Pigment epithelium-derived factor inhibits advanced glycation end-product-induced angiogenesis and stimulates apoptosis in retinal endothelial cells.
OBJECTIVE
The purpose of this study was to investigate the effect of pigment epithelium-derived factor (PEDF) on the signaling cascade in porcine retinal endothelial cells (PRECs) related to angiogenesis induced by advanced glycation end-products (AGEs).


METHODS
Endothelial cells were isolated from porcine retina by the enzymatic method. Immunocytochemistry was performed to confirm the identity of PRECs. The effect of AGEs and PEDF on cell viability was determined by the MTT assay. An in vitro wound-scratch assay was performed to study the migration of ECs, and in vitro tube formation was assessed by the on-gel assay system using an extracellular matrix. Inhibitor assays were carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt inhibitor VIII. PI3K/Akt activity was assessed by transient transfection and western blot analysis. Induction of apoptosis by PEDF was determined by caspase-3 colorimetric assay and DNA fragmentation analysis.


RESULTS
Treatment of PRECs with AGE-bovine serum albumin (AGE-BSA) significantly increased the cell proliferation, migration and tube formation compared to non-glycated BSA. AGE-BSA mediates cell survival via the PI3K/Akt/FKHR-dependent pathway as evidenced by transient transfection and western blot analyses. Furthermore, PEDF significantly inhibited the proliferation, migration and tube formation, both in the presence and absence of AGE-BSA in PRECs. PEDF inactivated the AGE-BSA-induced PI3K/Akt/FKHR activity and induced apoptosis via caspase-3.


CONCLUSIONS
The results reveal that PEDF inhibits AGE-BSA-induced PI3K/Akt/FKHR signaling in PRECs. Thus, PEDF has potent anti-angiogenic effects against AGE-induced angiogenesis and is suggested to be a promising molecule for the treatment of diabetic retinopathy.
Sheikpranbabu
Sardarpasha
S
Department of Biotechnology, Division of Molecular and Cellular Biology, Kalasalingam University, Anand Nagar, Krishnankoil, 626190, Tamil Nadu, India.
Haribalaganesh
Ravinarayanan
R
Banumathi
Elayappan
E
Sirishkumar
Namagiri
N
Lee
Kyung-Jin
KJ
Gurunathan
Sangiliyandi
S
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000502:physiology; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D053938:DNA Fragmentation; D004727:Endothelium / Q000502:physiology; D005136:Eye Proteins / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D017127:Glycation End Products, Advanced / Q000037:antagonists & inhibitors* / Q000502:physiology; D009025:Morpholines / Q000494:pharmacology; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D009414:Nerve Growth Factors / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D012160:Retina / Q000502:physiology*; D015843:Serpins / Q000502:physiology*; D012710:Serum Albumin, Bovine; D013552:Swine; D000099174:Pigment Epithelium-Derived Factor
D053148:Caspase 3; D002867:Chromones; D005136:Eye Proteins; D017127:Glycation End Products, Advanced; D009025:Morpholines; D009414:Nerve Growth Factors; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D015843:Serpins; D012710:Serum Albumin, Bovine; D000099174:Pigment Epithelium-Derived Factor; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one