Early in vivo anticoagulation inhibits the angiogenic response following hindlimb ischemia in a rodent model

Summary

Emerging findings have demonstrated the critical role of blood clotting factors in the formation and stabilization of embryonic blood vessels. Whether a similar role is true during post-natal angiogenesis remains to be determined. Here we sought to determine whether the suppression of thrombin generation with anticoagulant drugs at doses routinely used for therapeutic purposes would affect the angiogenesis pattern following hindlimb ischemia in rats. Animals were treated with r- hirudin or enoxaparin within six hours post induction of hindlimb ischemia, whereas two other groups received oral anticoagulation warfarin beginning at day 3 post-ischemia or saline (as control). The revascularization anatomical and functional responses were evaluated 30 days following tissue ischemia. Chronic administration of the drugs resulted in stable anticoagulation in all animals throughout the experiment. Animals that received drugs with fast anticoagulation effects (i.e. r-hirudin and enoxaparin) presented a significant decrease in capillary density and capillary-to-myocyte ratio compared to control animals. These effects were not associated with changes in relative perfusion of the hindlimb at steady state. These anti-angiogenic effects occur in a time-dependent manner, since delayed inhibition of coagulation (> 72 hours) presents no adverse effect on the angiogenic response. We conclude that the use of anticoagulant drugs immediately after tissue ischemia induction hampers in vivo angiogenic response in a rodent hindlimb ischemia model.

Keywords

Angiogenesis - warfarin - hirudin - heparin - anticoagulant

• References

• 1 Hughes GC, Annex BH. Angiogenic therapy for coronary artery and peripheral arterial disease. Expert Rev Cardiovasc Ther 2005; 03: 521-35.
• 2 Sun WY. et al. Prothrombin deficiency results in embryonic and neonatal lethality in mice. Proc Natl Acad Sci USA 1998; 95: 7597-602.
• 3 Cui J. et al. Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature 1996; 384: 66-8.
• 4 Toomey JR. et al. Targeted disruption of the murine tissue factor gene results in embryonic lethality. Blood 1996; 88: 1583-7.
• 5 Bugge TH. et al. Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation. Proc Natl Acad Sci USA 1996; 93: 6258-63.
• 6 Griffin CT. et al. A role for thrombin receptor signaling in endothelial cells during embryonic development. Science 2001; 293: 1666-70.
• 7 Rickles FR. et al. Tissue factor, thrombin, and cancer. Chest 2003; 124: 58S-68S.
• 8 Wang Y. et al. Vitamin k epoxide reductase: a protein involved in angiogenesis. Mol Cancer Res 2005; 03: 317-23.
• 9 Takeshita S. et al. Endothelium-dependent relaxation of collateral microvessels after intramuscular gene transfer of vascular endothelial growth factor in a rat model of hindlimb ischemia. Circulation 1998; 98: 1261-3.
• 10 Paek R. et al. Correlation of a simple direct measurement of muscle pO(2) to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia. J Vasc Surg 2002; 36: 172-9.
• 11 Takeshita S. et al. Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation 1994; 90: II228-34.
• 12 Mack CA. et al. Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion. J Vasc Surg 1998; 27: 699-709.
• 13 Ziada AM. et al. The effect of long-term vasodilatation on capillary growth and performance in rabbit heart and skeletal muscle. Cardiovasc Res 1984; 18: 724-32.
• 14 Rissanen TT. et al. Blood flow remodels growing vasculature during vascular endothelial growth factor gene therapy and determines between capillary arterialization and sprouting angiogenesis. Circulation 2005; 112: 3937-46.
• 15 Hudlicka O. et al. The effect of chronic skeletal muscle stimulation on capillary growth in the rat: are sensory nerve fibres involved?. J Physiol 2003; 546: 813-22.
• 16 Garcia JG. et al. Vascular endothelial cell activation and permeability responses to thrombin. Blood Coagul Fibrinolysis 1995; 06: 609-26.
• 17 Galis ZS. et al. Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1997; 17: 483-9.
• 18 Zucker S. et al. Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 1998; 75: 780-6.
• 19 Huang YQ. et al. Thrombin induces increased expression and secretion of VEGF from human FS4 fibroblasts, DU145 prostate cells and CHRF megakaryocytes. Thromb Haemost 2001; 86: 1094-8.
• 20 Huang YQ. et al. Thrombin induces increased expression and secretion of angiopoietin-2 from human umbilical vein endothelial cells. Blood 2002; 99: 1646-50.
• 21 Tsopanoglou NE, Maragoudakis ME. On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 1999; 274: 23969-76.
• 22 Senger DR. et al. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin. Am J Pathol 1996; 149: 293-305.
• 23 Nagy JA. et al. Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta 1989; 948: 305-26.
• 24 Lee CW. et al. Temporal patterns of gene expression after acute hindlimb ischemia in mice: insights into the genomic program for collateral vessel development. J Am Coll Cardiol 2004; 43: 474-82.
• 25 Tsopanoglou NE. et al. Thrombin promotes angiogenesis by a mechanism independent of fibrin formation. Am J Physiol 1993; 264: C1302-7.
• 26 Collen A. et al. Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro . Cancer Res 2000; 60: 6196-200.
• 27 Mousa SA, Mohamed S. Inhibition of endothelial cell tube formation by the low molecular weight heparin, tinzaparin, is mediated by tissue factor pathway in-hibitor. Thromb Haemost 2004; 92: 627-33.
• 28 Scholz D. et al. Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice. J Mol Cell Cardiol 2002; 34: 775-87.
• 29 Brevetti LS. et al. Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia. J Surg Res 2001; 98: 21-6.
• 30 Walder CE. et al. Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia. J Cardiovasc Pharmacol 1996; 27: 91-8.