Elicit: Aflibercept and VEGF: Impact on Vascular Dynamics

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Aflibercept and VEGF: Impact on Vascular Dynamics

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May 6, 2026

How does aflibercept's pharmacology relate to VEGF-driven vascular permeability and neovascularization?

Aflibercept's pharmacological properties—exceptionally high binding affinity, broad ligand specificity for VEGF-A/B and PlGF, and prolonged vitreal retention—directly produce superior and sustained suppression of vascular permeability and neovascularization through VEGF neutralization, secondary angiopoietin-2 suppression, and galectin-1 pathway inhibition, though effective VEGF blockade triggers compensatory HIF-1α/ANGPTL4 responses that can limit therapeutic efficacy.

Abstract

Aflibercept’s pharmacological properties directly determine its effects on VEGF-driven vascular permeability and neovascularization through multiple interconnected mechanisms. The molecule’s fusion protein structure, comprising VEGFR1/2 extracellular domains and IgG Fc, enables exceptionally high-affinity binding to VEGF-A (K D = 171.9 fM)—approximately 100-fold stronger than ranibizumab—and broader ligand specificity encompassing VEGF-A, VEGF-B, and PlGF. This superior binding translates to prolonged suppression of vascular permeability, with aflibercept maintaining leak inhibition to day 55 compared to day 35 for ranibizumab and completely restoring VEGF-A-disturbed endothelial barrier function at concentrations ≤25 μg/ml. The anti-neovascularization effects extend beyond direct VEGF neutralization: aflibercept suppresses angiopoietin-2 protein and mRNA through downstream pathway modulation, neutralizes galectin-1-mediated VEGFR2 activation independent of VEGF, and reduces subretinal mononuclear phagocyte recruitment through combined VEGF-A/PlGF inhibition. However, effective VEGF blockade triggers counterregulatory HIF-1α accumulation and ANGPTL4 expression in retinal pigment epithelium, which can limit therapeutic responses in some patients. The relationship between aflibercept’s pharmacology and vascular effects is further modulated by Fc-mediated cellular uptake and context-dependent dose thresholds, with therapeutic effects occurring at concentrations considerably lower than those interfering with normal cellular functions.

Methods

We analyzed 10 sources from an initial pool of 200, using 6 screening criteria. Each paper was reviewed for 7 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Aflibercept as Primary Intervention, VEGF-Driven Vascular Outcomes, Pharmacological Mechanisms, Pathological VEGF Activity, Appropriate Study Design, Measurable Outcomes with Mechanistic Data

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How does aflibercept’s pharmacology relate to VEGF-driven vascular permeability and neovascularization?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract aflibercept’s pharmacological properties and mechanism of action relevant to VEGF pathway modulation, including:

Extract findings on how aflibercept affects VEGF-driven vascular permeability, including:

Extract findings on how aflibercept affects VEGF-driven angiogenesis and neovascularization, including:

Extract data linking aflibercept plasma/tissue levels to biological effects on VEGF-driven vascular processes, including:

Extract mechanistic findings that explain how aflibercept’s pharmacological properties lead to effects on VEGF-driven vascular permeability and neovascularization, including:

Extract details about the experimental system and disease context that affect interpretation of aflibercept’s pharmacology-vascular effects relationship, including:

Extract comparative data showing how aflibercept’s pharmacological profile and vascular effects differ from other anti-VEGF agents (bevacizumab, ranibizumab, etc.), including:

Results

Characteristics of Included Studies

Study

Full text retrieved?

Disease Model/Condition

Experimental System

Aflibercept Dose/Regimen

Specific Vascular Bed

Key Focus

A. Lockhart et al., 2010

No

Refractory solid tumors or non-Hodgkin’s lymphoma

Human patients, Phase I trial

0.3-7.0 mg/kg IV every 2 weeks; recommended 4 mg/kg

Tumor vascularity

Safety, pharmacokinetics, pharmacodynamics

H. Deissler et al., 2014

No

Diabetic retinopathy, diabetic macular edema

In vitro, bovine retinal endothelial cells

≤25 μg/ml

Retinal microvascular endothelial cells

Barrier function, proliferation, migration

C. Lange et al., 2023

Yes

Retinal vascular hyperpermeability

In vivo, Dutch belted rabbits

1 mg (50% clinical dose)

Retina and choroid

ANG2 suppression, VEGF neutralization

Sergio Crespo-Garcia et al., 2017

No

Choroidal neovascularization (CNV)

In vivo and ex vivo

Intravitreal injection

Choroid

Mononuclear phagocyte recruitment, PlGF inhibition

Atsuhiro Kanda et al., 2015

Yes

Proliferative diabetic retinopathy (PDR)

Human (in vitro and surgical samples)

Not specified

Retina, neovascular tissues

Galectin-1 interaction, VEGFR2 signaling

Deepti Sharma et al., 2024

Yes

Neovascular age-related macular degeneration (nvAMD)

Human samples and mouse model

300 ng, 400 ng intravitreal

Retina, RPE, choroidal neovascularization

HIF-1α and ANGPTL4 expression

G. Lang et al., 2014

No

Retinal diseases

In vitro and in vivo monkey eyes

Not specified

Retina

Cellular uptake, PlGF inhibition

W. Schubert et al., 2022

Yes

Retinal vascular hyperpermeability

In vivo, Dutch belted rabbits

Not explicitly mentioned

Retina

Comparative binding affinity, pharmacokinetics

D. Lal et al., 2010

No

Human acute myeloid leukemia (AML)

Xenograft in immunodeficient mice

Not mentioned

AML xenografts and leukemia-associated vasculature

Antiangiogenic effects, chemotherapy enhancement

Orly Gal-Or et al., 2016

No

Corneal neovascularization (chemical burn)

In vivo, Sprague-Dawley rats

0.08 mL (25 mg/mL) subconjunctival

Cornea

Neovascularization inhibition

toof

Pageof

The included studies spanned multiple experimental systems, from in vitro retinal cell cultures to clinical trials, with the majority focusing on retinal vascular beds. Eight of ten studies examined retinal pathology (diabetic retinopathy, macular edema, choroidal neovascularization), while two investigated tumor and corneal vasculature. Full text was available for only three studies (C. Lange et al., 2023; Atsuhiro Kanda et al., 2015; Deepti Sharma et al., 2024; W. Schubert et al., 2022).

Aflibercept’s Molecular Structure and VEGF Binding Properties

Aflibercept’s molecular design comprises the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human immunoglobulin G, forming a glycoprotein fusion protein. This structure enables aflibercept to bind multiple VEGF family ligands with high affinity. The binding specificity extends to VEGF-A, VEGF-B, and placental growth factor (PlGF), distinguishing it from ranibizumab which only recognizes VEGF-A.

Binding affinity measurements revealed aflibercept’s exceptionally strong interaction with VEGF. In kinetic exclusion assays, aflibercept demonstrated a K D of 171.9 fM for VEGF-A, significantly lower than ranibizumab (K D = 21.8 pM) or brolucizumab (K D = 1.3 pM). This represents approximately 100-fold higher affinity than ranibizumab. The in vitro potency, measured by half-maximal inhibitory concentration (IC 50), was 2.42 nM for aflibercept, the lowest among compared anti-VEGF agents. Pharmacokinetic analysis in rabbit eyes showed a vitreal half-life of approximately 5.63 days, longer than both brolucizumab (3.10 days) and ranibizumab (3.15 days).

The mechanism of VEGF sequestration involves direct binding to VEGF ligands, preventing their interaction with endogenous VEGF receptors and thereby reducing downstream signaling. In clinical studies, maximum plasma concentration of free aflibercept increased proportionally with dose, while maximal VEGF-bound aflibercept complex levels were reached at doses ≥2.0 mg/kg. The recommended phase II dose for systemic administration was established at 4 mg/kg every 2 weeks.

