Microtubule Stabilization Protects Hypothermia-Induced Damage to the Cytoskeleton and Barrier Integrity of the Corneal Endothelial Cells

To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyos...

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Veröffentlicht in:Journal of ocular pharmacology and therapeutics 2021-09, Vol.37 (7), p.399-411
Hauptverfasser: Thanuja, Marasarakottige Y, Suma, Bangalore S, Dinesh, Divyasree, Ranganath, Sudhir H, Srinivas, Sangly P
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container_end_page 411
container_issue 7
container_start_page 399
container_title Journal of ocular pharmacology and therapeutics
container_volume 37
creator Thanuja, Marasarakottige Y
Suma, Bangalore S
Dinesh, Divyasree
Ranganath, Sudhir H
Srinivas, Sangly P
description To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyosin ring (PAMR), and ZO-1 was assessed by immunocytochemistry with and without pretreatment with a microtubule-stabilizing agent (Epothilone B; EpoB; 100 nM) and a p38 MAP kinase inhibitor (SB-203580; 20 μM). In addition, EpoB-loaded PLGA nanoparticles (ENPs) prepared by nanoprecipitation technique and coated with poly-L-lysine (PLL-ENPs) were administered one-time for sustained intracellular delivery of EpoB. Exposure to hypothermia led to microtubule disassembly concomitant with the destruction of PAMR and the displacement of ZO-1 at the cellular periphery, suggesting a loss in barrier integrity. These adverse effects were attenuated by pretreatment with EpoB or SB-203580. PLL-ENPs possessed a zeta potential of ∼26 mV and a size of ∼110 nm. Drug loading and entrapment efficiency were 5% (w/w) and ∼87%, respectively, and PLL-ENPs showed a biphasic release : burst phase (1 day), followed by a sustained phase (∼4 weeks). Pretreatment with PLL-ENPs (0.4 mg/mL) for 24 h stabilized the microtubules and opposed the hypothermia-induced damage to PAMR and the redistribution of ZO-1. Hypothermia induces microtubule disassembly via activation of p38 MAP kinase and subsequently breaks down the barrier function of the endothelium. Sustained intracellular delivery of EpoB using nanoparticles has the potential to overcome endothelial barrier failure during prolonged cold storage of donor cornea.
doi_str_mv 10.1089/jop.2021.0036
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subjects Animals
Cells, Cultured
Cytoskeleton - metabolism
Endothelium, Corneal - metabolism
Epothilones - chemistry
Epothilones - metabolism
Hypothermia - metabolism
Microtubules - metabolism
Nanoparticles - chemistry
Nanoparticles - metabolism
p38 Mitogen-Activated Protein Kinases - metabolism
Polylactic Acid-Polyglycolic Acid Copolymer - chemistry
Polylactic Acid-Polyglycolic Acid Copolymer - metabolism
Swine
title Microtubule Stabilization Protects Hypothermia-Induced Damage to the Cytoskeleton and Barrier Integrity of the Corneal Endothelial Cells
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