June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Trabecular Meshwork Exosomes: Fibronectin Binding and ECM homeostasis
Author Affiliations & Notes
  • W Michael Dismuke
    Ophthalmology, Duke University, Durham, NC
  • W Daniel Stamer
    Ophthalmology, Duke University, Durham, NC
  • Footnotes
    Commercial Relationships W Dismuke, None; W Stamer, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4852. doi:
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      W Michael Dismuke, W Daniel Stamer; Trabecular Meshwork Exosomes: Fibronectin Binding and ECM homeostasis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4852.

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

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Abstract

Purpose: Primary human trabecular meshwork (hTM) cells release extracellular nanovesicles called exosomes. While their function(s) in conventional outflow biology is unknown, hTM exosomes bind soluble fibronectin (Fn) and other extracellular matrix (ECM) components. We hypothesize that TM cells utilize this feature for efficient uptake of digested ECM. <br />

Methods: Exosomes were purified from media conditioned by mature hTM cell monolayers via serial ultracentrifugation. Protein composition was determined by LC-MS/MS and western blot. Vesicle size was measured by nanoparticle tracking analysis (NTA). Dipeptidyl peptidase IV (DPPIV) activity was assessed by fluorogenic substrate cleavage. Fn conformation was monitored by detergent solubility. Collagen uptake was assayed by internalization of fluorescent collagen-1 coated or carboxylated microspheres.<br />

Results: hTM cells release vesicles with size (mode = 75nm) and protein composition (CD9, CD81, CD63, AnxA2) characteristic of exosomes. These exosomes bind Fn in a calcium-independent manner. LC-MS\MS revealed a known Fn-binding protein DPPIV, whose activity was detected on purified exosomes. Bound Fn exists in several conformations, including a deoxycholate and SDS insoluble polymer, indicating the presence of both the plasma and cellular forms of Fn. Interestingly, a concurrent increase in hydrodynamic radius was measured in exosomes bound to Fn. Finally, while addition of excess hTM-derived exosomes to hTM cells had no effect on Fn and collagen-IV secretion, excess hTM-derived exosomes increased uptake of collagen-1-coated but not carboxylated microspheres by hTM cells.<br />

Conclusions: Data support the hypothesis that hTM cells release exosomes at sites of focal ECM degradation to coordinate the uptake of digested ECM components. We speculate that dysfunction in this system is involved in the aberrant accumulation of ECM in the conventional outflow tract and ocular hypertension seen in many forms of glaucoma.<br />

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