Study Of The Mechanisms And Its Clinical Effects And Safety Of Thalidomide Treatment For GIVM

BACKGROUDS: Gastrointestinal vascular malformation(GIVM)is the main course of obscure gastrointestinal bleeding.At present,its etiological factors are still unclear.Recent studies have demonstrated that high level of HIF 1α and HIF 2α under hypoxia condition is related with angiogenesis.However,there are few studies about the mechanism of HIF 1α and HIF 2α in gastrointestinal vascular malformation.Several studies have verified that thalidomide has a good clinical efficacy for GIVM bleeding patients,but it is still need large samples study with long term follow-up period to confirm the clinical effectiveness and safety of thalidomide treatment.Part Ⅰ The role of HIF 1α and HIF 2α in GIVM pathogenesis and thalidomide’s impact on their expressionAims: To investigate the role of HIF 1α and HIF 2α in GIVM pathogenesis and thalidomide’s impact on their expression.Methods: Immunohistochemistry of surgically resected GIVM tissues and normal colonic tissues were performed to examine the expression of HIF 1α and HIF 2α.In addition,in vitro expression of HIF 1α and HIF 2α protein in primary human umbilical vein endothelial cells(HUVEC)during normoxia and hypoxia were detected by Western blot.Mouse aortic ring angiogenesis test was used to know its angiogenesis during nomoxia and hypoxia condition.Also,the expression of HIF 1α and HIF 2α protein in HUVEC cultured under hypoxia condition treated with 200ug/ml thalidomide for different hours were detected by Western blot.The expression of HIF 1α and HIF 2α protein in HUVEC during hypoxia treated with different concentration of thalidomide for 48 h were also detected by immunofluorescence and Western blot.Results: 1.The expression of HIF 1α and HIF 2α in GIVM lesions were significantly higher than in normal intestinal tissues.2.The expression of HIF 1α and HIF 2α protein in HUVEC during hypoxia were significantly higher than in nomoxia.3.Mouse aortic rings cultured in hypoxia condition developped much more new blood vessels.4.Thalidomide could inhibit the expression of HIF 1α and HIF 2α protein in HUVEC during hypoxia.Conclusion: The increased expression of HIF 1α and HIF 2α during hypoxia may be related with GIVM pathogenesis.Thalidomide could inhibit the expression of HIF 1α and HIF 2α protein in HUVEC during hypoxia.PartⅡImpact of HIF 1α and HIF 2α on angiogenesis pathwaysAims: To investigate the imapct of HIF 1α and HIF 2α on angiogenesis pathways.Methods: Real-time PCR and Western blot were used to detect the mRNA and protein level of HIF 1α、HIF 2α、VEGF、Notch1、Dll4 and Ang2 in HUVEC overexpressed with HIF 1α or HIF 2α gene.Dual luciferase report gene assay was use to know the influence of HIF 1α and HIF 2α on VEGF promotor region activity.Matrigel tube formation test was also applied to know their effects on HUVEC angiogenesis.Results: After overexpressed with HIF 1α or HIF 2α gene,VEGF promotor region activity was obviously increased,the mRNA and protein level of VEGF、Notch1、Dll4 and Ang2 in HUVEC and number of capillary-like tubes were significantly increased.Conclusion: The overexpression of HIF 1α or HIF 2α of HUVEC could increase the expression of VEGF、Notch1、Dll4 and Ang2,result in more angiogenesis.Part Ⅲ Long term effectiveness and safety of thalidomide for treating refractory gastrointestinal bleeding due to GIVMAims: To study long term effectiveness and safety of thalidomide for treating refractory gastrointestinal bleeding due to GIVM.Methods: Prospectively collected 80 GIVM bleeding patients.Patients eligible for the study were treated with thalidomide and followed up at least 1 year.The primary end point was the overall effective response rate.The secondary end point included the rates of cessation of bleeding.We also evaluated the change of yearly blood transfusion amounts,yearly bleeding episodes,hemoglobin levels and yearly hospitalization numbers after thalidomide treatment.Results: 1.The overall effective response rate during the whole follow-up period was 79.49%.2.The rates of cessation of bleeding was 38.46%.After thalidomide treatment,yearly blood transfusion amounts,yearly bleeding episodes and yearly hospitalization numbers were significantly decreased,while the hemoglobin level was significantly increased(P<0.001).3.The rate of main adverse events was 31.25%.Conclusion: Thalidomide is an effective and relatively safety drug for patients with refractory gastrointestinal bleeding due to GIVM.