Up-regulation Of Brm Is Involoved In The Response Of Endothelial Cells To Hypoxia

Hypoxia pulmonary hypertension(HPH)plays a crucial role in pathogenesis of high altitude heart disease and pulmonary heart disease,featured by persistent pulmonary vascular contracting and vascular remodeling.HPH aggravates right heart overload,and further,causes right heart insufficiency.Those with server symptoms could develop right heart failure and even death.The mechanisms for HPH is complicated,and remain largely unclear.Recent studies have found that the pulmonary vascular inflammatory response and unbalance in the secretion of pulmonary vasoactive substances play essential roles in pathogenesis of HPH.In addition,cell adhesion molecules(CAMs)and Endothelin-1(ET-1)are two important factors mediating pulmonary vascular inflammatory response and increasing of vascular tension.Our previous studies found that hypoxia promoted the expression of Chromatin Remodeling proteins Brm(Brahma)which are involved in the epigenetic regulation of CAMs and ET-1 and mediated pulmonary vascular inflammation,pulmonary vascular structural remodeling and even right ventricular hypertrophy.However,the mechanisms of up-regulating Brm expression by hypoxia is still unclear.Brm is the core ATP subunit of chromatin remodeling complexes SWI/SNF(Switching/ sucrose non-fermentation),and acts as core proteins in regulating structural changes of nucleosomes and gene transcription.Brm achieves the remodeling or replacement of nucleosomes,depending on its ATP enzyme active structure domain to release energy after hydrolysis of ATP and forming a key role in the transcription regulation of eukaryotic genes.Under the condition of hypoxic stress,a large part of genes is regulated by hypoxia inducible factor-1 α(HIF-1 α).Further,the epigenetic regulation mechanism plays an important role in response to hypoxia.Specifically,histone modifications mediated by H3K4 methyl transfer enzyme complexes(COPASS)in H3K4 methylation are crucial in gene transcription activation.Therefore,in this study,we aim at exploring the mechanisms involved in enhancing Brm expression by HIF-1 α and H3K4 methylation under hypoxic environment;and their roles in response to hypoxia of endothelial cell,in combination with the bioinformatics analysis.Methods:1.Human Umbilical Vein Endothelial cells(HUVEC)are placed in hypoxic workstations(1%O2,5%CO2,94%N2)for a certain time period(12h,24 h,48h).The methods of RT-PCR and Western blot are employed to detect the mRNA and protein expression of Brm.Then,the liposome transfection method is performed to translate promoter plasmid of BRM gene into HUVECs.The promoter activity of Brm under hypoxic environment is detected by dual-luciferase reporter gene system.2.Exogenous HIF-1α plasmid or siRNA is transfected into the endothelial cells for interfering with endogenous HIF-1α expression,the promoter activity of Brm was detected by the dual-luciferase reporter gene system.Furthermore,the mRNA and protein expression of Brm was detected by RT-PCR and Western Blot.Chromatin Immunoprecipitation(ChIP)technique is performed to detect alterations in structures where HIF-1α combined on Brm promoter of HUVECs in hypoxic environment,and to clarify the mechanisms of promoting Brm transcription by HIF-1α.3.Exogenous plasmid or siRNA of Wdr5(WD Repeat Domain 5)or Ash2l(ASH2 Like Histone Lysine Methyltransferase Complex Subunit)which are the core subunits of H3K4 methyltransferase complex is transfected into the endothelial cells.The promoter activity of Brm was detected by dual-luciferase reporter gene system after translating over expression plasmid or siRNA of Wdr5 or Ash2 l into HUVECs for disturbing endogenous Wdr5 or Ash2 l expression.Accordingly,mRNA and protein expression of Brm is detected by RT-PCR and Western blot.The change of H3K4 methylation level in Brm promoter region in endothelial cells treated with hypoxia was detected by ChIP technique.Results:1.The Brm promoter activity in endothelial cells,which are cultured in the hypoxia(1%O2)environment after transfecting plasmid of Brm for 24 h,detected by the dual fluorescence enzyme reporting gene system.Comparing with control group in 1% O2 hypoxia could significantly increase the activities of Brm promoter.The expression of Brm mRNA and protein hypoxia environment(12h,24 h,48h)are significantly higher than that in normal oxygens control group.It poses remarkable relationship with the hypoxia time along with the culture time prolong in hypoxic environment.2.Comparing with normoxic controls,hypoxia significantly increased the ability of combination of HIF-1 and promoter region in Brm in endothelial cells.After transfection of exogenous HIF-1 α expression plasmids into endothelial cells successfully,HIF-1α mRNA and protein expression levels are significantly improved.After interfering endogenous HIF-1α by siRNA in endothelial cells successfully,mRNA and protein expression levels of HIF-1α are remarkably inhibited.Inhibiting HIF-1 α in endothelial cells could suppress promoter activity of the Brm,and decrease mRNA and protein expression of Brm.3.Compared with the control group,the level of H3K4me3 in Brm promoter region in the hypoxia group was significantly increased.After importing exogenous plasmid of Wdr5 and Ash2 l successfully in endothelial cells,the expression of mRNA and protein of Wdr5 and Ash2 l are up-regulated,while the according level of Brm in hypoxia was significantly higher than that in control group.Interfering endogenous Wdr5 and Ash2 l expression with siRNA in hypoxia environment inhibits the promoter activity of Brm,the mRNA and protein expression level of Brm comparing to control group.Conclusion:1.Hypoxia could significantly increase promoter activity and up-regulate the mRNA and protein expression of Brm in endothelial cells.These results indicate that transcription activation is an important mechanism of hypoxia up-regulating Brm expression in endothelial cells.2.Hypoxia could increase in the combination ability of HIF-1α and Brm promoter in endothelial cells,and promote the transcription and expression of Brm.It indicates that HIF-1α plays an important role in hypoxia induced up-regulation of the expression of Brm.3.The transcription activation of Brm is also affected by the level of H3K4me3 around its promoter region.Besides,hypoxia could increase the level of H3K4me3 in the promotor region of Brm,and futher regulate the transcription and translation of Brm.Increased H3K4me3 is also essential to the regulation of Brm by hypoxia.4.More studies are warranted to further clarify if there exists cooperation of HIF-1α and histone methyltransferase in regulating Brm during hypoxia.