Purchase this article with an account.
Ping Xu, Kristine Porter, Nalla Thambi Jeyabalan, Paloma Liton; MECHANICAL STRESS ACTIVATES AUTOPHAGY IN TRABECULAR MESHWORK CELLS. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3547.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the activation of autophagy with mechanical stress in trabecular meshwork (TM) cells.
Confluent cultures of human and porcine TM cells were subjected to static mechanical stress (30% stretching) for up to 16 hours. Autophagy dynamics was monitored by confocal microscopy using AdtfLC3; immunoblotting, by monitoring the LC3-I to LC3-II conversion; and electron microscopy. Protein expression levels were quantified by WB analysis using specific antibodies against Atgs, LAMP1, p62, LC3B, CTSB, p70S6K and pp70S6K. mRNA expression levels of beclin, Atg4, Atg5, Atg7, LC3, LAMP1, and LAMP2 were monitored by qPCR. Rapamycin, bafilomycin A1, chloroquine, and 3-MA were used to pharmacologically modulate the autophagic pathway. Activation of autophagy was also evaluated in enucleated whole globe porcine eyes subjected to perfusion pressures of 10, 20, and 40mmHg
Western-blot analysis showed increased levels of the autophagosome marker LC3-II in porcine and human TM cells as early as 30 minutes post-stretching compared to non-stretched cultures (1.35±0.07 fold). Elevated LC3-II peaked at 3 hours of continuous stretching (2.23±0.51 fold) and gradually decreased at 16 hours, concurrent with decreased levels of Atg12 (0.32±0.07 fold), a protein regulating LC3 lipidation. No changes in the mRNA levels for the genes tested were observed. The increase in LC3-II in the mechanically stressed cultures was prevented with the autophagy inhibitor 3-MA. Confocal microscopy showed increased number of autophagolysomes (8.9±1.1 versus 5.5±0.7 autophagosome/cell, p=0.0001), indicating the proper maturation of nascent autophagosomes. This data was confirmed by electron microcopy. A significant increased in LC3-II protein levels was also observed in the outflow pathway of whole globe porcine eyes subjected for one hour to perfusion pressure of 40 mmHg compared to eyes perfused at 10 mmHg.
Our results clearly indicate that autophagy is rapidly induced in TM cells in respond to mechanical stress. Based on this, we hypothesize that autophagy is part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment. We further hypothesize that this response mechanism is impaired in glaucoma, as a result of a decrease in the autophagic capacity with chronic oxidative stress and/or aging.
This PDF is available to Subscribers Only