Effects on VEGF-Driven Vascular Permeability

Aflibercept demonstrated robust effects on restoring and preventing VEGF-induced vascular permeability across multiple experimental systems. In bovine retinal endothelial cells, aflibercept completely restored VEGF-A-disturbed barrier function at concentrations ≤25 μg/ml without interfering with normal barrier function at higher concentrations. This restoration was measured using transendothelial resistance (TER) and expression of the tight junction protein claudin-1.

The mechanism underlying permeability reduction involves multiple pathways. Beyond direct VEGF neutralization, aflibercept suppressed angiopoietin-2 (ANG2) protein levels in vitreous and ANGPT2 mRNA in retinal tissue. ANG2 binds to TIE2 receptors, affecting endothelial barrier function, and anti-ANG2 antibodies have been shown to attenuate reductions in transendothelial electrical resistance (TEER). Aflibercept demonstrated the greatest inhibitory effect on ANG2 levels among anti-VEGF agents tested, correlating with strong, durable suppression of intraocular VEGF levels.

In the rabbit retinal vascular hyperpermeability model, aflibercept showed significantly greater efficacy in preventing retinal microvascular leakage compared to brolucizumab and ranibizumab. The duration of effective VEGF inhibition, as measured by prevention of vascular leakage, extended to day 55 for aflibercept, compared to day 44 for brolucizumab and day 35 for ranibizumab. These differences aligned with pharmacokinetic properties, with moderate to severe leakage occurring when more than 40% of injected VEGF was not captured by the anti-VEGF agent.

In tumor vasculature, dynamic contrast-enhanced MRI (DCE-MRI) measurements demonstrated dose-related changes in volume transfer constant at 24 hours after administration, indicating effects on tumor vascular permeability. Similarly, in choroidal neovascularization models, aflibercept treatment correlated with reduced leakage, though specific permeability measurements were not detailed.

Effects on VEGF-Driven Neovascularization

Aflibercept exerted potent anti-angiogenic effects across diverse models of pathological neovascularization. In the corneal neovascularization model induced by chemical burn, aflibercept significantly reduced the relative area of neovascularization compared to both control (12.27±9.91% vs. 29.66±9.96% at day 7, p<0.05) and bevacizumab-treated groups (12.27±9.91% vs. 21.27±8.19% at day 7, p<0.05). Histological analysis revealed blood vessels limited to the peripheral cornea in aflibercept-treated eyes, contrasting with central corneal extension in control and bevacizumab groups.

In choroidal neovascularization models, aflibercept reduced subretinal mononuclear phagocyte accumulation, particularly those expressing PlGF. The combined inhibition of VEGF-A and PlGF proved superior to specific anti-PlGF treatment alone in terms of subretinal mononuclear phagocyte recruitment. This effect correlated with reduced leakage associated with CNV. The mechanism involves the PlGF/VEGFR1 pathway in immune cells, which plays a pivotal role in early CNV progression.

In acute myeloid leukemia xenograft models, aflibercept reduced VEGF/VEGFR-positive AML colony formation growth in vitro and inhibited AML xenograft growth by up to 93%. This was associated with antiangiogenic and antiproliferative effects, hypoxia, and VEGF sequestration. Vascular changes included vascular narrowing, decreased vessel number, and perivascular apoptosis.

Aflibercept also inhibited proliferation and migration of retinal endothelial cells stimulated by VEGF-A and PlGF. At therapeutically achievable concentrations, VEGF-A-stimulated migration was reduced not only to baseline but driven below basal levels. However, this effect required careful interpretation, as high concentrations of IgG domain-containing proteins (including bevacizumab and the unrelated antibody rituximab) also inhibited basal or VEGF-A-stimulated migration at clinically relevant concentrations. Nevertheless, aflibercept specifically blocked VEGF-A-stimulated migration at lower concentrations without influencing basal processes.

Beyond VEGF: Galectin-1 Neutralization

Aflibercept demonstrated a novel anti-angiogenic mechanism independent of VEGF family binding. Immunoprecipitation and mass spectrometry identified galectin-1 as an aflibercept-interacting protein. Biolayer interferometry revealed aflibercept binding to galectin-1 with higher affinity than VEGFR1-Fc and VEGFR2-Fc, with binding abolished by deglycosylation with peptide:N-glycosidase F. This indicates the interaction occurs through aflibercept’s glycosylation rather than its protein backbone.

In proliferative diabetic retinopathy, retinal LGALS1/Galectin-1 mRNA expression was enhanced by hypoxic stimulation in vitro and by disease induction in vivo. Galectin-1 immunoreactivity co-localized with VEGFR2 in neovascular tissues surgically excised from human eyes with PDR. Intravitreal galectin-1 protein levels were elevated in PDR eyes compared to non-diabetic controls, showing no correlation with increased VEGF-A levels. Notably, preoperative bevacizumab injection reduced VEGF-A but not galectin-1 levels, demonstrating galectin-1’s independence from VEGF-A regulation.

Functionally, galectin-1 application to human retinal microvascular endothelial cells up-regulated VEGFR2 phosphorylation, which was eliminated by aflibercept. This suggests aflibercept can suppress both VEGF-driven and galectin-1-driven VEGFR2 activation, providing a mechanistic advantage over VEGF-A-specific inhibitors.

Cellular Uptake and Intracellular Effects

Aflibercept uptake by retinal cells was observed after 1 hour of treatment, with amounts increasing during prolonged incubation. Most internalized aflibercept localized to subcellular fractions of proteins assigned to membranes and organelles, with co-immunofluorescence showing localization in or close to the Golgi apparatus. The Fc antibody domain was implicated in facilitating this uptake, with aflibercept and bevacizumab showing similar internalization patterns while ranibizumab was internalized in smaller amounts.

This cellular uptake had functional consequences. At high concentrations, aflibercept interfered with normal retinal endothelial cell migration and pigment epithelial cell phagocytosis. The uptake and storage by ocular cells could have yet-unrecognized implications for long-term application, though aflibercept prevented and restored VEGF-A-induced disturbances at considerably lower concentrations than those causing interference with normal functions.

Counterregulatory Responses: HIF-1α and ANGPTL4

Anti-VEGF therapy with aflibercept triggered countertherapeutic mechanisms in neovascular age-related macular degeneration. Following aflibercept treatment, VEGF expression decreased but ANGPTL4 expression increased. This increase resulted from HIF-1α accumulation in the retinal pigment epithelium in response to VEGF/KDR inhibition. ANGPTL4, a HIF-regulated angiogenic factor, can promote neovascularization despite VEGF reduction.

In laser-induced CNV mouse models, aflibercept increased ANGPTL4 mRNA and protein expression. The combination of aflibercept with 32-134D, a HIF-1 inhibitor, prevented this countertherapeutic increase in HIF-1α and ANGPTL4. The combination therapy proved more effective than aflibercept alone in reducing CNV lesion size, suggesting that preventing HIF-1α accumulation may overcome inadequate responses to anti-VEGF monotherapy.

Enhancement of Chemotherapy Delivery

In acute myeloid leukemia models, aflibercept demonstrated an unexpected effect on chemotherapy pharmacokinetics. Sequential aflibercept followed by doxorubicin resulted in progressive anthracycline accumulation in marrow and extramedullary AML sites, with 2-fold higher drug levels 24 hours after administration compared to tissues treated with chemotherapy alone. This enhancement was associated with vascular narrowing, decreased vessel number, and perivascular apoptosis. The findings suggest that leukemia-associated vasculature inefficiency mediates chemoresistance, and VEGF blockade can improve drug delivery.

Synthesis

The relationship between aflibercept’s pharmacology and its effects on VEGF-driven vascular permeability and neovascularization operates through multiple interconnected mechanisms that extend beyond simple VEGF neutralization.

Binding Specificity and Affinity Drive Duration of Action

The superior and prolonged efficacy of aflibercept compared to other anti-VEGF agents derives primarily from two pharmacological properties: exceptionally high binding affinity (K D = 171.9 fM vs. 21.8 pM for ranibizumab) and broader ligand specificity (VEGF-A, VEGF-B, and PlGF vs. VEGF-A alone). This 100-fold difference in binding affinity translates directly to prolonged therapeutic effects, with aflibercept maintaining vascular leak inhibition to day 55 compared to day 35 for ranibizumab. The longer vitreal half-life (5.63 days vs. 3.15 days for ranibizumab) compounds this advantage, as more drug remains available to bind newly produced VEGF.

