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Janice A Vranka, Julia Staverosky, Ying Ying Sun, Yong-Feng Yang, John Bradley, Kate E Keller, Ted S Acott; Substrate stiffness triggers an extracellular matrix response in human trabecular meshwork cells and tissues. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4722. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The juxtacanalicular (JCT) region of the trabecular meshwork (TM) plays a key role in regulating outflow resistance and intraocular pressure (IOP) homeostasis, though the molecular mechanisms are poorly understood. Ultrastructural changes in the extracellular matrix (ECM) of the JCT have been shown to be associated with glaucoma. Moreover, glaucoma JCT is stiffer than normal TM and there are differences in the stiffness in segmental outflow regions. The purpose of this study was to investigate the molecular basis underlying these stiffness differences by culturing human trabecular meshwork cells on substrates of varying stiffness and measuring the ECM protein response.
Human TM (hTM) cells were cultured on hydrogels of varying stiffness mimicking normal (5 kPa) and glaucomatous (75 kPa) TM tissue. Western blots of cell lysates and media were used to assess ECM protein differences (N = 3 biological donors). MMP activity and hyaluronan levels were measured in cell lysates and media from hTM cells cultured on hydrogels of difference stiffness. Human anterior segments from human donor eyes were perfused at physiological pressure in organ culture. Anterior segments were labeled with fluorescent tracer to distinguish segmental flow regions. Immunohistochemistry of TM tissues was performed to identify ECM proteins found to be localized in the JCT region of the TM.
Western blots of hTM lysates and media showed differences in splice forms and amounts of versican, fibronectin, tenascin, type XII collagen, MMP14, and TIMP2. Hyaluronan levels and MMP activity were significantly increase as substrate stiffness increased. Immunohistochemistry in tissues shows specific localization of versican, fibronectin, tenascin, fibrillin, thrombospondin, nidogen, perlecan, and hyaluronan binding protein in the innermost JCT region in segmental regions of the TM suggesting their role in maintaining the structural integrity of the outflow pathway.
Our data show that substrate stiffness can influence the synthesis of ECM proteins in cultured hTM cells. These proteins may affect the overall compliance of the tissues reflecting the large stiffness differences measured in glaucomatous TM compared with normal TM tissue. Tissue stiffness is likely a contributing factor to impaired homeostasis and increased IOP seen in glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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