Abstract
Purpose :
Our goal is to determine the role of mechanotransduction in intraocular pressure (IOP) homeostasis within the Schlemm’s canal (SC). Abnormal mechanotransduction may result in elevated IOP and glaucoma. The adherens junction complex (AJC) which include VE-CADHERIN (VEC) and Src family kinases (SFK) are central players in mechanotransduction in blood vessels. We have previously shown that SC cells are a mixture of blood and lymphatic phenotypes, hence we have tested the hypothesis that the SC AJC responds to IOP elevation by phosphorylation of VEC by SFK to regulate adherens junction permeability.
Methods :
We determined the following changes in the SC in response to pressure elevation using immunofluorescence on whole mount anterior eye segments: 1) VEC phosphorylation using residue specific phospho-VEC antibodies, 2) activation of SFK using phospho tyrosine 418 SFK antibody 3) measured SC permeability using a fluorescent Alexa Fluor 594 lectin tracer. Each experiment was replicated 6 times. We elevated IOP by cannulating mouse eyes and setting one of three different pressures-16, 25, and 45 mmHg for 45 min, using a water column and pressure transducer. The eyes were then fixed in situ, while still at the set pressure, using an acrolein-based ultra-rapid fixation protocol that we have adapted for SC.
Results :
We observed a significant increase in VEC phosphorylation at tyrosine 658 with increasing IOP (in a representative experiment 1.5-fold (x) at 25 mmHg versus (vs) 16 mmHg, p=0.0002, 1.8x 45mmHg vs 25mmHg, p<0.0001 and 3x at 45mmHg vs 16mmHg, p<0.0001). We also found significant SFK activation at the AJ with increasing IOP (2.6x at 25 mmHg vs. mmHg, 1.2x 45mmHg vs. 25mmHg and 3.3x at 45mmHg vs.16mmHg, p<0.0001 for all). We observed a pressure dependent increase in lectin tracer at the AJ marked by VEC suggesting that permeability at SC AJ is increased (4.5x at 25 mmHg vs. mmHg, p=0.0002, 1.8x 45mmHg vs. 25mmHg, p<0.0001 and 8x at 45mmHg vs.16mmHg, p<0.0001). Results were similar in other replicates of all three studies.
Conclusions :
Together, our data indicates that the SC cells responded to an increase in IOP by activating SFK at the AJ with a concomitant increase in VEC phosphorylation. These phosphorylation changes correlated with increased junctional permeability. Thus, these results point to mechanotransduction signaling functioning in SC in response to IOP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.