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Nevena Vicic, Cindy Guo, John G. Flanagan, Jeremy M Sivak; The Role of Annexin A4 in Stabilizing Cell Membranes Undergoing Biomechanical Stress Relevant to Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2440.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanotransduction mechanisms by which inner retinal cells respond to biomechanical stress in glaucoma are not well understood. We have previously shown that annexin A4 (ANXA4), a calcium-dependent membrane binding protein, is significantly upregulated in an in vitro biomechanical insult model. Here, we determine ANXA4 expression and localization in the retina in vivo, and test its potential protective role in membrane stabilization in response to biomechanical strain in vitro.
Immunofluorescence microscopy was used to identify the pattern of ANXA4 protein in mouse, rat, and human retinas. Acute biomechanical and ischemic stress was induced in rodent retinas in vivo, and in primary rodent retinal astrocytes in vitro. ANXA4 localization and expression was examined using fluorescence imaging and quantitative RT-PCR, respectively. Fluorescence imaging of an astrocytic cell line transiently transfected with GFP-tagged ANXA4 was used to visualize its localization and membrane dynamics in response to elevated intracellular calcium.
ANXA4 antibody staining revealed conserved localization to the ganglion cell layer and optic nerve head (ONH) in healthy mouse, rat, and human retinas. Furthermore, there was a 6-fold increase in ANXA4 expression by 18 hours following elevation of intraocular pressure in vivo (p=0.039). Imaging of primary retinal astrocytes showed evidence of ANXA4 localization to cellular and nuclear membranes upon application of biomechanical stress. Furthermore, ANXA4 dramatically reduced membrane blebbing upon rapid elevation of intracellular calcium.
ANXA4 was localized to the cells lining the inner retina and ONH, and responded to calcium and biomechanical strain relevant to glaucoma. Results suggest that when translocated to the membrane, ANXA4 can increase membrane stability to improve cell survival under conditions of biomechanical strain.
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