Vascular endothelial growth factor enhances venous thrombus recanalisation and organisation

Summary

Vascular endothelial growth factor (VEGF) is a regulator of physiological and pathological angiogenesis and is found in naturally resolving experimental venous thrombi, where it may also regulate recanalisation. In this study VEGF protein was injected into venous thrombi to determine if this enhanced recanalisation and organisation. A rat model of inferior vena cava (IVC) thrombosis was used. Thrombi were formed in 3 groups (n = 3 per group). 10 μl ¹²⁵I-VEGF was directly injected into thrombus thirty minutes after induction. Three hours, 1 day and 6 days later thrombus, IVC, and other tissues were harvested. ¹²⁵I-VEGF was mostly distributed in the thrombus and the IVC, with smaller amounts in other tissues. Thrombi were formed in a further 4 groups (n = 6 per group).Thirty minutes after induction control solution or 1 ng, 10 ng or 100 ng recombinant human VEGF₁₆₅ was injected directly into the thrombus. Lumen recanalisation, thrombus organisation and monocyte content were measured on digitised sections by image analysis. In animals treated with 10 ng of VEGF there was a greater area of lumen recanalisation [mean 5492 pixels, standard error of mean (sem) 922] compared to controls (mean 2974, sem 385) (P = 0.005). There was a significant increase in the organisation score in all treated animals (1 ng: mean 70, sem 1.7, P = 0.0025; 10 ng: mean 70, sem 2.0, P = 0.0042; 100 ng: mean 72, sem 1.9, P = 0.0003) compared to controls (mean 63, sem 1.7). The monocyte content was lower in the animals treated with 1 ng VEGF (mean 3.8% of thrombus area, sem 0.3%) compared to controls (mean 5.5%, sem 0.4%) (P = 0.0008).The proportion of monocytes migrating to the centre of the thrombus increased in a dose-related manner. VEGF may prove to be of use in the treatment of venous thrombosis.

