell adhesion molecules. A truncated soluble Axl form is proteolytically generated but it remains unknown whether this soluble receptor acts as a competitive inhibitor for Gas6 by sequestering free ligand, is involved in binding Axl directly and originating a signal distinct from Gas6, or in inactivating the receptor on the cell surface (Costa et al., 1996). Gas6 is expressed in lung and intestine (Manfioletti et al., 1993). Sky is predominantly expressed in the brain, while Mer is expressed in monocytes (from peripheral blood and bone marrow) and in cells derived from epithelial tissues (lung, kidney) and reproductive tissues (ovary, prostate), but not in unstimulated granulocytes. Axl is also expressed in monocytes but not in unstimulated granulocytes or mature B or T lymphocytes. Gas6 and Axl are expressed by vascular endothelial cells (Manfioletti et al., 1993). The function of Gas6 remains largely undetermined and controversial. Gasδ has been reported to inhibit homophiiic Axl-mediated aggregation of myeloid cells (Avanzi et al., 1998), but cell-bound Gas6 may mediate aggregation of myeloid cells via interaction with Axl receptor on adjacent cells (McCloskey et al., 1997). Gas6 did not affect adhesion of granulocytes to resting endothelial cells, while it inhibited granulocyte adhesion to TNF-alpha activated endothelial cells, but only at high concentrations (Avanzi et al., 1998). Gas6 is mitogenic for fibroblasts (Goruppi et al., 1996) and Schwann cells (Li et al., 1996) but not for myeloid cells (Avanzi et al., 1997) or endothelial cells (Avanzi et al., 1998). Gas6, induced in injured vascular smooth muscle cells, induces Axl-mediated chemotaxis of smooth muscle cells (Fridell et al., 1998) and, although not mitogenic by itself, enhances the mitogenic activity of thrombin (Fridell et al., 1998). Gas6 also acts as a survival factor for serum-starved fibroblasts (Goruppi et al., 1996) and GnRH neuronal cells (Allen et al., 1999), presumably via activation of PI3-kinase and Akt kinase (Goruppi et al., 1997). Ark signalling protects against apoptosis induced by serum withdrawal or TNF-alpha, but the increased apoptosis of Ark deficient fibroblasts after serum- withdrawal could not be rescued by Gas6 (Bellosta et al., 1997). Gas6 may play a role in clearance of apoptotic cells by forming a bridge between phosphatidylserine on the surface of apoptotic cells and Axl on phagocytotic cells (Nakano et al., 1997b). Since growth-arrested genes are characteristically expressed when cells undergo growth arrest, the role of Gas6 in endothelial biology was examined. Indeed, endothelial cells become quiescent and enter a state of prolonged growth arrest (half-life estimated to be ~ 30 years in humans), once vascular development during embryogenesis and postnatal growth is accomplished, and abundantly express Gas6. However, in contrast to other growth-arrested cells (including terminally differentiated cardiomyocytes or neurons), endothelial cells exhibit a unique plasticity to rapidly proliferate and migrate, or to become activated to express prothrombotic factors or leukocyte adhesion molecules. Such responsiveness is essential to accommodate rapidly changing needs to vascularize ischemic tissues, to provide hemostasis upon vascular trauma, to mediate leukocyte extravasation in inflamed tissues or to provide adequate vasomotor control during abnormal tissue perfusion. Notably, the molecular mechanisms that allow quiescent endothelial cells to exhibit such rapid responsiveness remain undetermined. Collectively, despite the above-mentioned data, the precise role of Gas6 and its receptors, in particular in endothelial (patho)-biology in vivo, remains largely undetermined. In this invention it has been surprisingly found that Gas6 deficient endothelial cells are resistant to activation by endotoxin and TNF-α in vitro and in vivo and that leukocytes did not or much less extravasate in Gas6 deficient mice. This proves that Gas6 plays an essential role in the activation of the endothelium in response to inflammatory stimuli. Since numerous cardiovascular, cancerous and infectious disorders, as indicated above, implicate endothelial activation