Characterization and comparison of intercellular adherent junctions expressed by human corneal endothelial cells in vivo and in vitro.
OBJECTIVE
Human corneal endothelial cell (HCEC) proliferation is controlled by HCEC junctions, but the mechanism of proliferation remains unknown. The authors sought to characterize adherent junction components of in vivo HCECs and to compare their gene expression and their proliferative potential with those of in vitro counterparts.


METHODS
Stripped human Descemet membranes were digested with collagenase A, and the resultant HCEC aggregates were cultured for 7, 14, and 21 days in supplemented hormonal epithelial medium (SHEM). The growth of HCEC monolayers was monitored by BrdU labeling performed 24 hours before termination. In vivo and in vitro HCECs were subjected to immunostaining to FITC-phalloidin and antibodies to different junction components and BrdU. Their mRNA expressions were determined by RT-PCR.


RESULTS
In vivo HCECs expressed transcripts of N-, VE-, E-, and P-cadherins, alpha-, beta-, gamma-, and p120-catenins, and p190. In vitro HCEC counterparts also expressed all these mRNAs except P-cadherin. In vivo HCECs displayed continuous circular F-actin, N-cadherin, beta- and p120-catenins, and p190, discontinuous circular VE-cadherin bands at or close to cell junctions, and E-cadherin in the cytoplasm. Such an in vivo pattern was gradually achieved by in vitro HCECs at day 21 and was correlated with a progressive decline of BrdU labeling.


CONCLUSIONS
In vivo and in vitro HCECs displayed distinct protein cytolocalization of N-, VE-, and E-cadherins, beta- and p120-catenins, and p190. Progressive maturation of adherent junctions was associated with a decline of the proliferative potential. This information allows us to devise new strategies to engineer in vitro HCECs by targeting these components.
Zhu
Ying-Ting
YT
Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research and Education Foundation, Miami, Florida, USA.
Hayashida
Yasutaka
Y
Kheirkhah
Ahmad
A
He
Hua
H
Chen
Szu-Yu
SY
Tseng
Scheffer C G
SC
D015703:Antigens, CD / Q000235:genetics; D015820:Cadherins / Q000235:genetics / Q000502:physiology*; D051177:Catenins / Q000235:genetics / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology*; D018929:Cell Culture Techniques; D002469:Cell Separation; D017931:DNA Primers; D004728:Endothelium, Corneal / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D051176:beta Catenin / Q000235:genetics; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D051177:Catenins; D017931:DNA Primers; D051176:beta Catenin; D000099152:Cadherin 5