Aqueous Humor Outflow Pathway Research Articles

VIP Stabilizes the Cytoskeleton of Schlemm's Canal Endothelia via Reducing Caspase-3 Mediated ZO-1 Endolysosomal Degradation.

Objectives In glaucomatous eyes, the main aqueous humor (AH) outflow pathway is damaged by accumulated oxidative stress arising from the microenvironment, vascular dysregulation, and aging, which results in increased outflow resistance and ocular hypertension. Schlemm's canal (SC) serves as the final filtration barrier of the main AH outflow pathway. The present study is aimed at investigating the possible regulation of vasoactive intestinal peptide (VIP) on the cytoskeleton by stabilizing ZO-1 in SC. Methods Model of chronic ocular hypertension (COH) induced by episcleral venous cauterization was treated with topical VIP. The ultrastructure of junctions, ZO-1 levels, and permeability of the SC inner wall to FITC-dextran (70 kDa) were detected in the COH models. The F-actin distribution, F/G-actin ratio, and ZO-1 degradation pathway in human umbilical vein endothelial cells (HUVECs) and HEK 293 cells were investigated. Results ZO-1 in the outer wall of the SC was less than that in the inner wall. COH elicited junction disruption, ZO-1 reduction, and increased permeability of the SC inner wall to FITC-dextran in rats. ZO-1 plays an essential role in maintaining the F/G-actin ratio and F-actin distribution. VIP treatment attenuated the downregulation of ZO-1 associated with COH or H2O2-induced oxidative damage. In H2O2-stimulated HUVECs, the caspase-3 inhibitor prevents ZO-1 disruption. Caspase-3 activation promoted endolysosomal degradation of ZO-1. Furthermore, a decrease in caspase-3 activation and cytoskeleton redistribution was demonstrated in VIP + H2O2-treated cells. The knockdown of ZO-1 or the overexpression of caspase-3 blocked the effect of VIP on the cytoskeleton. Conclusion This study provides insights into the role of VIP in stabilizing the interaction between the actin cytoskeleton and cell junctions and may provide a promising targeted strategy for glaucoma treatment.

Targeted Delivery of Cell Softening Micelles to Schlemm's Canal Endothelial Cells for Treatment of Glaucoma.

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency. To address these issues, a targeted PEG-b-PPS micelle loaded with actin depolymerizer latrunculin A (tLatA-MC) is developed. Targeting of SC cells is achieved by modifying the micelle surface with a high affinity peptide that binds the VEGFR3/FLT4 receptor, a lymphatic lineage marker found to be highly expressed by SC cells relative to other ocular cells. During in vitro optimization, increasing the peptide surface density increased micellar uptake in SC cells while unexpectedly decreasing uptake by human umbilical vein endothelial cells (HUVEC). The functional efficacy of tLatA-MC, as measured by decreased SC cell stiffness compared to non-targeted micelles (ntLatA-MC) or targeted blank micelles (tBL-MC), is verified using atomic force microscopy. tLatA-MC reduced IOP in an in vivo mouse model by 30-50%. The results validate the use of a cell-softening nanotherapy to selectively modulate stiffness of SC cells for therapeutic reduction of IOP and treatment of glaucoma.

Erythrocyte-Mediated Angiography: Quantifying Absolute Episcleral Blood Flow in Humans

We introduce erythrocyte-mediated angiography (EMA) as a novel technique for quantifying absolute episcleral blood flow in humans. This technique is highly reproducible and may be a valuable quantitative tool for studying aqueous hemodynamics.

Aqueous Angiography in Normal Canine Eyes.

