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

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

image-explore-the-evidence-interactive@2x.png

Inflammation Vascular leakage Neovascularisation

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

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.

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

Why was this study done?

Ang-2 has been shown to act as a facilitator of cytokine-induced vascular instability and also primes the endothelium to TNF-α–induced leukocyte adhesion. Based on this, Benest et al. hypothesised that Ang-2 may also act as a facilitator to vascular leakage-inducing cytokines, such as VEGF, histamine, and bradykinin.⁵
How was this study performed?

A Miles assay was performed in Ang-2–deficient mice and compared with wildtype control mice. A Miles assay assesses the ability of a cytokine injected under the skin, e.g. VEGF, to induce vascular leakage relative to a control substance (i.e. saline), by measuring intravenously injected albumin-bound Evans blue dye leakage from the vasculature. This leakage can be observed visually as blue dye on the skin, and quantified by looking at the absorbance.
What was the outcome?

Compared with wildtype controls, VEGF-induced vascular leakage was reduced in Ang-2–deficient mice, with similar results observed for the cytokines histamine and bradykinin. These findings suggest that Ang-2 acts as an essential facilitator for vascular leakage induced by these cytokines.

SPONTANEOUS CNV MOUSE MODEL^†

Ang-2 inhibition promotes sustained reduction of vascular leakage from CNV lesions⁶

1 week after last antibody dose

5 weeks after last antibody dose

**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.

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

Why was this study done?

Choroidal neovascularisation (CNV) progressively damages the retina. Both VEGF and Ang-2 are indicated to play a role in neovascularisation, endothelial dysfunction, and vessel leakiness. This experiment aimed to further elucidate the roles of VEGF and Ang-2 in pathologic neovascularisation and vascular leakage.
How was this study performed?

This 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 via intraperitoneal injection, and fluorescein angiography (FA) was performed at baseline and the assessed time points (1, 3, and 5 weeks after treatment [Weeks 1 and 5 presented here]). Tissues were collected and CNV lesion size was defined based on FA measurements of CNV leakage to determine the immediate and long-term effects on lesion size, leakage, and activity.
What was the outcome?

Compared with VEGF inhibition, inhibition of Ang-2 resulted in a prolonged effect of reducing vascular leakage from CNV lesions. The effects of Ang-2 inhibition lasted at least 5 weeks after treatment, whereas VEGF inhibition lost its effects at 3 weeks (data not shown here). These findings suggest a more prominent role for Ang-2 in mediating sustained vascular leakage.

Test your knowledge

Check your understanding of key preclinical evidence for angiopoietins and vascular instability

PDGF-B–DEFICIENT MOUSE MODEL^†

Ang-2 inhibition reduces vascular leakage in retinas with depleted pericytes and BRB impairment⁷

Ang-2 genetic deletion in
PDGF-B–deficient mice

IVT anti–Ang-2 in
PDGF-B–deficient mice

*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.

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

Why was this study done?

Pericyte dropout or apoptosis is a common feature of DR, but the involvement of pericytes in BRB regulation is not fully understood. Park et al. investigated the role of platelet-derived growth factor (PDGF)-B signalling in pericyte recruitment, BRB integrity and vascular leakage in retinal vessels, and the interplay with the Ang–Tie signalling pathway.⁷
How was this study performed?

Mice with an endothelial cell–specific deficiency of PDGF-B or Ang-2/PDGF-B in retinal vessels were generated using cre/lox recombination and displayed a range of phenotypes similar to hallmarks of DR, such as impaired pericyte coverage, disrupted cell junctions, hypoxia with increased VEGF-A and increased inflammation. Vascular leakage was analysed using FITC-conjugated dextran at P12 (left panel). Wildtype mice treated with a PDGF-B–blocking antibody at P1 were subsequently injected with an IVT Ang-2–blocking antibody at P5, and vascular leakage was analysed at P8 (right panel).
What was the outcome?

This study found that inhibition of Ang-2 via genetic deletion or blocking antibodies reduced vascular leakage in retinas with depleted pericytes. These data suggest that inhibiting Ang-2 could be beneficial in ameliorating BRB breakdown and DR progression and therefore potentially DME pathology.

