Constriction of carotid arteries by urokinase-type plasminogen activator requires catalytic activity and is independent of NH₂-terminal domains

Summary

Urokinase-type plasminogen activator (uPA) is expressed at increased levels in stenotic, atherosclerotic human arteries. However, the biological roles of uPA in the artery wall are poorly understood. Previous studies associate uPA with both acute vasoconstriction and chronic vascular remodeling and attribute uPA-mediated vasoconstriction to the kringle – not the catalytic domain of uPA. We used an in-vivo uPA overexpression model to test the hypothesis that uPA-induced vasoconstriction is a reversible vasomotor process that can be prevented – and uPA fibrinolytic activity preserved – by: 1) removing the growth factor and kringle domains; or 2) anchoring uPA to the endothelial surface. To test this hypothesis we constructed adenoviral vectors that express: wild-type rabbit uPA (AduPA); a uPA mutant lacking the NH₂-terminal growth-factor and kringle domains (Adu-PAdel); a mutant lacking catalytic activity (AduPAS→A), and a cell-surface anchored mutant (AdTMuPA). uPA mutants were expressed and characterised in vitro and in carotid arteries in vivo. uPAS→A had no plasminogen activator activity. Activity was similar for uPA and uPAdel, whereas AdTMuPA had only cell-associated activity. AduPAS→A arteries were not constricted. AduPA, AduPAdel, and AdTM-uPA arteries were constricted (approximately 30% smaller lumens; p≤0.008 vs. AdNull arteries). Papaverine reversed constriction of AduPA arteries. uPA-mediated arterial constriction is a vasomotor process that is mediated by uPA catalytic activity, not by the NH₂-terminal domains. Anchoring uPA to the endothelial surface does not prevent vasoconstriction. uPA catalytic activity, generated by artery wall cells, may contribute to lumen loss in human arteries. Elimination of uPA vasoconstrictor activity requires concomitant loss of fibrinolytic activity.

Keywords

Gene therapy - gene transfer - vascular remodelling - endothelial cells - proteases

