VEGF164 is proinflammatory in the diabetic retina.
OBJECTIVE
The objectives of this study were to characterize the differential potency of two major VEGF isoforms, VEGF(120) and VEGF(164), for inducing leukocyte stasis (leukostasis) within the retinal vasculature and blood-retinal barrier (BRB) breakdown and to determine whether endogenous VEGF(164) mediates retinal leukostasis and BRB breakdown in early and established diabetes.


METHODS
Retinal leukostasis and BRB breakdown were simultaneously quantified by combining concanavalin A lectin (ConA) perfusion labeling with a fluorophotometric dextran leakage assay. CD45 immunohistochemistry was performed to confirm that ConA-stained cells within the vasculature were leukocytes. Retinal leukostasis and BRB breakdown were compared in nondiabetic rats receiving intravitreous injections of VEGF(120) or VEGF(164). Retinal intercellular adhesion molecule (ICAM)-1 and VEGF protein levels were studied by Western blot and ELISA, respectively. An anti-VEGF(164(165)) aptamer (EYE001) was administered by intravitreous injection to 2-week and 3-month diabetic rats, and the effect on retinal leukostasis and BRB breakdown was quantified.


RESULTS
Compared with VEGF(120), VEGF(164) more potently increased retinal ICAM-1 levels (2.2-fold), leukostasis (1.9-fold), and BRB breakdown (2.1-fold, P < 0.01 for all), despite negligible differences in vitreoretinal VEGF levels at the time of evaluation (P > 0.05). Retinal leukostasis and leakage increased with the duration of diabetes (P < 0.01) and correlated closely (P < 0.01, r = 0.889). The isoform-specific blockade of endogenous VEGF(164) with EYE001 resulted in a significant suppression of retinal leukostasis and BRB breakdown in both early (72.4% and 82.6%, respectively) and established (48.5% and 55.0%, respectively) diabetes (P < 0.01).


CONCLUSIONS
On an equimolar basis, VEGF(164) is at least twice as potent as VEGF(120) at inducing ICAM-1-mediated retinal leukostasis and BRB breakdown in vivo. The inhibition of diabetic retinal leukostasis and BRB breakdown with EYE001 in early and established diabetes indicates that VEGF(164) is an important isoform in the pathogenesis of early diabetic retinopathy.
Ishida
Susumu
S
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA.
Usui
Tomohiko
T
Yamashiro
Kenji
K
Kaji
Yuichi
Y
Ahmed
Ednan
E
Carrasquillo
Karen G
KG
Amano
Shiro
S
Hida
Tetsuo
T
Oguchi
Yoshihisa
Y
Adamis
Anthony P
AP
D000818:Animals; D001813:Blood-Retinal Barrier / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D003208:Concanavalin A / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000209:etiology / Q000378:metabolism* / Q000517:prevention & control; D003930:Diabetic Retinopathy / Q000209:etiology / Q000378:metabolism* / Q000517:prevention & control; D016228:Endothelial Growth Factors / Q000494:pharmacology / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D007150:Immunohistochemistry; D007267:Injections; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology / Q000502:physiology*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D018921:Leukostasis / Q000209:etiology / Q000378:metabolism* / Q000517:prevention & control; D008222:Lymphokines / Q000494:pharmacology / Q000502:physiology*; D009841:Oligonucleotides / Q000627:therapeutic use; D020033:Protein Isoforms; D051381:Rats; D020318:Rats, Long-Evans; D012171:Retinal Vessels / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014822:Vitreous Body
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D009841:Oligonucleotides; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C042784:fluorescein isothiocyanate-concanavalin A; D003208:Concanavalin A; D018799:Intercellular Adhesion Molecule-1; C430291:NX1838; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate