Abstract
Purpose :
The blood-aqueous barrier (BAB) of the eye is a critical vascular unit that regulates the production, composition, and outflow of aqueous humor (AH). Established and maintained by proteins of tight-, adherens-, and gap-junctions, the BAB is largely localized to the ciliary process nonpigmented epithelium, endothelium of the iris vasculature, and inner wall of Schlemm’s canal. Previous studies suggest that dysfunction of the BAB within the conventional outflow pathway contributes to elevated AH outflow resistance. Here, we investigated whether endothelial-like cells of the trabecular meshwork, which are distinct from the BAB, similarly express proteins associated with tight junctions.
Methods :
Human primary TM cells were conditioned in serum-free media overnight and subsequently incubated in the absence or presence of TGF-β2 (5 ng/ml) and GAPDH-relative changes in claudin-5, occludins, JAM-1, ZO-1, Nox4, and Col1α1 mRNA expression were quantified. In some experiments, TM cells were challenged with agents previously reported to alter mRNA expression of tight junction proteins.
Results :
As we have previously reported, TGF-β2 (5 ng/ml, 24h) elicited marked increases in both Nox4 (700-fold)- and Col1α1 (10-fold)-mRNA expression, consistent with oxidative remodeling of the TM extracellular matrix. TGF-β2 alone, however, did not significantly alter endogenous mRNA expression of tight junction proteins in quiescent TM cells. By comparison, TM cells incubated in the presence of db-cAMP (102 µM) / IBMX (90 µM) exhibited a 6-fold increase in claudin-5 mRNA expression with a concomitant reduction (35%) in ZO-1 mRNA expression. In contrast, co-incubating TM cells with TGF-β2 prevented cAMP / IBMX-mediated changes in claudin-5 and ZO-1 mRNA expression while concurrently potentiating Nox4 mRNA expression.
Conclusions :
Cultured human primary TM cells can be induced to selectively increase mRNA for claudin-5, a well-established tight junction protein implicated in endothelial cell barrier function. TGF-β2 dependent changes in claudin-5 and ZO-1 mRNA expression represents a novel mechanism by which this profibrotic cytokine increases Nox4-dependent aqueous humor outflow resistance.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.