低分子量肝素对VEGF-A介导的内皮细胞激活的影响
2016年05月20日 【健康号】 孙晨宇     阅读 5371

Abstract:

       It has been found that the risk of venous thromboembolism (VTE) is increased in cancer patients, however it is not fully understood how cancer leads to increased risk of thrombosis, which results in a worse outcome and survival due to more metastasis. The mechanism behind tumor cell-induced activation of coagulation is still not well known so far, despite of much effort has been put into this field. A very initial step in metastasis formation is that cancer cells interact with the endothelium in the circulating system to extravasate and metastasize. This interaction is characterized by tumor cell-induced endothelial cell (EC) activation followed by release of von Willebrand factor (VWF), a multimeric glycoprotein that is integral inmediating hemostasis and thrombosis. The VWF multimers form ultra large VWF(ULVWF) fibers in the luminal EC surface, which are strong binding partners for platelets mediating pathophysiological vessel occlusion. In this context it was previously shown that LMWHs are able to block EC activation and microthrombus formation associated with prolonged survival, less tumor burden and reduced tumor progression in a melanoma mouse model. LMWHs carry out these functions mainly through two pathways, including blockage of thrombin indirectly via blocking upstream coagulation factors and direct blockage of vascular endothelial growth factor-A (VEGF-A), which itself has been proven to be able toactivate endothelial cells. It also has been found heparin treatment inhibits heparanase activity and leads to reduction of metastasis. Thus, in order to find the effects of low molecular weight heparins (LMWHs) on tumor-induced EC activation, we used the ELISA technique showing decreased level of VWF release from human umbilical vein endothelial cells (HUVECs) demonstrating that treatment with Tinzaparin reduces Ret melanoma cell-induced EC activation. By contrast,treatment with Fondaparinux showed no effect on VWF release upon stimulation with Ret cell supernatant. We also showed via ELISA technique that VEGF-A induces EC activation in a concentration-dependent manner. Since VEGF-A is one main mediator of tumor cell-mediated activation of the endothelium, we conducted spectrophotometry to analyze molecular interactions of the growth factor with heparins which demonstrated high binding affinity of Tinzaparin andVEGF-A, while Fondaparinux shows extremely weak binding affinity to VEGF-A. For an in vivo proof of these findings we then analyzed primary tumors of intradermally inoculated Ret cells after treatment of Fondaparinux, Tinzaparin or vehicle control. Immunofluorescence studies showed a high amount of vessels with intraluminal VWF fibers in the tumor. Treatment with Tinzaparin reduced number of vessels with intraluminal VWF,while Fondaparinux had no significant effects.

       Apart from blocking thrombin, selectins and VEGF-A,LMWHs have also been shown to inhibit heparanase, leading to reduced metastasis formation in a mouse model. Therefore, we then analyzed the role of heparansein melanoma by performing ELISA showing a decreased level of VEGF insupernatant of heparanase knockdown (SiHPSE) Ret cells and a decreased level ofVWF release from HUVECs that have been treated with supernatant of heparanase knock down (SiHPSE) Ret cells. To further prove the in vivo relevance of these findings, primary tumors of  hepranase knock down (SiHPSE) Ret cells were analyzed by immunofluorescence studies of tumor vessels, showing a decreased number of vessels with intraluminal VWF fibers as well as less platelets within tumor microvessels in compared to control tumors. To summarize, as tumor cell-induced EC activation is associated with tumor-related coagulation and cancer progression, we postulate that anti-coagulant therapy of some LMWHs (such as Tinzaparin) suppress EC activation and reduce microthrombi formation, which could be potentially helpful in the treatment of melanoma.

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孙晨宇
主治医师/研究员
安徽医科大学第一附属...
普外科门诊(绩溪路)...
现主要研修内外妇儿常见病及普外科疾病诊治,可以中英文双语提供服务。
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