Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5.
OBJECTIVE
To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs.


METHODS
The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program.


RESULTS
Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay.


CONCLUSIONS
HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.
Yamaguchi
Masahiro
M
Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation, Chuo-ku, Kobe, Japan. hiro23net@yahoo.co.jp
Ebihara
Nobuyuki
N
Shima
Nobuyuki
N
Kimoto
Miwa
M
Funaki
Toshinari
T
Yokoo
Seiichi
S
Murakami
Akira
A
Yamagami
Satoru
S
D000368:Aged; D002448:Cell Adhesion / Q000187:drug effects*; D015815:Cell Adhesion Molecules / Q000378:metabolism / Q000494:pharmacology*; D002452:Cell Count; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism; D008875:Middle Aged; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D017931:DNA Primers; D039201:Integrin alpha3beta1; D011994:Recombinant Proteins; D000097794:Kalinin