PurposeTo conduct aqueous angiography (AA) using a clinically applicable technique in normal dogs and to compare findings to intravenous scleral angiography (SA).MethodsWe examined 10 canine cadaver eyes and 12 eyes from live normal dogs. A gravity-fed trocar system delivered 2% sodium fluorescein and 0.25% indocyanine green (ICG) intracamerally (IC) in cadaver eyes. In vivo AA was subsequently performed in one eye of each of the 12 dogs via IC bolus of ICG under sedation. The same 12 dogs received SA via intravenous ICG (mean ± SD) 10.7 ± 3.3 days later. Identical scleral sectors were imaged using a Spectralis confocal scanning laser ophthalmoscope.ResultsThe gravity-fed trocar system permitted visualization of the conventional aqueous humor outflow (CAHO) pathways in cadaver eyes, but not in vivo. Fluorescence was observed superonasally in four of the 10 cadaver eyes within 24.0 ± 3.6 seconds. A single IC bolus of ICG showed CAHO pathways in vivo, demonstrating sectoral outflow patterns in the superotemporal sclera in 10 of the 12 eyes within 35.0 ± 4.3 seconds; four of the 12 eyes exhibited pulsatile aqueous movement. SA exhibited fluorescence patterns comparable to AA with weak pulsatile aqueous humor outflow.ConclusionsAngiography (AA or SA) in dogs permits visualization of the CAHO pathway and its vascular components in vivo. AA may be a more useful modality to assess aqueous humor outflow.Translational RelevanceIntracameral AA has potential utility for evaluating CAHO in vivo in dogs, an important animal model species.

Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.

Targeting the vascular-specific phosphatase PTPRB protects against retinal ganglion cell loss in a pre-clinical model of glaucoma.

Elevated intraocular pressure (IOP) due to insufficient aqueous humor outflow through the trabecular meshwork and Schlemm's canal (SC) is the most important risk factor for glaucoma, a leading cause of blindness worldwide. We previously reported loss of function mutations in the receptor tyrosine kinase TEK or its ligand ANGPT1 cause primary congenital glaucoma in humans and mice due to failure of SC development. Here, we describe a novel approach to enhance canal formation in these animals by deleting a single allele of the gene encoding the phosphatase PTPRB during development. Compared to Tek haploinsufficient mice, which exhibit elevated IOP and loss of retinal ganglion cells, Tek+/-;Ptprb+/- mice have elevated TEK phosphorylation, which allows normal SC development and prevents ocular hypertension and RGC loss. These studies provide evidence that PTPRB is an important regulator of TEK signaling in the aqueous humor outflow pathway and identify a new therapeutic target for treatment of glaucoma.

A Chronic Ocular-Hypertensive Rat Model induced by Injection of the Sclerosant Agent Polidocanol in the Aqueous Humor Outflow Pathway.

Background: To induce a moderate chronic ocular hypertension (OHT) by injecting polidocanol, a foamed sclerosant drug, in the aqueous humor outflow pathway. Methods: Intraocular pressure (IOP) was monitored for up to 6 months. Pattern and full-field electroretinogram (PERG and ERG) were recorded and retinal ganglion cells (RGC) and retinal nerve fiber layer (RNFL) thickness were assessed in vivo with optical coherence tomography (OCT) and ex vivo using Brn3a immunohistochemistry. Results: In the first 3 weeks post-injection, a significant IOP elevation was observed in the treated eyes (18.47 ± 3.36 mmHg) when compared with the control fellow eyes (12.52 ± 2.84 mmHg) (p < 0.05). At 8 weeks, 65% (11/17) of intervention eyes had developed an IOP increase >25% over the baseline. PERG responses were seen to be significantly reduced in the hypertensive eyes (2.25 ± 0.24 µV) compared to control eyes (1.44 ± 0.19 µV) (p < 0.01) at week 3, whereas the ERG components (photoreceptor a-wave and bipolar cell b-wave) remained unaltered. By week 24, RNFL thinning and cell loss in the ganglion cell layer was first detected (2/13, 15.3%) as assessed by OCT and light microscopy. Conclusions: This novel OHT rat model, with moderate levels of chronically elevated IOP, and abnormal PERG shows selective functional impairment of RGC.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

Rapamycin Removes Damaged Mitochondria and Protects Human Trabecular Meshwork (TM-1) Cells from Chronic Oxidative Stress.

