Orthovanadate Alters The Cytoskeleton Of Trabecular Meshwork Cells And Increases Outflow Facility In Live Monkeys

Abstract

Purpose: : Vanadate compounds inhibit protein–phosphotyrosine phosphatases (PPTPase) which help regulate the actin cytoskeleton and cell adhesions. Vanadate is reported to reduce intraocular pressure (IOP) by affecting aqueous secretion, but whether it affects outflow facility (OF) is unclear.

Methods: : The effect of sodium orthovanadate (Na₃VO₄) on OF and IOP was studied in live cynomolgus monkeys. Total OF was measured by 2–level constant pressure perfusion of the anterior chamber (AC) before and for 1.5 hours after exchange with 1mM Na₃VO₄, or vehicle, followed by continuous AC infusion of corresponding drug/vehicle solution in opposite eyes. Topical 1% Na₃VO₄ (55mM) was given daily (4x25microL drops) for 4 days. Goldmann IOP measured over 6 hours on days 1 and 4 was compared between treatment and control groups, with analysis expressed as the mean±s.e.m. and range of differences of IOP over the entire 6–hour measurement periods. The effect of Na₃VO₄ on cultured trabecular meshwork (TM) cells was examined by immunofluorescence microscopy.

Results: : Na₃VO₄ increased OF by 29.3±8.8% (mean±s.e.m) over the 1.5–hour perfusion interval when adjusted for baseline and contralateral eye washout (p=0.01; n=12). On day 1, baseline IOP in pre–treated eyes was 16.2±1.5mmHg and in control eyes was 15.9±1.3mmHg. Following treatment on day 1, IOP was no different between treated eyes (16.6±0.2mmHg) and control eyes (17.4±0.3mmHg). On day 4, IOP was lower in treated eyes (13.5±0.8mmHg) than control eyes (16.1±0.2mmHg; p=0.009; range of differences: 2.0–5.3mmHg). IOP in the same treated eyes was lower on day 4 than on day 1 (p=0.0000; range of reduction: 3.0–6.5mmHg). In vitro, 1mM Na₃VO₄ disrupted the actin cytoskeleton and cells separated within 2 hours. Vinculin staining at focal adhesions was reduced. Cells reformed confluent monolayers within 24 hours of replacing Na₃VO₄–containing medium with control medium.

Conclusions: : Na₃VO₄ increases OF at least modestly and reduces IOP in monkeys. Increased OF appears to be mediated by PPTPase inhibition which reversibly alters the actin cytoskeleton and cell adhesions of TM cells.

Keywords: cytoskeleton • outflow: trabecular meshwork • trabecular meshwork