Abstract
Purpose :
The optic nerve head (ONH) shows the earliest signs of damage during glaucoma. An imbalance of extracellular matrix (ECM) protein formation and degradation in the human lamina cribrosa (LC) region leads to retinal ganglion cell (RGC) axon damage and eventual apoptosis. ONH astrocytes, LC cells, and microglia have been implicated in glaucomatous ONH damage, with early microglia activation releasing damage associated molecular patterns (DAMPs) that can activate toll-like receptor 4 (TLR4). Current literature shows significant crosstalk between TLR4 and TGFß2 signaling in the development of fibrogenesis and TGFß2 is known to be increased in the glaucomatous ONH. From this, we hypothesize that endogenous DAMPS activate TLR4 and augment TGFß2 signaling, increasing ECM production and pathogenic paracrine signaling between cells of the ONH.
Methods :
Normal and glaucomatous human donor eyes were received from the Lions Eye Banks of WI, fixed, cryo-embedded, and processed for immunohistochemistry. Primary ONH LC cells were isolated from fresh normal human donor eyes, cultured, and characterized by western blot and ICC. Immortalized microglia were purchased from ATCC (CRL-3304). Cells were treated with a TLR4 selective inhibitor TAK-242 in the presence or absence of TGFβ2, LPS, or cFN for 48 hours and changes to protein production was assessed by western blot and immunocytochemistry.
Results :
The ECM protein fibronectin (FN), as well as the endogenous DAMP fibronectin extra domain A (FN-EDA) were elevated in the LC region of glaucomatous (N=3) human donor eyes compared to normal controls (N=3). Primary human LC cells expressed alpha-SMA and were negative for astrocytic marker GFAP. Treatment with TGFβ2 elevated FN expression and the TLR4 inhibitor, TAK-242, blocked TGFβ2 induced FN expression (p<0.05). Immortalized microglia (ATCC CRL-3304) exposed to the TLR4 activator LPS in the presence or absence of TGFβ2 increased FN and Laminin protein expression by western blot and ICC.
Conclusions :
These data suggest that multiple cell types within the ONH show significant crosstalk between TLR4 and TGFβ2 signaling during the development of fibrogenesis in the LC region.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.