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Michael Lucio De Ieso, Mark McClellan, Iris D Navarro, Michael H Elliott, Daniel Stamer; Caveolin-1 mediates response of trabecular meshwork cells to mechanical stretch via Rho/ROCK signaling pathway.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2716.
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© ARVO (1962-2015); The Authors (2016-present)
The primary risk factor for primary open angle glaucoma (POAG) is elevated intraocular pressure (IOP). Polymorphisms at the Cav1/2 gene loci have been implicated in POAG, however the role of Cav1/2 in POAG is not fully understood. Caveolae are specialized cellular domains that form “cup-shaped” invaginations in the membrane, and caveolin-1 (Cav1) is required for caveolae biosynthesis. Transient knockdown of Cav1 (and subsequently caveolae) in the trabecular meshwork (TM) activates downstream effectors of the Rho/ROCK signaling pathway, which are known to regulate conventional outflow and thus IOP. We hypothesized that caveolae serve as mechanosensors in the TM, which respond to changes in IOP by modulating the Rho/ROCK signaling pathway.
Three independent human TM cell strains were isolated from donor eyes and used for all data sets. Adenoviruses encoding shRNA targeted to Cav1 were used to silence expression. Western blotting was used to determine relative protein levels. For cyclic stretch experiments, TM cells were plated on type IV collagen-coated flexible silicone bottom plates and subjected to 20% stretch, 1 Hz for 24 h. Data are expressed as mean ± SEM.
Compared to scrambled-shRNA, Cav1-shRNA-transduced TM cells resulted in 55% knockdown of Cav1 (100±5.8% vs. 45±4.5%, n=14, p<0.0001). In Cav1-deficient TM cells, RhoA expression was elevated compared with control (406±51.4% vs. 100±14.5%, n=3, p=0.0046). We then investigated how Cav1 expression and activation regulates Rho/ROCK signaling in the presence of cyclic stretch. Phosphorylated Cav1 levels were higher in stretched conditions compared to unstretched (126±8.2% vs. 100±5.4%, n=14, p=0.012), suggesting stretch activated caveolae in TM cells. Using phosphorylated myosin light chain (pMLC) as a positive indicator for Rho/ROCK activity and contractile tone, we found that control cells showed reduced pMLC/MLC levels in stretched verses unstretched conditions (59±13% vs. 100±20%, n=7, p=0.012). Interestingly, pMLC/MLC levels in Cav1-deficient cells appeared unaffected in stretched verses unstretched conditions (86.5±16% vs. 100±21%, n=7, p=0.45).
Cav1 negatively regulates RhoA expression, and is necessary for stretch-induced relaxation of TM cells.
This is a 2020 ARVO Annual Meeting abstract.
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