Combined PI3K/Akt and Smad2 Activation Promotes Corneal Endothelial Cell Proliferation.
The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2.



Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas.



Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na+/K+-ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction.



We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain 2Department of Ophthalmology, Clínica Universidad de Navarra, Pamplona, Navarra, Spain 3Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States.
Alfonso L
AL
Sabater
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
Enrique J
EJ
Andreu
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
María
M
García-Guzmán
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
Tania
T
López
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
Gloria
G
Abizanda
Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States.
Victor L
VL
Perez
Department of Ophthalmology, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
Javier
J
Moreno-Montañés
Area of Cell Therapy, Clínica Universidad de Navarra, Pamplona, Navarra, Spain.
Felipe
F
Prósper
D028341:Activins / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D065306:Corneal Injuries / Q000628:therapy; D004195:Disease Models, Animal; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004728:Endothelium, Corneal / Q000166:cytology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D020890:Neuregulin-1 / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014945:Wound Healing / Q000187:drug effects; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
D028341:Activins; D018031:Connexin 43; D007334:Insulin-Like Growth Factor I; D020890:Neuregulin-1; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051899:Smad2 Protein; D000254:Sodium-Potassium-Exchanging ATPase; D062826:Zonula Occludens-1 Protein; C422405:activin A; C087230:heregulin beta1