Abstract
Purpose :
In primary open angle glaucoma (POAG), the trabecular meshwork (TM) has been found to have decreased levels of cytosolic/plasma membrane phosphatidylserine (PS) and the ATPase phospholipid transporting 8B2 (ATP8B2) compared to controls. We hypothesize that PS transport is impaired in the POAG TM, and that the flippase ATP8B2 is specifically responsible for the transport of PS in the TM.
Methods :
Primary TM cells were derived from normal cadaveric eyes (n=20) with equal gender distribution. All collections followed the tenets of the Declaration of Helsinki. To evaluate PS transport, TM cells were incubated with nitrobenzoxadiazole (NBD)-PS (excitation=433 nm, emission=540 nm) and imaged with a confocal microscope. This transport method with suitable modifications was also adapted for POAG TM tissues (n=20) and control TM tissues (n=20) with equal gender distribution. POAG and control TM tissue ages were 75±5.5 and 68±4.5 years respectively. Knockout (KO) of the ATP8B2 gene was performed with a CRISPR/Cas9 lentivector in primary TM cells. To complement this experiment, artificial unilamellar vesicles embedded with ATP8B2 and control unilamellar vesicles were incubated with PS. Bligh and Dyer lipid extraction was performed on ATP8B2 KO TM cells, control TM cells, ATP8B2 embedded unilamellar vesicles, and control unilamellar vesicles. The extracted lipids were analyzed with TSQ Quantum Access MAX Triple Quadruple Mass Spectrometer with EquisplashTM internal standard to assay the specificity of ATP8B2. Statistical analysis was performed with a two-way ANOVA test.
Results :
The kinetics of NBD-PS transport from the extracellular matrix into primary TM cells occurred over the course of 15 minutes. These kinetics were consistent in control TM tissue, but occurred at a 3-5-fold slower rate in POAG TM tissue. A repertoire of PS lipids (acyl chain length 16-22) exhibited reduced transport in ATP8B2 KO TM cells compared to controls. Unilamellar vesicles embedded with ATP8B2 exhibited increased PS transport compared to controls.
Conclusions :
Our results are consistent with the occurrence of impaired PS transport in the POAG TM, and also with the transport of PS by ATP8B2 in the TM. These findings are consistent with the finding of decreased levels of ATP8B2 in the POAG TM, and could indicate that the decreased levels of ATP8B2 could be contributing to impaired PS transport in the POAG TM.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.