Abstract
Purpose :
Elevated intraocular pressure contributes to glaucomatous optic nerve degeneration by inducing mechanical strain upon the optic nerve head (ONH). ONH astrocytes (ONHA) respond to biomechanical signals through extracellular matrix (ECM) remodeling activities, promoting tissue fibrosis and consequent damage to retinal ganglion cell axons. Protein cross-linking enzymes, such as transglutaminase 2 (TGM2), are important mediators of ECM remodeling. TGM2 is known to contribute to the stiffening of connective tissues in the anterior segment of the eye during glaucoma. TGM2 may function similarly in the ONH, as TGM2 levels are elevated in ONHA after treatment with the pro-fibrotic cytokine TGFβ2, which is elevated in the ONH of glaucoma patients. In this study, we examined changes in TGM2 activity in response to a glaucomatous mechanical insult.
Methods :
Primary human ONHA strains (n=3 different donor eyes) were grown on collagen-coated synthetic rubber membranes and transfected with a small, fluorescent amine substrate of TGM2 (AlexaFluor 488-conjugated Cadaverine), and then exposed to 0-12% cyclic stretch for 24 hours using a FlexCell tension system. Live-imaging with a fluorescence microscope was used to observe transglutaminase-mediated binding of the fluorescent substrate to glutamine and lysine residues on cellular proteins. Conditioned medium and cell lysate samples were also collected after stretch for western blot analysis. Fibronectin (FN) was immunoprecipitated from conditioned media samples to probe for TGM2-mediated modifications.
Results :
In stretched ONHA, TGM2 activity was elevated by 2.4-fold (p=0.0070). Intracellular protein levels of TGM2 and FN were decreased by 2.5-fold (p=0.041) and 5.61-fold (p=0.0180), respectively. Extracellular levels of TGM2 were increased by 3.76-fold (p=0.0004), while extracellular levels of FN were unchanged. A 2.5-fold increase (p=0.0081) in the presence of a primary amine marker of TGM2-mediated protein modifications was observed on soluble, extracellular FN immunoprecipitated from the conditioned medium of stretched cells.
Conclusions :
Cyclic stretch increased TGM2 activity and extracellular TGM2 levels in human ONHA. TGM2-mediated modifications are also present on extracellular FN at higher levels in stretched ONHA. These findings support the notion that TGM2 contributes to pathological ECM remodeling of the ONH in response to glaucomatous pressure changes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.