On the Mechanism of Thrombin-induced Angiogenesis

Abstract

Many of the cellular actions of thrombin may contribute to the angiogenesis-promoting effect of thrombin reported previously. In this study, we investigated the interaction between thrombin and vascular endothelial growth factor (VEGF), the specific endothelial cell mitogen and key angiogenic factor. Exposure of human umbilical vein endothelial cells to thrombin sensitizes these cells to the mitogenic activity of VEGF. This thrombin-mediated effect is specific, dose-dependent and requires the activated thrombin receptor. Quantitative reverse transcription- polymerase chain reaction analysis reveals a time- and dose-dependent up-regulation of mRNA for VEGF receptors (KDR and flt-1). Optimal thrombin concentration for maximal expression of mRNA for KDR is 1.5 IU/ml (170% over controls) and appears 8–12 h after thrombin stimulation. Nuclear run-on experiments demonstrate that the up-regulation of KDR mRNA by thrombin occurred at the transcriptional level. In addition, functional protein of KDR receptor is increased to about 200% over control after 12 h of thrombin treatment. The up-regulation of KDR and flt-1 mRNA is also mimicked by the thrombin receptor activating peptide. These findings could explain at least in part the potent angiogenic action of thrombin. Many of the cellular actions of thrombin may contribute to the angiogenesis-promoting effect of thrombin reported previously. In this study, we investigated the interaction between thrombin and vascular endothelial growth factor (VEGF), the specific endothelial cell mitogen and key angiogenic factor. Exposure of human umbilical vein endothelial cells to thrombin sensitizes these cells to the mitogenic activity of VEGF. This thrombin-mediated effect is specific, dose-dependent and requires the activated thrombin receptor. Quantitative reverse transcription- polymerase chain reaction analysis reveals a time- and dose-dependent up-regulation of mRNA for VEGF receptors (KDR and flt-1). Optimal thrombin concentration for maximal expression of mRNA for KDR is 1.5 IU/ml (170% over controls) and appears 8–12 h after thrombin stimulation. Nuclear run-on experiments demonstrate that the up-regulation of KDR mRNA by thrombin occurred at the transcriptional level. In addition, functional protein of KDR receptor is increased to about 200% over control after 12 h of thrombin treatment. The up-regulation of KDR and flt-1 mRNA is also mimicked by the thrombin receptor activating peptide. These findings could explain at least in part the potent angiogenic action of thrombin. vascular endothelial growth factor thrombin receptor activating peptide protein kinase C mitogen-activated protein human umbilical vein endothelial cell bovine serum albumin fetal bovine serum reverse transcription polymerase chain reaction base pair(s) platelet-derived growth factor basic fibroblast growth factor glyceraldehyde-3-phosphate dehydrogenase polyacrylamide gel electrophoresis phosphate-buffered saline 3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide phenylalanyl-propyl-arginine chloromethyl ketone The original observation made by Trousseau in 1872 (1Trousseau A. Lectures in Clinical Medicine, Delivered in Hotel-Dieu, Paris. New Sydenham Society, London1872: 281-295Google Scholar), that there is frequent blood coagulation in cancer patients, has been verified by many investigators. Clinical, laboratory, histopathological, and pharmacological evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients (2Rickles F.R. Edwards R.L. Blood. 1983; 64: 14-31Crossref Google Scholar). This may be explained by the fact that many tumor cells elicit procoagulant activity either directly or through interaction with platelets, leukocyte and endothelial cells (3Sloan B.F. Rozhin J. Jonhson K. Taylor H. Crissman J.D. Honn K.V. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2483-2487Crossref PubMed Scopus (210) Google Scholar). Zacharsky et al. (4Zacharsky L.R. Memoli V.A. Morain W.D. Schlaeppi J.M. Rousseau S.M. Thromb. Haemostasis. 1995; 73: 793-797Crossref PubMed Scopus (83) Google Scholar) have shown recently the presence of thrombin in a variety of tumor types. The presence of thrombin in these tumors explains the hypercoagulability in cancer but does not answer the question whether thrombin contributes directly to the tumor promotion and metastasis. It has been shown in a recent large clinical study that primary thromboembolism increases by 3-fold the risk of overt cancer diagnosis within the next 6–12 months after thrombosis (5Sörensen H.T. Mellem K.L. Steffensen F.H. Olsen J.H. Nielsen G.L. N. Engl. J. Med. 1998; 338: 1169-1173Crossref PubMed Scopus (564) Google Scholar). These clinical observations are in line with animal experiments, where thrombin treatment of B16 melanoma cells increases dramatically the number of lung metastasis in rats (6Nierodzik M.L. Kajumo F. Karpatikin S. Cancer Res. 1992; 52: 3267-3272PubMed Google Scholar). Mor