The broader ligand specificity provides additional mechanistic advantages. PlGF, while mitogenic for retinal endothelial cells, plays a distinct role from VEGF-A in recruiting mononuclear phagocytes during the early inflammatory phase of choroidal neovascularization. Studies showing PlGF upregulation (but not VEGF-A) in early CNV and the superior efficacy of combined VEGF-A/PlGF inhibition over PlGF inhibition alone demonstrate that dual targeting addresses multiple drivers of pathological angiogenesis. This explains case reports of aflibercept benefit in patients unresponsive to VEGF-A-specific therapies.

Secondary Pathway Modulation Amplifies Direct Effects

Aflibercept’s effects extend beyond its direct binding targets through downstream pathway regulation. The suppression of ANG2 protein and mRNA was initially surprising given that anti-VEGF agents do not directly bind ANG2. However, this reflects regulatory crosstalk: VEGF signaling induces ANG2 expression, and VEGF neutralization reduces this stimulus. ANG2 itself promotes vascular permeability by binding TIE2 receptors and disrupting endothelial barrier function. Thus, aflibercept’s strong VEGF suppression creates cascading effects that amplify permeability reduction beyond what VEGF neutralization alone would achieve.

Similarly, aflibercept’s interaction with galectin-1 provides anti-angiogenic effects independent of VEGF. The elevation of galectin-1 in PDR eyes without correlation to VEGF-A levels, combined with galectin-1’s ability to activate VEGFR2 phosphorylation, indicates an alternative pathway driving angiogenesis. Aflibercept’s neutralization of both VEGF-A and galectin-1-mediated VEGFR2 activation provides dual pathway inhibition unavailable to VEGF-A-specific antibodies. This mechanistic distinction may explain superior outcomes in specific patient populations.

Context-Dependent Dose-Response Relationships

The relationship between aflibercept concentration and biological effects shows context-dependent thresholds. For barrier restoration in retinal endothelial cells, concentrations ≤25 μg/ml completely restored VEGF-A-disturbed function without affecting normal barrier integrity. However, at the same or lower concentrations, migration was reduced below basal levels. This apparent contradiction reflects distinct cellular mechanisms: barrier function depends on tight junction protein expression and organization, while migration involves cytoskeletal reorganization and matrix interactions.

The cellular uptake of aflibercept complicates dose-response interpretation. While therapeutic effects occur at low concentrations, accumulation in retinal cells over prolonged treatment could lead to interference with normal cellular functions like migration and phagocytosis. The Fc domain-mediated uptake suggests this effect would be shared with bevacizumab but not ranibizumab. However, the clinical significance remains uncertain, as therapeutic concentrations are considerably lower than those causing functional interference.

Countertherapeutic Mechanisms Limit Long-Term Efficacy

The HIF-1α/ANGPTL4 axis represents a resistance mechanism that emerges specifically from effective VEGF blockade. VEGF/KDR inhibition causes HIF-1α accumulation in the retinal pigment epithelium, which in turn drives ANGPTL4 expression. This creates a paradox: the more effectively aflibercept blocks VEGF, the stronger the compensatory pro-angiogenic signal through ANGPTL4. This mechanism explains why some nvAMD patients show inadequate response to anti-VEGF monotherapy.

Importantly, this counterregulation appears specific to sustained VEGF inhibition rather than being unique to aflibercept. The combination of aflibercept with HIF-1 inhibitors proved more effective than aflibercept alone, indicating that preventing the compensatory response restores efficacy. This suggests that aflibercept’s pharmacological properties (high affinity, long half-life) create sustained VEGF suppression sufficient to trigger HIF-1α accumulation, whereas shorter-acting agents might allow intermittent VEGF signaling that prevents full HIF-1α activation.

Vascular Normalization Enhances Drug Delivery

In the AML models, aflibercept’s effects on tumor vasculature improved chemotherapy delivery through vascular normalization. The 2-fold increase in doxorubicin levels 24 hours after sequential aflibercept-doxorubicin administration, combined with vascular narrowing and decreased vessel number, suggests that pathological tumor vessels are inefficient for drug delivery. VEGF blockade appears to prune the most abnormal vessels while normalizing remaining vasculature, improving perfusion and drug penetration. This mechanism has broader implications for anti-VEGF therapy, indicating that vascular effects beyond simple anti-angiogenesis may contribute to therapeutic benefit.

Model System Relevance and Translation

The heavy representation of rabbit retinal hyperpermeability models reflects their utility for pharmacokinetic and pharmacodynamic studies, given the ability to induce quantifiable vascular leakage with exogenous VEGF and measure drug concentrations in the vitreous. However, these models may not fully capture the chronic, multifactorial nature of human retinal diseases. The human PDR surgical samples and nvAMD patient tissues provide complementary evidence that mechanisms identified in animal models operate in human disease, including galectin-1 elevation and ANGPTL4 induction.

The in vitro bovine retinal endothelial cell studies offer mechanistic precision but may not fully represent human endothelial cell responses. The consistency between bovine cell findings (barrier restoration at ≤25 μg/ml) and rabbit in vivo efficacy suggests reasonable translation, though species differences in VEGF receptor expression or downstream signaling could affect quantitative extrapolation.

The tumor and corneal models demonstrate that aflibercept’s pharmacological principles apply across vascular beds, though tissue-specific factors (fenestration, basement membrane composition, resident cell types) create context-dependent variations in response magnitude and kinetics.

References

A. Lockhart, M. Rothenberg, J. Dupont, W. Cooper, P. Chevalier, and 12 more\ (2010).Phase I study of intravenous vascular endothelial growth factor trap, aflibercept, in patients with advanced solid tumors. Journal of Clinical Oncology

H. Deissler, G. Lang, G. Lang\ (2014).Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells. Experimental Eye Research

C. Lange, Reimo Tetzner, Tobias Strunz, K. Rittenhouse\ (2023).Aflibercept Suppression of Angiopoietin-2 in a Rabbit Retinal Vascular Hyperpermeability Model. Translational Vision Science & Technology

Orly Gal-Or, Eitan Livny, R. Sella, Y. Nisgav, D. Weinberger, and 2 more\ (2016).Efficacy of Subconjunctival Aflibercept Versus Bevacizumab for Prevention of Corneal Neovascularization in a Rat Model. Cornea

Sergio Crespo-Garcia, Caitlin Corkhill, C. Roubeix, Anja-Maria Davids, N. Kociok, and 3 more\ (2017).Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization. Investigative Ophthalmology and Visual Science

Atsuhiro Kanda, K. Noda, W. Saito, S. Ishida\ (2015).Aflibercept Traps Galectin-1, an Angiogenic Factor Associated with Diabetic Retinopathy. Scientific Reports

Deepti Sharma, Evan Lau, Yu Qin, Kathleen Jee, Murilo Rodrigues, and 9 more\ (2024).VEGF inhibition increases expression of HIF-regulated angiogenic genes by the RPE limiting the response of wet AMD eyes to aflibercept. Proceedings of the National Academy of Sciences of the United States of America

G. Lang, G. Lang, H. Deissler\ (2014).Grundlegende In-vitro-Untersuchungen zur VEGF-Inhibition mit Aflibercept: Gemeinsamkeiten und Unterschiede zu anderen VEGF-bindenden therapeutischen Proteinen. Klinische Monatsblätter für Augenheilkunde

W. Schubert, Carsten Terjung, A. Rafique, C. Romano, P. Ellinger, and 1 more\ (2022).Evaluation of Molecular Properties versus In Vivo Performance of Aflibercept, Brolucizumab, and Ranibizumab in a Retinal Vascular Hyperpermeability Model. Translational Vision Science & Technology

