FAS-mediated apoptosis and its relation to intrinsic pathway activation in an experimental model of retinal detachment.
OBJECTIVE
To determine whether the FAS-mediated apoptosis pathway becomes activated in the retina after retinal detachment and to investigate the temporal relationship between the activation of the FAS-pathway and the intrinsic apoptosis pathway involving caspase-9 and cytochrome c.


METHODS
Experimental retinal detachments were created in Brown-Norway rats by injecting 10% hyaluronic acid into the subretinal space. Retinal tissue was harvested at 2, 4, 8, 24, 72, and 168 hours after creation of the detachment. Immunoprecipitation was performed to assess for FAS-receptor/FAS-ligand complex formation, and activation of caspase-8 and BID (a member of the Bcl-2 family of proteins) was assessed by Western blot analysis. A caspase-9 activity assay and immunoprecipitation of the caspase-9/cytochrome c complex were performed at these same time points. Specific pathway inhibition was performed with the caspase-9 inhibitor zLEHD.fmk or neutralizing antibodies against either the FAS-receptor or FAS-ligand. Transcription levels of FAS and intrinsic pathway intermediates were assessed as a function of time after retinal detachment by using quantitative real-time polymerase chain reaction.


RESULTS
Retinal detachment resulted in the time-dependent formation of the FAS-receptor/FAS-ligand complex that preceded the peak of caspase-9 activity and caspase-9/cytochrome c complex formation. Cleavage of caspase-8 and truncation of BID were also observed. Injection of zLEHD.fmk into the subretinal space of a detached retina resulted in decreased caspase-9 activity, as did injection of anti-FAS-receptor antibody into either the subretinal space or the vitreous. Retinal detachment resulted in the transcriptional upregulation of the FAS-receptor, FAS-ligand, caspase-8 and BID, but not caspase-9 and cytochrome c.


CONCLUSIONS
The FAS-mediated apoptosis pathway becomes activated and transcriptionally upregulated after retinal detachment. The peak of FAS activation precedes that of the intrinsic pathway, and inhibition of FAS activation can decrease caspase-9 activity.
Zacks
David N
DN
Retina Service, Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, 1000 Wall Street, Ann Arbor, MI 48105, USA. davzacks@umich.edu
Zheng
Qiong-Duan
QD
Han
Ying
Y
Bakhru
Rita
R
Miller
Joan W
JW
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011914:Rats, Inbred BN; D012163:Retinal Detachment / Q000201:enzymology / Q000503:physiopathology*; D013997:Time Factors; D019014:fas Receptor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D051023:BH3 Interacting Domain Death Agonist Protein; C494816:Bid protein, rat; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D010446:Peptide Fragments; D012333:RNA, Messenger; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D019014:fas Receptor; D045304:Cytochromes c; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases