Overexpression of endothelin-1 elicits phenotypes of reactive astrocytosis and elastinopathy in primary optic nerve astrocytes

Abstract

Purpose : Elevated levels of the vasoactive peptide endothelin-1 (ET-1 ) are associated with the pathophysiology of various subtypes of glaucomatous optic neuropathy. Here we sought to investigate the effects of ET-1 overexpression in primary optic nerve head astrocytes (ONHA).

Methods : Primary rat ONHA were transduced with lentivirus encoding either ET-1 or Flag-V5 (control) to generate polyclonal ONHA cultures. Overexpression of Flag-V5 and ET-1 was confirmed by immunoblotting after selection with 3.5 µg/ml puromycin. Morphology was assessed by brightfield microscopy and rate of proliferation was quantified by Trypan blue cell viability assay. Cells were seeded in 96-well plates and exposed to a dose range of tert-butyl hydroperoxide (tBHP; 10 – 500 µM) for 4 h in the presence or absence of the prototypic antioxidant, Trolox (100 µM) and cell viability was quantified by lactate dehydrogenase (LDH) release assay. Cell lysates for processed for immunoblotting and expression of lysyl oxidase like-1 (LOXL1) and elastin were quantified.

Results : ET-1 overexpressing ONHA exhibited a less-differentiated morphology and significantly increased doubling times, compared with control or non-transduced ONHA. Exposure to oxidative stress resulted in a statistically significant sensitization to tBHP; EC₅₀ values for tBHP were 389.7 µM vs. 317.7 µM for control and ET-1 groups, respectively (n=6, p<0.001). Trolox completely prevented loss of cell viability even at 500 µM tBHP. Protein expression analysis revealed a statistically significant reduction of LOXL1 (48%, n=3; p<0.05) and elastin (62%; n=3; p<0.05).

Conclusions : Our results support increased ET-1 expression as a mediator of reactive astrocytosis in ONHA. Consistent with our previous findings, ONHA undergoing reactive astrocytosis exhibit increased levels of oxidative stress and signatures of elastinopathy associated with decreased LOXL1 and elastin expression. Stably ET-1 overexpressing ONHA may prove a useful model for mechanistic and drug discovery studies in glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.