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Jose Miguel Gonzalez, James C H Tan; Tissue-based caldesmon silencing by naked siRNA increases F-actin in the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5653. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Caldesmon overexpression downregulates actin fiber formation in human trabecular meshwork (TM) cells and increases outflow facility in cultured anterior segments. We tested the hypotheses that (1) caldesmon RNAi appreciably inhibits TM caldesmon expression, (2) and upregulates filamentous actin (F-actin) in situ.
Human donor corneoscleral tissues containing the intact TM were sectioned and incubated with Accell On-Target Plus siRNA for human Caldesmon (CALD-1) or a non-targeting control construct in siRNA delivery buffer (Life Technologies) for 3-5 days at 37°C and 8% CO2 then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and labeled with Hoechst 33342 and Alexa 568-conjugated phalloidin (Life Technologies) or anti-caldesmon antibodies (Abcam) and Alexa 568-conjugated secondaries. Tissues were imaged by 2-photon microscopy (TPM) using 800-850nm pulsed laser excitation and specific filters: 500-550nm bandpass (green; nuclei and structural autofluorescence) and 610-680nm (red; F-actin and caldesmon). Tissue viability was assessed by calcein AM and propidium iodide (PI) intravital co-labeling in situ.
1μM naked siRNA silencing of caldesmon reduced caldesmon immunolabeling in the human TM by 53.9±38.9% (p=0.03; n=5) relative to non-targeting control tissue. In situ redistribution of F-actin occurred following caldesmon RNAi as evidenced by enhanced F-actin cortical localization and a more prominent actin network relative to controls. F-actin fluorescence levels were increased by 61.0±46.8% with siRNA treatment (p=0.07; n=3). Structural matrix organization of collagen and elastin (via autofluorescence), cell counts, and viability were unchanged after caldesmon RNAi. 86.1% of cells were viable in siRNA-treated tissue compared with 85.8% in non-targeting controls.
Tissue-based caldesmon RNAi resulted in lower caldesmon expression, a redistribution of F-actin, and a more prominent actin network in the human TM. The TM actin reorganization represents increased actin polymerization and actomyosin contractility that is expected to affect aqueous outflow dynamics. We are now conducting in vivo studies to determine the effect of caldesmon RNAi on live animal outflow facility.
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