May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Characterization and Comparison of Intercellular Adherent Junctions in Human Cornea Endothelial Cells in vivo and in vitro Sphere Cultures
Author Affiliations & Notes
  • Y. Zhu
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • Y. Hayashida
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • H. He
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • S. Chen
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • Y. Chen
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • S. C. G. Tseng
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • Footnotes
    Commercial Relationships  Y. Zhu, Tissue Tech Inc, E; Y. Hayashida, None; H. He, Tissue Tech Inc, E; S. Chen, Tissue Tech Inc, E; Y. Chen, None; S.C.G. Tseng, Tissue Tech Inc, I; Tissue Tech Inc, P.
  • Footnotes
    Support  RO1 EY06819 and RO1 EY015735 (to SCGT) from National Eye Institute, National Institutes of Health
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3940. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Y. Zhu, Y. Hayashida, H. He, S. Chen, Y. Chen, S. C. G. Tseng; Characterization and Comparison of Intercellular Adherent Junctions in Human Cornea Endothelial Cells in vivo and in vitro Sphere Cultures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3940. doi: https://doi.org/.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Human corneal endothelial cell (HCEC) proliferation is controlled by their cell-cell junctions, of which the mechanism remains unknown. We recently reported a novel method of cultivating HCEC monolayer by collagenase digestion followed by expansion in the supplemented hormonal epithelial medium (SHEM). Herein we sought to characterize adherent junctional components of in vivo HCECs, compare their gene expression to that of in vitro counterparts, and correlate with their proliferative potential.

Methods: : Stripped human Descemet’s membranes containing HCECs were digested with 1 mg/mL collagenase A at 37 °C for 16 h. Afterwards, HCEC spheres were cultured in collagen IV-coated dishes for 7, 14, and 21 days in SHEM. BrdU labeling was performed 24 h before termination. Both in vivo HCEC flat mounts and HCEC monolayers cultured at different times were subjected to immunostaining with FITC-phalloidin and antibodies specific to different adherent junctional components and BrdU. Their mRNA expressions were determined by RT-PCR.

Results: : In vivo HCECs expressed mRNAs of N- (type I and II), VE-, E- (type I and II), P-cadherins, α-, β-, γ- and p120 catenins. In vitro HCEC counterparts also expressed mRNAs of all these genes except P-cadherin. In vivo HCECs displayed continuous circular N-cadherin, β-catenin, p120, p190 and F-actin as well as discontinuous circular VE-cadherin protein bands at the cell-cell junction, and E-cadherin in the cytoplasm. Immunostaining revealed that gradual maturation of adherent junctions of HCECs cultured for 21days to a pattern similar to that of HCECs in vivo, and such a process was coupled with progressive decline of BrdU labeling.

Conclusions: : Both in vivo and in vitro HCECs expressed mRNA and proteins of N-, VE-, E-cadherin, α-, β-, γ- and p120 catenins, and displayed distinct distribution of N-, VE-cadherin, β- and p120 catenins and F-actin at the cell-cell border and E-cadherin in the cytoplasm. Progressive maturation of intercellular adherent junctions was correlated with progressive decline of proliferative potential in HCEC cultures. This baseline information allows us to devise new strategies to engineer HCECs in vitro by targeting these components.

Keywords: cornea: endothelium • cell adhesions/cell junctions • gene/expression 
×
×

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.

×