Abstract
Tissue plasminogen activator, a serine protease encoded by the PLAT gene is present in the trabecular meshwork (TM) and other ocular tissues and has been reported to be downregulated by treatment with steroids in vitro. Steroids are known to cause changes in outflow facility of aqueous humor in many species. In the present study, we tested whether overexpression of PLAT can prevent and/or reverse the outflow facility of mouse eyes treated with steroids. Animals received bilateral injection with 20 µl of triamcinolone acetonide (TA) (40mg/ml) suspension subconjunctivally to induce outflow facility changes. Some animals received unilateral intracameral injection with 2 µl of adenoviral suspension [3-4x1012 virus genomes per milliliter (vg/ml)] carrying sheep PLAT cDNA (AdPLAT) either concurrently with TA injection or one week after TA injection, whereas others received bilateral intracameral injection with 2µl of adenoviral suspension (9x1012 vg/ml) carrying no transgene (AdNull) concurrently with TA injection. Animals were sacrificed one week after AdPLAT or AdNull treatment. Endogenous mRNA expression levels of mouse PAI-1 and MMP-2, -9 and -13 were also measured using qRT-PCR. Outflow facility one week after AdPLAT administration was increased by 60% and 63% respectively for animals that had not or had been pretreated with steroids. Overexpression of PLAT significantly upregulated expression of PAI-1, MMP-2, -9 and -13 compared to the levels found in TA only treated eyes. These findings suggest that overexpression of PLAT in TM of mouse eyes can both prevent and reverse the decrease in outflow facility caused by steroid treatment and is associated with upregulation of MMPs.
Highlights
Tissue plasminogen activator is a serine protease, localized in the uveal microvasculature, corneal endothelium, corneal epithelium and the trabecular meshwork (TM) of the eye [1]. tPA enzymatic activity has been measured in aqueous humor (AH) in dog, calf, monkey [2] and human eyes [3]
PLAT expression in AdPLAT injected eyes To verify PLAT expression, mCherry expression was visualized on flat-mounted TM immediately after outflow facility determination. mCherry expression was limited to the TM and distributed uniformly along the entire length of the TM
To further confirm PLAT expression and to determine whether expression occurs in eyes without significant visible mCherry expression, qPCR was performed on RNA from TM tissue
Summary
Tissue plasminogen activator (tPA) is a serine protease, localized in the uveal microvasculature, corneal endothelium, corneal epithelium and the trabecular meshwork (TM) of the eye [1]. tPA enzymatic activity has been measured in aqueous humor (AH) in dog, calf, monkey [2] and human eyes [3]. TPA enzymatic activity has been measured in aqueous humor (AH) in dog, calf, monkey [2] and human eyes [3]. It catalyzes the conversion of plasminogen (an inactive proenzyme of the fibrinolytic system), to plasmin, a protease involved in the degradation of a variety of proteins. It has been suggested that these enzymes are necessary to maintain normal (AH) outflow Decrease in these enzymes could result in the accumulation of ECM components in the TM leading to alteration in aqueous outflow [5,6]. Proper balance between tPA and its inhibitors is critical for homeostatic maintenance
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