Glaucoma is a chronic optic neuropathy that could lead to permanent vision loss. Primary open-angle glaucoma (POAG) is the most common type of glaucoma, with elevated intraocular pressure (IOP) as a major risk factor. IOP is mainly regulated by trabecular meshwork (TM), an important component of the conventional aqueous humor (AH) outflow pathway. TM cells are constantly subjected to oxidative stress. Long-term exposure to oxidative stress has been shown to cause elevation of AH outflow resistance, leading to higher IOP. In this study, we induced chronic oxidative stress in human trabecular meshwork (TM-1) cells with 1μM rotenone and investigated the levels of reactive oxygen species (ROS), autophagy, and mitochondrial functions. Protective effects of rapamycin, an inducer of autophagy, were also investigated. Our data indicated that rotenone significantly increased oxidative stress, but not autophagy, in TM-1 cells. Rapamycin at 10nM effectively suppressed the rotenone-induced cell apoptosis, as well as the ROS elevation. The protective effects of rapamycin could be associated to the induction of autophagy and removal of damaged mitochondria in TM-1 cells. Our results suggest autophagy has important roles in protecting TM-1 cells from oxidative stress, which could be further developed into a novel treatment to POAG.

Nd:YAG laser goniopuncture as a mandatory adjuvant procedure after non-penetrating deep sclerectomy (long-term observation results)

To evaluate the effect of Nd:YAG laser goniopuncture on the long-term hypotensive effectiveness of non-penetrating deep sclerectomy. The study included 114 patients after non-penetrating deep sclerectomy (NPDS). In the control group (n=58), Nd:YAG laser goniopuncture was performed within 3.4±1.9 (1.5-6.7) months, and in the main group (n=56) - within 1.12±0.08 (0.9-1.5) months after the surgery. Ultrasound biomicroscopy (UBM) was used to evaluate the semiotics of trabecular-Descemet's membrane (TDM), intrascleral canal (ISC) and filtering bleb. The follow-up period was 5 years. According to UBM data, the thickness (0.10±0.009 mm) and density (50±6%) of TDM (p=0.0001) increased before LGP in the control group, the height of ISC decreased to 0.49±0.19 (0.20-0.40) (p=0.03), the height of UBM scan - to 1.49±0.05 (1.41-2.9) (p=0.0001); IOP (P0) was 18.48±4.7 (11.2-22.9) mmHg (p=0.001). In the main group before LGP, TDM thickness was 0.08±0.006 mm, density was 40±5%, and IOP (P0) was 15.7±4.1 (9.1-18.5) mm Hg. Complete hypotensive success was achieved in 83.6% of cases in the control group and 96.2% in the main group in 6 months; 68.07% and 92.59% in 12 months; 41.3% and 75.8% in 24 months; 15.25% and 48.93% in 36; 15% and 34.8% in 60 months after the surgery, respectively (p=0.0001, 95% confidence interval). TDM is an additional level of retention of aqueous humor and plays key role in the formation of outflow pathways after NPDS. Performing LGP in the early postoperative period is an effective and safe adjuvant option, which excludes the influence of TDM on the formation of aqueous humor outflow pathways and significantly increases the long-term hypotensive efficacy of non-penetrating deep sclerectomy.

Visual Assessment of Aqueous Humor Outflow.

In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving aqueous humor outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.

Single step laser procedures combined technology in the treatment algorithm of advanced primary open-angle glaucoma stage after surgery

Results are presented of a combined laser treatment method of advanced primary open-angle glaucoma stagewith high trabecular meshwork pigmentation in anterior chamber angle and pseudoexfoliation syndrome stage 2, after non-penetrating deep sclerectomy at recurrent IOP increase in the post-op period. The laser treatment included a single-step modified selective laser activation of the trabeculum and a laser Descemets goniopuncture. The study results demonstrated more significant IOP-lowering effect as well as the integrity of surgically-formed aqueous humor outflow pathways during a follow-up period of 24 months compared with controls, in whom only classical Descemets goniopuncture (DGP) method was used.