Keywords

Thrombus resolution - angiogenesis - VEGF - recanalisation

• References

• 1 Payling Wright H, Kubik MM, Hayden M. Influence of anticoagulant administration on the rate of recanalisation of experimentally thrombosed veins. Br J Surg 1952; 40: 163-6.
• 2 Killewich LA, Bedford GR, Beach KW, Strandness Jr DE. Spontaneous lysis of deep venous thrombi: rate and outcome. J Vasc Surg 1989; 9: 89-97.
• 3 van Ramshorst B, van Bemmelen PS, Hoene-veld H, Faber JA, Eikelboom BC. Thrombus regression in deep venous thrombosis. Quantification of spontaneous thrombolysis with duplex scanning. Circulation 1992; 86: 414-9.
• 4 Franzeck UK, Schalch I, Bollinger A. On the relationship between changes in the deep veins evaluated by duplex sonography and the postthrombotic syndrome 12 years after deep vein thrombosis. Thromb Haemost 1997; 77: 1109-12.
• 5 Meissner MH, Manzo RA, Bergelin RO, Markel A, Strandness Jr DE. Deep venous insufficiency: the relationship between lysis and subsequent reflux. J Vasc Surg 1993; 18: 596-605.
• 6 O’Shaughnessy AM, Fitzgerald DE. Natural history of proximal deep vein thrombosis assessed by duplex ultrasound. Int Angiol 1997; 16: 45-9.
• 7 Meissner MH, Caps MT, Zierler BK, Polissar N, Bergelin RO, Manzo RA, Strandness Jr DE. Determinants of chronic venous disease after acute deep venous thrombosis. J Vasc Surg 1998; 28: 826-33.
• 8 Wakefield TW. Treatment options for venous thrombosis. [Review]. J Vasc Surg 2000; 31: 613-20.
• 9 Armon MP, Hopkinson BR. Thrombolysis for acute deep vein thrombosis. Br J Surg 1996; 83: 580-1.
• 10 Wakefield TW, Linn MJ, Henke PK, Kadell AM, Wilke CA, Wrobleski SK, Sarkar M, Burdick MD, Myers DD, Strieter RM. Neovascularization during venous thrombosis organization: a preliminary study. J Vasc Surg 1999; 30: 885-92.
• 11 Virchow RR. Cellular Pathology. 1860
• 12 Burnand KG, Gaffney PJ, McGuinness CL, Humphries J, Quarmby JW, Smith A. The role of the monocyte in the generation and dissolution of arterial and venous thrombi. [Review]. Cardiovasc Surg 1998; 6: 119-25.
• 13 McGuinness CL, Humphries J, Waltham M, Burnand KG, Collins M, Smith A. Recruitment of labelled monocytes by experimental venous thrombi. Thromb Haemost 2001; 85: 1018-24.
• 14 Cox JST. The maturation and canalization of thrombi. Surg Gynecol Obstet 1963; 116: 593-9.
• 15 Sevitt S. Organic canalisation and vascularisation of deep vein thrombi studied with dyed-micropaque injected at necropsy. J Pathol 1970; 100: i.
• 16 Humphries J, McGuinness CL, Smith A, Waltham M, Poston R, Burnand KG. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg 1999; 30: 894-9.
• 17 Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 1999; 5: 1359-64.
• 18 Folkman J. Therapeutic angiogenesis in ischemic limbs. Circulation 1998; 97: 1108-10.
• 19 Folkman J. Angiogenic therapy of the human heart. Circulation 1998; 97: 628-9.
• 20 Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246: 1306-9.
• 21 Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13: 9-22.
• 22 Waltham M, Burnand KG, Collins M, Smith A. Vascular endothelial growth factor and basic fibroblast growth factor are found in resolving venous thrombi. J Vasc Surg 2000; 32: 988-96.
• 23 McGuinness CL, Humphries J, Smith A, Burnand KG. A new model of venous thrombosis. Cardiovasc Surg 1997; 5: 123.
• 24 Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161: 851-8.
• 25 Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989; 246: 1309-12.
• 26 Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J. Human vascular permeability factor. Isolation from U937 cells. J Biol Chem 1989; 264: 20017-24.
• 27 Pepper MS, Ferrara N, Orci L, Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 1992; 189: 824-31.
• 28 Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996; 87: 3336-43.
• 29 Hobson MI. Increased vascularisation enhances axonal regeneration within an acellular nerve conduit. Ann R Coll Surg Engl 2002; 84: 47-53.
• 30 Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis. A single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994; 93: 662-70.
• 31 Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg 1995; 21: 314-24.
• 32 Folkman J, Klagsbrun M, Sasse J, Wadzinski M, Ingber D, Vlodavsky I. A heparin-binding angiogenic protein – basic fibroblast growth factor – is stored within basement membrane. Am J Pathol 1988; 130: 393-400.
• 33 Sahni A, Francis CW. Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation. Blood 2000; 96: 3772-8.
• 34 Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992; 267: 26031-7.
• 35 Barleon B, Siemeister G, Martiny-Baron G, Weindel K, Herzog C, Marme D. Vascular endothelial growth factor up-regulates its receptor fms-like tyrosine kinase 1 (FLT-1) and a soluble variant of FLT-1 in human vascular endothelial cells. Cancer Res 1997; 57: 5421-5.
• 36 Pepper MS, Ferrara N, Orci L, Montesano R. Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasmino-gen activator inhibitor-1 in microvascular endothelial cells. Biochem Biophys Res Commun 1991; 181: 902-6.
• 37 Northeast AD, Soo KS, Bobrow LG, Gaffney PJ, Burnand KG. The tissue plasminogen activator and urokinase response in vivo during natural resolution of venous thrombus. J Vasc Surg 1995; 22: 573-9.
• 38 Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3’-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 1998; 273: 30336-43.
• 39 Asahara T, Murohara T, Sullivan A, Silver M, van der ZR, Li T, Witzenbichler B, Schatte-man G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964-7.
• 40 Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999; 18: 3964-72.
• 41 Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221-8.
• 42 Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983; 219: 983-5.
• 43 Soo KS, Northeast AD, Happerfield LC, Burnand KG, Bobrow LG. Tissue plasminogen activator production by monocytes in venous thrombolysis. J Pathol 1996; 178: 190-4.
• 44 Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan YC, Olander JV, Connolly DT, Stern D. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte pro-coagulant activity, and promotes monocyte migration. J Exp Med 1990; 172: 1535-45.