The Preliminary Research On The Role And Mechanism Of MYSM1 In Angiogenesis

BACKGROUNDDevelopment and formation of blood vessels, which is a complex process that various cells and molecules are involved in, goes through every stage of embryo development and individual growth. Besides, angiogenesis is regulated by multiple mechanisms. In recent years, epigenetic regulations in vascular development have attracted more and more attentions and become a hot topic in this field.Histone modification, a critical part of epigenetics, occurs in nucleosomal histone H2 A, H2 B, N- or C-terminal of H3 and H4. Common Forms of histone modification include methylation, acetylation, phosphorylation, ubiquitination, et al. These modifications are vital for conformation of chromatin and genetic transcription. Histone ubiquitination mainly occurs in H2 A and H2 B. Ubiquitined Histone H2A?ub H2A? approaches 5-15% of total H2 A, which is the highest ubiquitinated degree of histones in nucleus. Besides, histone ubiquitination is also critically involved in the process of cell cycle and DNA repair. Histone ubiquitination and deubiquitination are reversible, mainly catalyzed by ubiquitin ligase and deubiquiting enzymes, respectively.MYSM1, as a deubiquitinase, whose main function is to remove the ubiquitin from K119 of ub H2 A, is a vital epigenetics molecule. It is reported that the MYSM1-/- mice appear significantly abnormal phenotypes in many systems such as hematopoiesis, immune, skin, adipose tissue, vision and skeleton. The present relevant researches on MYSM1 chiefly focused on its function on hematopoiesis and immune system. Recently, an available research showed the pathogenesis of diabetic retinopathy?DR? in Taiwanese was associated with 2 SNP loci in MYSM1 gene. Microangiopathy is the main pathological feature of DR, indicating that MYSM1 may have profound effects on angiogenesis. This program is aimed to explore the role and mechanism of MYSM1 in angiogenesis. In this study, we measured the expression of MYSM1 in endothelial cells. We clarified the function of MYSM1 via in vivo and in vitro experiments mainly using the MYSM1-/- mice and HUVEC cells. Besides, we obtained the endothelial cell-specific MYSM1 knockout mice, Cdh5-Cre⁺-MYSM1^f/f mice, in order to better investigate these findings. We also figured out the potential signaling pathway and target genes of MYSM1 according to the DNA microarray results. Our findings showed that MYSM1 was able to promote angiogenesis, which laid the foundation to explore further mechanism and provided new clues to systematically understanding the process of angiogenesis.OBJECTIVESTo clarify the possible functions and potential mechanism of MYSM1 in angiogenesis.METHODS1. Immunological Histological Chemistry?IHC?, immunofluorescent staining?IF? assays, q RT-PCR and Western-blot were applied to detect the expression of MYSM1 in vascular tissues and HUVEC cells.2. Using Flow cytometry?FCM?, Wound healing assays, transwell assays and angiogenesis experiments to detect the effects of MYSM1 on cell cycle, apoptosis, cell migration and invasion as well as the capacity of angiogenesis.3. Vascular formation abilities of MYSM1 were detected through the mouse's aortic rings assay and retinal vessels staining assay. The integrity of vessel was detected by H&E in mouse main organs.4. Cdh5-Cre mice were crossed with MYSM1^f/f mice and finally we obtained the Cdh5-Cre⁺-MYSM1^f/f mice.5. Based on the gene microarray study to explore possible mechanism of MYSM1 in angiogenesis.RESULTS1. MYSM1 was globally high in vascular tissues and mainly expressed in nucleus other than cytoplasm under the normal conditions by Immunological Histological Chemistry?IHC? and immunofluorescent staining?IF? assays. Western blot and q RT-PCR showed that MYSM1 was significantly down-regulated by its specific si RNA and up-regulated via the stimulation of hypoxia or LPS.2. Flow cytometry?FCM? displayed that MYSM1 had no effect on cell cycle or apoptosis; Wound healing and transwell assays exhibited that cell migratory and invasive abilities were decreased by down-regulating the expression of MYSM1; angiogenesis experiments and fibrin bead assay showed that knockdown of MYSM1 might lead to a descend in the capacity of angiogenesis. All the experiments above regarded HUVEC cells as the study objects.3. We found that the sprouting ability of the mouse's aortic rings was markedly impaired when the expression of MYSM1 was knockout or decreased. Simultaneously, retinal vessels of MYSM1-/- mice were dramatically reduced and more inflammatory exudation around the blood vessels could be detected compared with the wild-type mice.4. Cdh5-Cre mice were crossed with MYSM1^f/f mice and finally we obtained the Cdh5-Cre⁺-MYSM1^f/f mice.5. The results of gene microarray showed that MYSM1 could regulate the signaling pathway of angiogenesis and the expression of angiogenesis related factors. In addition, the expression of two anti-angiogenesis factors, Arresten and Canstatin, were elevated when MYSM1 was knockdown.CONCLUSION1. MYSM1 is globally high in vascular tissues and mainly expressed in nucleus other than cytoplasm under the normal conditions. Stimulation by hypoxia or LPS up-regulates the expression of MYSM1 in HUVEC cells.2. MYSM1 has no effect on cell cycle and apoptosis; but it promotes the HUVEC cells' abilities of migration, invasion and angiogenesis.3. MYSM1 facilitates the sprouting ability of mouse's aortic rings, the intensity of retinal vessels and the integrity of vascular tissue.4. We managed to obtain the endothelial cell-specific MYSM1 knockout Cdh5-Cre⁺-MYSM1^f/f mice.5. MYSM1 participates in the signaling pathway of angiogenesis and regulates the expression of angiogenesis related factors.