Connexin43 hemichannel block protects against retinal pigment epithelial cell barrier breakdown

Aims The retinal pigment epithelium (RPE) is an important component of the outer blood-retinal barrier (BRB) that separates the choroid from the rest of the retina. Loss of RPE-mediated BRB integrity is a key feature of diabetic macular oedema (DME), a chronic pathology resulting from diabetic retinopathy (DR). Recent studies have shown that connexin43 hemichannel opening mediates key inflammatory pathways in DR, though its effect on the barrier properties of RPE cells remains unknown. Therefore, RPE breakdown was induced by exposing a monolayer of ARPE-19 cells to high glucose (HG) and 10 ng/mL each of the pro-inflammatory cytokines IL-1 beta and TNF-alpha. The role of connexin43 hemichannels was assessed using a connexin43 hemichannel blocker, Peptide5. Methods Transepithelial resistance (TEER) and FITC-dextran dye leak across the ARPE-19 monolayer were used to measure RPE layer permeability. Immunohistochemistry was used to assess changes in connexin43, collagen IV and ZO-1 expression. ATP and lactate dehydrogenase (LDH) release were measured using commercially available kits. Results Connexin43 hemichannel block with Peptide5 prevented TEER reduction and FITC-dextran dye leak induced by a combination of HG and inflammatory cytokines. Peptide5 also blocked LDH and ATP release induced by the addition of HG and inflammatory cytokines. ZO-1 and connexin43 disruption and internalisation as well as upregulated secretion of collagen IV following HG and inflammatory cytokine exposure were also prevented. The addition of exogenous ATP into the culture medium was able to reverse Peptide5 protection against LDH release and change in connexin43 localisation, indicating that the initiating pathway in RPE disruption is connexin43 hemichannel-mediated ATP release. Conclusion These findings support the idea that connexin43 hemichannels may mediate RPE disruption (and its role within the BRB) that occurs in DME through an ATP release/inflammasome pathway activation dependent manner. Connexin43 hemichannels are therefore a potential therapeutic target for the treatment of DME.