Suppression of Integrin-Linked Kinase Inhibits SPARC’s Effect on Extracellular Matrix and Intraocular Pressure

Abstract

Purpose : Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein shown to regulate intraocular pressure (IOP). Integrin-Linked Kinase (ILK) is a membrane-bound kinase that interacts with SPARC and affects the extracellular matrix (ECM) and actin cytoskeleton. We overexpressed SPARC and then inhibited ILK in different models to investigate the pathway of how SPARC regulates ECM proteins in the trabecular meshwork (TM). We hypothesize that SPARC mediates its effect through ILK.

Methods : Primary human TM cells, perfused human cadaveric anterior segments, and live mice eyes were treated with adenovirus carrying cDNA of human SPARC (Ad.SPARC) to overexpress SPARC and lentivirus carrying shRNA targeting human ILK (shILK) to inhibit ILK. IOP measurements were taken on mice eyes and human anterior segments. ECM proteins were analyzed by immunoblotting and immunohistochemistry. Statistical analysis was performed in Prism.

Results : SPARC overexpression in mice increased IOP compared to baseline by 1.61±0.94mmHg (p=0.047, n=10). Subsequent shRNA-mediated ILK inhibition reduced IOP by 3.51±0.92mmHg (p=0.009, n=8) compared to SPARC overexpression. In human anterior segments, SPARC overexpression elevated IOP 2.10±0.25fold (p=0.021, n=4), however, addition of shILK attenuated SPARC’s effect and decreased IOP 0.61±0.31fold (p=0.015, n=4). In human TM cells, SPARC overexpression increased levels 3.2±2.5fold (p=0.029, n=12) and also induced elevated laminin 1.45±0.37fold (p=0.004, n=10) compared to controls. ILK inhibition reduced levels 0.54±0.17fold (p<0.001, n=12) and also reduced levels of collagen I 0.60±0.17fold (p<0.001, n=8) and collagen VI 0.67±0.17fold (p<0.001, n=8).

Conclusions : ILK inhibition by shRNA attenuated the effects of SPARC overexpression on IOP and ECM proteins, implicating ILK signaling as a major molecular pathway of SPARC-mediated regulation of ECM homeostasis in TM. ILK is important in the downstream cascade of SPARC.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Mouse IOP measurements with injection of Ad.SPARC and either a shRNA targeting ILK (“shILK”, n=8) or control (“shCtrl”, n=10). The contralateral eye served as uninjected controls. (**) Represents p<0.01, comparing Ad.SPARC shCtrl and Ad.SPARC shILK. (#) Represents p<0.05, comparing No Treatment and Ad.SPARC shCtrl.

 

ECM protein fold changes with injection of Ad.SPARC and shILK compared to virus control “Ad.Ctrl shCtrl” in human TM cells.