with subsequent binding and extravasation of leukocytes, Gas6 antagonists and Gas6 receptor inhibitors can be used as compounds for the manufacture of a medicament for the treatment of said disorders. In a first aspect the present invention concerns an animal deficient in Gas6 function. Preferably, said animal is a transgenic animal in which the Gas6 alleles are inactivated (Gas6 'A ). More preferably, said animal is a Gas6 "Λ mouse. In another aspect the invention relates to the use of said Gas6 deficient animal to study the role of Gas6. Said Gas6 deficient transgenic animals can be used for screening compounds having an effect, positive or negative, on said endothelial dysfunction. In another aspect the invention provides a method to screen for inhibitors of Gas6 function and/or inhibitors of a Gas6 receptor that result in an inhibition of the endothelial activation. Preferably, inhibitors of Gas6 function are soluble forms of the Gas6 receptor and/or Gas6 neutralising antibodies. Alternatively, Gas6 inhibitors may be compounds that prevent the binding of Gas6 to its receptor and/or the activation of said receptor. In still another aspect, the invention provides the use of an inhibitor of the Gas6 function and/or an inhibitor of a Gas6 receptor for the manufacture of a medicament to treat endothelial dysfunction. Definitions The following definitions are set forth to illustrate the meaning and scope of the various terms used to describe the invention therein. Activation of Gas6 receptor: depends on the type of receptor and is known to the people skilled in the art. For Gas6 tyrosine kinase type receptors, it has been extensively described in W09628548 and refers to the step of causing the intracellular domain of the receptor to phosphorylate tyrosine residues in a substrate polypeptide. Inhibitor of Gas6 receptor: any compound that prevents the activation of said receptor. Gas6 neutralising antibody: any antibody that prevents Gas6 to execute its function. Inhibitor of Gas6 function: any compound that prevents Gas6 to execute its function. This inhibition may be due to actions such as chemical modification, denaturation of the tertiary structure, complex formation or proteolysis. Gas6 function: binding of Gas6 with a Gas6 receptor. Bind(ing) means any kind of interaction, be it direct (direct interaction of Gas6 with the extracellular domain of said receptor) or indirect (interaction of Gas6 with one or more identical and/or non- identical compounds resulting in a complex of which one or more compounds can interact with the extracellular domain) that results in activation of the Gas6 receptor. The extracellular domain means the domain as it occurs in the natural Gas6 receptors, or a fragment thereof, eventually fused to other amino acid sequences, characterised in that it is still able to bind Gas6. Compound: any chemical or biological compound, including simple inorganic or organic molecules, peptides, peptido-mimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof. Examples Example 1: inhibition of endothelial activation by Gas6 gene-inactivation in mice In order to study the role of Gas6 in vivo, the Gas6 gene was inactivated in embryonic stem cells via homologous recombination, and Gas6 deficient mice were generated. Surprisingly, these knockout mice developed normally, survived, and were apparently healthy. However, when the Gas6 deficient mice were challenged with inflammatory stimuli, they were found to be protected against endothelial activation. Eight to twelve week old WT and Gas6 "Λ mice of either sex with a genetic background of 75% C57BI6 and 25%129/Sv, and weighing 20-30 g, were used. Animals were maintained in an open animal facility and experiments were performed according to the guidelines of the institutional animal care committee. Adult mice were intraperitoneally injected with endotoxin (0, 10 and 20 mg/kg) and after 12 h, mice were anesthetized by intraperitoneal injection of 60 mg/kg sodium pentobarbital. Blood was collected in citrate via vena cava puncture with a 24-gauge needle. Following centrifugation at 3000 rpm for 10 min, plasma was collected and stored at -20° C until analysis. Factor