Experimental Study Of The Effect Of CYLDa On Angiogenesis

Background:Angiogenesis represents one of essential ways of blood vessel formation in adult body, and it refers to vascular endothelial cells sprout from small vein at the existing vascular bed. Angiogenesis plays important roles not only in many physiological processes, but also in many diseases, such as tumor growth, pathological scar, rheumatic diseases, ischemic heart disease and so on. Thus it is of clinical significance to study the mechanism of angiogenesis for the treatment of angiogenesis-related diseases.Effect of cylindromatosis (CYLD) on angiogenesis and its underling mechanisms remain unclear. Cylindromatosis (CYLD) is a tumor suppressor gene, mutant of which will cause human cylindromatosis. CYLD inhibit NFkB pathway by removing the k63 ubiquitin of, and thus deactivate upstream molecules of NFkB pathway, such as TRAF2, TRAF6, NEMO, and bcl-3. Studies have reported that knock-down of CYLD in vascular endothelial cells (VECs) using RNAi inhibited migration of VECs. However, there are still some question need to be addressed to further understand the effecto of CYLDa on agiogenesis:1. the effect of CYLD type a (CYLDa) on angiogenesis remains unclear. CYLD has three subtypes (CYLDa, CYLDb, sCYLD) with different structures, which indicates the function of each subtype may be different. There is no literature which is focused on the function of the specific subtype.2. It is still unknown whether CYLDa regulates angiogenesis via its deubiquitinating activity.In this study, we created the adenovirus of CYLDa and CYLDamut (deubiquitinating activity dead), and created transgenic mice of CYLDa and CYLDamut, and then we used the VECs migration model, tumor angiogenesis model, matrigel angiogenesis model, and aorta ring assay to investigate the effect of CYLDa in angiogenesis and to determine whether CYLDa regulate angiogenesis via its deubiquinating activity by experimental methods of histology, cell biology, and molecular biology.Research objectives:Explore the effect of CYLDa over-expression on VECs proliferation, migration and the formation of vascular lumen in vitro, and verify the effect of CYLDa on angiogenesis in CYLDa transgenic mice. Meanwhile, the CYLDamut group was included in this study (does not have deubiquitinating enzyme activity, thus cannot inhibit NFκB pathway), in order to determine whether the effect of CYLDa on angiogenesis is related to deubiquitinating enzyme activity.Research methods and results:1) Through adenovirus infection, the CYLDa and CYLDamut were over-expressed in VECs. The proliferation and migration of VECs were investigated by using CCK-8 method and wound healing assay, the tube formation of VECs was detected using three-dimensional collagen assay.2) After creation of breast cancer model and lung cancer model in CYLDa transgenic mice, CYLDamut transgenic mice and wild type mice, the effect of CYLDa on angiogenesis in breast caner tissue and in lung cancer tissue were observed using immunohistochemical staining of CD31, which is the cell marker of VECs. In addition, the effect of CYLDa oo angiogenesis was further confirmed through matrigel assay and mouse aortic ring assay.Conclusion:1) Over-expression of CYLDa inhibits angiogenesis both in vitro and in vivo;2) The inhibitory effect of CYLDa over-expression on angiogenesis is not related to deubiquitinating enzyme activity.