Explore the evidence

Experimental evidence has shown that Ang-2 and VEGF work together to drive vascular instability characterised by vascular leakage, inflammation, and neovascularisation.1,2
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Overview

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

Hear from the experts:
It’s all in the balance:
Ang-1 vs Ang-2

Anat Loewenstein, MD

Tel Aviv Medical Center

Clinical relevance

Preclinical evidence

Studies have shown that Ang-2 increases inflammation and vascular leakage, and facilitates the effects of VEGF.2,4-7 Review the evidence below.

Hear from the experts:
Ang-2 and vascular instability

Charles Wykoff, MD, PhD

Retina Consultants of Houston

Hear from the experts:
Ang-2 and vascular instability

Charles Wykoff, MD, PhD

Retina Consultants of Houston

Vascular leakage
and neovascularisation

MOUSE SKIN MODEL MILES ASSAY

Ang-2 may act as a facilitator of VEGF-induced vascular leakage5
Visualisation of vascular leakage
Miles assay showing vascular leakage in wild type and Ang-2 deficient mice
Quantification of vascular leakage
Chart / Vascular leakage 0 0.1 0.2 0.3 0.4 Control VEGF (25 ng) Absorbance (620-405 nm) ** **

Wild type Ang-2–deficient mice

**p<0.01. Error bars represent means±SD.

Adapted from Benest AV, et al. PLoS One. 2013;8:e70459.

© 2013 Benest et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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VEGF-induced vascular leakage is attenuated in Ang-2–deficient mice.

SPONTANEOUS CNV MOUSE MODEL

Ang-2 inhibition promotes sustained reduction of vascular leakage from CNV lesions6
1 week after last antibody dose
Chart / CNV leakage / 1 week 0 100,000 50,000 150,000 Control Anti–VEGF-A Anti–Ang-2 CNV leakage area (FFA; pixels) *** ***
5 weeks after last antibody dose
Chart / CNV leakage / 5 weeks 0 100,000 50,000 150,000 Control Anti–VEGF-A Anti–Ang-2 CNV leakage area (FFA; pixels) ** ***

**p<0.01; ***p<0.001. Error bars represent means±SD.
†The experiment involved 7-week-old JR5558 mice (5–10 mice per group). Figures adapted from Canonica J, et al. poster [no. 628] presented at EURETINA, October 2020, Virtual Meeting. 

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Reduction in CNV leakage area is more prolonged with Ang-2 inhibition than with VEGF inhibition.

PDGF-B–DEFICIENT MOUSE MODEL

Ang-2 inhibition reduces vascular leakage in retinas with depleted pericytes and BRB impairment7
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Ang-2 genetic deletion in 
PDGF-B–deficient mice
75D51DBE-1448-4390-892A-1DC211535B43 0 20 10 30 PDGF-B KO EC Ang-2/PDGF-B KO EC Dextran leakage (%) *
IVT anti–Ang-2 in
PDGF-B–deficient mice
chart / anti-ang-2 0 10 5 15 Fc control IVT anti–Ang-2 Vascular leakage (%) **

*p<0.05 versus pericyte depleted retina. **p<0.01 versus Fc control. Error bars represent means±SD. 

†Mouse model of pericyte depletion in postnatal retina with PDGF-B deficiency. 

Figures adapted from Park DY, et al. Nat Commun. 2017;16:15296.

© 2017 Park et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Vascular leakage is reduced in retinas with depleted pericytes following Ang-2 inhibition.

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Inflammation

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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

Ang-2 promotes leukocyte adhesion and transmigration into inflamed tissues.9
Wild type
Chart / Wild type 0 1 2 3 0 10 60 120 TNF-α stimulation (min) Adherent cells/mm 2 (x10 −3 )
Ang-2–deficient
Chart / ang-2 deficient 0 1 2 3 0 10 60 120 TNF-α stimulation (min) Adherent cells/mm 2 (x10 −3 )

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. 

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Leukocyte adhesion is defective in Ang-2–deficient mice, suggesting an important role of Ang-2 in the inflammatory response.

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.

Explore the roles of VEGF and Ang-2 in DME and nAMD through an interactive Eye Travel simulation…

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References

  1. Saharinen P, et al. Nat Rev Drug Discov. 2017;16:635–61
  2.  Joussen AM, et al. Eye. 2021;35:1305–16
  3. Regula JT, et al. EMBO Mol Med. 2019;11:e10666
  4. Regula JT, et al. EMBO Mol Med. 2016;8(11):1265–88
  5. Benest AV, et al. PLoS One. 2013;8:e70459
  6. Canonica J, et al. poster [no. 628] presented at EURETINA, October 2020, Virtual Meeting
  7. Park DY, et al. Nat Commun. 2017;16:15296
  8. Augustin HG, et al. Nat Rev Mol Cell Biol. 2009;10:165–77
  9. Fiedler U, et al. Nat Med. 2006;12:235–9
Ang-2
Ang-2 levels in human vitreous samples
1 4 16 64 1024 256 4096 16,384 (pg/mL) Log scale Control nAMD R VO DR PDR **** 1625 **** 302 **** 1 140 * 139 68.4

Median values

*p<0.05; ****p<0.0001.

A nonparametric Kruskal–Wallis analysis followed by Dunn’s method for multiple comparisons was used to show significant differences of the groups to control.

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

This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited

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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

Ang-1
Ang-1 levels in human vitreous samples
1 4 16 64 1024 256 4096 16,384 (pg/mL) Log scale Control nAMD R VO DR PDR 34.8 107.6 107.3 86.2 65.4

Median values

Adapted from Regula JT, et al. EMBO Mol Med. 2019;11:e10666.

© 2019 F. Hoffmann‐La Roche AG Published under the terms of the CC BY 4.0 license

This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited

icon-info.png

Ang-1 levels were similar in the vitreous samples from patients with retinal or choroidal vascular diseases and in samples from healthy individuals, suggesting stable expression in normal and pathologic conditions.2,3

1 week
1 week after last antibody dose
Chart / CNV leakage / 1 week 0 100,000 50,000 150,000 Control Anti–VEGF-A Anti–Ang-2 CNV leakage area (FFA; pixels) *** ***
5 weeks
5 weeks after last antibody dose
Chart / CNV leakage / 5 weeks 0 100,000 50,000 150,000 Control Anti–VEGF-A Anti–Ang-2 CNV leakage area (FFA; pixels) ** ***