VII activity was measured via the Coatest FVII assay, as described by the manufacturer (Chromogenix, Brussels, Belgium). The coagulant activity of the other factors was determined as a clotting time after mixing the murine plasma with human plasma, deficient in the specific factor and addition of thromboplastin (for Fll, FV, FVII, FX) or kaolin (FIX) in the University Hospital (UZ Gasthuisberg, Leuven, Belgium). All procoagulant activities were expressed as a percent of the procoagulant activity in a plasma pool of adult wild type mice. Plasma fibrinogen was determined by a coagulation rate assay (Vermylen et al., 1963). For hematological analysis, blood was collected in 0.01 volume 4% trisodium citrate, and cells were counted using an automated analyzer (Cell-Dyn 610U-Hematology Analyzer; Sequioa-Turner Co, Mountain View, CA). Cell counts are expressed per millilitres of whole blood. In order to obtain mouse capillary endothelial cells, anaesthetized mice were injected s.c. with 500 μl of ice-cold matrigel containing VEGF (100 ng/ml) and heparin (300 μg/ml). After 7 days, the pellet containing neovessels was dissected and enzymatically dispersed using 0.1% type II collagenase (Sigma, St Louis, Mo). Mouse endothelial cells were routinely cultured in T75 flasks coated with 0.1% gelatin in M131 medium supplemented with 5% MVGS (ref S005025; Gibco-BRL). For passage, cells were detached by trypsin/EDTA (0.02-0.05%). For TNF-α stimulation, endothelial cells were refed with fresh culture medium containing 100 ng/ml TNF-α (R&D, Abingdon, UK). After 24 hr, the conditioned medium was harvested and analyzed for the following cytokines: IL-1 β and IL-6 (Quantikine™ M, R&D, Abindon, UK); ELAM, ICAM, VCAM and tissue factor (all from R&D). Recombinant Gas6, produced in 293 monkey kidney cells stably transfected with the pcDNA3 expressing vector encoding the mouse Gas6 cDNA (Grinnell et al., 1990), was supplemented at 200 ng/ml during the experiment as indicated. For the determination of cytokine concentration after endotoxin injection, adult mice were intraperitoneally injected with endotoxin (20 mg/kg) and after 90 or 180 min, the mice were anesthetized by intraperitoneal injection of 60 mg kg sodium pentobarbital. Blood was collected in citrate by vena cava puncture with a 24-gauge needle. Following centrifugation at 3000 rpm for 10 min, plasma was collected and stored at -20° C until analysis. Cytokines were measured using Quantikine™ M assays for IL-1 β, IL-6, IL-10 and TNF-α according to the manufacturer's instructions (all from R&D, Abindon, UK). For apoptosis studies, mouse endothelial cells were cultured in T75 flasks coated with 0.1 % gelatine in RPMI 1640 medium containing 10% foetal calf serum (Life Technologies, Paisley, UK), 100 IU penicillin, 100 μg/ml streptomycin, 2 mM glutamine, heparin (100 μg/ml) and endothelial cell growth supplement (30 μg/ml). Apoptosis was induced by (i) supplementation of TNF-α (100 ng/ml; R&D), Fas- ligand (100 ng/ml; R&D), or withdrawal from growth factors (0.1 % foetal calf serum). Apoptosis was quantified by measuring cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) using a photometric enzyme immunoassay (Cell Detection ELISA, Boehringer Mannheim, Mannheim, Germany). Protection against endothelial activation was concluded based on the following criteria: - Intravenous injection of endotoxin in wild type mice induced strong activation of the coagulation cascade, resulting in reduced levels of factor II, factor V, factor VIII, factor X, and fibrinogen (Table 1). In addition, platelet counts were reduced after endotoxin, indicating formation of platelet-rich thrombi (Table 1 ). In contrast, endotoxin completely failed to activate coagulation and consume platelets in Gas6 deficient mice (Table 1 ), despite comparable increase in plasma levels of IL-6, IL- 1 β, IL-10 and TNF-α (Table 2). Upon local injection of endotoxin (50μg) in the footpad, significantly fewer veins developed occluding thrombosis in Gas6 v" than in Gasδ 47"1" mice (Table 3). When cultured endothelial cells from wild type mice were treated with TNF-α, they expressed increased levels of tissue factor (the initiator of blood