Research ArticleClinical Studies
Open Access

VEGF-C Aqueous Humor Levels in Patients With Primary Open Angle Glaucoma

GEORGIOS S. DIMTSAS, ALEXANDRA IERONYMAKI, KLIO I. CHATZISTEFANOU, GERASIMOS SIASOS and MARILITA M. MOSCHOS
In Vivo January 2026, 40 (1) 382-388; DOI: https://doi.org/10.21873/invivo.14202

Abstract

Background/Aim: This study aimed to quantitatively analyze and compare aqueous humor concentrations of vascular endothelial growth factor-C (VEGF-C) in patients with primary open-angle glaucoma (POAG) versus non-glaucomatous controls while evaluating potential significant correlations.

Patients and Methods: We conducted an observational cross-sectional study. At surgery initiation, anterior chamber paracentesis was performed under sterile conditions, and 50-100 μl of aqueous humor samples were collected. VEGF-C quantification employed a multiplex magnetic bead immunoassay platform.

Results: The study involved the collection of aqueous humor samples from 76 participants: 39 samples were collected from the POAG group and 37 from the control group (age-related cataract). Quantitative analysis revealed mean VEGF-C concentrations of 26.41±26.016 pg/ml in POAG eyes compared to 16.70±8.60 pg/ml in controls (p=0.277), demonstrating no statistically significant difference. Receiver operating characteristic (ROC) curve analysis showed limited prognostic ability for POAG detection (AUC=0.60; p=0.278).

Conclusion: This study represents the first large-scale evaluation of aqueous humor VEGF-C levels in patients with POAG. Our results provide evidence against VEGF-C up-regulation in POAG.

Keywords:

Introduction

Glaucoma ranks as the second most common cause of permanent vision loss, with its global prevalence in individuals over the age of 40 years projected to rise to approximately 111.8 million by the year 2040 (1). Primary open-angle glaucoma (POAG) is the most common type of glaucoma, accounting for approximately 74% of all glaucoma cases worldwide (1). It is characterized by progressive optic neuropathy and retinal ganglion cell (RGC) degeneration, leading to irreversible vision loss, typically beginning with peripheral visual field defects (2).

Unlike angle-closure glaucoma, POAG is characterized by an open iridocorneal angle, with elevated intraocular pressure (IOP) arising from impaired balance between aqueous humor (AH) production and outflow through the trabecular meshwork (TM) and Schlemm’s canal (3). The exact mechanisms of TM dysfunction remain unclear but may involve extracellular matrix remodeling, oxidative stress, and reduced cellularity, which increase outflow resistance (4). Schlemm’s canal constitutes the major resistance to aqueous humor outflow. The endothelium lining of the inner wall of Schlemm’s canal has both blood and lymphatic vascular characteristics (5). The primary modifiable risk factor for POAG is elevated IOP, though some patients develop glaucoma despite normal IOP (normal-tension glaucoma) (6).

The vascular endothelial growth factor (VEGF) family comprises six structurally related members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF) (7). The VEGF family plays a fundamental role in angiogenesis, lymphangiogenesis and vasculogenesis. Among VEGF family members, VEGF-A the most studied, particularly for its well-characterized involvement in pathological neovascularization (8). Additionally, our prior investigations have further demonstrated significantly elevated VEGF-A concentrations in the aqueous humor of patients with POAG, establishing its potential significance as a prognostic biomarker (9, 10). VEGF-C, first identified in 1996, has emerged as a key regulator of both lymphangiogenesis and angiogenesis during embryonic development and tumor progression (11, 12). VEGF-C exerts its biological actions by binding as a ligand to VEGFR-2 and VEGFR-3, and inducing tyrosine autophosphorylation of the cytoplasmic tail of its receptors (13). While both VEGFR-2 and VEGFR-3 are expressed on vascular endothelial cells, VEGFR-3 shows particularly high expression in lymphatic endothelium (7). VEGFR-2 activation can modulate vascular endothelial survival, proliferation, migration and the formation of vascular tubes, while VEGFR-3 activation promotes the development of blood and lymphatic vasculature (14).

