Abstract
Purpose :
Interleukin 6 (IL-6) plays a role in the endothelial barrier dysfunction seen in diabetic retinopathy (DR). Our recent studies have shown that the primary IL-6 signaling modality in retinal endothelial cells is IL-6 “trans-signaling”, which utilizes a soluble form of the IL-6 receptor (sIL-6R). The molecular mechanisms underlying this dysfunction, however, have not been fully elucidated. Adherens junctions play a critical role in the formation of cellular barriers, and the purpose of this study is to evaluate the contribution of adherens junction components to IL-6 trans-signaling induced barrier dysfunction.
Methods :
Expression of VE-cadherin, β-catenin, α-catenin, and p120-catenin were measured in human retinal endothelial cells (HRECs) by RT-PCR following overnight treatment with IL-6 (50 ng/mL) ± sIL-6R (150 ng/mL). Protein expression was measured by western blotting and immunofluorescence (IF). β-catenin gene knockdown in HRECs was performed using siRNA, and endothelial barrier function was measured using electric cell-substrate impedance sensing (ECIS).
Results :
IL-6 trans-signaling significantly decreased expression of VE-cadherin (0.7-fold) and β-catenin (0.6-fold), but not α-catenin or p120-catenin. IF showed that β-catenin was primarily junctional, and trans-signaling led to gaps in membrane coverage. Activation of IL-6 trans-signaling significantly decreased endothelial barrier function (0.76-fold vs. untreated). β-catenin knockdown with siRNA resulted in a baseline decrease in barrier function (0.85-fold vs. wildtype), and IL-6 trans-signaling activation further reduced barrier function (0.73-fold vs. wildtype untreated), suggesting that β-catenin loss can partially explain IL-6 trans-signaling induced barrier dysfunction. Furthermore, β-catenin knockdown did not alter expression of β-catenin-regulated genes involved in tight junctions and the formation of capillary fenestrae. IL-6 trans-signaling, however, induced changes in expression of several permeability-associated genes, including CLDN1 (0.28-fold), CLDN3 (0.58-fold), OCLN (0.69-fold), and PLVAP (1.4-fold).
Conclusions :
Our findings suggest that loss of β-catenin and adherens junctions partially explain the mechanism of IL-6 trans-signaling-induced endothelial barrier dysfunction and provide further support for the role of IL-6 trans-signaling in the pathology of diabetic retinopathy.
This is a 2021 ARVO Annual Meeting abstract.