Abstract
Purpose :
The pathophysiology of glaucoma is multifactorial,however,oxidative stress and biomechanical stress are considered crucial for the initiation of fibrotic changes in the anterior segment and neurodegeneration of the retina and optic nerve.The purpose of this Study was to quantify clinically-relevant biomarkers associated with pathology.
Methods :
Primary human TM obtained from healthy human donors (Eversight) and ARPE-19 cells (ATCC) were cultured and seeded in collagen I-coated BioFlex® plates.Once confluent, TM and ARPE-19 cells were transferred to serum-free and serum-starved conditions,respectively, and exposed to mechanical strain (10% static strain) in the presence or absence of recombinant TGFB2 (5 ng/ml) for 48h and tert-butylhydroperoxide (tBHP) and glucose.The supernatant was collected and secreted proteins were concentrated.Osteopontin (OPN) was quantified by ELISA.Total RNA was extracted from cell lysates, and cDNA was prepared.Gene expression levels of osteopontin (SPP1),matrix metalloproteinase 9(MMP9),tumor necrosis factor alpha,vascular endothelial growth factor A,TGFB1 and TGFB2 were quantified by quantitative polymerase chain reaction (qPCR).
Results :
Human TM cells secreted baseline levels of OPN, which increased ~5-fold in response to recombinant TGFB2 treatment (n=3, P<0.01).The combination of mechanical strain and TGFB2 treatment resulted in an >350-fold increase in OPN secretion (n=3; P<0.01).In ARPE-19 cells,the combination of mechanical strain and pathophysiologically-relevant stressors (tBHP and Glucose) resulted in~20-fold decrease in OPN gene expression (n=3; P<0.01).In addition,mechanical strain significantly increased OPN and MMP9 gene expression (2-fold, n=3; P<0.01).
Conclusions :
TGFB2 exposure significantly increased OPN levels in TM cells,which was exacerbated by simultaneous exposure to mechanical strain.Mechanical strain exacerbates the fibrotic effects of recombinant TGFB2, likely due to upregulation of TGFB1 and TGFB2 gene expression.Mechanical strain exposure to ARPE-19 cells resulted in significantly increase of OPN and MMP9 expression,which were significantly decreased by simultaneous exposure to tBHP and glucose.Our data highlight the complex signaling crosstalk in ocular tissues and the need to identify tissue-specific biomarkers for glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.