June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
A peptide that disrupts fibronectin fibril assembly also affects collagen, fibrillin, and laminin extracellular matrix (ECM) assembly in human trabecular meshwork (HTM) cells.
Author Affiliations & Notes
  • Mark S Filla
    Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Donna M Peters
    Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Mark Filla, None; Donna Peters, None
  • Footnotes
    Support  NEI grants EY017006, EY026009, P30 EY016665; Brightfocus Foundation Award G2014051
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3490. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Mark S Filla, Donna M Peters; A peptide that disrupts fibronectin fibril assembly also affects collagen, fibrillin, and laminin extracellular matrix (ECM) assembly in human trabecular meshwork (HTM) cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3490.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : To determine if disrupting fibronectin (FN) fibril assembly, which is a critical mediator of the ECM, affects the assembly of other matrix proteins in HTM cells.

Methods : FN fibril formation was disrupted using a recombinant FN-binding peptide, derived from the Streptococcus pyogenes F1 adhesin protein, called FUD (pUR4). A mutated, inactive FUD peptide was used as a control. HTM cells were treated ± FUD for 1-14 days depending upon the experiment. In some experiments, FN expression was induced with dexamethasone (DEX) during FUD treatment. Both newly confluent cells lacking a mature ECM and cell cultures possessing a preformed, mature matrix were treated with FUD. FN fibril formation was determined using a differential extraction of cells with deoxycholic acid in an On-cell western (OCW) assay. Assembly of FN, fibrillin (FBN), type IV collagen (Col IV) and laminin (LN) into matrices was assessed using immunofluorescence (IF) microscopy.

Results : HTM monolayers treated for 2 days with AlexaFluor®488-tagged FUD demonstrated a matrix of FN fibrils that co-localized with tagged FUD. By 4 days, however, FUD-treated cultures demonstrated a striking loss of FN fibrils. This was observed in both newly confluent cultures and cultures with a mature ECM. FUD also caused a reduction in FN fibrils in HTM cultures treated with DEX. OCW analysis showed that FUD decreased DEX-induced FN fibrils by 59% (100 nM DEX) and 77% (500 nM DEX). By IF microscopy FUD also appeared to disrupt Col IV, FBN and LN matrices in newly confluent cultures. However in cultures with a mature ECM, FUD had no effect on these other matrices even though FUD effectively removed de novo and existing FN fibrils from these cultures.

Conclusions : FN plays an important role in mediating the assembly of the ECM within the TM. Disruption of pre-existing and de novo FN fibril assembly by FUD in vitro also affected the de novo assembly of other proteins like FBN, Col IV and LN into the TM ECM. This suggests that under in vivo conditions, FUD would disrupt FN fibrils and de novo assembly of other ECM proteins but not existing ECM protein matrices. These data also suggest that targeting FN fibril assembly may be a viable treatment for POAG and possibly other glaucomas.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×