Within ocular tissues, VEGF-C was reported to be expressed by the Schlemm’s canal endothelial cells and collecting ducts from patients with POAG and neovascular glaucoma (NVG) (15). VEGF-C and its receptor, VEGFR-3, are involved in Schlemm’s canal development and enlargement. Especially in adult mice, an injection of recombinant VEGF-C in the anterior chamber was associated with a sustained decrease in IOP (16). Additionally, a recent study in rabbits showed that adding VEGF-C to standard 5-fluorouracil antimetabolite treatment, improved glaucoma filtration surgery outcome (17). The above data suggest a potentially crucial role of VEGF-C in the treatment of POAG.

This study aimed to compare aqueous humor VEGF-C concentrations between individuals with POAG and those without glaucoma, and to identify any significant correlations that may exist.

Patients and Methods

Design. This cross-sectional study was conducted at the First Department of Ophthalmology, National and Kapodistrian University of Athens, General Hospital of Athens “G. Gennimatas” in Athens, Greece. The research protocol received approval from the Institutional Review Board of “G. Gennimatas” General Hospital (Approval Code: 1831/23.01.2020). All study procedures strictly adhered to the ethical principles outlined in the Declaration of Helsinki for medical research involving human subjects.

Cohort description and sample acquisition. For the purposes of this study, two distinct cohorts were recruited. The first group included patients with a confirmed diagnosis of POAG who were scheduled to undergo either trabeculectomy or cataract extraction. All patients in this group were receiving topical antiglaucoma therapy. The control group comprised age-matched individuals with age-related cataracts, free of any other ocular pathology, who were scheduled for routine cataract surgery. To reduce potential demographic bias, participants in both groups were matched for age and sex. Exclusion criteria for both groups included a history of ocular surgery, diabetes mellitus, any form of glaucoma other than POAG, or other ocular conditions such as uveitis or age-related macular degeneration (AMD).

Aqueous humor samples (50–100 μl) were collected under sterile conditions via limbal paracentesis using a 30-gauge anterior chamber cannula connected to an one ml syringe. Sampling was performed at the outset of the surgical procedure–either trabeculectomy or phacoemulsification. All specimens were immediately frozen at −80 °C and stored until further analysis.

Evaluation of VEGF-C concentrations. VEGF-C levels in aqueous humor were analyzed using the MILLIPLEX MAP Human Angiogenesis Magnetic Bead Panel – Multiplex Assay (HAGP1MAG-12K, Merck Millipore, Darmstadt, Germany), a high-sensitivity multiplex immunoassay system. This advanced platform utilizes fluorescently-coded magnetic microspheres, enabling simultaneous quantification of multiple analytes across 96-well plates. 25 μl of each sample was dispensed into the microwells of each platform and read simultaneously with a Luminex 200 reader (Luminex Corporation, Austin, TX, USA). Data acquisition and analysis were performed with xPONENT software (Diasorin, Saluggia, Italy).

Statistical analysis. Continuous variables are summarized as means with standard deviations or as medians with interquartile ranges (IQR), depending on the distribution of the data. Categorical variables are presented as absolute frequencies and corresponding percentages. Comparisons of proportions between groups were performed using the chi-square test or Fisher’s exact test, as appropriate. For continuous variables, intergroup comparisons were conducted using the Student’s t-test for normally distributed data and the Mann–Whitney U test for non-normally distributed data, particularly in comparisons between participants with and without POAG. Receiver operating characteristic (ROC) curve analysis was employed to assess the prognostic performance of VEGF-C for POAG. Optimal cut-off values were used to compute sensitivity, specificity, and both positive and negative predictive values. The area under the ROC curve (AUC) was also calculated as a measure of overall diagnostic accuracy. Multivariate logistic regression models were subsequently constructed to further evaluate the prognostic utility of VEGF-C, with results expressed as adjusted odds ratios (aORs) and corresponding 95% confidence intervals (CIs). All statistical tests were two-sided, and a p-value of less than 0.05 was considered indicative of statistical significance. Statistical analyses were performed using SPSS software, version 26.0 (IBM Corp., Armonk, NY, USA).