Donadores de óxido nítrico como hipotensores en glaucoma

IntroductionAn elevated intraocular pressure (IOP) remains the main risk factor for progression of glaucoma upon which we can efficiently act. Pharmacological strategies to reduce IOP are directed towards the reduction of aqueous humour (AH) production and/or the increase in AH drainage through the uveoscleral pathway. However, there are no drugs currently available as first-line treatment to increase AH outflow primarily via the conventional route. Ocular nitric oxide (NO) production takes place in AH outflow pathways and in the ciliary muscle, modulating the cellular response to elevated IOP. MethodsThis review describes the mechanism of action of endogenous NO and NO-donating compounds that are under research. It includes information regarding pre-clinical and clinical studies previously conducted with these compounds, discussing their role and therapeutic potential in the pharmacological treatment of ocular hypertension in glaucoma. ResultsThe topical ocular administration of NO-donating compounds significantly lowered IOP and maintained it in animal models of glaucoma and subjects with ocular hypertension. ConclusionsThe mechanism of action of these compounds is novel and scientific evidence that shows promising results. However, there is a need for more comprehensive studies to assess long-term safety and tolerability in order to properly evaluate their use in chronic therapies.

Impact of the clinical use of ROCK inhibitor on the pathogenesis and treatment of glaucoma.

Rho-associated protein kinase (ROCK), a ubiquitously expressed signaling messenger and downstream effector of Rho, is activated by several bioactive factors in the aqueous humor (AH). Rho-ROCK signaling regulates a wide spectrum of fundamental cellular events, including cell adhesion, motility, proliferation, differentiation, and apoptosis. Previous studies, including our own, found that ROCK inhibitor lowers intraocular pressure (IOP) via a direct effect on the conventional AH outflow pathway, by regulation of contractile properties, fibrotic activity, and permeability of the trabecular meshwork (TM) and Schlemm's canal (SC) tissues, influencing extracellular matrix (ECM) production. Recently, a novel ROCK inhibitor, ripasudil, has been introduced in Japan. Other ROCK inhibitors are now in clinical trials as new IOP-lowering drugs for glaucoma patients. To date, ripasudil, administered together with other glaucoma medications, has proved safe and efficient in lowering IOP as well as additional effects such as prostaglandin analogs, beta-blockers, and carbonic anhydrase inhibitors, all of which help lower IOP by different mechanisms. In addition, we found that long-term treatment with ripasudil exerted an additional IOP-lowering effect, especially in eyes with high IOP, suggesting that late-onset remodeling of the ECM in glaucomatous eyes may elicit mild and delayed changes in IOP levels. ROCK inhibitors have also shown several additional effects, including increased retinal blood flow, direct protection of neurons against various types of stress, and regulation of wound healing; these benefits may potentially be useful in glaucoma treatment.

Structural and functional imaging of aqueous humour outflow: a review.

Maintaining healthy aqueous humour outflow (AHO) is important for intraocular cellular health and stable vision. Impairment of AHO can lead to increased intraocular pressure, optic nerve damage and concomitant glaucoma. An improved understanding of AHO will lead to improved glaucoma surgeries that enhance native AHO as well as facilitate the development of AHO-targeted pharmaceuticals. Recent AHO imaging has evolved to live human assessment and has focused on the structural evaluation of AHO pathways and the functional documentation of fluid flow. Structural AHO evaluation is predominantly driven by optical coherence tomography, and functional evaluation of flow is performed using various methods, including aqueous angiography. Advances in structural and functional evaluation of AHO are reviewed with discussion of strengths, weaknesses and potential future directions.

Automated circumferential construction of first-order aqueous humor outflow pathways using spectral-domain optical coherence tomography.

