Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of Choroidal Neovascularization.
Inhibition of chemokine C-C motif receptor 3 (CCR3) signaling has been considered as treatment for neovascular age-related macular degeneration (AMD). However, CCR3 is expressed in neural retina from aged human donor eyes. Therefore, broad CCR3 inhibition may be harmful to the retina. We assessed the effects of CCR3 inhibition on retina and choroidal endothelial cells (CECs) that develop into choroidal neovascularization (CNV). In adult murine eyes, CCR3 colocalized with glutamine-synthetase labeled Műller cells. In a murine laser-induced CNV model, CCR3 immunolocalized not only to lectin-stained cells in CNV lesions but also to the retina. Compared to non-lasered controls, CCR3 mRNA was significantly increased in laser-treated retina. An intravitreal injection of a CCR3 inhibitor (CCR3i) significantly reduced CNV compared to DMSO or PBS controls. Both CCR3i and a neutralizing antibody to CCR3 increased TUNEL+ retinal cells overlying CNV, compared to controls. There was no difference in cleaved caspase-3 in laser-induced CNV lesions or in overlying retina between CCR3i- or control-treated eyes. Following CCR3i, apoptotic inducible factor (AIF) was significantly increased and anti-apoptotic factor BCL2 decreased in the retina; there were no differences in retinal vascular endothelial growth factor (VEGF). In cultured human Műller cells exposed to eotaxin (CCL11) and VEGF, CCR3i significantly increased TUNEL+ cells and AIF but decreased BCL2 and brain derived neurotrophic factor, without affecting caspase-3 activity or VEGF. CCR3i significantly decreased AIF in RPE/choroids and immunostaining of phosphorylated VEGF receptor 2 (p-VEGFR2) in CNV with a trend toward reduced VEGF. In cultured CECs treated with CCL11 and/or VEGF, CCR3i decreased p-VEGFR2 and increased BCL2 without increasing TUNEL+ cells and AIF. These findings suggest that inhibition of retinal CCR3 causes retinal cell death and that targeted inhibition of CCR3 in CECs may be a safer if CCR3 inhibition is considered as a therapy for neovascular AMD.
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
Haibo
H
Wang
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.|Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Shenyang, P.R. China.
Xiaokun
X
Han
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
Deeksha
D
Gambhir
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
Silke
S
Becker
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
Eric
E
Kunz
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
Angelina Jingtong
AJ
Liu
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
M Elizabeth
ME
Hartnett
D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D054413:Chemokine CCL11 / Q000494:pharmacology; D002829:Choroid / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D020256:Choroidal Neovascularization / Q000209:etiology / Q000235:genetics* / Q000473:pathology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D063928:Ependymoglial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D058449:Intravitreal Injections; D007834:Lasers / Q000009:adverse effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology*; D061251:Primary Cell Culture; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D054397:Receptors, CCR3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
D057134:Antibodies, Neutralizing; D051033:Apoptosis Inducing Factor; C515457:Ccl11 protein, mouse; C515351:Ccr3 protein, mouse; D054413:Chemokine CCL11; C494830:AIFM1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D054397:Receptors, CCR3; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C052617:Bcl2 protein, mouse; D010649:Phenylalanine; C000592875:Kdr protein, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; C505409:Casp3 protein, mouse; D053148:Caspase 3