Study Of Role And Mechanism Of Brg1and Brm Epigenetically Regulate CAMs And ET-1Activation During Hypoxic Pulmonary Hypertension

Background：Hypoxia Pulmonary Hypertension is a chronic progressive disease which caused bylow oxygen, characterized by elevated pulmonary hypertrophy and pulmonary vascularremodeling and associated with right ventricular hypertrophy. The formation of HPH is acomplex pathophysiological process. Pulmonary vasoconstriction, inflammation, cellproliferation and transformation, all of them were involved in HPH. Hypoxic pulmonaryvasoconstrictions mediated by endothelin, which secrete from vascular endothelial cells,play an important role in the development of HPH. In addition, inflammation may play animportant role in this process. Elevated inflammation is invariably initiated by theaggregation and adhesion of immune cells on the vessel wall. Pivotal to this process is theadhesion of leukocytes to the vessel wall facilitated by the up-regulation of cell adhesionmolecules (CAMs) in vascular endothelial cells. Therefore, accelerated transcription ofCAMs genes in hypoxic endothelial cells represents a key event that foretells enhancedleukocyte adhesion, perpetuated vascular inflammation, and eventually the development ofHPH. As we know，CAMs and ET-1transactivation were regulated by nuclear factor kappaB (NF-kB), hypoxia inducible factor1α (HIF-1α) and megakaryocytic leukemia1(MKL1).These transcription factors combined with specific promoter sequence under hypoxia, tostart CAMs and ET-1transcription and expression. But the epigenetic switch that dictatesCAMs and ET-1transactivation in response to hypoxia in endothelial cells leading up toHPH is not fully appreciated.Unlike the prokaryotic organisms, eukaryotic genes are wrapped by histones intoindividual nucleosomes to form chromatin that needs to be unfolded in order for the basictranscription machinery to gain access. Brahma-related gene1(Brg1) and brahma (Brm)are the catalytic components of the mammalian chromatin remodelling complex playingimportant roles in maintaining vascular homoeostasis in embryogenesis and promoting a pathogenic agenda under stress conditions. This remodelling complex utilizes ATP tomobilize nucleosomes and alter transcription. Brg1and Brm playing important roles inmaintaining vascular homeostasis in embryogenesis and promoting a pathogenic agendaunder stress conditions. Brg1and Brm were also involved in cellular hypoxia reactionwhich was mediated by HIF-1alpha. However, whether Brg1/Brm is upregulated byhypoxia stress and accelerated the CAMs and ET-1genes in hypoxic endothelial cells. Andthe transcription mechanism of CAMs and ET-1involved in HPH development is not fullyunderstood.Therefore, this study was undertaken to observe the expression levels of Brg1and Brmin isolated pulmonary arteries of HPH rats and cultured human endothelial cells. Andobserve the regulation of transactivation of CAMs and ET-1genes in endothelial cells inresponse to hypoxic stress. In addition, we constructed the endothelial-specific vector thatencodes shRNA targeting both Brg1and Brm, to test the role of Brg1and Brm in thehypoxia-induced vascular inflammation and pulmonary vascular remodeling. As such,targeting Brg1/Brm in endothelial cells may yield promising strategies in the interventionand/or prevention of HPH.Methods：1. SD rats were housed in a hypobaric hypoxia chamber (Simulated altitude:5000m)for4weeks to induce hypoxic pulmonary hypertension. Pulmonary arteries were thenisolated to measure the expression of Brg1and Brm by RT-qPCR and Western blot. HPEC、HUVEC were exposed to1%O2, the expressions of Brg1and Brm were also detected.2. Over-expression of exogenous Brg1and Brm or knocked down the expression ofendogenous Brg1and Brm using siRNA in endothelial cells. The promoter activities ofCAM genes induced by hypoxia were detected by reporter assays. The endogenous CAMsmessages induced by hypoxia were also detected by RT-qPCR and Western blot. In vitro,adhesion assay was used to detect the interaction between endothelial cells and leukocytes.To further explore the interplay between p65and Brg1/Brm in hypoxia-inducedtransactivation of CAMs genes, we knocked down the expression of p65or Brg1/Brm withsiRNA in endothelial cells. ChIP assay to detect the combination of NF-kB/p65orBrg1/Brm with CAMs promoter. We also detect the histone modifications surrounding theCAMs promoters by ChIP assay 3. Over-expression of exogenous Brg1and Brm or knocked down the expression ofendogenous Brg1and Brm using siRNA in endothelial cells. The promoter activitie of ET-1gene induced by hypoxia was detected by reporter assays. The endogenous ET-1messagesinduced by hypoxia were also detected by RT-qPCR and ELISA. ChIP assay was used todetect the combination of Brg1/Brm with ET-1promoter. The histone modificationssurrounding the ET-1promoter was also detectd by ChIP assay.4. Male C57/BL6mice were randomly divided into normoxia group, hypoxia group,control virus group and interfere virus group. The last three group were housed in ahypobaric hypoxia chamber (Simulated altitude:5000m) for4weeks to induce hypoxicpulmonary hypertension. In the meantime, these mice were injected