Abnormal Angiogenesis Of Bisphenol A Analogues Via The Disruption Of TR/ER-PI3K-eNOS Signaling Pathway

Abnormal angiogenesis is a hallmark and key event of cardiovascular diseases,which is regulated by nuclear receptor pathway.It remains to be studied whether bisphenol A(BPA)analogues could induce abnormal angiogenesis through nuclear receptor signaling.This study focused on the disruption of thyroid receptor(TR)and estrogen receptor(ER)induced by BPA,bisphenol S(BPS),tetrabromobisphenol A(TBBPA)and tetrabromobisphenol S(TBBPS),revealed the effects of endothelial nitric oxide synthase(e NOS)pathway mediated by TR/ER and abnormal angiogenesis of BPA analogues.The main results and conclusions are as follows:(1)BPA analogues disturbed the conformation of TR?ligand binding domain(LBD),induced TR?antagonistic effect and thyroid dysfunction in male C57BL/6 mice.BPA analogues caused the red shift of the maximum fluorescence emission wavelength of TR?LBD and static fluorescence of the protein and larger distance between the helix H11 and H12 of TR LBD,affected the secondary structure of TR?LBD.BPA analogues had diverse TR?antagonistic activities,TBBPA and TBBPS showed stronger antagonistic activity.Exposure to BPA,BPS,TBBPA and TBBPS(2 mg/kg BW/d)caused thyroid stimulating hormone(TSH)levels increased from 24.50 pg/m L to 28.02,31.00,25.26 and 28.87 pg/m L,respectively.(2)BPA disturbed the TR/ER-phosphatidylinositol 3-kinase(PI3K)-e NOS pathway.BPA and BPS(10^-7 M,10^-6 M)increased the interaction of ER and PI3K in human umbilical vein endothelial cells(HUVECs)based on the ER agonistic effect,while TBBPA and TBBPS attenuated interaction of TR with PI3K.BPA and BPS(10^-7 M,10^-6 M)increased the phosphorylation of e NOS Ser1177 mainly through the ER/PI3K pathway,and the e NOS enzyme activity increased to 1.13-,1.07-and 1.43-,1.28-fold of the control,respectively.TBBPA and TBBPS(10^-7 M,10^-6 M)decreased the phosphorylation of e NOS Ser1177 mainly through the TR/PI3K pathway,and the e NOS activity decreased to 0.56-,0.61-and 0.58-,0.62-fold of the T3 group,respectively.(3)BPA analogues induced endothelial dysfunction and caused transcriptome aberration of mouse aorta.NO levels in mice serum were increased to 1.74-and 1.42-fold of control after exposure to BPA and BPS(20 mg/kg BW/d)for five weeks,and decreased to 0.61-fold after TBBPS exposure,indicating that TBBPS significantly reduced serum NO level in mice and induced endothelial dysfunction.BPA analogues caused aberrant expression of differentially expressed genes(DEGs)of endothelial function,it was found that BPA,BPS,TBBPA and TBBPS(2 mg/kg BW/d)induced54,82,167 and 196 DEGs related to endothelial function.(4)BPA analogues disrupted pathways related to angiogenesis and caused abnormal angiogenesis in vivo and in vitro.The enrichment analysis of GO and KEGG pathways showed that DEGs induced by BPA analogues were related to endothelial cell proliferation,migration and angiogenesis factor response.BPA and BPS significantly increased the HUVECs tube formation(10^-9 M-10^-5M),and induced the formation of vascular network of Matrigel plug,while TBBPS significantly inhibited tube formation and sprouting of aorta,suggesting that BPA analogues significantly induced abnormal angiogenesis.This study interpreted that BPA analogues induced abnormal angiogenesis via the TR/ER-PI3K-e NOS pathway,which is conducive to a more comprehensive assessment of the health risks and safety of BPA analogues.The study provided an idea for exploring the potential cardiovascular disease risk of nuclear receptor disrupting chemicals.