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Mini Aga, Kate Keller, John Bradley, Ted Acott; Caveolins modulate ECM turnover by Trabecular Meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3560.
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© ARVO (1962-2015); The Authors (2016-present)
Caveolins (CAVs) are the principal structural components of caveolae. One role of CAVs is the endocytosis and recycling of extracellular matrix (ECM) components. SNPs identified near the CAV1/CAV2 gene loci, have been associated with primary open-angle glaucoma. Therefore, CAV-silencing lentivirus was generated to evaluate ECM uptake in TM cells and to measure the effect on outflow facility.
Studies were conducted using cultured primary human and porcine TM cells and perfused anterior segments. Short hairpin CAV1 and CAV2 silencing (shRNA) and control lentivirus were generated. Effects of CAV silencing on podosome- or invadopodia-like structures (PILS) component localization and ECM degradation were determined by confocal microscopy. Perfused anterior segments were subjected to CAV shRNA to determine the effect on outflow facility.
Both CAV1 and CAV2 colocalized with cortactin at PILS, areas of focal ECM degradation. Interestingly CAVs also colocalized with various ECM components including fibronectin (FN) and MMP2. CAV silencing effectively reduced protein expression of their respective CAVs in TM cells. A distinct increase in FN fibrils was observed upon CAV silencing as compared to control. Modest FN uptake vesicles were associated with CAV1 or CAV2 at 30 and 90 minutes after exposing TM cells to exogenous Rhodamine-labeled FN (rhodFN), and associated more strongly with clathrin and RAB7. CAV1 or CAV2 silencing also resulted in decreased expression of integrin beta1. Increased matrix degradation was observed in both CAV1 and CAV2 knockdown cells. MMP2 was colocalized with CAVs in TM cells. More ADAMTS4 vesicles were observed in CAV2-silenced cells and clustering of ADAMTS4-stained vesicles was seen in CAV1 knockdown cells. Outflow facility increased significantly in CAV1-silenced anterior segments, whereas a significant decrease was observed with CAV2-silenced eyes.
CAVs are co-localized with numerous ECM components at PILS in TM cells. CAV silencing changed the ECM composition and organization at PILS, which may explain in part the observed changes in outflow facility in perfusion culture following CAV silencing.
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