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. 2020 May;177(9):1949-1966.
doi: 10.1111/bph.14962. Epub 2020 Feb 4.

The effects of anti-VEGF and kinin B1 receptor blockade on retinal inflammation in laser-induced choroidal neovascularization

Soumaya Hachana  1   2 , Olivier Fontaine  1   3 , Przemyslaw Sapieha  3 , Mark Lesk  3 , Réjean Couture  2 , Elvire Vaucher  1
Affiliations

The effects of anti-VEGF and kinin B1 receptor blockade on retinal inflammation in laser-induced choroidal neovascularization

Soumaya Hachana et al. Br J Pharmacol. 2020 May.

Abstract

Background and purpose: Age-related macular degeneration (AMD) is a complex neurodegenerative disease treated by anti-VEGF intravitreal injections. As inflammation is potentially involved in retinal degeneration, the pro-inflammatory kallikrein-kinin system is a possible alternative pharmacological target. Here, we investigated the effects of anti-VEGF and anti-B1 receptor treatments on the inflammatory mechanisms in a rat model of choroidal neovascularization (CNV).

Experimental approach: Immediately after laser-induced CNV, Long-Evans rats were treated by eye-drop application of a B1 receptor antagonist (R-954) or by intravitreal injection of B1 receptor siRNA or anti-VEGF antibodies. Effects of treatments on gene expression of inflammatory mediators, CNV lesion regression and integrity of the blood-retinal barrier was measured 10 days later in the retina. B1 receptor and VEGF-R2 cellular localization was assessed.

Key results: The three treatments significantly inhibited the CNV-induced retinal changes. Anti-VEGF and R-954 decreased CNV-induced up-regulation of B1 and B2 receptors, TNF-α, and ICAM-1. Anti-VEGF additionally reversed up-regulation of VEGF-A, VEGF-R2, HIF-1α, CCL2 and VCAM-1, whereas R-954 inhibited gene expression of IL-1β and COX-2. Enhanced retinal vascular permeability was abolished by anti-VEGF and reduced by R-954 and B1 receptor siRNA treatments. Leukocyte adhesion was impaired by anti-VEGF and B1 receptor inhibition. B1 receptors were found on astrocytes and endothelial cells.