D. Lal, Jennifer A. Park, K. Demock, J. Marinaro, Amanda M. Perez, and 8 more\ (2010).Aflibercept Exerts Antivascular Effects and Enhances Levels of Anthracycline Chemotherapy In vivo in Human Acute Myeloid Leukemia Models. Molecular Cancer Therapeutics

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VEGF inhibition increases expression of HIF-regulated angiogenic genes by the RPE limiting the response of wet AMD eyes to aflibercept

Deepti Sharma, Evan Lau, Yu Qin, Kathleen Jee, Murilo Rodrigues, Chuanyu Guo, Aumreetam Dinabandhu, Emma McIntyre, Shaima Salman, Yousang Hwang, A. Moshiri, G. Semenza, S. Montaner, Akrit Sodhi

Proceedings of the National Academy of Sciences of the United States of America·

2024·

14 citations

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Aflibercept Pharmacology

- Molecular structure and design: Not mentioned - Binding specificity and affinity: Not mentioned - Mechanism of VEGF sequestration/blockade: Aflibercept blocks VEGF, leading to a countertherapeutic increase in HIF-1α and ANGPTL4. - Pharmacokinetic properties: Not mentioned - Optimal dosing regimens and administration routes: Intravitreal injection; doses used in the study were 300 ng and 400 ng. - Unique pharmacological features: Increases HIF-1α accumulation, which is countertherapeutic.

Vascular Permeability Effects

- Permeability measurements: Not mentioned - Effects on tight junction proteins and endothelial barrier integrity: Not mentioned - Dose-response relationships for permeability reduction: Not mentioned - Time course of permeability effects: Not mentioned - Specific permeability outcomes in different vascular beds: Not mentioned - Mechanism by which aflibercept restores/prevents VEGF-induced permeability: Aflibercept decreases VEGF levels, but increases ANGPTL4 expression; combining with 32-134D may prevent this countertherapeutic effect.

Neovascularization Effects

- Aflibercept reduces VEGF levels but increases ANGPTL4 expression, which can counteract anti-angiogenic effects. - Combination with 32-134D is more effective than aflibercept alone in reducing CNV lesion size. - Specific measurements of vessel density, vessel formation, or sprouting assays are not provided. - Effects on pathological vs. physiological angiogenesis, vessel maturation, and stability are not detailed. - Dose-response relationships and time course of neovascularization inhibition are not explicitly mentioned.

Pharmacokinetic-Pharmacodynamic Relationships

Not mentioned (the paper does not provide specific pharmacokinetic-pharmacodynamic relationships for aflibercept, such as free vs. bound concentrations, complex formation kinetics, threshold concentrations, duration of VEGF suppression, tissue penetration, or correlation between systemic exposure and vascular effects)

Mechanistic Insights

- Downstream signaling pathway modulation: Inhibition of VEGF/KDR signaling leads to accumulation of HIF-1α, increasing ANGPTL4 expression. - Effects on endothelial cell behavior: Not explicitly mentioned. - Impact on inflammatory cell recruitment and activation: Not explicitly mentioned. - Secondary effects on other angiogenic factors: Increased ANGPTL4 expression is a countertherapeutic effect. - Cellular uptake and intracellular fate of aflibercept: Not explicitly mentioned. - Molecular basis for superior/different efficacy vs. other anti-VEGF agents: Combination with 32-134D prevents countertherapeutic increase in HIF-1α and ANGPTL4.

Study Model Context

- Disease model or clinical condition studied: Neovascular age-related macular degeneration (nvAMD), specifically choroidal neovascularization (CNV). - Species and experimental system: Human samples and mouse model (C57BL/6 and HIF-1α heterozygous mice). - Baseline VEGF levels and vascular pathology: Not explicitly mentioned, but changes in VEGF and ANGPTL4 levels are discussed. - Presence of other angiogenic stimuli or confounding factors: HIF-1α and ANGPTL4 are influenced by VEGF inhibition. - Relevance to human VEGF-driven vascular diseases: Relevant to nvAMD. - Specific vascular bed or tissue studied: Retina, focusing on retinal pigment epithelium (RPE) and choroidal neovascularization.

Comparative Pharmacology

- Head-to-head comparisons: Not mentioned - Comparative effects on permeability and angiogenesis outcomes: Aflibercept increases HIF-1α and ANGPTL4, potentially counteracting its therapeutic effects - Differences in pharmacokinetic properties and dosing requirements: Not mentioned - Relative potency and duration of action: Not mentioned - Mechanistic advantages or disadvantages: Aflibercept's increase in HIF-1α and ANGPTL4 is a mechanistic disadvantage - Clinical implications of pharmacological differences: Combination with a HIF inhibitor (32-134D) may enhance efficacy

Significance Many patients with neovascular age-related macular degeneration (nvAMD) respond inadequately to therapies targeting vascular endothelial growth factor (VEGF). Following treatment of patients with nvAMD with anti-VEGF therapy, we report decreased expression of VEGF but increased expression of a second angiogenic mediator, angiopoietin-like 4 (ANGPTL4). Increased ANGPTL4 expression is a consequence of accumulation of hypoxia-inducible factor (HIF)-1α in response to VEGF/kinase insert domain receptor (KDR) inhibition in the retinal pigment epithelium. By preventing the increase in HIF-1α accumulation in response to anti-VEGF therapy, combining 32-134D with aflibercept was more effective for choroidal neovascularization (CNV) treatment in mice than either drug alone. This suggests that combining 32-134D with current therapies may overcome the inadequate response of patients with nvAMD to anti-VEGF monotherapy.

Choroidal neovascularization (CNV), the growth of abnormal leaky blood vessels under and within the retina, is a hallmark of patients with neovascular (nv) or "wet" age-related macular degeneration (nvAMD). Untreated, CNV leads to rapid and often irreversible vision loss from leakage of fluid (causing macular edema), bleeding, and scarring ( 1 , 2 ) . A single angiogenic mediator, vascular endothelial growth factor (VEGF), has been shown to play a central role in the development of CNV. Expression of VEGF in the eyes of patients with nvAMD is regulated by the transcription factor, hypoxia-inducible factor (HIF)-1 ( 3 ). Increased expression of VEGF by HIF-1, in turn, promotes the growth of the abnormal leaky vessels that compromise vision in patients with nvAMD.

The introduction of therapies targeting VEGF (i.e., anti-VEGF therapies) has revolutionized the treatment of nvAMD with almost half of treated patients demonstrating a clinically significant improvement of their vision ( 4 , 5 ) . Nonetheless, most patients with nvAMD will demonstrate persistent intraretinal or subretinal fluid despite strict adherence to the recommended treatment regimens ( 6 ). Why many patients with nvAMD treated with anti-VEGF therapies fail to respond adequately to treatment ( 7 ) remains unclear.

Using a protocol designated treat-and-extend (TAE), pause and monitor (TEP/M), a hybrid of the TAE, and pro re nata (PRN) protocols, designed to wean patients with nvAMD off anti-VEGF therapy, it was reported that after 1 y of treatment, patients fall into subgroups based on their sensitivity to anti-VEGF therapy ( 8 ). While their response to anti-VEGF therapy did not correlate with the aqueous levels of VEGF, they did correlate with the aqueous levels of a second HIF-regulated angiogenic mediator, angiopoietin-like 4 (ANGPTL4) ( 9 ). Expression of ANGPTL4 synergized with VEGF to promote endothelial cell tubule formation in vitro and CNV in vivo ( 9 ). Accordingly, simultaneously targeting both ANGPTL4 and VEGF in the laser CNV mouse model was more effective than targeting either factor alone ( 9 ). These observations support a role for ANGPTL4 in the development of CNV and the resistance to anti-VEGF therapy in patients with nvAMD. Here, we use a combination of human samples, mouse models, and cell-based studies to explore the mechanism whereby ANGPTL4 expression influences the response of patients with nvAMD to anti-VEGF therapy and provide evidence that a recently developed HIF-inhibitor, 32-134D, in combination with anti-VEGF therapy, may be a more effective approach to treat patients with nvAMD than anti-VEGF therapy alone.