• References

• 1 Padró T, Emeis JJ, Steins M. et al. Quantification of plasminogen activators and their inhibitors in the aortic vessel wall in relation to the presence and severity of atherosclerotic disease. Arterioscler Thromb Vasc Biol 1995; 15: 893-902.
• 2 Kienast J, Padro T, Steins M. et al. Relation of urokinase-type plasminogen activator expression to presence and severity of atherosclerotic lesions in human coronary arteries. Thromb Haemost 1998; 79: 579-586.
• 3 Bachmann F. Plasminogen-Plasmin Enzyme System. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Fourth ed. Lippincott, Williams, and Wilkins, Philadelphia; 2001: 275-320.
• 4 Cohen AT, Agnelli G, Anderson FA. et al. Venous thromboembolism (VTE) in Europe. The number of VTE events and associated morbidity and mortality. Thromb Haemost 2007; 98: 756-764.
• 5 Verso M, Agnelli G. Prophylaxis of upper limb deep vein thrombosis in cancer patients with central vein catheter. Thromb Haemost 2008; 100: 167-168.
• 6 Zreiqat H, James B, Brieger D. et al. Acute coronary stent thrombosis: toward insights into possible mechanism using novel imaging methods. Thromb Haemost 2008; 99: 976-977.
• 7 Haj-Yehia A, Nassar T, Sachais BS. et al. Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo . FASEB J 2000; 14: 1411-1422.
• 8 Falkenberg M, Tom C, DeYoung MB. et al. Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth. Proc Natl Acad Sci USA 2002; 99: 10665-10670.
• 9 Plekhanova O, Berk BC, Bashtrykov P. et al. Oligonucleotide microarrays reveal regulated genes related to inward arterial remodeling induced by urokinase plasminogen activator. J Vasc Res 2008; 46: 177-187.
• 10 Nassar T, Haj-Yehia A, Akkawi S. et al. Binding of urokinase to low density lipoprotein-related receptor (LRP) regulates vascular smooth muscle cell contraction. J Biol Chem 2002; 277: 40499-40504.
• 11 Adams DS, Griffin LA, Nachajko WR. et al. A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor. J Biol Chem 1991; 266: 8476-8482.
• 12 Dichek DA, Anderson J, Kelly AB. et al. Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors. Circulation 1996; 93: 301-309.
• 13 Lijnen HR, Nelles L, Holmes WE. et al. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-5598.
• 14 Stump DC, Stassen JM, Demarsin E. et al. Comparative thrombolytic properties of single-chain forms of urokinase-type plasminogen activator. Blood 1987; 69: 592-596.
• 15 Mostov KE, Friedlander M, Blobel G. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 1984; 308: 37-43.
• 16 Reich V, Mostov K, Aroeti B. The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains. J Cell Sci 1996; 109: 2133-2139.
• 17 Vines DJ, Lee SW, Dichek DA. et al. Receptor-mediated regulation of plasminogen activator function: plasminogen activation by two directly membrane-anchored forms of urokinase. J Pept Sci 2000; 06: 432-439.
• 18 Wen S, Schneider DB, Driscoll RM. et al. Secondgeneration adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the arterial wall. Arterioscler Thromb Vasc Biol 2000; 20: 1452-1458.
• 19 Bett AJ, Haddara W, Prevec L. et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA 1994; 91: 8802-8806.
• 20 Ginsberg HS, Lundholm-Beauchamp U, Horswood RL. et al. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci USA 1989; 86: 3823-3827.
• 21 Lee SW, Trapnell BC, Rade JJ. et al. In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries. Circ Res 1993; 73: 797-807.
• 22 Dichek DA, Lee SW, Nguyen NH. Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors. Blood 1994; 84: 504-516.
• 23 Estreicher A, Wohlwend A, Belin D. et al. Characterization of the cellular binding site for the urokinase-type plasminogen activator. J Biol Chem 1989; 264: 1180-1189.
• 24 Schneider DB, Vassalli G, Wen S. et al. Expression of Fas ligand in arteries of hypercholesterolemic rabbits accelerates atherosclerotic lesion formation. Arterioscler Thromb Vasc Biol 2000; 20: 298-308.
• 25 Lee SW, Kahn ML, Dichek DA. Expression of an anchored urokinase in the apical endothelial cell membrane. Preservation of enzymatic activity and enhancement of cell surface plasminogen activation. J Biol Chem 1992; 267: 13020-13027.
• 26 Langille BL, Bendeck MP, Keeley FW. Adaptations of carotid arteries of young and mature rabbits to reduced carotid blood flow. Am J Physiol 1989; 256: H931-939.
• 27 Luttrell IP, Swee M, Starcher B. et al. Erectile dysfunction in the type II diabetic db/db mouse: impaired venoocclusion with altered cavernosal vasoreactivity and matrix. Am J Physiol Heart Circ Physiol 2008; 294: H2204-2211.
• 28 Ellis V, Behrendt N, Danø K. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor. J Biol Chem 1991; 266: 12752-12758.
• 29 Zeiher AM, Drexler H, Wollschlager H. et al. Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 1991; 83: 391-401.
• 30 Drew AF, Tucker HL, Kombrinck KW. et al. Plasminogen is a critical determinant of vascular remodeling in mice. Circ Res 2000; 87: 133-139.
• 31 Levi M, Moons L, Bouche A. et al. Deficiency of urokinase-type plasminogen activator-mediated plasmin generation impairs vascular remodeling during hypoxia-induced pulmonary hypertension in mice. Circulation 2001; 103: 2014-2020.
• 32 Lafont A, Guzman LA, Whitlow PL. et al. Restenosis after experimental angioplasty. Intimal, medial, and adventitial changes associated with constrictive remodeling. Circ Res 1995; 76: 996-1002.
• 33 Gulba DC, Bode C, Runge MS. et al. Thrombolytic agents--an overview. Ann Hematol 1996; 73: S9-S27.
• 34 Early effects of tissue-type plasminogen activator added to conventional therapy on the culprit coronary lesion in patients presenting with ischemic cardiac pain at rest. Results of the Thrombolysis in Myocardial Ischemia (TIMI IIIA) Trial. Circulation 1993; 87: 38-52.
• 35 Gorich J, Rilinger N, Sokiranski R. et al. Mechanical thrombolysis of acute occlusion of both the superficial and the deep femoral arteries using a thrombectomy device. AJR Am J Roentgenol 1998; 170: 1177-1180.
• 36 Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983; 49: 208-215.
• 37 Clowes AW, Clowes MM, Au YPT. et al. Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery. Circ Res 1990; 67: 61-67.
• 38 Backstrom JR, Tokes ZA. The 84-kDa form of human matrix metalloproteinase-9 degrades substance P and gelatin. J Neurochem 1995; 64: 1312-1318.
• 39 Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res 1999; 85: 906-911.
• 40 Fernandez-Patron C, Stewart KG, Zhang Y. et al. Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res 2000; 87: 670-676.
• 41 Hirano K. The roles of proteinase-activated receptors in the vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2007; 27: 27-36.
• 42 Akkawi S, Nassar T, Tarshis M. et al. LRP and alphavbeta3 mediate tPA activation of smooth muscle cells. Am J Physiol Heart Circ Physiol 2006; 291: H1351-1359.
• 43 Dichek DA, Neville RF, Zwiebel JA. et al. Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 1989; 80: 1347-1353.
• 44 Santo SD, Tepper OM, von Ballmoos MW. et al. Cell-based therapy facilitates venous thrombus resolution. Thromb Haemost 2009; 101: 460-464.