Conclusion and implications: B1 receptor and VEGF pathways were both involved in retinal inflammation and damage in laser-induced CNV. The non-invasive, self-administration of B1 receptor antagonists on the surface of the cornea by eye drops might be an important asset for the treatment of AMD.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of anti‐VEGF on the expression of retinal inflammatory mediators, retinal vascular permeability, and retinal leukocyte adhesion. In the control and CNV retina treated with IgG or anti‐VEGF are shown (a–l) mRNA levels of B1 receptors, B2 receptors, VEGF‐A, VEGF‐R2, HIF‐1α, IL‐8, IL‐1β, TNF‐α, COX‐2, ICAM‐1, CCL2 and VCAM‐1, (m) Evans blue dye extravasation, (n) representative images of adherent leukocytes in retinal vessels. Scale bar = 100 μm. (o) Number of adherent leukocytes. Data are means ± SEM of values obtained from six rats per group. Ctl, control without lesion; CNV, laser‐induced choroidal neovascularization; IgG, immunoglobulin; *P ≤ .05, significantly different from control + IgG. + P ≤ .05, significantly different from CNV + IgG. B1R, B2R; B1 receptors, B2 receptors
Figure 2
Figure 2
Effect of R‐954 or B1 receptor siRNA on the expression of retinal inflammatory mediators, retinal vascular permeability, and retinal leukocyte adhesion. In the control and CNV retina treated with vehicle or R‐954, scrambled siRNA or B1 receptor siRNA are shown (a–l) mRNA levels of B1 receptors, B2 receptors, VEGF‐A, VEGF‐R2, HIF‐1α, IL‐8, IL‐1β, TNF‐α, COX‐2, ICAM‐1, CCL2, and VCAM‐1, (m) Evans blue dye extravasation, (n) representative images of adherent leukocytes in retinal vessels. Scale bar = 100 μm. (o) Number of adherent leukocytes. Data are means ± SEM of values obtained from six rats per group. Ctl, control without lesion; CNV, choroidal neovascularization; veh, vehicle; * P ≤ .05, significantly different from control + veh or control + scrambled siRNA. + P ≤ .05, significantly different from CNV + veh or CNV + scrambled siRNA. B1R, B2R; B1 receptors, B2 receptors
Figure 3
Figure 3
Micrographs of cellular distribution of B1 receptors on glial and endothelial cells. Representative micrographs of immunolabeling for B1 receptors (green), GFAP (orange), and RECA1 (red) in control (Ctl) and CNV retinas. Sections are counterstained for DAPI (blue) which labels cell nuclei. Note that B1 receptors co‐localized (yellow, arrows) with GFAP (panel c′) and RECA‐1 (panel f′) in CNV retina and to some extent in control retina (c, f). Data are from four rats. Scale bar: 75 μm. B1R, B1 receptors
Figure 4
Figure 4
Micrographs of cellular distribution of B1 receptors on microglia. Representative micrographs of immunolabeling for B1 receptors (green) and Iba‐1 (red) in control (Ctl) and CNV retinas. When appropriate, sections are counterstained for DAPI (blue) which labels cell nuclei. Iba‐1(+) cells were sparsely distributed in the control inner retina (panel b, arrows) but more intensively in CNV inner retina (panels b′, b‴, arrows) RPE and choroid (panels b″, b‴). No visible co‐localization was seen in most sections for B1 receptors and Iba‐1 in control (panel c). Note that B1 receptors partly co‐localized (yellow, arrows) with Iba‐1 in inner retina, subretinal space, RPE, and of CNV (panels c′, c″, c‴). CNV rat retina shows a network of ramified and ameboid Iba‐1 microglia in the ONL (panel d). Counting of ramified and ameboid‐shaped microglia in the ONL, subretinal space, and RPE (panel e). Data are from four rats. Scale bar: 75 μm (panels a–c‴) and 150 μm (panel d). B1R, B1 receptors
Figure 5
Figure 5
Micrographs of cellular distribution of B1 receptors on VEGF‐R2. Representative micrographs of immunolabelling for B1 receptors (green) and VEGF‐R2 (red) in control (Ctl) and CNV retinas. B1 receptors and VEGF‐R2 are co‐expressed partly (yellow) in the GCL layer of CNV retina (panel c′). Data are from four rats. Scale bar: 75 μm. B1R, B1 receptors
Figure 6
Figure 6
Effect of anti‐VEGF, R‐954, and B1 receptor siRNA on CNV lesion. CNV is shown as visible hyperfluorescence in the late phase of rat eyes with occult CNV on fluorescein angiography (panel a). The lesion area was more visible in the untreated eyes (left pictures, indicated by red circles and arrows) than in the treated eyes with IVT injection of anti‐VEGF (right pictures). Effects of IVT anti‐VEGF or B1 receptor siRNA and ocular administration of R‐954 on retinal vascular leakage are shown in representative micrographs of the FITC‐dextran (panel b) and quantified by the number of green pixels (panel c). The area of retinal lesions is identified in red in flat‐mounted CNV‐retinas. Data are means ± SEM of values from six rats (five laser spots for each group). * P ≤ .05, significantly different from CNV without treatment. Microphotographs of immunolocalization of B1 receptors (panel d, green) are shown on endothelial cells (panel d, red) in choroid (upper panels) and retina (bottom panels). Scale bar: 200 μm (a), 50 μm (b), 75 μm (d). B1R, B1 receptors
Figure 7
Figure 7
Possible interaction between kinin‐B1 receptor (B1R) and VEGF signalling in retinal inflammation. Data suggest that the pro‐inflammatory mediators (IL‐1β, CCL2, TNF‐α, ICAM‐1, VCAM‐1, and COX‐2) are differently up‐regulated (↑) by the B1 receptors and VEGF‐A, which can bind to both VEGF‐R1 and VEGF‐R2. These two main systems could favour pathological effects in a complementary but distinct manner in the vascular and inflammatory processes. B1 receptors and VEGF may work in concert as a loop of auto‐amplification mediated by TNF‐α known to up‐regulate B1 receptors and VEGF. In that scenario, the B1 receptor is instrumental for the inflammatory effects of VEGF. Anti‐VEGF IVT injection and topical application of R‐954 were both able to reduce TNF‐α expression in CNV retina. Thus, anti‐VEGF therapy prevents the up‐regulation of B1 receptors. Conversely, the anti‐B1 receptor antagonist does not affect the expression of the VEGF system and may exert its effects independently of VEGF
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