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
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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-2.1

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.

Hear from the experts:
Ang-2 and vascular instability

Charles Wykoff, MD, PhD

Retina Consultants of Houston

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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.

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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 lesions5
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Reduction in CNV leakage area is more prolonged with Ang-2 inhibition than with VEGF inhibition.

TEER ASSAY* USING HUMAN ENDOTHELIAL CELLS

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

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

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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

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Inflammation

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

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

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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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References

  1. Saharinen P, et al. Nat Rev Drug Discov. 2017;16:635–61
  2. Regula JT, et al. EMBO Mol Med. 2019;11:e10666
  3. Regula JT, et al. EMBO Mol Med. 2016;8:1265–88 
  4. Benest AV, et al. PLoS One. 2013;8:e70459
  5. Chakravarthy U, et al. Presented at ARVO May 2020, Virtual Meeting
  6. Augustin HG, et al. Nat Rev Mol Cell Biol. 2009;10:165–77
  7. Fiedler U, et al. Nat Med. 2006;12:235–9
Ang-1
Ang-1 levels in human vitreous samples
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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
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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.

1 WEEK
1 week after last antibody dose
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**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
chart-cnv-leakage-3-weeks@2x.png

**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
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**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).