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. 2009 Feb;53(2):175-81.
doi: 10.1161/HYPERTENSIONAHA.108.117663. Epub 2009 Jan 5.

Plasma Kallikrein Mediates Angiotensin II Type 1 Receptor-Stimulated Retinal Vascular Permeability

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Plasma Kallikrein Mediates Angiotensin II Type 1 Receptor-Stimulated Retinal Vascular Permeability

Joanna A Phipps et al. Hypertension. .
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Hypertension is a leading risk factor for the development and progression of diabetic retinopathy and contributes to a variety of other retinal diseases in the absence of diabetes mellitus. Inhibition of the renin-angiotensin system has been shown to provide beneficial effects against diabetic retinopathy, both in the absence and presence of hypertension, suggesting that angiotensin II (Ang II) and the Ang II type 1 receptor may contribute to retinal vascular dysfunction. We investigated the effects of the Ang II type 1 receptor antagonist candesartan on retinal vascular permeability (RVP) in normotensive rats with streptozotocin-induced diabetes mellitus and in rats with Ang II-induced hypertension. We showed that candesartan treatment decreased diabetes mellitus- and Ang II-stimulated RVP by 58% (P<0.05) and 79% (P<0.05), respectively, compared with untreated controls, suggesting that activation of the Ang II type 1 receptor contributes to blood-retinal barrier dysfunction. We found that plasma kallikrein levels are increased in the retina of rats with Ang II-stimulated hypertension and that intravitreal injection of either plasma kallikrein or bradykinin is sufficient to increase RVP. We showed that a novel small molecule inhibitor of plasma kallikrein, 1-benzyl-1H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide, delivered systemically via a subcutaneous pump, decreased Ang II-stimulated RVP by 70% (P<0.05) and ameliorates Ang II-induced hypertension, measured from the carotid artery by telemetry, but did not reduce Ang II-induced retinal leukostasis. These findings demonstrate that activation of the Ang II type 1 receptor increases RVP and suggest that systemic plasma kallikrein inhibition may provide a new therapeutic approach for ameliorating blood-retinal barrier dysfunction induced by hypertension.

Conflict of interest statement

Conflict of Interest/Disclosure(s): S. Sinha and T.J. Chilcote, ActiveSite Pharmaceuticals, “Inhibitors of plasma kallikrein”, PCT Publication WO/2008/016883, 2008.


Figure 1
Figure 1. Effect of AT1-receptor blockade on RVP and blood pressure in diabetic rats
(A) NDM (non-diabetic, n=8), NDM+Cand (non-diabetic + candesartan, n=5), DM (2-week diabetes, n=12), DM+Cand (2-week diabetes + candesartan, n=11). Bars represent mean ±SEM, a.u. (arbitrary units). (B) Systolic (SBP) and diastolic (DBP) blood pressure measurements (mean ± SEM) using telemetry in diabetic rats before and after treatment with candesartan. P values indicate comparisons with Day 0.
Figure 2
Figure 2. Effects of chronic AngII-infusion and AT1-receptor blockade on RVP
(A) The effect of systemic AngII infusion on RVP measured by vitreous fluorescein intensity at 1, 3 and 6 days post-treatment in AngII (unfilled bars, n=4) and saline control animals (filled bars, n=5). (B) Representative fluorescein angiograms from 6-day saline- and AngII-treated rats. Upper panels show the arterial phase, with arrows indicating arterial caliber abnormalities. Lower panels show diffuse vascular leakage of the AngII animal in the venous phase compared with the saline control. (C) Effect on RVP following 6-days of saline (n=19), AngII (n=16), or AngII + candesartan (n=10) treatment. Bars represent mean ± SEM, a.u. (arbitrary units).
Figure 3
Figure 3. Western blot analysis of KKS components in retinal lysates
(A) Representative western blots showing immunoreactivity of proteins in saline (S)- and AngII (A)-treated animals. B1-R (bradykinin B1-receptor), B2-R (bradykinin B2-receptor), HK (cleaved high molecular weight kininogen). (B) Quantification of western blot results in saline (n=4 B-1R, n=3 B-2 R, n=11 kallikrein, n=12 HK) and AngII (n=4 B-1R, n=4 B-2 R, n=12 kallikrein, n=11 HK) animals infused for 6 days. Bars represent mean ± SEM.
Figure 4
Figure 4. Role of the kallikrein-kinin pathway in RVP and SBP
(A) Effect of intravitreal injection of BSS (balanced salt solution, n=4) or purified activated plasma kallikrein (n=5) on RVP measured by VFP. (B) Effect of intravitreal injection of BSS (n=5) or bradykinin (n=8) on RVP in the absence or presence of systemic infusion with HOE-140. (C) Fluorescein angiograms from representative animals treated for 3 days with saline, AngII and AngII + ASP-440 in the arterial (top panels) and venous (bottom panels) phases. Arrow indicates area of arterial caliber abnormalities in the AngII treated animal. (D) Effect of 3-day treatment with ASP-440 on AngII-mediated RVP increases. Saline (n=5), AngII (n=7), AngII + ASP-440 (n=7). (E) Effect of 3-day treatment with HOE-140 on AngII mediated RVP increases. Saline (n=8), AngII (n=7), AngII + HOE-140 (n=8). Bars represent mean ± SEM. (F) Time course of SBP measured using telemetry in rats infused with AngII in the absence or presence of ASP-440. * and † indicate P<0.05 for Day 0 vs AngII alone (n=4) and AngII+ASP-440 (n=7), respectively. ‡ indicates P<0.05 for AngII alone vs AngII+ASP-440.

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