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Takeshi Kita, Allen C. Clermont, Kimihiko Fujisawa, Tatsuro Ishibashi, Lloyd P. Aiello, Edward P. Feener; Identification of a VEGF-independent and Plasma Kallikrein-Kinin-dependent pathway of retinal vascular permeability in Diabetic Macular Edema. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2431.
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We previously demonstrated that levels of plasma kallikrein-kinin system (KKS) components including plasma kallikrein (PK) were increased in the vitreous of patients with diabetic macular edema (DME) and identified subgroups of DME patients with high PK and lower VEGF (vit↑PK), or high VEGF and lower PK (vit↑VEGF) in their vitreous. In this study, we evaluated the direct effects of vit↑PK and vit↑VEGF on retinal vascular permeability (RVP) following intravitreal injection in rats. Since the effects of PK are mediated in large part by bradykinin peptides, we also examined the role of bradykinin (BK), des-Arg9-bradykinin (DABK), and B1 receptor (B1R) and B2 receptor (B2R) antagonists on RVP in both diabetic and nondiabetic rats and retinal endothelial cells.
Cultured primary bovine retinal endothelial cells (REC) were stimulated with BK (B2R agonist) or DABK (B1R agonist). vit↑PK and vit↑VEGF samples were obtained from patients with DME but without proliferative diabetic retinopathy and injected intravitreally into streptozotocin (STZ)-induced diabetic rats. Effects on rat RVP following intravitreal injection of BK, DABK or vitreous samples [with or without co-injection of bevacizumab or systemic administration of des-Arg-Hoe140 (B1R antagonist) and Hoe140 (B2R antagonist)] were analyzed by vitreous fluorophotometry. RVP following intravitreal injection of BK in eNOS-deficient mice was measured using Evans blue.
Intravitreal injection of vit↑VEGF increased RVP within 40min, while vit↑PK did so only after 48hrs. Hoe140/des-Arg10-Hoe140 inhibited vit↑PK induced RVP but not vit↑VEGF induced RVP. Conversely, bevacizumab inhibited vit↑VEGF induced RVP but not vit↑PK induced RVP. BK and DABK increased RVP in diabetic rat at 40min and 48hrs, respectively, both of which were significantly suppressed by systemic administration of L-NAME (a nitric oxide synthesis inhibitor) and bradykinin receptor antagonists. Bevacizumab did not inhibit BK- or DABK-induced RVP. In vitro, BK rapidly increased eNOS phosphorylation in RECs. In addition, BK significantly increased RVP in wild type mice, but not in eNOS deficient mice. Intravitreal injection of DABK increased mRNA and protein expression of iNOS in diabetic rat retina, which co-localized with glial fibrillary acidic protein (GFAP) and retinal vessels.
The effects of the KKS on RVP in diabetes involve the combined actions of B1R and B2R, and are mediated at least in part, by nitric oxide. These mechanisms are VEGF-independent. Heterogeneity of DME vitreous with regard to VEGF and PK concentrations might account in part for variability of clinical response to anti-VEGF agents.
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