Schlemm's Canal Endothelial Cells Research Articles

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

The Factors Affecting the Stability of IOP Homeostasis.

Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

Comparison of Schlemm's Canal Morphology Parameters Between Propensity Score-Matched Primary Open-Angle Glaucoma and Exfoliation Glaucoma.

This study aimed to histologically compare the status of Schlemm's canal (SC) and Schlemm's canal endothelial (SCE) cells between trabeculectomy specimens from patients with primary open-angle glaucoma (POAG) and exfoliation glaucoma (EXG). A total of 182 eyes from 152 patients with POAG and 138 eyes from 116 patients with EXG underwent immunohistochemical staining for thrombomodulin. Equal numbers of cases were selected from both groups using propensity score matching. The following parameters were evaluated: total SC length, staining positive and negative SC length (PSC and NSC, respectively), opened and closed SC length, staining positive and opened SC length, staining positive and closed SC length, staining negative and opened SC length (NOSC), and staining negative and closed SC length. After matching for age and gender, 87 cases were selected in each group. The EXG group had significantly higher preoperative IOP and medication scores. PSC was significantly longer in the POAG group, while NSC and NOSC were longer in the EXG group. Multiple regression analysis of these 174 cases revealed that PSC was significantly shorter in the EXG group. After matching for age, gender, preoperative IOP, and medication score, 64 cases were selected in each group, and NOSC was significantly longer in the EXG group. These findings suggest that in EXG, SCE loss occurs independently of background factors such as aging and medication use. The loss of SCE may have a more critical impact on IOP elevation in EXG compared to POAG.

Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy.

Primary open-angle glaucoma is associated with elevated intraocular pressure (IOP) that damages the optic nerve and leads to gradual vision loss. Several agents that reduce the stiffness of pressure-regulating Schlemm's canal (SC) endothelial cells, in the conventional outflow pathway of the eye, lower IOP in glaucoma patients and are approved for clinical use. However, poor drug penetration and uncontrolled biodistribution limit their efficacy and produce local adverse effects. Compared to other ocular endothelia, FLT4/VEGFR3 is expressed at elevated levels by SC endothelial cells and can be exploited for targeted drug delivery. Here, we validate FLT4 receptors as clinically relevant targets on SC cells from glaucomatous human donors and engineer polymeric self-assembled nanocarriers displaying lipid-anchored targeting ligands that optimally engage this receptor. Targeting constructs were synthesized as lipid-PEGx-peptide, differing in the number of PEG spacer units (x), and were embedded in micelles. We present a novel proteolysis assay for quantifying ligand accessibility that we employ to design and optimize our FLT4-targeting strategy for glaucoma nanotherapy. Peptide accessibility to proteases correlated with receptor-mediated targeting enhancements. Increasing the accessibility of FLT4-binding peptides enhanced nanocarrier uptake by SC cells while simultaneously decreasing the uptake by off-target vascular endothelial cells. Using a paired longitudinal IOP study in vivo, we show that this enhanced targeting of SC cells translates to IOP reductions that are sustained for a significantly longer time as compared to controls. Confocal microscopy of murine anterior segment tissue confirmed nanocarrier localization to SC within 1 h after intracameral administration. This work demonstrates that steric effects between surface-displayed ligands and PEG coronas significantly impact the targeting performance of synthetic nanocarriers across multiple biological scales. Minimizing the obstruction of modular targeting ligands by PEG measurably improved the efficacy of glaucoma nanotherapy and is an important consideration for engineering PEGylated nanocarriers for targeted drug delivery.

ITGA8 positive cells in the conventional outflow tissue exhibit Schlemm's canal endothelial cell properties