coagulation) (Table 4). In contrast, tissue factor could not be induced by TNF-α in Gas6 deficient endothelial cells. - Intravenous injection of endotoxin (10 to 40 mg/kg) in wild type mice reduced circulating numbers of leukocytes, which extravasated in peripheral tissues such as the lung, kidney and heart (Table 1 ). In contrast, leukocyte numbers in the peripheral blood remained high in Gas6 deficient mice, and leukocytes failed to extravasate. In contrast to cultured wild type endothelial cells, Gas6 deficient endothelial cells failed to express upregulated levels of VCAM, ICAM, and ELAM in response to TNF-α. Further, secretion of IL-1 β and IL-6 was abrogated in Gas6 deficient cells (Table 4). Upon local injection of endotoxin (50 μg) in the footpad significantly fewer leukocytes adhered to the arterial endothelial cells in Gasδ " " than in Gas6 +/+ mice (Table 3) - Anti-Fas antibodies induced severe apoptotic liver damage leading to fulminant hepatitits, liver haemorrhage, and death in wild type mice, whereas Gas6 deficient mice were almost completely protected (number of TUNEL positive cells per optical field: 28 ± 7 for Gas6 +/+ versus 8 ± 1 for Gase 7" ). Gas6 deficient endothelial cells, unlike wild type cells, are also protected against apoptosis, induced by a cocktail of cytokines (Table 5). Upon local injection of endotoxin (50 μg) in the footpad, significantly fewer apoptotic (TUNEL-positive) endothelial cells were present in Gas6 " ' ' than in Gas6 + + mice (Table 3) Collectively, these data unveil a novel role for Gasδ. Gas6 plays a role in activation of endothelial cells and subsequent binding and extravasation of leukocytes in response to various inflammatory or noxious stimuli. These data illustrate the role of Gas6 in mediating adhesion between activated endothelial and leukocytes. Example 2: in vivo role of Gas6 in arterial stenosis A mouse model of arterial stenosis based on ligation is used. Previous studies have indicated that following ligation, the endothelium becomes activated, resulting in adhesion of leukocytes to the endothelium, with subsequent infiltration in the intima and media (Kockx et a/., 1993). Due to leukocyte production of growth factors and cytokines, medial smooth muscle cells become activated, proliferate and migrate backwards into the intima, thereby forming a neointima, rich in smooth muscle cells. Wild type and Gas6 "Λ mice were subjected to ligation of the right common carotid artery, just proximal to the bifurcation, with silk 4/0. Two weeks after ligation, the luminal stenosis, expressed in percent of the area within the internal elastic lamina was 37 ± 5.8% in wild type and 18 ± 3.5% in Gas6 "Λ mice (mean ± SEM, n= 8, p= 0.012). From the results, it is clear that Gas6 deficient mice develop less arterial intimal thickening. Example 3: in vivo role of Gas6 in stroke A growing body of evidence indicates that inflammatory mechanisms contribute to secondary neuronal injury after acute cerebral ischemia (DeGabra, 1998; Hallenbeck, 1997). Ischemia followed by reperfusion rapidly leads to the expression of inflammatory cytokines, particularly TNF-α and I L- 1 β , which stimulate a complex cascade of events involving endothelial cells, neurons, astrocytes, and perivascular cells. A secondary response involves activation of the coagulation system and upregulation of cell adhesion molecules. The net effect of these events is transformation of the local endothelium to a prothrombotic/proinfiammatory state and induction of leukocyte migration to the site of injury within hours of reperfusion. Leukocytes cause tissue injury by several mechanisms, including occlusion of microvasculature, generation of oxygen free radicals, release of cytotoxic enzymes, alteration of vasomotor reactivity, and increase in cytokine and chemoattractant release. A mouse model of stroke, based on ligation of the middle cerebral artery, was developed to study the role of plasminogen system proteinases in vivo (Nagai et al., 1999) and was used to the study the role of Gas6. Ischemic brain injury was achieved by permanently ligating and sectioning the middle cerebral artery. Infarct size was morphometrically measured