MOUSE SKIN MODEL MILES ASSAY
Experimental evidence has shown that Ang-2 and VEGF work together to drive vascular instability characterised by vascular leakage, inflammation, and neovascularisation.1,2
In the retina, under pathologic conditions, an angiogenic switch occurs, shifting the balance from anti- to pro-angiogenic factors, including the upregulation of Ang-2 as well as VEGF.1
What happens to Ang-1 and Ang-2 values in retinal diseases?
*p < 0.05; ***p < 0.0001. Kruskal–Wallis non-parametric analysis according to Dunn's method of multiple comparisons was used to show significant differences in the control groups. Adapted from Regula JT, et al. EMBO Mol Med. 2016;8(11):1265–88.© 2019 F. Hoffmann – La Roche AG Published under the terms of the CC BY 4.0 license. Open access article under the terms of the Creative Commons Attribution 4.0 license under which use, distribution, and reproduction in any medium is permitted provided the original work is properly cited.
Ang-2 levels are increased in the vitreous of patients with retinal or choroidal vascular diseases, including AMD, DR, and RVO, supporting a role for Ang-2–Tie2 signalling in these pathologic conditions.3,4
Studies have shown that Ang-2 increases inflammation and vascular leakage, and facilitates the effects of VEGF.2,4-7 Review the evidence below.
Dr. Charles Wykoff
Retina Consultants of Houston Texas, U.S.
M-IL-00001671
**p < 0.01. Error bars represent mean ± standard deviation. Adapted from Benest AV, et al. PLoS One. 2013.;8:e70459.© 2013. Benest et al. Open access article distributed under the terms of the Creative Commons Attribution license under which unrestricted use, distribution, and reproduction in any medium is permitted provided the original author and source are properly cited.
VEGF-induced vascular leakage is attenuated in Ang-2–deficient mice.
Ang-2 has been shown to enhance cytokine-induced vascular instability and sensitize TNF-α-mediated leukocyte adhesion. Based on this, Benest et al. hypothesized that Ang-2 may also support cytokines that promote vascular leakage, such as VEGF, histamine, and bradykinin.5
The Miles study was performed on Ang-2-deficient mice and compared to wild-type control mice. The Miles test analyzes the ability of a subcutaneously injected cytokine, e.g., VEGF, to stimulate vascular leakage relative to a controlled substance (i.e., saline) by measuring the leakage of intravenously injected Evans blue dye that binds to albumins from the vasculature. This leakage can be visually observed as a blue color on the skin and quantified by observing absorption.
Compared with wild-type controls, VEGF-induced vascular leakage was reduced in Ang-2-deficient mice with similar results observed for the cytokines histamine and bradykinin. These results point to the fact that Ang-2 is crucial in the increase in vascular leakage induced by these cytokines.
SPONTANEOUS CNV MOUSE MODEL†
*p < 0.01; **p < 0.001. Error bars represent mean ± standard deviation.†Seven-week-old JR5558 mice (5–10 mice per group) were included in the experiment. Images adapted from Canonica J, et al. poster [no. 628] presented at EURETINA in October 2020, virtual meeting.
Reduction in CNV leakage area is more prolonged with Ang-2 inhibition than with VEGF inhibition.
Choroidal neovascularization (CNV) progressively damages the retina. Both VEGF and Ang-2 have been implicated as factors that play a role in neovascularization, endothelial dysfunction, and vessel leakage. The aim of this experiment is to further clarify the role of VEGF and Ang-2 in pathological neovascularization and vascular leakage.
The experiment was performed in a spontaneous CNV mouse model (JR5558) that develops spontaneous leaky neovascular lesions. Antibodies for VEGF-A and Ang-2 were administered by intraperitoneal injection, and fluorescein angiography (FA) was performed at baseline and at the analyzed time points (one week, three weeks, and five weeks after treatment [weeks 1 and 5 shown here]). . Tissues were collected and CNV lesion size determined based on FA measurements of CNV leakage to determine immediate and long-term effects on lesion size, leakage and activity..
Compared to VEGF inhibition, Ang-2 inhibition resulted in a prolonged effect of reducing vascular leakage from CNV lesions. The effects of Ang-2 inhibition lasted for at least five weeks after treatment, while VEGF inhibition ceased to be effective after three weeks. The results indicate a greater role of Ang-2 in increasing vascular permeability.
PDGF-B DEFICIENT MOUSE MODEL†
*p < 0.05 compared to retina without pericytes. **p < 0.01 compared to control Fc. Error bars represent mean ± standard deviation.†A pericyte-depleted mouse model in PDGF-B-deficient postnatal retina. Figures adapted from Park DY, et al. Nat Commun. 2017.;16:15296.© 2017. Park et al. Open access article distributed under the terms of the Creative Commons Attribution 4.0 license under which unrestricted use, distribution, and reproduction in any medium is permitted provided the original author and source are properly cited.
Vascular leakage in the retina with pericyte loss is reduced by Ang-2 inhibition.
Pericyte loss or apoptosis is a common feature of DR, but the involvement of pericytes in regulating BRB is not entirely clear. Park et al. investigated the role of platelet-derived growth factor (PDGF)-B in signalling pericyte recruitment, BRB integrity, and vascular leakage in retinal vessels and interaction with the Ang–Tie signalling pathway.7
Retinal vascular endothelial cell-specific PDGF-B or Ang-2/PDGF-B deficient mice were generated using cre/lox recombination and displayed a range of phenotypes similar to the hallmarks of DR such as impaired pericyte coverage, impaired cell junctions, hypoxia with increased VEGF-A and increased inflammation. Vascular leakage was analyzed using FITC-conjugated dextran at P12 (left panel). Wild-type mice treated with PDGF-B blocking antibody at P1 were subsequently injected with IVT Ang-2 blocking antibody at P5, and vascular leakage was analyzed at P8 (right panel).
This study demonstrated that inhibiting Ang-2 via genetic deletion or blocking antibodies reduced retinal vascular leakage with pericyte loss. The obtained data indicate that inhibition of Ang-2 may be useful for preventing BRB damage and DR progression, and thus potentially DME pathology.
During inflammation, cytokines such as TNF-α induce the expression of adhesion molecules on the endothelial cell surface, mediating leukocyte tethering and rolling. Ang-2 acts as an amplifier to activate and sensitise vascular endothelial cells to inflammatory cytokines, regulating the transition from leukocyte rolling to firm adhesion and facilitating leukocyte migration across the vascular endothelium, into tissues such as the retina. 8,9
MOUSE DORSAL SKINFOLD CHAMBER MODEL
Error bars represent means±SD.
Figures reprinted from Nature Medicine, 12(2), Fiedler U, et al., Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation, 235–9, Copyright (2006), with permission from Springer Nature.
Leukocyte adhesion is defective in Ang-2–deficient mice, suggesting an important role of Ang-2 in the inflammatory response.
During inflammation, exogenous stimuli, such as TNF-α, activate endothelial cells by stimulating the expression of adhesive molecules, resulting in leukocyte adhesion and then firm adhesion, resulting in transendothelial migration of leukocytes. The aim of this study is to understand the role of Ang-2 in the regulation of these inflammatory processes.
A titanium frame was implanted into the dorsal skin fold of control wild-type and Ang-2-deficient mice to surround the skin bilayer. One layer of skin was removed, leaving a layer consisting of striated muscle, subcutaneous tissue, and epidermis, which is covered with a glass slide to allow microscopic observation of the vasculature.
Control wild-type and Ang-2-deficient mice were intravenously injected with a fluorescent dye to allow visualization of leukocytes in the vasculature. TNF-α was applied to the skin and the same vessels were observed after 10 minutes, one hour and two hours. Leukocytes were considered adherent if they remained in place for at least 30 seconds. Moving leukocytes were scored/marked as the percentage of non-adherent cells observed for more than 30 seconds.
After TNF-α stimulation in wild-type mice, the number of moving leukocytes initially increased before returning to baseline values, and the number of adherent leukocytes increased over time. In Ang-2-deficient mice after TNF-α stimulation, leukocyte adhesion increased over time and fewer adherent cells were observed compared to wild-type mice. These findings suggest a role for Ang-2 in controlling the transition from adhesion to firm adhesion of leukocytes or the overall sensitivity of endothelial cells to inflammatory stimuli (such as TNF-α).
AMD, age-related macular degeneration; Ang, angiopoietin; BRB, blood–retinal barrier; CNV, choroidal neovascularisation; DME, diabetic macular edema; DR, diabetic retinopathy; FA, fluorescein angiography; Fc, fragment crystallisable; FFA, fundus fluorescein angiography; FITC, fluorescein isothiocyanate; IVT, intravitreal; KOEC, endothelial cell-specific knockout; nAMD, neovascular age-related macular degeneration; Px, postnatal day x; PDGF, platelet-derived growth factor; PDR, proliferative diabetic retinopathy; RVO, retinal vein occlusion; Tie, tyrosine kinase with immunoglobulin-like domains; TNF-α, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.
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Reference:
- Saharinen P, et al. Nat Rev Drug Discov. 2017.;16:635.–61.
- Joussen AM, et al. Eye. 2021.;35;1305.–16.
- Regula JT, et al. EMBO Mol Med. 2019.;11:e10666
- Regula JT, et al. EMBO Mol Med. 2016.;8(11):1265.–88.
- Benest AV, et al. PLoS One. 2013.;8:e70459
- Canonica J, et al. poster [br. 628] prezentiran na EURETINI u listopadu 2020., virtualni sastanak
- Park DY, et al. Nat Commun. 2017.;16:15296
- Augustin HG, et al. Nat Rev Mol Cell Biol. 2009.;10:165.–77.
- Fiedler U, et al. Nat Med. 2006.;12:235.–9.