Results

Treatment of Patients with nvAMD with Anti-VEGF Therapy Results in a Countertherapeutic Increase in the Expression of ANGPTL4. Aqueous levels of ANGPTL4-but not VEGF-have been reported to correlate inversely with the response of patients with nvAMD to treatment with anti-VEGF therapy: The higher the levels of ANGPTL4, the poorer the response of patients to anti-VEGF therapy (9,10). To better understand the relationship between ANGPTL4 expression in patients with nvAMD and their response to anti-VEGF therapy, we compared the expression of VEGF and ANGPTL4 in the aqueous of patients with treated and untreated nvAMD as well as patients with non-neovascular (nnv) or "dry" AMD and non-AMD (control) patients (SI Appendix, Table S1 ). Close inspection of the scatter plot demonstrated that the levels of ANGPTL4 and VEGF in the aqueous fluid of patients with nvAMD could be used to distinguish them from patients with nnvAMD and from control patients (Fig. 1A and SI Appendix, Fig. S1 ). VEGF levels were markedly lower in the aqueous fluid from patients who underwent their first treatment with anti-VEGF therapy (either bevacizumab or aflibercept) 4 to 6 wk prior to sample collection (nvAMD first treatment; Fig. 1A ; vertical arrow), as expected. Conversely, in patients who had a remote (>12 wk) history of receiving anti-VEGF therapy and evidence of recurrent CNV, the aqueous levels of VEGF were similar to untreated patients with nvAMD (Fig. 1A ). By contrast, in patients with nvAMD who had a prior history of anti-VEGF therapy, the levels of ANGPTL4 were further increased both in patients with active CNV who had a recent (within 4 to 6 wk; nvAMD first treatment) or remote (>12 wk; nvAMD recurrent) history of treatment compared to untreated patients with nvAMD (nvAMD Untreated; Fig. 1A ; horizontal arrows).

To further explore the expression of ANGPTL4 following treatment with anti-VEGF therapy in patients with nvAMD, we examined its expression in the aqueous fluid from patients with nvAMD with no prior history of anti-VEGF therapy (nvAMD No Tx) or from a second group of patients with nvAMD who received their first treatment with anti-VEGF therapy within 4 to 6 wk of sample collection (nvAMD 1st Tx). We observed lower VEGF levels ( Fig. 1B ), but higher ANGPTL4 levels ( Fig. 1C ) in the aqueous fluid of patients with nvAMD who had recently been treated with anti-VEGF therapy compared to untreated patients.

We next analyzed aqueous fluid samples from 8 patients with newly diagnosed (i.e., treatment-naïve) nvAMD for the levels of VEGF and ANGPTL4 and then treated them with anti-VEGF therapy ( Fig. 1D ). When we reexamined aqueous levels of VEGF and ANGPTL4 in these patients 4 wk later, we observed a decrease in VEGF levels following treatment with anti-VEGF therapy in 8/8 patients with nvAMD ( Fig. 1E ). Conversely, we observed an increase in ANGPTL4 levels in 8/8 patients with nvAMD 4 wk after a single treatment with anti-VEGF therapy ( Fig. 1F ). Interestingly, the increase in ANGPTL4 mirrored the decrease in VEGF in 6/8 patients ( Fig. 1G ). Collectively, these results demonstrate that treatment of patients with nvAMD with anti-VEGF therapy results in a therapeutic decrease in VEGF, but a countertherapeutic increase in the expression of ANGPTL4, supporting a role for ANGPTL4 in limiting the treatment response of patients with nvAMD to anti-VEGF therapy.

Treatment with Anti-VEGF Therapy Results in Increased ANGPTL4 mRNA and Protein Expression in a Mouse Model for nvAMD. We next employed a mouse model of CNV, in which a laser is used to rupture Bruch's membrane (11), to explore the mechanism whereby treatment with anti-VEGF therapy results in an increase in ANGPTL4. The laser CNV model has proven to be a powerful tool to examine molecular events underlying the development of CNV (12) and the role of ANGPTL4 in its promotion (9). We treated mice with an intravitreal injection of aflibercept (which is effective in humans and mice) or vehicle control (PBS), 1 d following laser treatment (Fig. 2A ) and examined the expression of ANGPTL4 protein; we used a low dose of aflibercept (300 ng) to allow for the development of the CNV lesion despite treatment. On day 3, when ANGPTL4 expression is not yet detected in the laser CNV model, we observed a marked increase in the expression of ANGPTL4 in the outer retina of CNV lesions of animals treated with aflibercept compared to vehicle control (Fig. 2B ). By day 7, expression of ANGPTL4 is detected in the outer retina of CNV lesions but is further increased in animals treated with aflibercept (Fig. 2C ), similar to what was observed in patients with nvAMD following treatment with anti-VEGF therapy. This correlated with an increase in Angptl4 mRNA expression in the RPE/choroid as measured by qPCR (Fig. 2D ). These results were corroborated by RNAscope when mice were pretreated with a low dose of aflibercept (300 ng) 3 d prior to laser treatment to allow sufficient time to detect the accumulation of Angptl4 mRNA (Fig. 2E ). Angptl4 mRNA was detected in the outer retina of CNV lesions in animals pretreated with aflibercept, but not with vehicle, on day 3 (Fig. 2F ). By day 7, Angptl4 mRNA was observed in CNV lesions in animals pretreated with vehicle control but was increased in animals pretreated with aflibercept (Fig. 2G ), similar to what was observed with ANGPTL4 protein. Collectively, these observations demonstrate that anti-VEGF therapy promotes increased ANGPTL4 mRNA and protein expression in a mouse model for CNV.

Hypoxia Promotes the Accumulation of HIF-1α and VEGF in the Laser-Induced Mouse Model of CNV. To further interrogate the mechanism whereby treatment of CNV with anti-VEGF therapy results in an increase in ANGPTL4 mRNA and protein expression, we shifted our attention upstream from ANGPTL4 to HIF-1α, the transcription factor that has been previously implicated in regulating ANGPLTL4 expression in ocular disease (13)(14)(15)(16)(17)(18)(19)(20)(21)(22), including nvAMD (3,18). To this end, we explored HIF-1α expression in the laser CNV mouse model. Following laser treatment, we observed an increase in outer retinal hypoxia, as measured using HypoxyProbe™ that peaked at day 1, and largely resolved by day 3 (SI Appendix, Fig. S2 A and B ). This correlated with an increase in HIF-1α protein accumulation as early as day 1, which was still present at day 3 but was no longer detectable We next examined whether the expression of HIF-1α is influenced by treatment with aflibercept. Treatment with a moderate dose of aflibercept (400 ng), 1 d after laser treatment, resulted in an increase in HIF-1α accumulation 24 h later (SI Appendix, Fig. S3A ) that localized to the outer retina of CNV lesions (SI Appendix, Fig. S3B ). These results suggested that the increase in ANGPTL4 expression following treatment with anti-VEGF therapy may be due to an increase in HIF-1α accumulation.

