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Sindhu Saraswathy, Thania Bogarin, Francis Brian, James C H Tan, Robert N Weinreb, Alex Huang; TGF-beta Pathway Alternations in High and Low Flow Trabecular Meshwork Regions as Defined by Aqueous Angiography in Human Eyes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4709. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate for differences in TGF-beta pathway and related proteins in high and low flow trabecular meshwork (TM) regions using aqueous angiography, conducted in post-mortem human eyes. Previously, we had shown elevated alpha-smooth muscle actin and versican extracellular matrix protein in low flow, compared to high flow, TM regions.
Post-mortem human eyes (n=6) (One Legacy Eye Bank) without a history of glaucoma were obtained within 48 hours of death. The eyes were perfused with Balanced Salt Solution (BSS) followed by tracer (fluorescein [2.5%] or indocyanine green [0.4%]) diluted in BSS at ~10 mm Hg. After angiographic imaging with the Spectralis HRA+OCT (Heidelberg Engineering), regions of peri-limbal low and high angiographic flow were marked. Eyes were fixed with 4% paraformaldehyde or prepared for Western blot (WB). Wedges including the angle were cut from angiographically positive and negative regions, paraffin embedded, and processed for sectioning. The sections were immunostained and viewed under a Keyence BZ-X700 digital imaging microscope (IF). Separately, the TM near positive and negative angiographic regions were dissected for WB. For WB, antibodies were used against TGF beta-2, TGF beta receptor, and fibronectin with GAPDH as control. For IF, antibodies against TSP-1 and fibronectin-EDA were used.
There was increased TGF beta-2 (n=3, 1.6-fold p=0.01) and a trend for increased TGF beta receptor protein (n=3, 1.5-fold; p=0.07) expression in low flow compared to high flow TM by WB. Increased TSP-1 expression was seen by IF in low flow TM (n=4, 3-fold increase; p<0.01). Even though total fibronectin was similar in both low and high flow TM by WB, increased expression of fibronectin-EDA was elevated in low flow compared to high flow TM by IF (n=4, 2.3-fold increase; p<0.01).
TM near low flow angiographic outflow displayed increased TGF-beta activity measured by increased ligand pool, receptor (trend), regulator (TSP-1) and a downstream fibrosis-related fibronectin isoform (fibronectin-EDA). These findings, in addition to prior versican and alpha-smooth muscle results, suggest that high TGF-beta pathway activity is associated with low flow TM.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Fig.1: Increased expression of EDA-FN (2.3 fold) in the Low flow regions of TM compared to High flow.
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