AimsElevated intraocular pressure is primarily induced by the increased resistance of conventional outflow of aqueous humor. Dysfunction of the juxtacanalicular region of trabecular meshwork (TM) and Schlemm's canal (SC) endothelium, as the main conventional outflow tissue, have been implicated as the major reasons for the increased resistance. Integrins are widespread in these tissues, especially alpha8 integrin (ITGA8). We aim to investigate the properties of cells expressing ITGA8 in the conventional outflow tissue. Main methodsFluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to detect the mRNA and protein levels of ITGA8 in human conventional outflow tissue. ITGA8-positive cells were isolated from the cultured human TM cells through a magnetic bead-based approach. Flow Cytometry was used to determine the purification efficiency. The expressions of TM and SC biomarkers and dexamethasone-induced myocilin secretion capacity of ITGA8-positive cells was assessed by Real-time PCR, IF and Western blot. A gel contraction assay was performed to evaluate contractility of ITGA8-positive cells after endothelin 1 treatment. Key findingsITGA8 was found with robust expression near the inner wall of SC endothelium. After purification, the proportion of ITGA8-positive cells were increased by about 10%. ITGA8-positive cells were identified with the properties as SC endothelial cells, such as more robust expressions of SC biomarkers, less dexamethasone-inducible myocilin expression, and stronger contractility. SignificanceThis study demonstrated that cells expressing ITGA8 in SC region possess more properties as SC endothelial cells. Our data implicate a crucial role of ITGA8 in aqueous humor (AH) outflow resistance regulation.

Morphological factors associated with giant vacuoles with I-pores in Schlemm's canal endothelial cells of human eyes: A serial block-face scanning electron microscopy study

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm's canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2–6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV's maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.

A Small Molecule Inhibitor of VE-PTP Activates Tie2 in Schlemm's Canal Increasing Outflow Facility and Reducing Intraocular Pressure.

PurposeTyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function.MethodsAKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC.ResultsAKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/− mice.ConclusionsThis is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

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.

Effect of Electrical Stimulation of Cervical Sympathetic Ganglia on Intraocular Pressure Regulation According to Different Circadian Rhythms in Rats.

PurposeThe purpose of this study was to investigate the relationship between circadian rhythm and intraocular pressure (IOP), and to explore whether electrical stimulation of cervical sympathetic ganglia (SCG) can regulate IOP via neurotransmitter distribution around the Schlemm's canal (SC) in rats.MethodsSprague Dawley rats were housed under normal (N-normal), constant dark (N-dark), and constant light (N-light) rhythms (n = 6 per group). Electrical stimulation (intermittent wave [20 hertz {Hz}, 2 mA, 10 minutes]) was used to stimulate the SCG. Atropine sulfate eye gel was applied three times a day. DiI was injected into the SCG and anterior chamber. The cross-sectional area and circumference of SC were evaluated using hematoxylin-eosin staining. Immunofluorescence staining was used to evaluate dopamine-β-hydroxylase (DβH) expression in SC endothelial (SCE) cells.ResultsN-Dark increased the IOP, decreased the cross-sectional area of SC, and increased DβH levels in SCE cells. Nerve projection between SC and SCG was detected, and electrical stimulation of SCG upregulated DβH expression in SCE cells. Under normal and constant light rhythms, electrical stimulation of SCG increased DβH and decreased the cross-sectional area and circumference of SC, while simultaneously increasing IOP and decreasing IOP fluctuations. After paralyzing the ciliary muscles, electrical stimulation of SCG decreased the cross-sectional area and circumference of SC under normal and constant light rhythms.ConclusionsN-Dark increased DβH in SCE cells, reduced the cross-sectional area of SC, and increased IOP. Under the normal and light rhythms, electrical stimulation of SCG increased DβH in SCE cells, reduced the cross-sectional area and circumference of SC, and in turn elevated IOP and decreased IOP fluctuations.

An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment.

PurposeDue to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes.MethodsWe harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators. To compare the cellular responses to candidate regulators of these mechanoresponsive genes, AAP and human SC cells (n = 3) were treated with 5 or 10 ng/mL transforming growth factor beta-2 (TGFβ2) for 24 or 48 hours, followed with expression profiling using real-time PCR or ddPCR.ResultsWe found that the isolated AAP cells displayed uniform cobblestone-like morphology and positive expression of two endothelial markers. In stretched SC cells, nine glaucoma-related genes were upregulated, and IPA implicated TGFβ as a potential upstream regulator. The effects of TGFβ2 treatment were similar for both AAP and SC cells in a dose- and time-dependent manner, activating TGFBR1 and SMAD2, inhibiting BMP4, and altering expression of three glaucoma-related genes (DCN, EZR, and CYP1B1).ConclusionsBovine AAP cells may serve as an alternative cellular model of human SC cells.Translational RelevanceThese AAP cells may be used to study the functional pathways related to the outflow facility.