To interrogate the contribution of HIF-1α accumulation to the increase in ANGPTL4 mRNA and protein expression following treatment with anti-VEGF therapy, we used mice that were heterozygous for a knockout allele at the Hif1a locus (Hif1a +/-) ( 24 ). We took advantage of prior observations that basal levels of HIF-1α are relatively normal in Hif1a +/-mice, whereas in response to ischemia, HIF-1α expression is largely unchanged in Hif1a +/- mice but potently stimulated in wild type (wt) littermate controls ( 25 ). Following laser treatment (SI Appendix, Fig. S3C ), both HIF-1α protein expression in the RPE/choroid (day 2; SI Appendix, Fig. S3D ) as well as VEGF and ANGPTL4 protein expression (day 7; SI Appendix, Fig. S3 E and F ) were markedly reduced in Hif1a +/-mice compared to wt littermate controls. In wt littermate controls, intravitreal injection with a moderate dose of aflibercept (400 ng) 1 d after laser treatment stimulated an increase in HIF-1α protein accumulation at day 2 (SI Appendix, Fig. S3D ). Conversely, in Hif1a +/-mice, intravitreal injection with aflibercept resulted in only a modest increase in HIF-1α accumulation compared to PBS control. Interestingly, while Vegf mRNA expression at day 7 was unchanged in wt mice following intravitreal injection with aflibercept, expression of Vegf mRNA in the RPE/choroid was decreased in Hif1a +/-mice (SI Appendix, Fig. S3G ), suggesting that the increase in HIF-1α accumulation in response to inhibition of VEGF is required to maintain stable Vegf mRNA expression. Conversely, intraocular injection with aflibercept stimulated a marked increase in Angptl4 mRNA expression in the RPE/choroid in wt animals; this increase was not observed in Hif1a +/-mice (SI Appendix, Fig. S3H ). Collectively, these results suggest that inhibition of VEGF with anti-VEGF therapy could result in a compensatory increase in HIF-1α accumulation and, in turn, ANGPTL4 expression in treated patients.

RPE Cells Up-Regulate HIF-1α Expression in the Laser CNV Mouse

Model. RPE cells have been reported to drive the expression of the angiogenic mediators that stimulate the development of CNV in patients with nvAMD (26). In patients geographic atrophy (GA), CNV develops in areas with preserved RPE surrounding the GA or within the spared foveal island (27), consistent with prior histopathological (28) and clinical studies (29,30). These studies suggest that a viable RPE is required for the development of CNV. We therefore examined whether RPE cells contribute to the expression of HIF-1α in the laser CNV mouse model. On day 1 (Fig. 3A ), we observed coexpression of the RPE-specific marker, RPE65 in cells expressing HIF-1α in the outer retina in mice following treatment with laser (Fig. 3 B-F ). On day 3 (Fig. 3G ), expression of HIF-1α in the outer retina increased, but remained localized primarily to the outer retina, and again included RPE65expressing cells (Fig. 3 H-K ). The diffuse expression of HIF-1α, predominantly in RPE65-expressing cells, was confirmed in CNV lesions on choroidal flat mounts at day 3 (Fig. 3 L-N ). Expression of HIF-1α was no longer detected by day 7 following laser treatment (SI Appendix, Fig. S4 ). HIF-1α expression was not observed in the outer retina of adjacent (nonlasered) control tissue at day 1 or 3 (Fig. 3 B and H), nor in the outer retina of the contralateral (nonlasered) control eyes (SI Appendix, Fig. S5 ).

Collectively, these results support a role for expression of HIF-1α in RPE cells in the development of CNV.

Increased HIF-1α Expressing RPE Cells in Laser CNV Mice Following

Treatment with Aflibercept. Administration of either aflibercept or ranibizumab to cocultures of RPE and endothelial cells has been reported to result in a dose-response increase in RPE cell viability and increased RPE cell migration and proliferation (31).

We therefore next examined the response RPE cells following treatment with aflibercept in laser CNV mice. To ensure sufficient time for the RPE to respond to treatment with anti-VEGF therapy, we performed intraocular injections with aflibercept 3 d prior laser treatment (SI Appendix, Fig. S6A ) and observed an increase in RPE65-expressing cells at day 1 (SI Appendix, Fig. 6 B and C ). This was not observed in adjacent (nonlasered) control tissue (SI Appendix, Fig. S6D ). These RPE cells demonstrated an increase in HIF-1α expression (Fig. 4 A-D ). RPE cells have been reported to express VEGF receptor 2 (KDR) ( 32 ) and that secreted VEGF may play a role as an autocrine survival for RPE in AMD ( 33 , 34 ) . Based on our observations, we postulated that expression of KDR on RPE cells may also allow these cells to detect (and respond to) reduced levels of angiogenic mediators critical for the survival of the underlying choriocapillaris. To interrogate the response of RPE cells to reduced VEGF/KDR signaling, we isolated primary RPE cells from mice. Expression of KDR in these cells was detected under physiologic (20% O 2 ) culture conditions and was slightly reduced under hypoxic (1% O 2 ) culture conditions ( Fig. 4E ). Inhibition of the KDR using the pharmacologic inhibitor, SU1498, resulted in an increase in HIF-1α protein expression ( Fig. 4F ) and, in turn, Angptl4 mRNA expression ( Fig. 4G ); expression of Vegf mRNA was not affected. These results were corroborated following knockdown of KDR expression in primary mouse RPE cells using RNA interference ( Fig. 4 H and I ), and in the immortalized human RPE cell line, ARPE-19 using the KDR inhibitor, SU1498 ( Fig. 4 J -L ). Collectively, these results support a role for the VEGF/KDR signaling axis in regulating HIF-1α protein expression (and the expression of HIF-regulated genes) in RPE cells.

RPE Cells Secrete ANGPTL4 and VEGF, but Not ANGPT2 or EPO.

HIF-1α regulates the expression of numerous angiogenic mediators, in addition to VEGF and ANGPTL4. We therefore set out to examine whether the increase in HIF-1α in response to anti-VEGF therapy resulted in an increase in other key HIF-regulated angiogenic mediators. Expression of the HIF-regulated angiogenic mediator ANGPT2 has been reported in surgically excised CNV membranes from patients with nvAMD (35). This has led to the development of therapies targeting ANGPT2 and its receptor, TIE2. Interestingly, unlike ANGPTL4, expression of ANGPT2 was not elevated in patients who had recently been treated with anti-VEGF therapy compared to untreated patients (SI Appendix, Fig. S7A ). Similar results were observed for another HIF-regulated gene, erythropoietin (EPO; SI Appendix, Fig. S7B ), which has also been previously implicated in the pathogenesis of nvAMD (36,37). We hypothesized that the reason why expression of ANGPTL4but not ANGPT2 or EPO-was increased following treatment of nvAMD patients with anti-VEGF therapy could be a consequence of the ability of RPE cells to express only a subset of HIF-regulated angiogenic mediators. To interrogate this hypothesis, we examined the expression of HIF-regulated angiogenic factors in ARPE-19 cells cultured in hypoxia. Expression of VEGF , ANGPTL4 , and ANGPT2 and EPO mRNA were all increased in ARPE-19 cells cultured in hypoxia (SI Appendix, Fig. S7 C -F ). However, we observed secretion of VEGF and ANGPTL4, but not ANGPT2, by ARPE-19 cells cultured in hypoxia, consistent with prior studies demonstrating that ANGPT2 was expressed specifically by vascular endothelial cells ( 38 ). Similar results were obtained with EPO. These results help explain why expression of ANGPTL4-but not ANGPT2 and EPO-is increased in patients following treatment with anti-VEGF therapy.

Inhibition of HIF-1α with 32-134D Effectively Treats CNV Lesions

in Mice. Collectively, these studies identify a potential limitation of current anti-VEGF therapies for the treatment of patients with nvAMD. To avoid the countertherapeutic increase in HIF-1α and ANGPTL4 expression by RPE cells following treatment with anti-VEGF therapies, we set out to evaluate the therapeutic efficacy of targeting HIF-1α for the treatment of CNV. While preclinical studies of the pharmacologic HIF inhibitor, acriflavine (23), for the treatment of ocular vascular disease have been promising (39), recent studies demonstrate that its safety profile following intraocular administration makes it a less desirable candidate to translate to the clinic (22). Instead, we took advantage of a recently developed pharmacologic HIF inhibitor, 32-134D (40), which was reported to effectively inhibit HIF-1α and HIF-2α protein accumulation, resulting in a modest but broad reduction of dozens of HIF-regulated vasoactive mediators following intraocular administration at doses that are well tolerated in mouse models for diabetic eye disease (22). 32-134D has been shown to be more effective than aflibercept at preventing retinal neovascularization in a mouse model for ischemic retinal neovascularization (22). We first evaluated the efficacy of intraocular administration of 32-134D in the laser CNV model. Treatment with 32-134D (70 ng) on day 3 (to allow CNV lesion to develop prior to treatment) resulted in a marked reduction in the size of CNV lesion size in mice (Fig. 5A ). Examination of the CNV lesion demonstrated a reduction in Vegf mRNA expression in the outer retina and RPE (Fig. 5B ). This, in turn, resulted in a reduction of VEGF protein expression in the RPE and choroid as well as the neurosensory retina to the levels observed in nonlasered (control) animals (Fig. 5 C and D), similar to what was observed following treatment with aflibercept (SI Appendix, Fig. S8 ). While VEGF expression and CNV lesion size were both markedly reduced with treatment with 32-134D, VEGF expression was observed within the smaller CNV lesions, albeit significantly less than that observed in the untreated CNV lesions (Fig. 5E ). Expression of ANGPTL4 following treatment with 32-134D was also markedly reduced even within these smaller CNV lesions (Fig. 5 F and G ). These results suggest that 32-134D monotherapy, which reduces expression of both VEGF and ANGPTL4, could be an effective approach for the treatment of CNV.

