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Khaled Hussein, Fang Wang, Ahmed S Ibrahim, Heba Mohamed Saleh, Khaled Elmasry, Nehal M El-Sherbiny, Paul Yu, Mohamed Al-Sayed Al-Shabrawey; BMP2 and 4 Contribute to Retinal Endothelial Cell Barrier Dysfunction in Diabetic Retinopathy: Role of p38 MAPK Pathway. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5448.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy (DR) is a leading cause of blindness among working-age individuals. Breakdown of the blood-retinal barrier and subsequent diabetic macular edema (DME) is the major cause of vision loss in DR. Recently, we reported, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas. In addition, we revealed the pro-inflammatory and pro-permeability effects of BMP2 on human retinal endothelial cells (HRECs). The goal of this study was to investigate the level of BMP4 during diabetes, and the underlying mechanism by which BMP2 and 4 impact the retinal endothelial cell barrier function.
Retinal level of BMP4 was evaluated in diabetic human and mouse retinas by immunofluorescence (IF) and Western Blot (WB). In vitro, the effect of BMP2 or 4 (50ng/ml) on the barrier function of HRECs was studied by assessment of the changes in transcellular electrical resistance (TER) using Electrical Cell-substrate Impedance Sensing (ECIS) and the changes in the tight junction proteins (TJPs); ZO1 and Claudin5 by WB and IF. To investigate the underlying mechanism of BMPs-mediated changes in HREC barrier function, we measured the activity of MMP2 and 9 using ELISA, nuclear translocation of NF-κB and the phosphorylation of p38 MAPK using WB. Furthermore, we assessed the effect of inhibition of BMP receptors (ALK2 and ALK3 using 100nM of LDN193189 and LDN212854) or p38 MAPK (using 10µM of SB202190) on BMP2/4-induced barrier dysfunction of HRECs.
Similar to BMP2, BMP4 was upregulated in diabetic human and mouse retinas. In addition, BMP2 and 4 disrupted the barrier function of HRECs as documented by significant decrease in TER and dysregulation of ZO-1. There was also a significant increase in the activity of MMP2 and 9 in conditionned media of HRECs treated with BMP2 or BMP4 for 48 hours. Moreover, phosphorylation of p38 MAPK and nuclear translocation of NF-κB were evident after 10 mins of BMP2 or 4 treatement. Inhibition of ALK3 (using LDN193189 but not LDN212854), as well as, p38 (using SB202190) significantly attenuated the permeability effect of BMP2 and 4.
BMP2 and 4 contribute to retinal endothelial cell barrier dysfunction in DR via signaling pathway that includes activation of ALK3, p38MAPK, NF-κB and MMP2/9. Thus, BMP2/4 signaling system is a potential therapeutic target to prevent the development of DME.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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