ABCA1 Regulates IOP by Modulating Cav1/eNOS/NO Signaling Pathway

PurposeThis study aimed to investigate the role and pathophysiological mechanism of ATP binding cassette transporter A1 (ABCA1) in regulating the IOP and aqueous humor outflow.MethodsABCA1 expression was measured in trabecular meshwork samples obtained from patients with POAG and human donor eyes by Western blot. To further evaluate the functional significance of ABCA1, porcine angular aqueous plexus (AAP) cells, which are equivalent to human Schlemm's canal endothelial cells, were either treated with ABCA1 agonist GW3965 or transduced with lentivirus expressing ABCA1-shRNA. Transendothelial electrical resistance, protein expression, and nitric oxide (NO) concentration were measured. GW3965 was administered by intracameral injection. IOP and aqueous humor outflow facility were also measured.ResultsABCA1 expression was significantly higher in the trabecular meshwork tissue of patients with POAG compared with controls. ABCA1 upregulation in angular aqueous plexus cells decreased the transendothelial electrical resistance in the angular aqueous plexus monolayers accompanied by a 0.56-fold decrease in caveolin-1 expression and a 2.85-fold and 1.17-fold increase in endothelial NO synthase expression and NO concentration, respectively (n = 3, P < 0.05). Conversely, ABCA1 downregulation increased transendothelial electrical resistance and caveolin-1 expression and decreased endothelial NO synthase expression and NO production (n = 3, P < 0.05). GW3965 decreased IOP and significantly increased conventional outflow facility (P < 0.05).ConclusionsRegulation of aqueous humor outflow via the caveolin-1/endothelial NO synthase/NO pathway is a newly defined function of ABCA1 that is different from its traditional role in mediating cholesterol efflux. ABCA1 is a compelling, novel therapeutic candidate for the treatment of glaucoma and ocular hypertension.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

VIP Regulates Morphology and F-Actin Distribution of Schlemm's Canal in a Chronic Intraocular Pressure Hypertension Model via the VPAC2 Receptor.

To investigate the roles of vasoactive intestinal peptides (VIPs) in regulating the morphology and F-actin distribution of Schlemm's canal (SC) of rat eyes. Chronic intraocular pressure (IOP) hypertension models with episcleral venous cauterization (EVC) were treated with topical VIP or PG99-465 (vasoactive intestinal peptide receptors 2 [VPAC2] antagonist). IOPs were measured with Tono-Pen, and the SC parameters, including the cross-section area, circumference, and length, were statistically evaluated by hematoxylin-eosin and CD31 immunohistochemical staining. Immunofluorescence was performed to detect the distribution of F-actin in the SC. Moreover, the distribution of filamentous actin (F-actin) and globular actin (G-actin) in human umbilical vein endothelial cells (HUVECs) was studied under a pressure system by immunofluorescence and Western blotting. Increased expressions of VIP and VPAC2 receptors, as well as a disordered distribution of F-actin were found in SC endothelial cells (SCEs) in the EVC model. Moreover, topical VIP maintained the normal distribution of F-actin in SCEs, expanded the collapsed SC, and induced a significant decrease in IOP in the EVC model. In in vitro HUVECs, the F-actin/G-actin ratio increased significantly under stress stimulation for 30 minutes. A total of 50 μM VIP helped maintain the normal F-actin/G-actin ratio of HUVECs against stress stimulation. VIP regulates the distribution of F-actin in SCEs via the VPAC2 receptor in order to induce a decrease in IOP. VIP may represent a new target for antiglaucoma drugs.

Interaction Between Pilocarpine and Ripasudil on Intraocular Pressure, Pupil Diameter, and the Aqueous-Outflow Pathway.

To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on IOP and pupil diameter in human eyes, and morphological and functional changes in outflow tissues in vitro. IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which were topically applied in healthy subjects. Human trabecular meshwork (HTM) cells were used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemm's canal endothelial (SCE) cells was evaluated by measuring transendothelial electrical resistance and fluorescein permeability. Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Ripasudil significantly inhibited gel contraction, TGFβ2-induced stress fiber formation, α-smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFβ2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced miosis. Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow.

Modulation of Schlemm's canal endothelial cell stiffness via latrunculin loaded block copolymer micelles.