via tail veinphysiological saline or lentiviral particles carrying a SCR or endothelial-specific vector thatencodes shRNA targeting both Brg1and Brm. Hemodynamic and pulmonarypathomorphology data were gathered. The hypertrophy of right ventricle was evaluated bythe ratio of weight of the right ventricle (RV) to the weight of the left ventricle (LV) plusventricular septal (VS) and RV weight over body weight. The remodelling of pulmonaryvasculature, as measured by vessel wall thickness, was detected by HE or IHC. Therecruitment of immune cells to the vessels was detected by IF. The activities of cytokineIL-1β、IL-6、MCP-1and TNF-α in lung tissue homogenate extracts was detected by ELISA.The combination of Brg1/Brm with ICAM-1promoter in lung tissue was also detectd byChIP assayResults：1. The levels of Brg1and Brm were significantly elevated in HPH rats as opposed toage-and sex-matched control rats. Exposure to1%O2caused an up-regulation in themessage levels of Brg1and Brm in both HUVEC and HPECs.2. Brg1and Brm were able to enhance the activation of CAMs promoters by hypoxia.Point mutations (ED, enzyme dead) that interfere with the catalytic domain deprived Brg1and Brm of their ability to activate CAMs promoters under hypoxic conditions. Moreimportantly, over-expression of Brg1and Brm led to a marked increase in the levels ofendogenous adhesion molecules. When knocked down the expression of endogenous Brg1and Brm using siRNA, induction of CAMs promoter activities by hypoxia was severelycrippled. Furthermore, the induction of endogenous CAMs messages by hypoxia was also limited in both HUVEC and HPEC. In vitro adhesion assay also revealed that theinteraction between endothelial cells and leukocytes was weakened by the elimination ofBrg1and Brm. ChIP assay indicates there was a dramatic increase in the occupancies ofBrg1and Brm on the promoter region of the CAMs genes spanning the NF-kB site underhypoxic conditions. Consistent with these observations, re-ChIP assays demonstrate thathypoxia promoted the interaction between Brg1/Brm and NF-kB/p65on the CAMspromoters. Reciprocally, depletion of Brg1or Brm negatively affected the binding of p65on all three CAMs promoters. Ablation of Brg1and Brm greatly reduced the levels of AcH3and H3K4Me3, all of which herald transcriptional activation, on the CAMs promoterregions. As a result, the recruitment of RNA polymerase II (Pol II) was hampered probablyleading to a decelerated transcription rate.3. Brg1and Brm were able to enhance the activation of ET-1promoters by hypoxia,which require the enzymatic activity. When knocked down the expression of endogenousBrg1and Brm using siRNA, induction of ET-1promoter activities by hypoxia was severelycrippled. Furthermore, the induction of endogenous ET-1messages was also limited inendothelial cells. ChIP assay indicates there was a dramatic increase in the occupancies ofBrg1and Brm on the promoter region of the ET-1gene under hypoxic conditions. Ablationof Brg1and Brm greatly reduced the levels of AcH3, H3K4Me2and H3K4Me3around theET-1promoter.4. In the mice HPH model, Immunofluorescence staining revealed that the expressionof Brg1and Brm was effectively reduced in the pulmonary endothelium by the viralparticles which contains endothelial-specific vector that encodes shRNA targeting bothBrg1and Brm. Correction of HPH by Brg1/Brm depletion was accompanied by thedown-regulation of all three CAM molecules in pulmonary arteries. As a result, elevation ofright ventricular pressure (RVSP) was significantly relieved by endothelial-specificBrg1/Brm silencing, as was as the hypertrophy of right ventricle. And, there was decreasedrecruitment of immune cells to the vessels. Furthermore, remodelling of pulmonaryvasculature, as measured by vessel wall thickness, was suppressed in mice injected withendo-siBrg1/siBrm compared with mice injected with control shRNA. Also the activities ofIL-1β, IL-6and MCP-1were inhibited remarkably. More importantly, hypoxia recruitedmore Brg1and Brm to the ICAM-1promoter in pulmonary arteries isolated from HPH mice when compared with the control mice.Conclusion：1. The expression of Brg1and Brm fluctuates in HPH rats and endothelial cells inresponse to oxygen tension indicative of a potential role for Brg1/Brm in endothelialmalfunction and pathogenesis of HPH.2. Brg1and Brm are both sufficient and necessary for hypoxia-induced CAMstransactivation and leukocyte adhesionin vitro. Brg1/Brm/p65complex forms on the CAMspromoters in response to hypoxia to activate transcription in endothelial cells. Brg1andBrm can form a crosstalk with the histone modification machinery to influencehypoxia-induced CAMs transactivation.3. Brg1and Brm are indispensable for hypoxia-induced transactivation of the ET-1gene. They can form a crosstalk with the histone modification machinery to influencehypoxia-induced ET-1transactivation.4. Endothelial-specific Brg1/Brm silencing stalled remodelling of pulmonaryvasculature and development of HPH, likely owing to the normalization of endothelialfunction and pulmonary inflammation in vivo.5. As such, targeting Brg1and Brm in endothelial cells may yield promising strategiesin the intervention and/or prevention of HPH.