June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Caveolae and conventional outflow: Proteomic profiling of outflow tissue caveolae and evidence of mechanosensing
Author Affiliations & Notes
  • Michael H Elliott
    Ophthalmology, OUHSC, Oklahoma City, OK
    Dean McGee Eye Institute, Oklahoma City, OK
  • Stefanie M Hauck
    Department of Protein Science, Helmholtz Center Munich, Neuherberg, Germany
  • Mikhail G Dozmorov
    Department of Biostatistics, Virginia Commonwealth University, Richmond, VA
  • Jin Liang
    Department of Ophthalmology, Duke University, Durham, NC
    Duke Eye Center, Durham, NC
  • Mark E Mcclellan
    Ophthalmology, OUHSC, Oklahoma City, OK
    Dean McGee Eye Institute, Oklahoma City, OK
  • Jonathan D Wren
    Arthritis & Clinical Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK
  • W Daniel Stamer
    Department of Ophthalmology, Duke University, Durham, NC
    Duke Eye Center, Durham, NC
  • Footnotes
    Commercial Relationships Michael Elliott, None; Stefanie Hauck, None; Mikhail Dozmorov, None; Jin Liang, None; Mark Mcclellan, None; Jonathan Wren, None; W Stamer, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3256. doi:
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      Michael H Elliott, Stefanie M Hauck, Mikhail G Dozmorov, Jin Liang, Mark E Mcclellan, Jonathan D Wren, W Daniel Stamer; Caveolae and conventional outflow: Proteomic profiling of outflow tissue caveolae and evidence of mechanosensing. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3256.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Polymorphisms at the CAV1/2 gene locus increase risk of primary open angle glaucoma. The CAV1 gene product is essential to form caveolae, membrane domains abundant in Schlemm’s canal (SC) and trabecular meshwork (TM). We have shown that absence of caveolae results in elevated intraocular pressure (IOP), reduced conventional outflow, and outflow pathway pathology. We hypothesize that caveolae are mechanosensors, participating in the regulation of conventional outflow and IOP. The goals of these studies were to provide evidence of caveolae mechanosensation and the first caveolae proteome for outflow tissue.

Methods: Caveolae were prepared from freshly dissected porcine iridocorneal angle tissue by detergent-free and detergent-based methods. Caveolae and parent membranes were analyzed by quantitative, label-free mass spectrometry. Proteomic datasets were subjected to bioinformatic analyses. To test if caveolae respond to mechanical, human TM and SC cells were subjected to a cyclic stretch paradigm (15%, 1 Hz for 24 h) and analyzed for caveolin-1 phosphorylation by western blotting and for interaction with PTRF/cavin-1, a protein necessary for the formation of caveolae, by co-immunoprecipitation.

Results: Proteomic analysis revealed dramatic enrichment of Cav-1 in both detergent-free and detergent-based preparations compared to parent membranes. Mass spectrometry identified 325 and 331 proteins significantly co-enriched with Cav-1 by both methods, respectively. Bioinformatics revealed that more than 50% of canonical pathways significantly overrepresented in caveolae were shared in both preparations. Of these, pathways relevant to glaucoma including nitric oxide, sphingolipid, Rho family GTPase, RhoGDI, Interleukin-8, and Integrin signaling pathways were significantly overrepresented. Mechanical stimulation of TM cells resulted in significantly increased of phosphorylation of Cav-1 (P < 0.05, unpaired t-test) and reduced association of PTRF/Cavin-1 with Cav-1. In SC cells, mechanical stimulation did not induce Cav-1 phosphorylation although reduced interaction of PTRF/cavin-1 was observed.

Conclusions: These results provide strong evidence that caveolae play important roles in membrane mechanosensation in the outflow pathway. The proteomic analyses provide a starting point to identify caveolae-dependent mechanisms governing outflow regulation.

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