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

Clinical relevance

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, supporting a role for Ang-2–Tie2 signalling in these pathologic conditions.^3,4

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,
Houston, Texas, United States

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.

Figures 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.⁵
How was this study performed?

A Miles assay was performed in Ang-2–deficient mice and compared with wild-type 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 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 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.

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The role of pericytes in retinal vascular stability

Healthy retina

Diseased retina

Healthy retina^7,8

In the healthy retina, pericytes line the outside of endothelial cells and are critical for retinal vessel formation, stability, and generation and maintenance of the blood retinal barrier.

Diseased retina^7,8

In the diseased retina, a deficiency of pericytes is associated with reduced barrier function and vascular instability.

Pericyte dropout or apoptosis is a common feature of diabetic retinopathy.

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.

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). Wild-type 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.^9,10

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MOUSE DORSAL SKINFOLD CHAMBER MODEL

Ang-2 promotes leukocyte adhesion and transmigration into inflamed tissues.¹⁰

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 wild-type 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.

Wild-type 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 wild-type 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 wild-type 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).

SPONTANEOUS CNV MOUSE MODEL^†

Ang-2 inhibition causes sustained prevention of subretinal macrophage infiltration from CNV lesions¹¹

1 week after last antibody dose

5 weeks after last antibody dose

****p<0.0001. Error bars represent means±SD.

^†The experiment involved 7-week old JR5558 mice (6–18 mice per group). Figures adapted from Canonica J, et al. Poster presented at ARVO, May 2021, Virtual Meeting.

The anti-inflammatory effect appears to be driven by Ang-2 inhibition.

Why was this study done?

Choroidal neovascularisation (CNV) progressively damages the retina. Both VEGF and Ang-2 are known to play a role in inflammatory activity; this experiment aimed to elucidate the roles of VEGF-A and Ang-2 inhibition in inflammatory cell infiltration associated with CNV in the subretinal space.
How was this study performed?

This experiment was performed in a CNV mouse model (JR5558) that develops spontaneous, leaky, neovascular lesions. Antibodies against VEGF-A and Ang-2 were administered via intraperitoneal injection, and tissues were collected and assessed via immunohistochemistry using flat-mounted retinal pigment epithelium/choroid at different time points (1, 3, and 5 weeks after treatment [Weeks 1 and 5 presented here]) to determine the immediate and long-term effects on the number of inflammatory cells around CNV lesions.
What was the outcome?

Only Ang-2 inhibition prevented significant subretinal infiltration of macrophages relative to control, at least 5 weeks after treatment; the effects of VEGF-A inhibition alone were not significant. These findings suggest a prominent role for Ang-2 in mediating and driving inflammatory activity associated with CNV in the subretinal space.

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.

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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
Santos GSP, et al. Eye. 2018;32:483–6
Augustin HG, et al. Nat Rev Mol Cell Biol. 2009;10:165–77
Fiedler U, et al. Nat Med. 2006;12:235–9
Canonica J, et al. Poster presented at ARVO, May 2021, Virtual Meeting

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

Clinical relevance

Ang-2 levels in human vitreous samples

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

The role of pericytes in retinal vascular stability

Healthy retina7,8

Diseased retina7,8

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.10

Wild type

Ang-2–deficient

Ang-2 inhibition causes sustained prevention of subretinal macrophage infiltration from CNV lesions11

1 week after last antibody dose

5 weeks after last antibody dose

Eye Travel Systems 2.0

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

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

Healthy retina^7,8

Diseased retina^7,8

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.¹⁰

Ang-2 inhibition causes sustained prevention of subretinal macrophage infiltration from CNV lesions¹¹