title: Mechanisms for sickle red blood cell retention in choroid.
abstract: OBJECTIVE
  Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited.
  
  
  METHODS
  Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-alpha intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flatmounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of alpha4beta1 (VLA-4) and alpha4beta7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells.
  
  
  RESULTS
  Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-alpha significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-alpha-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of monoclonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4.
  
  
  CONCLUSIONS
  The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-alpha-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4's ligands, the CS-1 portion of fibronectin.
authors.affiliation: Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, MD, USA. glutty@jhmi.edu
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Lutty
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Otsuji
authors.forename: Makoto
authors.initials: M
authors.lastname: Taomoto
authors.forename: Carol
authors.initials: C
authors.lastname: Merges
authors.forename: D Scott
authors.initials: DS
authors.lastname: McLeod
authors.forename: Sahng Yeon
authors.initials: SY
authors.lastname: Kim
authors.forename: Peter
authors.initials: P
authors.lastname: Vanderslice
authors.forename: Sandra
authors.initials: S
authors.lastname: Suzuka
authors.forename: Mary E
authors.initials: ME
authors.lastname: Fabry
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Nagel
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood*; D000818:Animals; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion; D002829:Choroid / Q000098:blood supply*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D004913:Erythrocytes, Abnormal / Q000187:drug effects / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D039041:Integrin alpha4beta1 / Q000037:antagonists & inhibitors; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D012171:Retinal Vessels / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D039041:Integrin alpha4beta1; D014409:Tumor Necrosis Factor-alpha; D016650:Fluorescein-5-isothiocyanate