32-134D Prevents the Countertherapeutic Increase in HIF-1α

Observed Following Treatment with Aflibercept. We next set out to determine whether 32-134D could prevent the increase in HIF-1α observed following treatment with aflibercept. To examine the effect of 32-134D on the early expression of HIF-1α, we pretreated mice with 70 ng of 32-134D 1 d prior to laser treatment and then collected eyes 1 d after laser treatment (Fig. 6A ) and examined HIF-1α accumulation. We observed a marked reduction in HIF-1α accumulation in the RPE/choroid, as well as the neurosensory retina in animals pretreated with 32-134D compared to vehicle control (Fig. 6 B and C ). Pretreatment with a low dose of aflibercept (300 ng) 1 d prior to laser treatment resulted in augmentation of HIF-1α accumulation at day 1; this was prevented in animals pretreated with 32-134D (Fig. 6D ). Interestingly, this was independent of the increase in RPE65-expressing cells, which was not affected by treatment with 32-134D (SI Appendix, Fig. S9 A and B ). This effect was more notable when animals were pretreated with both 32-134D and aflibercept 3 d prior to laser treatment (Fig. 6 E-G and SI Appendix, Fig. S9 C and D ). This suggests that the addition of 32-134D could prevent the countertherapeutic increase of HIF-1α accumulation (and ANGPTL4 expression) observed in patients treated with anti-VEGF therapy.

Simultaneous Inhibition of Both HIF-1 and VEGF Is More Effective than Inhibition of Either Alone for the Treatment of CNV in Mice.

To determine the therapeutic potential of simultaneous inhibition of HIF-1 and VEGF for the treatment of CNV, we first performed a dose-response for aflibercept and 32-134D delivered on day 3 (Fig. 7A ) to identify the threshold dose of each for the treatment of CNV. We observed that 300 ng of aflibercept and 30 ng of 32-134D (SI Appendix, Fig. S10 ) was sufficient to observe a small but significant reduction in CNV lesion size in treated animals. We then combined a subthreshold (100 ng) or threshold (300 ng) dose of aflibercept with a subthreshold (10 ng) or threshold (30 ng) dose of 32-134D (Fig. 7B ) to determine whether targeting HIF-1α influences the response of CNV lesions size in mice treated with anti-VEGF therapy. There was a trend toward increased efficacy when combining a subthreshold dose of aflibercept (100 ng) and/or 32-134D (10 ng) with a subthreshold (Fig. 7B ; highlighted in Fig. 7C ) or threshold (Fig. 7B ; highlighted in Fig. 7 D and E) dose of the other therapy; however, this did not reach statistical significance. When we combined a threshold dose of aflibercept (300 ng) with a threshold dose of 32-134D (30 ng), we observed a significant improvement in the efficacy of the combination of the two compared to either treatment alone (Fig. 7B ; highlighted in Fig. 7F ).

We next examined whether the addition of 32-134D could improve upon an effective dose of aflibercept. To this end, we performed a dose-response of aflibercept and 32-134D to identify the dose that was 50% as effective as the maximal effective dose (i.e., the IC 50 ) and determined that the IC 50 for aflibercept was approximately 300 ng and for 32-134D was 70 ng (SI Appendix, Fig. S11 ). Combining 300 ng of aflibercept with 70 ng of 32-134D was more effective than either drug alone ( Fig. 7G ). Collectively, these results demonstrate that 32-134D is effective for the treatment of CNV in mice and provide the foundation for clinical trials assessing its efficacy when used alone or in combination with current therapies targeting VEGF for the treatment of patients with nvAMD.

Discussion

Results from multiple clinical trials consistently demonstrate that the majority of patients with nvAMD fail to achieve a clinically significant improvement in vision (i.e., an increase of 3 line or more on the ETDRS vision chart) despite monthly or bimonthly treatment over 2 y ( 5 ). Follow up studies on these clinical trials have further demonstrated that the vision gains experienced by patients in the initial 2 y are often lost over subsequent years despite continued treatment ( 6 ). While this may be due, in part, to inadequate treatment, these observations raise concerns that anti-VEGF monotherapy may not provide a long-term solution for the treatment of most patients with nvAMD.

There are also concerns that a subpopulation of patients with nvAMD are vulnerable to continued vision loss despite monthly treatment with anti-VEGF therapy. In a post hoc analysis of CATT, a subset of patients (approximately 6%) developed sustained vision loss despite ongoing treatment with anti-VEGF therapy ( 41 ). Another 10% of patients in CATT developed a transient, but pnas.org significant decrease in visual acuity (i.e., a decrease of 3 line or more on the ETDRS vision chart; termed sporadic vision loss), despite treatment with anti-VEGF therapy ( 42 ). Analyses of the VIEW 1/2 and LUCAS trials similarly reported vision loss in some patients despite continued treatment during the trials ( 43 , 44 ) . Collectively, these studies highlight the importance of understanding why some patients respond inadequately, transiently, or not at all, despite treatment with therapies that effectively neutralize VEGF.

In this regard, it has previously been reported that expression of a second HIF-regulated vasoactive mediator, ANGPTL4, is expressed in the eyes of patients with nvAMD, and localizes to CNV lesions ( 9 ). In mice, ANGPTL4 synergized with VEGF to promote CNV, markedly increasing CNV lesions size and leakage, as assessed by fluorescein angiography ( 9 ). It was further observed that simultaneously targeting both ANGPTL4 and VEGF in the laser CNV mouse model was more effective than targeting either factor alone ( 9 ). These results suggest that the expression of ANGPTL4 may influence how patients with nvAMD will respond to anti-VEGF therapy.

Here, we report that aqueous fluid from patients with no history-or a remote history (i.e., >12 wk)-of anti-VEGF therapy fell into two categories: high levels of both VEGF and ANGPTL4 (90%) or low levels of VEGF and high levels of ANGPTL4 (10%). We speculate that the latter group could help identify the subpopulation of patients with nvAMD who are most vulnerable to continued vision loss despite treatment with anti-VEGF monotherapy. We further observed an increase in ANGPTL4 levels in patients following initiation of anti-VEGF therapy. This unexpected observation may further explain why some patients experience a return of fluid with deterioration in their vision following an initial response to treatment with anti-VEGF therapy ( 6 ).

Consistent with these observations, in patients with nvAMD, aqueous levels of ANGPTL4 at the time of treatment initiation correlated inversely with the response to anti-VEGF therapy at the end of 12 mo of treatment ( 9 ). In another study, aqueous levels of ANGPTL4 in patients with nvAMD were shown to correlate directly with the frequency of treatment with anti-VEGF therapy at 12 mo ( 10 ). Collectively, these studies provide a possible explanation for why patients with nvAMD respond inadequately to anti-VEGF therapy upon treatment initiation (i.e., in treatment-naïve eyes) as well as following an initial response to treatment (i.e., over time).

