Elevated intraocular pressure induces Rho GTPase mediated contractile signaling in the trabecular meshwork

Abstract

Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4-5 h at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway.

Keywords: Intraocular pressure; Mechanotransduction; Outflow resistance; Rho GTPase; Trabecular meshwork.

Fig. 1

Elevated IOP in enucleated porcine eyes increases aqueous outflow rate and decreases aqueous outflow facility. A and B shows percent change in aqueous outflow rate and aqueous outflow facility from baseline in eyes perfused at 50 and 15 mm Hg, respectively. The eyes perfused at 50 mm Hg showed a significant increase in aqueous outflow rate (µl/min) from the base line value compared to eyes perfused at 15 mm Hg (A). On the other hand, the aqueous outflow facility (µl/min/mm Hg) was found to be decreased significantly from base line facility in eyes perfused at 50 mm Hg compared with eyes perfused at 15 mm Hg (B). Values represent mean ± standard error. n = 7, *p < 0.05, **p < 0.01. C. Porcine eyes perfused at 50 mm Hg for 5 hours revealed collapsed aqueous plexi (AP) and disorganized/compressed TM compared with eyes perfused at 15 mm Hg, based on histological evaluation by transmission electron microscopy. Bar indicates 10µm image magnification.

Fig. 2

Activation of Rho GTPase regulated contractile activity by elevated IOP in the trabecular meshwork of porcine eyes. A. TM derived from eyes perfused under elevated pressure (50 mm Hg) showed significantly increased levels of activated RhoA (RhoA-GTP form) compared to TM derived from eyes perfused at basal pressure (15 mm Hg). Levels of activated RhoA were normalized to total RhoA protein levels. B. TM tissue derived from eyes perfused at 50 mm Hg showed a significant increase in levels of phosphorylated-MLC, MYPT1, MARCKS and Paxillin compared to TM tissue from eyes perfused at 15 mm Hg. pMLC immunoblots were normalized to the total MLC loading control, while other immunoblot data were normalized to the β-actin loading control. Histograms depict values derived from densitometric analysis of immunoblots. Values represent mean± Standard deviation. n = 3, *p < 0.05.