DataSheet_1_Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells.pdf

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFβ ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-β) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

糖尿病视网膜病变（DR）的发病机制尚未完全明了。我们先前已证实，在实验性糖尿病及糖尿病人类受试者的视网膜中，视网膜骨形态发生蛋白-2（BMP2）的表达上调。本研究的目的是探讨非典型炎症通路在BMP2诱导的视网膜内皮细胞（REC）屏障功能障碍中的作用。为此，我们采用实时定量PCR和蛋白质印迹技术，评估了培养的人视网膜内皮细胞（HRECs）在BMP2（50ng/ml）作用下最多24小时内的BMP2信号传导组分（BMP2、BMP4、BMP受体）、VEGF、磷酸化p38 MAPK和NFκB以及氧化应激标志物的水平。同时，我们还检查了高葡萄糖（HG，30mM D-葡萄糖）对HRECs中BMP2及其下游基因表达的影响。H2-DCF是一种荧光染料，用于测量细胞内活性氧（ROS）的水平，以此评估BMP2的促氧化作用。此外，我们通过电细胞底物阻抗传感（ECIS）技术测量跨内皮细胞电阻（TER），以评估抑制p38和VEGF信号对BMP2诱导的HRECs屏障功能障碍的影响。我们还在存在或不存在BMP2信号通路抑制剂的情况下，测试了HG对HRECs屏障完整性的影响。我们的数据显示，BMP2和高葡萄糖上调了BMP信号通路中的BMP组分（SMAD效应器、BMP受体以及TGF-β配体本身），并诱导p38 MAPK和NFκB的磷酸化，以及NFκB的核转位。抑制p38或NFκB可以减轻BMP2诱导的VEGF表达和HRECs屏障功能障碍。此外，VEGFR2的抑制可减轻BMP2诱导的屏障功能障碍。BMP2可诱导HRECs中ROS的产生以及内皮型一氧化氮合酶（eNOS）的表达和活性。最后，HG上调了HRECs中BMP2及其下游基因（SMAD、BMP4、ALKs和TGF-β）的表达，而BMP2抑制剂可减轻HG诱导的HRECs屏障功能障碍。我们的结果表明，除了常规的典型SMAD信号传导外，BMP2通过激活p38/NFκB通路在HRECs中诱导非典型炎症通路，这导致VEGF的上调和HRECs的破坏。抑制BMP2信号传导是保护DR中内皮细胞屏障功能的一种潜在治疗干预措施。