May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Keratocyte Hyaluronan Synthesis Stimulates Their Migration
Author Affiliations & Notes
  • M. L. Funderburgh
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • N. Guo
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • M. M. Mann
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • E. Rao
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • J. L. Funderburgh
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships M.L. Funderburgh, None; N. Guo, None; M.M. Mann, None; E. Rao, None; J.L. Funderburgh, None.
  • Footnotes
    Support NIH Grants EY09368, P30-EY08098, Research to Prevent Blindness , Eye and Ear Foundation of Pittsburgh. JLF is a Jules and Doris Stein RPB Professor
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1487. doi:https://doi.org/
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    • Get Citation

      M. L. Funderburgh, N. Guo, M. M. Mann, E. Rao, J. L. Funderburgh; Keratocyte Hyaluronan Synthesis Stimulates Their Migration. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1487. doi: https://doi.org/.

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

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Abstract

Purpose:: Hyaluronan (HA) is a ubiquitous component of the extracellular matrix in many tissues. In the corneal stroma, keratocytes produce transparent extracellular matrix devoid of hyaluronan; after corneal wounding, however, stromal HA is abundant. HA appears in wounds concurrent with migration and division of keratocytes. We have previously shown cultured keratocytes to secrete matrix similar to that of normal stroma when quiescent, but in the presence of serum, secretion of fibrotic matrix components including HA was induced. This study examined to role of HA in keratocyte motility.

Methods:: Primary bovine keratocytes were cultured as substratum-free spheroids and HA synthesis was downregulated using siRNA against hyaluronan synthase 2 (HAS2) or the HAS inhibitor 4-methylumbelliferone (4-MU). Spheroids were transferred to tissue-culture plastic and cultured for 24 hr to initiate keratocytes migration. The rate of migration was determined by morphometry and by time-lapse micrography.

Results:: Keratocyte HA biosynthesis was rapidly induced by exposure to fetal bovine serum, reaching a maximum rate within 12 hr. Transfection of cells with HAS2 siRNA or treatment with 4-MU resulted in a marked reduction in HA secretion in response to serum. After transfer to attachment-permissive substrata, spheroids became anchored to the surface within 30 min and within one hour cells were observed migrating in a radial pattern away from the site of spheroid attachment. Migration occurred both in serum-free conditions and after exposure to fetal bovine serum. HA staining revealed cell-associated HA in migrating keratocytes as well as HA deposited on the substratum. Migration of keratocytes exposed to serum was markedly inhibited by transfection with HAS2 siRNA, but cells migrating under serum-free conditions were less strongly inhibited. The HAS inhibitor 4-MU inhibited migration both with and without serum.

Conclusions:: These experiments show that HA secreted by keratocytes in response to serum remains cell- and matrix-associated, stimulating keratocyte migration on permissive substrata. Such results suggest the rapid synthesis of HA by keratocytes in response to wounding in vivo serves to stimulate keratocyte migration toward the site of injury.

Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • wound healing 
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