To provide a molecular explanation for these observations, we examined the expression of ANGPTL4 and VEGF in the laser CNV mouse model for nvAMD following treatment with anti-VEGF therapy (i.e., aflibercept). We observed an increase in ANGPTL4 protein expression following intraocular administration of aflibercept, consistent with our observations in patients in the clinic. We further observed that enhanced ANGPTL4 protein expression following treatment with anti-VEGF therapy was a consequence of an increase in Angptl4 mRNA expression and that this correlated with an increase in HIF-1α accumulation in the RPE. Prior work implicates the accumulation of HIF-1α in the RPE, caused by localized ischemia ( 45 ) and oxidative stress ( 3 ) as an early event in the development of CNV in patients with AMD. Preclinical studies lend additional support for a role for HIF-1 in the regulation of VEGF expression and the development of CNV in patients with nvAMD ( 9 , 46 -50 ) . Our results, demonstrating that the use of anti-VEGF therapy may cause a countertherapeutic increase in HIF-1α (and, in turn, ANGPTL4) in RPE, could explain the diminished efficacy of anti-VEGF therapy monotherapy for the treatment of CNV in many patients with nvAMD.

We next explored whether a recently described HIF-1 inhibitor, 32-134D ( 40 ), would be an effective treatment for patients with nvAMD. Intraocular delivery of 32-134D effectively inhibits HIF-1α and HIF-2α protein accumulation, normalizes the expression of HIF-regulated vasoactive genes, and prevents the development of retinal NV and vascular hyperpermeability in mouse models of diabetic eye disease without affecting retinal histology or function ( 22 ). We therefore examined the efficacy of a single intraocular injection of 32-134D for the treatment of CNV in mice. 32-134D effectively prevented the increase in VEGF mRNA and protein in the laser CNV mouse model of nvAMD, similar to aflibercept. However, unlike with aflibercept, treatment with 32-134D did not result in a countertherapeutic increase in ANGPTL4 expression. Rather, expression of ANGPTL4 was also markedly diminished following treatment with 32-134D. Accordingly, treatment of mice with 32-134D effectively treated CNV in mice, demonstrating that 32-134D could be an effective monotherapy for the treatment of patients with nvAMD.

The observation that 32-134D effectively blocked HIF-1α accumulation as well as the expression of ANGPTL4 in the laser CNV model prompted us to explore whether the addition of 32-134D could prevent the countertherapeutic increase in HIF-1α (and, in turn, ANGPTL4) observed following treatment with aflibercept. We report that the addition of 32-134D to treatment with aflibercept effectively prevented the increase in both HIF-1α and ANGPTL4 and had more effective for the prevention of CNV in mice than either therapy alone, demonstrating the potential of simultaneous inhibition of HIF-1α and VEGF as a therapeutic approach for the treatment of CNV. Collectively, these studies provide the foundation for a clinical study assessing 32-134D, alone or in combination with anti-VEGF therapies, for the treatment of patients with nvAMD.

Materials and Methods

Mice. All animal procedures were performed under the guidelines of the Johns Hopkins University School of Medicine Animal Care and Use Committee and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Ten-to twelve-week-old pathogenfree female mice were used for all experiments. C57BL/6 mice and HIF-1α heterozygous mice were obtained from Jackson Laboratories (JAX).

Cell Culture and Reagents. Cell culture of ARPE19 and primary mouse RPE cells was as previously described (3). Cell lines were routinely tested for Mycoplasma contamination by PCR. DMSO (472301-500ML) was purchased from Sigma-Aldrich. 4-(6-bromo-1H-indol-3-yl)-2-(7-bromo-1H-indol-3-yl)thiazole, designated 32-134D, was synthesized as previously described (40). Acriflavine and SU1498 were obtained from MilliporeSigma. Aflibercept was obtained from the Johns Hopkins University Pharmacy.

siRNA. Predesigned control (scrambled or scr), Kdr siRNA sequences were obtained from Qiagen. Kdr (20 µM) and scr siRNA delivery was performed using HiPerFect (Qiagen).

Western Blot. Antibodies are listed in SI Appendix, Table S2 . Western blots were performed as previously described (3). Western blot scans are representative of at least 3 independent experiments. Intraocular Injections. Intraocular injections were performed as previously described (22).

Laser Treatment. Laser CNV was used to rupture Bruch's membrane and cause neovascularization as previously described (9). Four lesions were made using a diode laser photocoagulator (IRIS medical) and a slit lamp delivery system (532 nm wavelength, 300 mW power, 100 ms duration, and 100 μm spot size). Animals were killed at indicated time points and eyes were enucleated and further studies conducted of laser CNV lesions.

Immunofluorescence Assays. All antibodies and dilutions are listed in SI Appendix, Table S2 . Immunofluorescence in RPE/choroid flat mounts and cross sections of the eye were performed as previously described (9). Images were captured at high magnification (20×) with a Zeiss fluorescent microscope (Carl Zeiss Inc.), and the area of CNV lesions was calculated as mm 2 by ImageJ software (NIH) by keeping the parameters the same for all the spots. 3D images of flat mounts of RPE/choroid were prepared using Imaris software. The quantitation of signal intensity in a region of interest (ROI) using the mean fluorescence intensity (MFI) method was calculated using ImageJ. Briefly, the original confocal image of a section and channel of interest to be quantified was selected. The ROI was selected by tracing the tissue using a high-resolution drawing, and the output from MFI was measured. MFI of the background from a rectangular area of the image that lacks tissue was also measured. The final MFI was the calculated by subtracting the background MFI from the ROI MFI. The percent final MFI of the sample compared to control was then calculated using Excel.

In Situ Hybridization. RNA in situ hybridization was performed as previously described (21).

Reverse Transcription and Quantitative Real-Time PCR. Primers are listed in SI Appendix, Table S3 . Quantitative real-time PCR was performed as previously described (21).

Patient Samples. All studies were in accordance with the Declaration of Helsinki. Institutional Review Board approval from the Johns Hopkins University School of Medicine was obtained for all patient tissue used in this study (NA_00075565). Consent was written and voluntary without stipend. All human samples used were deidentified prior to use. Aqueous samples were collected, and ELISA kits were performed as previously described (9). ELISAs are representative of at least three independent experiments. Statistical Analysis. Results are shown as a mean value ± SD from at least 3 independent experiments. Data from clinical samples are shown as mean ± SEM. Statistical analysis was done using Excel and Prism 9 software (GraphPad). Statistical differences between two or more heterogeneous groups were determined using the Kruskal-Wallis with Dunn's multiple-comparison test, unpaired Student's t test with Welch's Correction, and the one-way or two-way ANOVA with Bonferroni's multiple-comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns = not significant.

annex

Author contributions: A.S. designed research; D.S., E.L., Y.Q., K.J., M.R., C.G., A.D., E.M., and A.S. performed research; S.S., Y.H., and G.L.S. contributed new reagents/analytic tools; D.S., E.L., Y.Q., K.J., M.R., C.G., A.D., A.M., S.M., and A.S. analyzed data; D.S., E.L., Y.Q., A.M., G.L.S., and S.M. reviewed/edited manuscript; and A.S. wrote the paper.

Competing interest statement: G.L.S. and A.S. are co-founders of and hold equity in HIF Therapeutics, Inc. S.S., Y.H., G.L.S., and A.S. are inventors on provisional patent application PCT/US2022/039883. This arrangement has been reviewed and approved by the Johns Hopkins University in accordancewith its conflict of interest policies.

Acknowledgements

Author affiliations: a Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287; b Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Eye Hospital of China Medical University, Key Lens Research Laboratory of Liaoning Province, Shenyang 110005, China; c Department of Oncology and Diagnostic Sciences, School of Dentistry, Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201; d Armstrong Oxygen Biology Research Center, Vascular Program, Institute for Cell Engineering, Department of Vascular Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; e Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287; f Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; g Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; h Department of Radiation, Oncology Johns Hopkins University School of Medicine, Baltimore, MD 21205; i Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; j Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and k Department of Ophthalmology and Vision Science, School of Medicine, University of California at Davis, Sacramento, CA 95817 10 of 10 https://doi.org/10.1073/pnas.2322759121pnas.org

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