Abstract
Purpose :
Cross-linked actin networks (CLANs) are a type of F-actin structure frequently found in glaucomatous trabecular meshwork (TM) cells and tissues. Also, TM cells form more CLANs after being treated with glaucoma-associated agents including TGFβ2 and glucocorticoids. Our recent studies showed that CLANs increase TM cell stiffness, decrease actin dynamics, and inhibit phagocytosis, which are very likely to contribute to elevated intraocular pressure. We previously showed that overexpression of a subset of calcium binding proteins increased CLANs formation. In this study, we determined the effect of gene knockdown of these proteins in primary human TM (pHTM) cells.
Methods :
Primary HTM cells were first transfected with different siRNAs against caldesmon, calponin, tropomyosin and/or myosin light chain overnight. Non-targeting (NT) siRNA was used as a control. After siRNA transfection, the cells were treated with 5ng/ml TGFβ2 to induce CLAN formation for 6-7 days. At the end of treatment, the cells were fixed for immunostained with phalloidin-Alexa-568 and DAPI. The CLAN-positive cells (CPCs) over total number of cells per well was calculated and one-way ANOVA was used for analysis.
Results :
The percentage of CPCs in the NT-siRNA control group was 13.5% and 21.5% in two different cell strains, respectively (N=4 for each strain). Knocking down of caldesmon, calponin, myosin light chain, or all four proteins (combo) showed a trend of inhibition of CLAN formation but it was not significant (likely due to small Ns). In contrast, the CPCs almost doubled in the tropomyosin knockdown group (24.6% and 45.9%, respectively; P<0.05 for both strains).
Conclusions :
Our previous gain-of-function study showed that overexpression of tropomyosin did not affect CLANs. In this loss-of-function study, we found that tropomyosin knockdown enhanced CLANs. Therefore, tropomyosin is likely to be necessary, but not sufficient, for CLANs formation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.