Explore the evidence

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

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-2.¹

Hear from the experts:

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

Anat Loewenstein, MD

Tel Aviv Medical Center

Clinical relevance

Ang-2 levels are elevated in retinal and choroidal vascular diseases, whereas Ang-1 levels remain balanced.^2,3

Preclinical evidence

Studies have shown that Ang-2 increases inflammation and vascular leakage, and facilitates the effects of VEGF.^3-5 Review the evidence below.

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

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

Quantification of
vascular leakage

Graph quantifying vascular leakage in wild type and Ang-2 deficient mice

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.

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.
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 lesions⁵

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). 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. These findings suggest a more prominent role for Ang-2 in mediating sustained vascular leakage.

TEER ASSAY* USING HUMAN ENDOTHELIAL CELLS

Inhibition of Ang-2 or addition of Ang-1 improves endothelial cell barrier integrity³

chart-integrity-of-endothelial-cells-key@2x.png

*Quantitative technique to measure the integrity of tight junction dynamics of endothelial (and epithelial) monolayers.

Adapted from Regula JT, et al. EMBO Mol Med. 2016;8(11):1265-88.

VEGF-A reduces the integrity of endothelial cell tight junctions.

Inhibition of Ang-2 or addition of Ang-1 improves barrier integrity.

Hear from the experts:

Ang-2 promotes endothelial permeability

Pipsa Saharinen, PhD

University of Helsinki

Why was this study done?

Breakdown of the endothelial barrier and resulting vascular leakage causes edema and perturbs physiological retinal functioning. VEGF-A induces the loss of endothelial barrier function; however, the role of angiopoietins in endothelial barrier integrity remains unclear.
How was this study performed?

TEER is a quantitative technique to measure barrier function in endothelial cell layers. Electrodes are placed on both sides of a cell layer on a semipermeable membrane, and an alternating current signal is applied. TEER measurements assess the electrical resistance across a cellular monolayer, indicating the integrity of endothelial cell tight junctions and permeability of the endothelial monolayer. In this study, human endothelial cells plated on filters were assessed for endothelial barrier function over time after the addition of VEGF-A alone or in combination with Ang-1, anti–Ang-2, or both Ang-1 and anti–Ang-2.
What was the outcome?

When cells were treated with VEGF-A, endothelial cell barrier function was dramatically reduced. Addition of either Ang-1 or anti–Ang-2 together with VEGF-A slightly improved barrier integrity, whereas addition of both anti–Ang-2 and Ang-1 together with VEGF-A resulted in even more improvement. These findings suggest that both VEGF-A and Ang-2 drive endothelial barrier breakdown, whereas Ang-1 promotes barrier integrity.

Inflammation

Ang-2 promotes leukocyte adhesion and transmigration.^6,7

Adapted from Augustin HG, et al. Nat Rev Mol Cell Biol. 2009;10:165–77.

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.

MOUSE DORSAL SKINFOLD CHAMBER MODEL

Leukocyte adhesion

Wild type

Error bars represent means±SD.

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.

Ang-2–deficient

Error bars represent means±SD.

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; CNV, choroidal neovascularisation; DR, diabetic retinopathy; EC, endothelial cell; FA, fluorescein angiography; FFA, fundus fluorescein angiography; nAMD, neovascular age-related macular degeneration; RVO, retinal vein occlusion; TEER, transendothelial electrical resistance; Tie, tyrosine kinase with immunoglobulin-like domains; TNF-α, tumour necrosis factor alpha; VEGF, vascular endothelial growth factor.

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

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Eye Travel Systems 2.0

References

Saharinen P, et al. Nat Rev Drug Discov. 2017;16:635–61
Regula JT, et al. EMBO Mol Med. 2019;11:e10666
Regula JT, et al. EMBO Mol Med. 2016;8:1265–88
Benest AV, et al. PLoS One. 2013;8:e70459
Chakravarthy U, et al. Presented at ARVO May 2020, Virtual Meeting
Augustin HG, et al. Nat Rev Mol Cell Biol. 2009;10:165–77
Fiedler U, et al. Nat Med. 2006;12:235–9

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

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.

Ang-2 levels are increased in the vitreous of patients with retinal or choroidal vascular diseases, including AMD, DR, and RVO,^2,3 supporting a role for the Ang-2–Tie2 pathway in these pathologic conditions.

References

1 WEEK

1 week after last antibody dose

**p<0.01; ***p<0.001; ****p<0.0001. Error bars represent means±SD.
^†The experiment involved 7-week-old JR5558 mice (5–10 mice per group).

3 WEEKS

3 weeks after last antibody dose

**p<0.01; ***p<0.001; ****p<0.0001. Error bars represent means±SD.
^†The experiment involved 7-week-old JR5558 mice (5–10 mice per group).

5 WEEKS

5 weeks after last antibody dose

**p<0.01; ***p<0.001; ****p<0.0001. Error bars represent means±SD.
^†The experiment involved 7-week-old JR5558 mice (5–10 mice per group).

Explore the evidence

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

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

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 lesions5

Inhibition of Ang-2 or addition of Ang-1 improves endothelial cell barrier integrity3

Hear from the experts:

Ang-2 promotes endothelial permeability

Inflammation

Ang-2 promotes leukocyte adhesion and transmigration.6,7

Leukocyte adhesion

Wild type

Ang-2–deficient

Eye Travel Systems 2.0

Ang-1 levels in human vitreous samples

Ang-2 levels in human vitreous samples

1 week after last antibody dose

3 weeks 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, neovascularisation, and inflammation.¹

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

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 lesions⁵

Inhibition of Ang-2 or addition of Ang-1 improves endothelial cell barrier integrity³

Ang-2 promotes leukocyte adhesion and transmigration.^6,7