Abstract
Purpose :
Aberrant trabecular meshwork (TM) extracellular matrix (ECM) remodeling is an important causal risk factor for ocular hypertension. The molecular consequences of remodeled ECM or their role in mediating cellular responses to pro-fibrotic cytokines in TM pathobiology is unknown. Given the well-established role of TGFβ2 signaling in POAG, we sought to determine how cell derived matrices modulate human TM (hTM) cell behavior and whether this occurs via Smad- and/or Non-Smad-dependent TGFβ2 signaling pathways.
Methods :
Primary hTM cells (n=3 donors) were cultured for 4 weeks in the absence/presence of dexamethasone to obtain vehicle control (VehM) and glaucoma-mimetic (GMM) cell derived matrices respectively. Subsequently, a fresh batch of hTM cells from the same donor was seeded on the VehM and GMM decellularized matrices in media containing 1% FBS ± 5ng/ml TGFβ2 treatment for up to 7 days. Changes in protein expression were quantified at 4 individual timepoints.
Results :
Smad pathway: In the absence of TGFβ2, Smad2 expression was increasingly elevated at 5d (~1.4 fold) and 7d (~1.65 fold, p<0.05 ANOVA), while pSmad2 was elevated at 7d (~2.45 fold, p<0.05 ANOVA) in hTM cells plated on GMM compared with VehM. In the presence of TGFβ2, Smad2/3 and pSmad2 protein levels were all downregulated (~0.5 fold, p<0.05 ANOVA) in hTM cells cultured on both VehM and GMM at all time points, although the inhibition was more pronounced for Smad3 and in cells seeded on GMM. Non-Smad pathway: In the absence of TGFβ2, pERK1/2 was elevated at all timepoints; in the presence of TGFβ2, it was increased from 1d to 5d in hTM cells on GMM. Concurrently, differential temporal expression patterns were noticed for P38, pP38, JNK2 and pJNK. In contrast, RhoA was first inhibited at 1d in the absence and presence of TGFβ2, and subsequently upregulated significantly with increasing time; although changes via GMM in the absence of TGFβ2 were greater than in its presence.
Conclusions :
These results strongly demonstrate that cell derived ECM potently modulates intrinsic signaling in hTM cells, as well as differentially regulating cellular responses to exogenous TGFβ2. Understanding the consequences of the altered ratio of Smad- to Non-Smad-dependent signaling in hTM cells will require further investigations. These results are important, since TGFβ2 levels are elevated in the aqueous humor in POAG whilst the TM tissue is being actively remodeled.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.