June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Transit-amplifying cell genes CAV1 and CAV2 are required to maintain corneal epithelial phenotype.
Author Affiliations & Notes
  • Yuzuru Sasamoto
    Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, United States
    Transplant Research Program, Boston Children's Hospital, Boston, Massachusetts, United States
  • Shoko Kiritoshi
    Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Catherine Lee
    Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, United States
    Transplant Research Program, Boston Children's Hospital, Boston, Massachusetts, United States
  • Bruce Ksander
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Markus Hermann Frank
    Transplant Research Program, Boston Children's Hospital, Boston, Massachusetts, United States
    Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States
  • Natasha Frank
    Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, United States
    Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Yuzuru Sasamoto None; Shoko Kiritoshi None; Catherine Lee None; Bruce Ksander Ticeba GmbH, Code P (Patent), Rheacell GmbH & Co. KG, Code P (Patent); Markus Frank Ticeba GmbH, Code C (Consultant/Contractor), Rheacell GmbH & Co. KG, Code C (Consultant/Contractor), Ticeba GmbH, Code P (Patent), Rheacell GmbH & Co. KG, Code P (Patent); Natasha Frank Ticeba GmbH, Code P (Patent), Rheacell GmbH & Co. KG, Code P (Patent)
  • Footnotes
    Support  NIH/NEI grants 5K99EY031741 to Y.S., 5R01EY025794 and 5R24EY028767 to N.Y.F., B.R.K and M.H.F, NIH/NEI Schepens Core grant P30EY003790 to B.R.K., NIH/NIBIB grant 2T32EB016652-06 to C.A.A.L, Alcon Young Investigator Grant to Y.S., Japan Eye Bank Overseas Grant to Y.S., VA R&D Merit Review Award 1I01RX000989 and a Harvard Stem Cell Institute seed grant award to N.Y.F.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3638 – A0203. doi:
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      Yuzuru Sasamoto, Shoko Kiritoshi, Catherine Lee, Bruce Ksander, Markus Hermann Frank, Natasha Frank; Transit-amplifying cell genes CAV1 and CAV2 are required to maintain corneal epithelial phenotype.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3638 – A0203.

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

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Abstract

Purpose : Caveolin (CAV) 1 and 2 are integral membrane proteins that constitute the major components of small membrane pouches termed caveolae. Despite the recognized pleiotropic functions in diverse tissues, their roles in the ocular surface compartments are not fully understood. In the cornea, CAV1 expression was previously found to increase with aging and was negatively associated with wound healing. However, the exact molecular roles of CAV1 and CAV2 in corneal epithelium remain to be elucidated. In the current study, we investigated the contribution of CAV1 and CAV2 to the maintenance of the corneal epithelial phenotype.

Methods : Human donor corneas were obtained from the Saving Sight eye bank, Kansas City, MO and CorneaGen, Seattle, WA. Immunostaining was performed to determine the distribution of CAV1 and CAV2 expression in the cornea. Limbal epithelial cell cultures were transfected with CAV1- and CAV2-targeting siRNAs designated as CAV1 KD #1, CAV1 KD #2, CAV2 KD #1 and CAV2 KD #2. Western blotting, colony-forming assays and RNA-seq were performed using CAV1 and CAV2 knockdown cells and their controls.

Results : Immunohistochemical analyses revealed high CAV1 and CAV2 expression by the transit-amplifying cells (TACs) located in the basal epithelial layer of the limbus and to a lesser degree of the central cornea. siRNA-induced CAV1 and CAV2 KD in cultured limbal TACs resulted in significant reduction of their self-renewal potential as evidenced by attenuated
colony-forming efficiency (66.6 +/- 16.8% decrease by CAV1 KD #1 and 36.8 +/- 21.4% decrease by CAV1 KD #2; 72.4 +/- 13.2% decrease by CAV2 KD #1 and 34.8 +/-14.7% decrease by CAV2 KD #2; all compared to controls, Ps = 0.0002, 0.0118, <0.0001 and 0.0024, respectively). RNA-seq analyses showed that loss of CAV1 and CAV2 led to significant downregulation of genes involved in corneal epithelial formation (e.g., IVL, MUC21, and KRT78) and induction of genes involved in stromal development (e.g., FBN1 and SMPD1).

Conclusions : Our study reveals novel functional roles of CAV1 and CAV2 expressed by TACs in the human cornea. Specifically, CAV1 and CAV2 are essential for TAC self-renewal and the maintenance of the corneal epithelial phenotype.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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