after TTC staining of 1 mm thick brain slices within 24 hrs after ligation as described by Nagai et al. (1999). Ischemic brain injury was significantly reduced in Gas6 'Λ as compared to Gas6 + mice after ligation of the middle cerebral artery (Table 6). Example 4: in vivo role of Gas6 in angiogenesis Unlike their active growth and remodeling during embryonic development, endothelial cells in the adult are quiescent, and need to become activated when new blood vessels are formed. Over the last decade, remarkable progress has been made in the identification of a variety of molecules that stimulate or inhibit angiogenesis in vivo. Angiogenesis inhibitors have attained significant interest for the treatment of cancer, inflammatory disorders and diabetic retinopathy, as endothelial cells that invade tumors are non-tumoral host-derived cells, that would not become resistant to chemotherapy (Folkman, 1998). Ingrowth of capillaries in matrigel was performed as described by Passanti et al. (1992). Briefly, 500 μl ice-cold matrigel containing heparin (300 μg/ml) and rVEGFies (100ng/ml) was injected subcutaneaously into the groin. After 7 days, the matrigel pellet with the neovessels was dissected for analysis of neovascularisation: one part was homogenized to determine the hemoglobin content determined using Drabkin's reagent (Sigma, St. Quentin Fallavier, France), whereas the other part was fixed in 1 % paraformaldehyde for histological analysis. Subcutaneous injection of matrigel containing VEGF induced a strong angiogenic ingrowth of capillaries in wild type mice, while this angiogenic response was inhibited for more than 80% in Gas6 deficient mice (Table 7). The significant suppression of angiogenesis in Gas6 deficient mice suggests that Gas6, somehow, is required for endothelial activation during angiogenesis, and that Gas6 antagonists may be useful angiogenesis inhibitors. References Ahmed, N. & Christou, N. Systemic inflammatory response syndrome: interactions between immune cells and the endothelium. Shock 6, S39-42 (1996). Allen, M.P. et al. Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signaling promotes gonadotropin-releasing hormone neuronal survival via extracellular signal-regulated kinase (ERK) and Akt [In Process Citation]. Mol Endocrinol 13, 191 -201 (1999). Avanzi, G.C. et al. GAS6, the ligand of Axl and Rse receptors, is expressed in hematopoietic tissue but lacks mitogenic activity. Exp Hematol 25, 1219-1226 (1997). Avanzi, G.C. et al. GAS6 inhibits granulocyte adhesion to endothelial cells. Blood 91 , 2334-2340 (1998). Bajou, K. et al. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med *, 923-928 (1998). Beliosta, P., Costa, M., Lin, D.A. & Basilico, C. The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding. Mol Cell Biol 15, 614-625 (1995). Beliosta, P., Zhang, Q., Goff, S.P. & Basilico, C. Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation. Oncogene 15, 2387-2397 (1997). Boyle, E.M., Jr., Lille, ST., Allaire, E., Clowes, A.W. & Verrier, E.D. Endothelial cell injury in cardiovascular surgery: atherosclerosis. Ann Thorac Surg 63, 885-894 (1997). Carmeliet, P., Moons, L., Ploplis, V., Plow, E. & Collen, D. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest 99, 200-208 (1997a). Carmeliet, P. et al. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice [see comments]. Circulation 96, 3180-3191 (1997b). Carmeliet, P. et al. Urokinase-type but not tissue-type plasminogen activator mediates arterial neointima formation in mice. Circ Res 81 , 829-839 (1997c). Carmeliet, P. et al. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet 17, 439-444 (1997d). Carmeliet, P., Stassen, J.M., Meidell, R., Collen, D. & Gerard, R. Adenovirus- mediated gene transfer of rt-PA restores thrombolysis in t-PA deficient mice. Blood 90, 1527-1534 (1997e) Carmeliet, P. & Collen, D. Vascular development and disorders: molecular analysis and pathogenetic insights. Kidney Int 53, 1519-1549 (1998). Carmeliet, P. et al. Receptor-independent role of urokinase-type plasminogen activator in arterial wound healing and intima formation in mice. J Cell Biol 140, 233-245 (1998a). Carmeliet, P. et al. Role of HIF-1 alpha in hypoxia-mediated apoptosis, cell proliferation, and tumor angiogenesis. Nature 394, 485-490 (1998b). Cavanagh, S.P., Gough, M.J. & Homer-Vanniasinkam, S. The role of the neutrophil in ischaemia-reperfusion injury: potential therapeutic interventions. Cardiovasc Surg β, 112-118 (1998). Cosentino, F. & Luscher, T.F. Endothelial dysfunction in diabetes mellitus [In Process Citation]. J Cardiovasc Pharmacol 32, S54-61 (1998). Costa, M., Beliosta, P. & Basilico, C. Cleavage and release of a soluble form of the receptor tyrosine kinase ARK in vitro and in vivo. J Cell Physiol 168, 737-744 (1996). Cotran, R.S. & Mayadas-Norton, T. Endothelial adhesion molecules in health and disease. Pathol Biol (Paris) 46, 164-170 (1998). Couffinhal, T. et al. Mouse model of angiogenesis. Am J Pathol 152, 1667-1679 (1998). Crosier, K.E. & Crosier, P.S. New insights into the control of cell growth; the role of the Axl family. Pathology 29, 131-135 (1997). DeGraba, T.J. The role of inflammation after acute stroke: utility of pursuing anti- adhesion molecule therapy. Neurology 5Λ , S62-68 (1998). Dinerman, J.L. & Mehta, J.L. Endothelial, platelet and leukocyte interactions in ischemic heart disease: insights into potential mechanisms and their clinical relevance [see comments]. J Am Coll Cardiol 16, 207-222 (1990). Dong, Z.M. & Wagner, D.D. Leukocyte-endothelium adhesion molecules in atherosclerosis. J Lab Clin Med 132, 369-375 (1998). Fernandes, P. B. (1998). Technological advances in high-throughput screening. Curr Opin Chem Biol 2, 597-603 Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1 , 27-31 (1995). Folkman, J. Antiangiogenic gene therapy. Proc Nat! Acad Sci U S A 95, 9064-9066 (1998). Fridell, Y.W., Villa, J., Jr., Attar, E.C. & Liu, E.T. GAS6 induces Axl-mediated chemotaxis of vascular smooth muscle cells. J Biol Chem 273, 7123-7126 (1998). Frenette, P.S. & Wagner, D.D. Insights into selectin function from knockout mice. Thromb Haemost 78, 60-64 (1997). Golino, P. et al. Inhibition of leucocyte and platelet adhesion reduces neointimal hyperplasia after arterial injury. Thromb Haemost 77, 783-788 (1997). Goruppi, S., Ruaro, E. & Schneider, C. Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene 12, 471-480 (1996). Goruppi, S., Ruaro, E., Varnum, B. & Schneider, C. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol 17, 4442- 4453 (1997). Griendling, K.K. & Alexander, R.W. Endothelial control of the cardiovascular system: recent advances. Faseb J 10, 283-292 (1996). Hallenbeck, J.M. Cytokines, macrophages, and leukocytes in brain ischemia. Neurology 49, S5-9 (1997). Ikeda, U., Takahashi, M. & Shimada, K. Monocyte-endothelial cell interaction in atherogenesis and thrombosis. Clin Cardiol lΛ , 11 -14 (1998). Kenny, B. A., Bushfield, M., Parry-Smith, D. J., Fogarty, S., and Treherne, J. M. (1998). The application of high-throughput screening to novel lead discovery. Prog Drug Res 51, 245-69. Kockx, M.M. et al. The endothelium during cuff-induced neointima formation in the rabbit carotid artery. Arterioscler Thromb 13, 1874-1884 (1993). Li, R. et al. Identification of Gas6 as a growth factor for human Schwann cells. J Neurosci ΛQ, 2012-2019 (1996). Libby, P., Schwartz, D., Brogi, E., Tanaka, H. & Clinton, S.K. A cascade model for restenosis. A special case of atherosclerosis progression. Circulation 86, III47- 52 (1992). Libby, P., Sukhova, G., Lee, R.T. & Liao, J.K. Molecular biology of atherosclerosis. Int J Cardiol 62 Suppl 2, S23-29 (1997). Lusher, T.F., Tanner, F.C., Tschudi, M.R. & Noll, G. Endothelial dysfunction in coronary artery disease. Annu Rev Med 44, 395-418 (1993). Manfioletti, G., Brancolini, C, Avanzi, G. & Schneider, C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K- dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 13, 4976-4985 (1993). McCloskey, P. et al. GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase Axl. J Biol Chem 272, 23285-23291 (1997). McGill, S.N., Ahmed, N.A. & Christou, N.V. Endothelial cells: role in infection and inflammation. World J Surg 22, 171 -178 (1998). Moons, L. et al. Reduced transplant arteriosclerosis in plasminogen deficient mice. J Clin Invest 102, 1788-1797 (1998). Murohara, T., Guo, J.P. & Lefer, A.M. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury. J Pharmacol Exp Ther 274, 1246-1253 (1995). Nagai, N., De Mol, M., Lijnen, H.R., Carmeliet, P. & Collen, D. Role of plasminogen system components in focal cerebral ischemic infarction. A gene targeting and gene transfer study in mice. Circulation (in press)(1999). Nagata, K. et al. Identification of the product of growth arrest-specific gene 6 as a common ligand for Axl, Sky, and Mer receptor tyrosine kinases. J Biol Chem 271 , 30022-30027 (1996). Nakano, T. et al. Requirement of gamma-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells. Biochem J 323, 387-392 (1997a). Nakano, T. et al. Cell adhesion to phosphatidylserine mediated by a product of growth arrest-specific gene 6. J Biol Chem 272, 29411-29414 (1997b). Pajkrt, D. & van Deventer, S.J. The cellular response in sepsis. Curr Top Microbiol Immunol 216, 119-132 (1996). Ross, R. Atherosclerosis--an inflammatory disease. N Engl J Med 340, 115-126 (1999). Schaper, W. & Ito, W.D. Molecular mechanisms of coronary collateral vessel growth. Circ Res 79, 911-919 (1996). Schneider, C, King, R.M. & Philipson, L. Genes specifically expressed at growth arrest of mammalian cells. Ce//54, 787-793 (1988). Stitt, T.N. et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 80, 661-670 (1995). Vadas, M.A., Gamble, J.R., Rye, K. & Barter, P. Regulation of leucocyte-endothelial interactions of special relevance to atherogenesis. Clin Exp Pharmacol Physiol 24, A33-35 (1997).. Tables Table 1 : Coagulation and hematological parameters after endotoxin injection (20 mg/kg). Data represent mean ± SD (5 mice) of plasma concentrations of coagulation factors (expresses in % of normal pool), platelets and leukocytes in mice within 12 h after injection of endotoxin (0-10 and 20 mg/kg). Table 2: Serum cytokine concentration after endotoxin injection (20 mg/kg). Data represent mean ± SD (7 mice) of cytokine serum concentrations in mice after injection of endotoxin (20 mg/kg). Table 3: Endotoxin injection in the footpad All values in Gas6 * mice are significantly different from those in Gasδ 4 ^ mice (p<0.05) Table 4: Activation of endothelial cells in vitro. Data represent mean ± SD (9) of cytokine concentrations (ng/10 cells). Table 5: Apoptosis of cultured endothelial cells Data represent mean ± SD (n=9) of oligonucleosomes/10 cells. Table 6: Ischemic brain injury (stroke) All values in Gas6 "A mice are significantly different from those in Gas6 ,+/+ mice (p<0.05) Table 7: Matrigel Angiogenesis p<0.05 versus Gas6 +/+

Claims: Claims 1. Non-human transgenic animal deficient in Gas6 function. 2. Non-human transgenic animal according to claim 1 in which said animal is Gas6 "A . 3. Non-human transgenic animal according to claim 1 or 2 in which said animal is a mouse. 4. Use of an animal according to claim 1 -3 to study the role of Gas6 in endothelial activation. 5. Use of an inhibitor of the Gas6 function and/or an inhibitor of a Gas6 receptor for the manufacture of a medicament for the treatment of endothelial dysfunction. 6. Use according to claim 5 in which endothelial dysfunction results in a cardiovascular disorder such as, but not limited to, stroke, arterial restenosis, myocardial infarction. 7. Use according to claim 5 in which endothelial dysfunction results in abnormal angiogenesis. 8. Use according to claim 5-7 in which the Gas6 receptor is AXL, MER and/or RSE. 9. Use according to claim 8 in which the inhibitor is a soluble form of the AXL, MER or RSE receptor. 10_Use according to claim 5-8 in which the inhibitor is a Gas6 neutralising antibody.

Inventors: APARICIO CRISTINA, CARMELIET PETER, COLLEN DESIRE, DAHLBACK BJORN, GARCIA DE FRUTOS PABLO, APARICIO, CRISTINA, CARMELIET, PETER, COLLEN, DESIRE, DAHLBACK, BJORN, GARCIA DE FRUTOS, PABLO
Applicants: APARICIO CRISTINA, CARMELIET PETER, COLLEN DESIRE, DAHLBACK BJORN, GARCIA DE FRUTOS PABLO, VLAAMS INTERUNIV INST BIOTECH, APARICIO, CRISTINA, CARMELIET, PETER, COLLEN, DESIRE, DAHLBACK, BJORN, GARCIA DE FRUTOS, PABLO, VLAAMS INTERUNIVERSITAIR INSTITUUT VOOR BIOTECHNOLOGIE VZW
Application year: 2000
Main classification: A01K67
IPC codes: C12N15