Thrombin Triggers Angiogenesis In Rat Brains Following Experimental Intracerebral Hemorrhage

Background and purpose:Intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype, with high morbility, mortality and mutilation rate. However, currently there is no medical therapy available for these patients to improve neurological deficit caused by ICH except blood pressure reduction and neurosurgical evacuation of the hematoma. Our previous studies have demonstrated that angiogenesis referred to the outgrowth of new vessels from pre-existing vasculature can occur in rat brains with collagenase-induced ICH, which may create a favorable micro-environment for repairing damaged neurons and axonal sprouting and synaptogenesis, and facilitate the drugs directly to lesions to exert their effect fully. However, the triggering mechanism of angiogenesis after ICH is not clear. Thrombin, a multifunctional serine protease, has been confirmed to inducing tumor angiogenesis in vitro and in vivo and up-regulating the expression of many angiogenic molecules. However, so far there has been few report about the effect of thrombin on ICH-induced angiogenesis. Accordingly, the purpose of the present study is to determine whether thrombin can trigger angiogenesis and affect the expression of many angiogenic molecules including hypoxia-inducible factor-la (HIF-la), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), Ang-2, matrix metalloproteinase-2(MMP-2), MMP-9 and integrinαvβ3 following ICH, to clarify the mechanism of thrombin triggerring angiogenesis after ICH, and to provide new concepts and ideas for exploring the clinical and basic treatment of ICH.Methods:This study was divided into two parts. (1) The rats were administered 100μL autologous blood (ICH) with or without hirudin (5 U) stereotaxically into the right globus pallidus. Sham controls received the same volume of 0.9% sterile saline. They then received intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU,50 mg/kg). Brains were perfused to identify BrdU+/von Willebrand factor+(vWF) nuclei, and the spatial and temporal distribution of HIF-la, VEGF, Ang-1, Ang-2, MMP-2, MMP-9 and integrinαvβ3 was evaluated by immunohistochemistry. The expression of HIF-1α, VEGF, Ang-1, Ang-2, MMP-2, MMP-9 and integrin av(33 was evaluated by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. (2) The rats received either 1 U (50μL) thrombin or 50μL 0.9% sterile saline (sham) by direct infusion into the right globus pallidus and then received intraperitoneal injections of BrdU (50 mg/kg). Brains were perfused to identify BrdU+/vWF+ nuclei, and the spatial and temporal distribution of HIF-la, VEGF, Ang-1, Ang-2, MMP-2, MMP-9 and integrinαvβ3 was evaluated by immunohistochemistry. Results:Immunohistochemistry showed that the sham operation group at different time points no BrdU, HIF-1α, Ang-2, MMP-2, MMP-9 and integrinαvβ3 immunoreactivity was found, occasional delicate positive vessels with VEGF or Ang-1 immunoreactivity scattered. After ICH induction, BrdU+ nuclei in vWF+ dilated vessels could be observed around the hematoma at 3 days, and peaked at 14 days. Moreover, numerous HIF-1α-, VEGF-Ang-1-, Ang-2-, MMP-2-,MMP-9-, and integrinαvβ3- positive microvessels of the enlarged profile were detected in the perihematomal tissue. And immunofluorescence double labeling confirmed that HIF-1α, VEGF, Ang-1, Ang-2, MMP-2, MMP-9 and integrinαvβ3 were localized in vWF+ vessels around the hematoma. RT-PCR demonstrated that notable up-regulation of VEGF, Ang-1, Ang-2, MMP-2, MMP-9, and integrinαvβ3 mRNA could be detected at 3 days after ICH, compared to sham control. The up-regulation of VEGF, Ang-1, integrin av andβ3 mRNA persisted until 14 days, while the expression of Ang-2, MMP-2 and MMP-9 mRNA decreased hereafter. No change was detected in HIF-la mRNA in any group and any time point. Western blot displayed that the expression of HIF-1α, VEGF, Ang-1, and Ang-2 protein was also significantly increased at 3 days after ICH, compared to sham control. The HIF-1αand Ang-2 protein levels decreased obviously, while the VEGF and Ang-1 protein levels increased till 14 days. However, in hirudin-treated brains, the BrdU+/vWF+ nuclei decreased significantly, and the expression of HIF-1α, VEGF, Ang-1, Ang-2, MMP-2, MMP-9, and integrin avβ3 around the clot was also down-regulated notably. After one unit thrombin was injected into the right globus pallidus, BrdU+ nuclei could be found in vWF+ vessels till 7 days Furthermore, many HIF-la-, VEGF-, Ang-1-, Ang-2-, MMP-2-, MMP-9-, and integrin av(33- immunoreactive microvessels of the dilated outline were detected around the thrombin-affected region till 7 days.Conclusions:1. Thrombin can induce angiogenesis in rat brains and may be an important trigger for ICH-related angiogenesis.2. Thrombin may trigger ICH-related angiogenesis by up-regulating the expression of HIF-la, VEGF, Ang-1, Ang-2, MMP-2, MMP-9 and integrin av(33.