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Danielle Desjardins, Yueying Liu, Jie Fan, Craig E Crosson, Zsolt Ablonczy; Glycated-albumin induced RPE barrier breakdown is mediated by HDAC6. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Macular edema is a major cause of vision loss due to diabetes. Though edema is often tied to endothelial dysfunction of the inner blood-retina barrier, recent studies show that dysfunction of the retinal pigment epithelium (RPE), which comprises the outer blood-retina barrier (BRB), also plays an important role in this disease. In this study, we investigate the role histone deacetylases (HDACs) play in advanced glycation-end product induced disruption of the RPE barrier.
Monolayers of ARPE-19 or human fetal RPE (hfRPE) cells were treated with bovine albumin (BSA) or glycated bovine albumin (gBSA) (100 µg/mL) in the presence of vehicle (DMSO, 0.01%), Trichostatin A (TSA; pan-HDAC inhibitor; 100 nM), or Tubastatin A (TubA; HDAC6 inhibitor; 1 µM). Transepithelial electrical resistance (TEER) was measured at 0h and 6h post administration. Lysates were collected and immunoblotted for acetylated-α-tubulin, and HDAC activity was assessed using the fluorophore-conjugated synthetic substrate Boc-Lys(Ac)-AMC.
Administration of gBSA resulted in significant reduction in TEER compared to controls in both hfRPE (24.8±0.7%) and ARPE19 (11.9±1.9 %). In cultures treated with TSA or TubA, the addition of gBSA did not significantly alter TEER in ARPE19 or hfRPE cells. In ARPE19 cells the addition of gBSA produced a significant (34±11 %) decrease in acetylated-α-tubulin, 6h post-treatment, compared to the same concentration of BSA. This decrease in acetylated- α-tubulin was associated with a 1.8±0.6 fold increase in HDAC6 activity after 6h exposure to gBSA. Treatment with TSA or TubA increased acetylated-α-tubulin levels by 2.4±1.3 fold and 7.9±0.6 fold, respectively and reduced HDAC6 activity levels below levels of detection.
Inhibition of HDAC6 suppressed gBSA-induced RPE barrier breakdown. Breakdown was linked to decreased acetylation of α-tubulin. As the acetylation of α-tubulin increases microtubule stability, these results provide initial evidence that increasing stability of microtubules may prove useful in the treatment of retinal edema. However, the change in acetylation status of other proteins may also contribute to suppressing the RPE’s response to gBSA.
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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