FAK and p38-MAP kinase-dependent activation of apoptosis and caspase-3 in retinal endothelial cells by alpha1(IV)NC1.
OBJECTIVE
To determine the impact of the antiangiogenic factor alpha1(IV)NC1 on vascular endothelial growth factor-mediated proangiogenic activity in mouse retinal endothelial cells (MRECs).


METHODS
Primary culture of MRECs was established as previously described and was used to determine the effects of alpha1(IV)NC1 on the proangiogenic activity of VEGF. Cell proliferation was evaluated using [(3)H]-thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assays. Cell migration was determined using modified Boyden chamber and scratch wound assays and tube formation was assessed on basement membrane matrix (BMM). Intracellular signaling events Bcl-2/Bcl-x(L) and caspase-3/poly (ADP-ribose) polymerase (PARP) activities were evaluated in cells stimulated with VEGF and plated on type IV collagen-coated dishes. Apoptosis was assessed by measuring caspase activity and by performing quantitative fluorescence analysis using fluorescence-activated cell sorting assay. Subcutaneously injected VEGF induced in vivo neovascularization was studied with the BMM plug assay.


RESULTS
VEGF-induced subconfluent MREC proliferation, migration, and tube formation were significantly inhibited by alpha1(IV)NC1 at 1 muM (P < 0.001). alpha1(IV)NC1 induced MREC apoptosis is mediated by inhibition of Bcl-2 and Bcl-x(L) expression and activation of caspase-3/PARP through FAK/p38-MAPK signaling. In addition, alpha1(IV)NC1 dose dependently inhibited VEGF-mediated neovascularization in vivo.


CONCLUSIONS
alpha1(IV)NC1 inhibited VEGF-mediated angiogenesis by promoting apoptosis and caspase-3/PARP activation and by negatively impacting FAK/p38-MAPK phosphorylation, Bcl-2, and Bcl-x(L) expression leading to MREC death. The endothelial-specific inhibitory actions of recombinant alpha1(IV)NC1 may be of benefit in the treatment of a variety of eye diseases with a neovascular component.
Boosani
Chandra S
CS
Cell Signaling and Tumor Angiogenesis Laboratory, Department of Genetics, Boys Town National Research Hospital, Omaha, Nebraska 68131, USA.
Nalabothula
Narasimharao
N
Munugalavadla
Veerendra
V
Cosgrove
Dominic
D
Keshamoun
Venkateshwar G
VG
Sheibani
Nader
N
Sudhakar
Akulapalli
A
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D024141:Collagen Type IV / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D000939:Epitopes; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D051379:Mice; D009389:Neovascularization, Pathologic / Q000209:etiology / Q000517:prevention & control; D010766:Phosphorylation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D051020:bcl-X Protein / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D020533:Angiogenesis Inhibitors; C494812:Bcl2l1 protein, mouse; D024141:Collagen Type IV; D000939:Epitopes; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D051020:bcl-X Protein; C467480:vascular endothelial growth factor A, mouse; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3