Study On The Action Mechanisms Of HMBOX1 In Apoptosis And Autophagy And The Regulation Mechanisms Of HMBOX1 In Vascular Endothelial Cells

The endothelium,composed of vascular endothelial cells(VECs),is the thin layer of cells that lines the interior surface of blood vessels.It forms an interface between circulating blood in the lumen and vessel wall.VECs,which directly contact with blood,are involved in many aspects of vascular biology,including barrier function,blood elotting,inflammation,angiogenesis and vasoconstriction.Apoptosis,defined as programmed cell death,plays an important role in maintaining cellular homeostasis by regulating cell death under stress.VEC apoptosis could represent a main form of VEC injury,which leads to inflammation in the vessel wall and has been associated with many diseases such as atherosclerosis,hypertension,diabetes mellitus and thrombosis.Autophagy is also known for cellular maintenance by specific degradation and the recycling of damaged organelles and misfolded proteins.Thus,apoptosis and autophagy are two evolutionarily conserved processes that maintain VEC homeostasis.However,the mechanisms underlining apoptosis and autophagy of VECs need to be further studied.Homeobox containing 1(HMBOX1),first identified and isolated from human pancreatic cDNA library,was first known as a transcription repressor.Further studies showed that HMBOX1 depressed the activation of NK cells through inhibiting interferon-? production and suppressing NKG2D/DAP10 signaling pathway.Besides,HMBOX1 participated in telomere elongation in telomerase-dependent and ALT cells by directly and specifically binding to double-stranded telomere repeats.A recent research demonstrated that HMBOX1 was a downstream target of miR-222,which played a vital role in modulating cardiomyocyte phenotypes.Recent studies by our group showed that HMBOX1 was essential for the differentiation of BMSCs and ESCs into VECs induced by a small molecule 6-amino-2,3-dihydro-3-hydroxymethy1-1,4-benzoxazine(ABO).However?the function of HMBOX1 in VECs is still unknown.In this study,we found that HMBOX1 was abundantly expressed in the cytoplasm of human umbilical vascular endothelial cells(HUVECs).Knockdown of HMBOXI induced apoptosis and inhibited autophagy.Overexpression of HMBOX1 inhibited apoptosis induced by fibroblast growth factor 2 deprivation and promoted autophagy.Metallothionein 2A(MT2A)was identified as an interaction protein with HMBOX1 by yeast two-hybrid assay,and confirmed by co-imnunoprecipitation.Overexpression of HMBOX1 elevated intracellular free zinc level.Knockdown of MT2A inhibited this phenomenon.Moreover,TPEN,a zinc chelator,reversed the anti-apoptosis and pro-autophagy effects of HMBOX1.In conclusion,HMBOX1 regulated intracellular free zinc level by interacting with MT2A to inhibit apoptosis and promote autophagy in VECs.Besides,in order to clarify the role of HMBOX1 in the aortic endothelial cells in vivo,we generated a knockout mouse for HMBOX1 using TALEN technology and found that HMBOX1 participated in the function maintenance of mouse aortic endothelial cells,the aortic endothelial cells of HMBOX1 knockout mouse showed increased apoptosis and decreased autophagy with LPS treatmentHMBOX1 is essential for the survival of HUVECs.However,the regulatory meehanism of HMBOX1 expression is still unclear.We reeently found that a small molecule 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine(ABO)directly targeted annexin A7(ANXA7)and inhibited its GTPase activity.In addition,both HMBOX1 and ANXA7 participated in the autophagy and apoptosis of HUVECs.However,their relationship in the regulation of HMBOX1 expression is unclear.In this study,we found that ABO could elevate HMBOX1 at translation level through inhibiting ANXA7 GTPase activity.ABO failed to increase HMBOX1 protein level in ANXA7-deficient HUVECs.TGFB2 overlapping transcript 1(TGFB2-OT1)that was increased by ABO facilitated HMBOX1 expression by increasing La-related protein 1(LARP1)expression.Furthermore,the protein level of HMBOX1 was decreased under oxidized low-density lipoprotein(oxLDL)treatment in HUVECs and in the aortic endothelium of apolipoprotein E-deficient(apoE-/-)mice,which could be reversed by ABO in vitro and in vivo.In conclusion,ANXA7 was an endogenous regulator of HMBOX1,and ABO promoted HMBOX1 translation by inhibiting ANXA7 GTPase activity and enhancing TGFB2-OT1 expression.Besides,our data suggested that HMBOX1 might be a novel diagnostic marker and therapeutic target of atherosclerosis.