The purpose was to create a three-dimensional (3-D) model of circumferential aqueous humor outflow (AHO) in a living human eye with an automated detection algorithm for Schlemm’s canal (SC) and first-order collector channels (CC) applied to spectral-domain optical coherence tomography (SD-OCT). Anterior segment SD-OCT scans from a subject were acquired circumferentially around the limbus. A Bayesian Ridge method was used to approximate the location of the SC on infrared confocal laser scanning ophthalmoscopic images with a cross multiplication tool developed to initiate SC/CC detection automated through a fuzzy hidden Markov Chain approach. Automatic segmentation of SC and initial CC’s was manually confirmed by two masked graders. Outflow pathways detected by the segmentation algorithm were reconstructed into a 3-D representation of AHO. Overall, only <1% of images (5114 total B-scans) were ungradable. Automatic segmentation algorithm performed well with SC detection 98.3% of the time and <0.1% false positive detection compared to expert grader consensus. CC was detected 84.2% of the time with 1.4% false positive detection. 3-D representation of AHO pathways demonstrated variably thicker and thinner SC with some clear CC roots. Circumferential (360 deg), automated, and validated AHO detection of angle structures in the living human eye with reconstruction was possible.

Ультрабиомикроскопические параметры адекватного формирования путей оттока внутриглазной жидкости после непроникающей хирургии глаукомы

Purpose. To evaluate the ultra-bio-microscopic semiotics of internal fistula, which determines the time and indications for laser descemetopuncture after non-penetrating deep sclerectomy (NPDS), as well as a long-term hypotensive efficacy of this type of interventions. Material and methods. A full and recognized hypotensive effect was evaluated in 1000 patients with primary open-angle glaucoma after non-penetrating deep sclerectomy followed by laser descemetogoniopuncture. The group 1 included 500 patients (retrospective study), the group 2 – 500 patients (prospective study). All patients in the postoperative period underwent an ultrabiomicroscopic monitoring with an evaluation of parametric (mm) and morphometric (acoustic density) indices of internal fistula, intrascleral canal, and filtering bleb. Clinical groups were relevant to sex, age, stages of glaucoma and initial regimen of local hypotensive therapy. Follow-up period was 3 years. Results. The UBM semiotics for validity of the internal fistula are determined: a height of 0.8±0.09mm and a presence of micro-fistula after laser descemetopuncture. In the group 1: time of laser descemetopuncture was 3.4±1.9 months, a full hypotensive effect was observed in 59.6% 1 year later, in 24.8% after 3 years. In the group 2: time of laser descemetopuncture was 1.12±0.08 months, a total hypotensive success was determined in 84.8% after 12 months, up to 3 years – in 60.4% of cases. In its turn, an impact of NPDS hypotensive effect was associated with laser descemetogoniopuncture in 100% of cases 1-12 months after surgical stage of operation. Conclusion. The two-stage technology of non-penetrating deep sclerectomy with laser descemetopuncture in the absolute number of cases allows to create a penetrating internal micro-fistula and to exclude its influence on morphogenesis of newly formed aqueous humor outflow pathways. In its turn, it allows to increase a full and recognized hypotensive effect of fistulizing interventions of non-penetrating type.

Microstructure visualization of conventional outflow pathway and finite element modeling analysis of trabecular meshwork.

BackgroundThe intraocular pressure (IOP) is maintained through a dynamic equilibrium between the production and drainage of aqueous humor. Elevation of intraocular pressure is mainly caused by the blocking of aqueous humor outflow pathway. Therefore, it is particularly important to study the structure of drainage pathway and the effect of ocular hypertension at the process of aqueous humor outflow.MethodsConventional drainage pathway of aqueous humor, including trabecular meshwork (TM), Schlemm’s canal (SC) and aqueous vein, were imaged by using trans-scleral imaging method with lateral resolution of 2 μm. For quantitative assessment, the morphological parameters of the TM were measured with different IOP levels via a combination of measurements and simulations.ResultsImages of the TM and the adjacent tissues were obtained. The porosity of TM with normal intraocular pressure varies from 0.63 to 0.74 as the depth increases, while in high IOP it is changed from 0.44 to 0.59. The diameter of aqueous vein varies from 32 to 43 μm, and is smaller than that of SC, which varies from 48 to 64.67 μm.ConclusionsOur research provides a non-contact method to visualize the microstructure of tissue for clinical examination associated with the blocking of the outflow pathway of aqueous humor in humans. The three-dimensional (3D) microstructures of limbus and the results of finite element modeling analysis of the TM model will serve for the future evaluation of new glaucoma surgical techniques.

