April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Proliferation induced by lysophosphatidic acid via Hippo-YAP pathway in contact-inhibited human corneal endothelial cells
Author Affiliations & Notes
  • Yi-Jen Hsueh
    Stem Cell Laboratory of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan
  • Hung-Chi Chen
    Stem Cell Laboratory of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan
  • Sung-En Wu
    Department of Physiology, Chang Gung University College of Medicine, Taoyuan, Taiwan
  • Jan-Kan Chen
    Department of Physiology, Chang Gung University College of Medicine, Taoyuan, Taiwan
  • David Hui-Kang Ma
    Stem Cell Laboratory of Ophthalmology, Chang Gung Memorial Hospital, Linkou, Taiwan
  • Footnotes
    Commercial Relationships Yi-Jen Hsueh, None; Hung-Chi Chen, None; Sung-En Wu, None; Jan-Kan Chen, None; David Ma, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2041. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Yi-Jen Hsueh, Hung-Chi Chen, Sung-En Wu, Jan-Kan Chen, David Hui-Kang Ma; Proliferation induced by lysophosphatidic acid via Hippo-YAP pathway in contact-inhibited human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2041. doi: https://doi.org/.

      Download citation file:


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

      ×
  • Supplements
Abstract
 
Purpose
 

Unlike in vivo contact-inhibited cells, human corneal endothelial cells (HCEC) were successfully ex vivo isolated and expanded, for which the methodologies are constantly evolving and improving. Suppressed Hippo pathway (increased expression of nuclear YAP-1) and enhanced cell proliferation were shown in either primary HCEC by downregulation of p120-catenin or cancer cell lines by administration of lysophosphatidic acid (LPA). We thus wonder if LPA may be used to unlock mitotic block in contact-inhibited HCEC via suppressing Hippo pathway.

 
Methods
 

Collagenase-isolated HCEC aggregates from stripped Descemet’s membrane were cultured to 14 days, and treated by 100 nM control or YAP-1 siRNA. Cells were further treated with 20 μM LPA and 10 μM BrdU . Likewise, post-confluent b4g12 cells (HCEC cell line) cultured to 4 days under the similar condition. Immunofluorescent staining was performed to demonstrate YAP-1, ZO-1, Na/K-ATPase, SMA and BrdU labeling. Western blot analysis was applied to evidence the regulation of proliferation by targeting p21 and cyclin D1 as well as the induction of endothelial-mesenchymal transition (EMT) by targeting ZO-1, Na/K-ATPase, and SMA.

 
Results
 

In post-confluence, cell proliferation of HCEC and b4g12 was inhibited by contact inhibition, as evidenced by abolished BrdU labeling. In both cell models, LPA promoted nuclear expression of YAP-1 and BrdU labeling, suggestive of increased proliferation. Furthermore, HCEC remained hexagonal in addition to unchanged expression of ZO-1, Na/K-ATPase, and SMA, suggestive of preserved normal phenotype without EMT. In b4g12 cells, enhanced cyclin D1 and suppressed p21 were detected upon administration of LPA, which were neutralized by silencing of YAP-1 using siRNA.

 
Conclusions
 

LPA may suppress Hippo pathway in contact-inhibited HCEC and b4g12 cells without inducing EMT, providing an innovative strategy to ex vivo cultivate HCEC for further transplantation or cell therapy. Moreover, investigation of how YAP-1 signaling regulate cell proliferation may unravel the controlling mechanisms of contact inhibition in HCEC.

 
 
(Immunofluorescent staining)
 
(Immunofluorescent staining)
 
Keywords: 481 cornea: endothelium • 654 proliferation  
×
×

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.

×