Results

Demographic characteristics of the study population. Aqueous humor samples were collected from 76 participants (76 eyes), comprising 39 individuals in the POAG group and 37 individuals in the control group. The demographic and clinical characteristics of the study population are summarized in Table I. Regarding antiglaucomatous therapy within the POAG group, the most frequently administered medication was Latanoprost (61.5%), followed by Timolol (46.2%) and Dorzolamide (43.6%) (Table II).

View this table:
Table I.

Sample characteristics in control and primary open angle glaucoma (POAG) group.

View this table:
Table II.

Treatment received in primary open angle glaucoma eyes (N).

Prognostic value of aqueous humor VEGF-C levels. VEGF-C was detected in all 76 aqueous humor samples examined. Especially, we found that the VEGF-C levels in the aqueous humor of patients with POAG and in the control group were 26.41±26.16 pg/ml and 16.7±8.6 pg/ml, respectively, and the difference was not statistically significant (p =0.277) (Table III). The ROC analysis did not reveal any significant prognostic ability of VEGF-C for POAG (AUC: 0.60; p=0.278) (Figure 1 and Table IV).

View this table:
Table III.

VEGF-C levels in the primary open angle glaucoma and control groups.

Figure 1.
Figure 1.

Receiver operating characteristic (ROC) curve of VEGF-C for primary open angle glaucoma.

View this table:
Table IV.

Receiver operating characteristic curve analysis results for VEGF-C.

Discussion

This cross-sectional study investigated VEGF-C levels in aqueous humor samples obtained from patients with POAG and control subjects. A total of 76 participants (76 eyes) were included, allocated into two well-defined groups: 39 patients comprised the POAG group, and 37 participants constituted the control group. Inclusion criteria for the POAG group included a confirmed diagnosis of POAG, ongoing topical antiglaucoma therapy, well-controlled IOP, and the absence of any coexisting ocular pathology. In contrast, the control group consisted of individuals with no ocular disease other than age-related cataract.

The study shows that the VEGF-C aqueous humor levels did not differ significantly between the POAG and the control group. Subsequently, the ROC analysis did not reveal any prognostic significance for VEGF-C.

Lately, the research interest concerning the pathophysiology of POAG, has been focused on the Schlemm’s canal. The Schlemm’s canal is a specialized vascular structure that preserves fluid homeostasis by channeling aqueous humor from the eye into the systematic circulation. Schlemm’s canal constitutes the major resistance to aqueous humor outflow. Schlemm’s canal inner wall endothelium displays both blood and lymphatic vascular characteristics (5). Schlemm’s canal closely resembles lymphatic vessels, featuring a continuous endothelial monolayer without pericytes on a discontinuous basement membrane and functioning to return fluid to the systemic circulation under flow stress (5).

Aspelund et al., found that a single, low-dose injection of intraocular VEGF-C in mice increased the sprouting and proliferation of Schlemm’s canal endothelial cells and showed a trend towards normalized IOP. On the contrary, adenoviral or adeno-associated virus (AAV) delivery of VEGF-A, induced a massive increase in IOP and obliterated Schlemm’s canal (16). The above results can be explained by the fact that the preferential VEGF-C receptor, VEGFR-3, is expressed by the Schlemm’s canal endothelial cells, while the canonical VEGF-A receptor, VEGFR-2, is expressed by the vascular structures of the eye (18). The research of Aspelund et al., depicted the potential role of VEGF-C in lowering IOP (16).

Hase et al., described a lower mean concentration of VEGF-C in the aqueous humor of patients with POAG compared to the control group, but they examined only six patients with POAG and nine samples from the control group. Additionally, they found VEGF-C immunoreactivity in the Schlemm’s canal endothelial cells and the collecting ducts in one sample tissue of one patient with POAG who underwent trabeculectomy. Moreover, they described that VEGF-C concentration in supernatants or cell lysates of Trabecular meshwork cells cultured under oxidative stress and hypoxia was significantly elevated compared with those cultured under normal conditions (15).

Additionally, Wu et al., demonstrated that the co-administration of VEGF-C and 5-Fluorouracil can enhance the bleb survival in a rabbit glaucoma filtering surgery model (17). In particular, VEGF-C combined with 5-Fluorouracil led to larger blebs with markedly prolonged survival and a progressive decrease in IOP (17). Furthermore, Lee et al., found that VEGF-C successfully promoted lymphatics without any change in blood vessels (19).

Recently, Lu et al., developed a human ocular fluid outflow on-chip, composed of a 3D lymphatic-like SC endothelium that is surrounded by TM cells and drains interstitial fluid. In both, in vitro and in vivo studies, they showed that dexamethasone-treated TM cells down-regulate VEGF-C expression through activin receptor-like kinase 5 (ALK5), and the lack of VEGF-C affects SC cells through VEGFR3, impairing SC junctions and fluid outflow (20).

Generally, the role of VEGF-C in POAG has not been extensively examined, but the data mentioned above show a potentially crucial role of VEGF-C in the formation and function of Schlemm’s canal. To the best of our knowledge, our study is the first in the literature to examine the aqueous humor levels of VEGF-C in a large cohort of patients with POAG and a well-balanced control group. Theoretically, and taking into account the previous research, we would expect to find elevated levels of VEGF-C in the POAG group but we did not find any significant difference between the two groups. A possible explanation could be that the patients in the POAG group were patients under treatment, and their IOP was within normal limits. In the future, we should add a third group of patients with newly diagnosed POAG without receiving any treatment and high IOP. It would be interesting to examine whether VEGF-C levels are elevated under stress conditions and high IOP. In that case, VEGF-C would theoretically promote the sprouting and proliferation of Schlemm’s canal endothelial cells. In addition, we should examine the levels of VEGFR-3. Specifically, we should perform immunohistochemistry in patients with POAG who underwent trabeculectomy, so as to evaluate the VEGF-C/VEGFR-3 ratio. In that case, it would be extremely difficult to have a control group. We could compromise using an enucleated eyeball from a patient with a malignant melanoma, as used by other researchers (15).

Conclusion

To the best of our knowledge, our study is the first to examine VEGF-C levels in the aqueous humor in a large cohort of patients with POAG. We did not reveal any statistically significant difference in the VEGF-C levels between the two groups. The role of VEGF-C in POAG has not been extensively examined yet, and additional studies are required to reveal its potential role in POAG.

Footnotes

  • Authors’ Contributions

    Conceptualization, G.S.D. and M.M.M.; methodology, G.S.D., A.I and M.M.M.; software, G.S.D and A.I.; validation, G.S.D., A.I. and M.M.M.; formal analysis, G.S.D. and A.I; investigation, G.S.D.; resources, G.S.D. and M.M.M.; data curation, G.S.D., A.I and M.M.M.; writing – original draft preparation, G.S.D.; writing – review and editing, G.S.D., K.C, G.S and M.M.M.; visualization, G.S.D. and M.M.M.; supervision, K.C, G.S and M.M.M.; project administration, G.S.D. and M.M.M.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Funding

    This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received September 11, 2025.
  • Revision received September 29, 2025.
  • Accepted October 1, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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VEGF-C Aqueous Humor Levels in Patients With Primary Open Angle Glaucoma
GEORGIOS S. DIMTSAS, ALEXANDRA IERONYMAKI, KLIO I. CHATZISTEFANOU, GERASIMOS SIASOS, MARILITA M. MOSCHOS
In Vivo Jan 2026, 40 (1) 382-388; DOI: 10.21873/invivo.14202
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