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Tuna Ustunkaya, Nivetha Murugesan, Allen C Clermont, Edward P Feener; Increased FXII activity in vitreous of patients with Diabetic Macular Edema (DME) and in animal models of VEGF- and bradykinin-induced retinal edema. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4183.
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© ARVO (1962-2015); The Authors (2016-present)
The Kallikrein-kinin system (KKS) has been implicated in contributing to DME, however the activators of this pathway in the vitreous has not been elucidated. Factor XII is a primary activator of the KKS and cleaves plasma prekallikrein (PPK) to its active form plasma kallikrein (PK). In this study, we characterize FXII activity in vitreous obtained from DME and macular hole (MH) patients and from animal models of retinal edema.
Sprague Dawley rats received intravitreal (IVT) injection of bradykinin (BK) (2uM), VEGF (10ng/eye), or saline vehicle. Vitreous was obtained at 4 and 24 hours post injection. An assay to measure FXII-mediated activation of PPK was developed using control, PPK-, and FXII-depleted human plasma. FXII-mediated activation of PPK was quantified by addition of samples (1uL of 1:10 diluted vitreous from human DME or MH, undiluted rat vitreous, or 1:600 diluted plasma) to 20 nM PPK and measurements of the rate of PK activity generated using fluorogenic substrate D-Pro-Phe-Arg-AFC (MP Biomedicals). Enzymatic activity was calculated as slopes based on change of fluorescence over time. PPK and FXII levels in vitreous samples were quantified by western blot.
Intravitreal injection of BK in rats increased FXII activity in vitreous by 4.6 fold (n=4, 358 + 179 vs 1652 + 399, p<0.05) at 4 hours and 21 fold (277 vs 5899) at 24 hours compared with vitreous from saline-injected eyes. Intravitreal injection of VEGF increased FXII activity in the vitreous by 8 fold (n=3, 609 + 402 vs 5053 + 2473) at 24 hour. FXII activity correlated with FXIIa immunoreactivity in vitreous from eyes injected with BK(r=0.79, p <0.05).Vitreous from DME patients (n=19) show 48 fold increased FXII catalytic activity compared to vitreous from MH patients (n=13) (2320 + 680 vs 48 + 33, p<0.01). The FXII catalytic activity levels observed in vitreous from DME patient correlate with vitreous FXII (n=16, r=0.86, p<0.001), PK (n=19, r=0.85, p<0.001) levels but not with VEGF (n=19, r=0.07, p=0.75) levels.
Our data suggest that FXII catalytic activity is increased in the vitreous of DME subjects and in rodent models of BK- and VEGF- induced retinal edema. Extravasation of FXII across the retinal endothelium may contribute to the activation of the intraocular KKS.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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