Inflammation

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

Test your knowledge

How well do you understand the role of Ang-2 in inflammation?

Thanks for completing our quiz. Click here to view your results

MOUSE DORSAL SKINFOLD CHAMBER MODEL

Ang-2 promotes leukocyte adhesion and transmigration into inflamed tissues.⁹

Wild type

Ang-2–deficient

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.

Why was this study done?

During inflammation, exogenous stimuli such as TNF-α activate endothelial cells by inducing the expression of adhesion molecules, leading to leukocyte rolling and subsequent firm adhesion, which results in leukocyte transendothelial migration. This study aimed to understand the role of Ang-2 in regulating these inflammatory processes.
How was this study performed?

A titanium frame was implanted into the dorsal skinfold of wildtype control and Ang-2–deficient mice to sandwich a double layer of skin. One layer of skin was removed, leaving a layer consisting of striated muscle, subcutaneous tissue, and epidermis, which was covered with a glass cover slip to allow microscopic observations of the vasculature.

Wildtype control and Ang-2–deficient mice were intravenously injected with a fluorescent dye to allow visualisation of leukocytes in the vasculature. TNF-α was administered onto the skin, and the same vessels were observed after 10 minutes, 1 hour, and 2 hours. Leukocytes were considered adherent if they remained stationary for at least 30 seconds. Rolling leukocytes were reported as the percentage of nonadherent cells observed over 30 seconds.
What was the outcome?

In wildtype mice, following TNF-α stimulation, the number of rolling leukocytes initially increased before returning to baseline levels and the number of adherent leukocytes increased over time. In Ang-2–deficient mice, after TNF-α stimulation, leukocyte rolling increased over time and fewer adherent cells were observed compared with wildtype mice. These findings suggest a role for Ang-2 in controlling either the transition from rolling to firm leukocyte adhesion, 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; KO^EC, 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…

Continue to

Eye Travel Systems 2.0

References

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 [no. 628] presented at EURETINA, October 2020, Virtual Meeting
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

Ang-2

Ang-2 levels in human vitreous samples

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.

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

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

Median values

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

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

5 weeks

5 weeks after last antibody dose

References

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

Overview

Hear from the experts:

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

Clinical relevance

Preclinical evidence

Hear from the experts:

Ang-2 and vascular instability

Hear from the experts:

Ang-2 and vascular instability

Vascular leakage and neovascularisation

Ang-2 may act as a facilitator of VEGF-induced vascular leakage5

Visualisation of vascular leakage

Quantification of vascular leakage

Ang-2 inhibition promotes sustained reduction of vascular leakage from CNV lesions6

1 week after last antibody dose

5 weeks after last antibody dose

Ang-2 inhibition reduces vascular leakage in retinas with depleted pericytes and BRB impairment7

Ang-2 genetic deletion in PDGF-B–deficient mice

IVT anti–Ang-2 in PDGF-B–deficient mice

Inflammation

How well do you understand the role of Ang-2 in inflammation? Thanks for completing our quiz. Click here to view your results

Ang-2 promotes leukocyte adhesion and transmigration into inflamed tissues.9

Wild type

Ang-2–deficient

Eye Travel Systems 2.0

Ang-2 levels in human vitreous samples

Ang-1 levels in human vitreous samples

1 week after last antibody dose

5 weeks after last antibody dose

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

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

Vascular leakage
and neovascularisation

Ang-2 may act as a facilitator of VEGF-induced vascular leakage⁵

Ang-2 inhibition promotes sustained reduction of vascular leakage from CNV lesions⁶

Ang-2 inhibition reduces vascular leakage in retinas with depleted pericytes and BRB impairment⁷

Ang-2 genetic deletion in
PDGF-B–deficient mice

IVT anti–Ang-2 in
PDGF-B–deficient mice

How well do you understand the role of Ang-2 in inflammation?

Thanks for completing our quiz. Click here to view your results

Ang-2 promotes leukocyte adhesion and transmigration into inflamed tissues.⁹