title: Upregulation of CCR3 by age-related stresses promotes choroidal endothelial cell migration via VEGF-dependent and -independent signaling.
abstract: OBJECTIVE
  To explore the molecular mechanisms by which the C-C chemokine receptor type 3 (CCR3) and chemokine (C-C motif) ligand 11 (CCL11) regulate choroidal endothelial cell (CEC) migration and the interactions with the vascular endothelial growth factor (VEGF) signaling pathway.
  
  
  METHODS
  Human retinal sections from young and aged donor normal eyes were immunolabeled. By real-time PCR, CCR3 mRNA was measured in retinal pigmented epithelium (RPE)/choroids obtained from young and aged human donor eyes and in cultured CECs exposed to hydrogen peroxide. CCR3 ligand and CCL11- or VEGF-stimulated CEC migration was also measured in the presence of the CCR3 inhibitor or control using fluorescence microscopy. Activation of Rac1, phosphorylated Akt as a readout for phosphoinositol 3-kinase signaling, and VEGFR2 activation were measured in CECs incubated with CCL11, VEGF, or combined CCL11/VEGF.
  
  
  RESULTS
  CCR3 was expressed to a greater level in older compared with that in younger human retinas or RPE/choroids. Ligand-activated CCR3 increased CEC migration, which was inhibited by the CCR3 inhibitor. Rac1 activity, p-Akt, and p-VEGFR2 were significantly increased in CECs incubated with CCL11. The CCR3 inhibitor prevented VEGF-induced CEC migration and Rac1 activation in CECs. Rac1 activity was additively increased in CECs treated with CCL11 and VEGF compared with that in cells with CCL11 or VEGF treatment alone. Ligand-activated CCR3 caused VEGFR2 phosphorylation and coimmunoprecipitation of VEGFR2 and CCR3.
  
  
  CONCLUSIONS
  Activated CCR3 promotes CEC migration and Rac1 activation and causes an association with and activation of VEGFR2. Cross-talk between CCR3 and VEGF signaling exists and may be important in choroidal neovascularization in human age-related macular degeneration.
authors.affiliation: Department of Ophthalmology, The John Moran Eye Center, University of Utah, Salt Lake City, Utah, USA.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: Erika S
authors.initials: ES
authors.lastname: Wittchen
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Jiang
authors.forename: Balamurali
authors.initials: B
authors.lastname: Ambati
authors.forename: Hans E
authors.initials: HE
authors.lastname: Grossniklaus
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Hartnett
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D054413:Chemokine CCL11 / Q000378:metabolism; D002829:Choroid / Q000166:cytology / Q000502:physiology*; D002830:Choroid Neoplasms / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D020239:Receptor Cross-Talk / Q000502:physiology; D054397:Receptors, CCR3 / Q000235:genetics / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013312:Stress, Physiological / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D055815:Young Adult; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: C515456:CCL11 protein, human; C515350:CCR3 protein, human; D054413:Chemokine CCL11; C107953:RAC1 protein, human; D054397:Receptors, CCR3; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D020830:rac1 GTP-Binding Protein