Aqueous Angiography-Mediated Guidance of Trabecular Bypass Improves Angiographic Outflow in Human Enucleated Eyes.

PurposeTo assess the ability of trabecular micro-bypass stents to improve aqueous humor outflow (AHO) in regions initially devoid of AHO as assessed by aqueous angiography.MethodsEnucleated human eyes (14 total from 7 males and 3 females [ages 52–84]) were obtained from an eye bank within 48 hours of death. Eyes were oriented by inferior oblique insertion, and aqueous angiography was performed with indocyanine green (ICG; 0.4%) or fluorescein (2.5%) at 10 mm Hg. With an angiographer, infrared and fluorescent images were acquired. Concurrent anterior segment optical coherence tomography (OCT) was performed, and fixable fluorescent dextrans were introduced into the eye for histologic analysis of angiographically positive and negative areas. Experimentally, some eyes (n = 11) first received ICG aqueous angiography to determine angiographic patterns. These eyes then underwent trabecular micro-bypass sham or stent placement in regions initially devoid of angiographic signal. This was followed by fluorescein aqueous angiography to query the effects.ResultsAqueous angiography in human eyes yielded high-quality images with segmental patterns. Distally, angiographically positive but not negative areas demonstrated intrascleral lumens on OCT images. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways. Trabecular bypass but not sham in regions initially devoid of ICG aqueous angiography led to increased aqueous angiography as assessed by fluorescein (P = 0.043).ConclusionsUsing sequential aqueous angiography in an enucleated human eye model system, regions initially without angiographic flow or signal could be recruited for AHO using a trabecular bypass stent.

Vascular Endothelial Growth Factor-A Increases the Aqueous Humor Outflow Facility.

PurposeAnti-vascular endothelial growth factor (VEGF) antibody therapy is an effective treatment for ocular angiogenesis. Although the intraocular pressure of some patients increases after anti-VEGF therapy, the effects of VEGF-A on the aqueous humor outflow pathway remain unknown. This study investigated the effects of VEGF-A on the aqueous humor outflow pathway.MethodsWe used human recombinant VEGF121 and VEGF165. Trabecular meshwork (TM) and Schlemm’s canal endothelial (SCE) cells were isolated from the eyes of cynomolgus monkeys. Expression of mRNA coding four VEGF receptors, VEGFR1 (FLT1), VEGFR2 (KDR), neuropilin-1, and neuropilin-2, was examined by RT-PCR. To evaluate the permeability of cell monolayers, we measured transendothelial electrical resistance (TEER). The outflow facility was measured in perfused porcine anterior segment organ cultures treated with 30 ng/mL VEGF121 for 48 h.ResultsFour VEGF-A-related receptor mRNAs were expressed in TM and SCE cells. The TEER of TM cells was not significantly affected by VEGF121 or VEGF165 treatment. In contrast, the TEER of SCE cells was significantly lower 48 h after treatment with 30 ng/mL VEGF121 to 69.4 ± 12.2% of baseline (n = 10), which was a significant difference compared with the control (P = 0.0001). VEGF165 (30 ng/mL) decreased the TEER of SCE cells at 48 h after treatment to 72.3 ± 14.1% compared with the baseline (n = 10), which was not a significant difference compared with the control (P = 0.0935). Ki8751, a selective VEGFR2 inhibitor, completely suppressed the effect of VEGF121 on SCE cell permeability, although ZM306416, a selective VEGFR1 inhibitor, did not affect the VEGF121-induced decrease in TEER. Perfusion with 30 ng/mL of VEGF121 for 48 h significantly increased the outflow facility compared with the control (47.8 ± 28.5%, n = 5, P = 0.013).ConclusionsThese results suggest that VEGF-A may regulate the conventional aqueous outflow of SCE cells through VEGFR2.