Increased stiffness of Schlemm's canal endothelial cells (SC cells) is a major contributing factor to the increased pressure characteristic of primary open-angle glaucoma. New treatments for glaucoma are being developed using actin depolymerizers and rho kinase inhibitors to address this increased stiffness. However, these agents have off-target effects and are not as potent as had been hoped. We have developed a micellar nanocarrier assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers capable of encapsulating latrunculin A (Lat A) with the goal of modulating SC cell stiffness. Lat A-loaded nanocarriers were similar in size and morphology to unloaded poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) micelles, loaded Lat A at 62% encapsulation efficiency, and retained loaded Lat A for at least 22 days. The continued functional activity of Lat A following encapsulation within micelles was verified in murine macrophages, which are known to display decreased endocytosis in response to Lat A-dependent cytoskeletal disruption. Endocytic inhibition remained unchanged when comparing equal concentrations of micelle-loaded versus free form Lat A. Uptake of Lat A-loaded micelles by human SC cells was verified in vitro with no sign of cytotoxicity, and modulation of SC cell stiffness was measured by atomic force microscopy. Lat A-loaded micelles significantly decreased SC cell stiffness, which resulted in visible changes in cell morphology as observed by confocal microscopy. Our results demonstrate that PEG-bl-PPS micelles represent a tunable platform for the controlled intracellular delivery of latrunculin. These self-assembled polymeric nanobiomaterials may support the rational design and engineering of delivery systems for the treatment of glaucoma. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1771-1779, 2018.

The progress of studies on aqueous humor dynamics abnormality induced by trabecular meshwork and Schlemm canal endothelial cell senescence and its relation with glaucoma

Glaucoma is the second leading cause of blindness in the world next to cataract. Aging is a strong risk factor leading to elevated intraocular pressure (IOP). IOP is associated with aqueous humor circulation. Trabecular meshwork cells and Schlemm canal endothelial cells, which form the conventional outflow pathway, play an important role in maintaining the IOP. Cell senescence induces abnormalities of the aqueous humor dynamics, leading to elevated IOP. Trabecular meshwork cells cause increased intrinsic stiffness, autophagy dysfunction, abnormal expression of microRNA and mitochondrial dysfunction with senescence. The senescence of Schlemm canal endothelial cells decreases cell permeability and mechanotransduction and disrupts the endothelial nitric oxide synthase signaling pathway. The changes will increase aqueous humor outflow resistance and elevate IOP. This review discusses how cell senescence induces aqueous humor dynamics abnormalities and the relation with glaucoma. (Chin J Ophthalmol, 2017, 53: 868-873).

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.

Bevacizumab clearance through the iridocorneal angle following intravitreal injection in a rat model

Antivascular endothelial growth factor (Anti-VEGF) agents have been widely used for a variety of ocular disorders. The etiology of sustained ocular hypertension following intravitreal administration of anti-VEGF agents is yet to be unraveled.Our study investigates and characterizes the presence of intravitreally injected bevacizumab in the aqueous outflow channels of a rat model. Choroidal neovascularization (CNV) was induced by diode laser photocoagulation to the right eye of twelve Brown Norway rats. Bevacizumab (25 mg/ml) was injected intravitreally after 3 days. Immediately after bevacizumab injection, and 3, 6, 24 and 48 h later, animals were euthanized for immunofluorescence staining. Donkey anti-human IgG labeled with Alexa Fluor® 488 was used for bevacizumab immunoreactivity detection. Anti-CD31 antibody was used as a marker for Schlemm's canal endothelial cells. Untreated eyes were used as negative controls. The intensity of the immunostaining was analyzed qualitatively. Bevacizumab immunoreactivity was found in the aqueous outflow channels including the trabecular meshwork and Schlemm's canal immediately after injection, and declined incrementally within the following hours. Forty-eight hours after the injection, no bevacizumab staining was detected in the aqueous outflow channel structures. Our manuscript demonstrates the presence of bevacizumab in the trabecular meshwork and Schlemm's canal structures after intravitreal injection in a CNV induced rat model. Bevacizumab molecules passed through the aqueous outflow channels within 48 h after intravitreal bevacizumab injection.

Morphological Abnormalities of Schlemm's Canal in Primary Open-Angle Glaucoma From the Aspect of Aging.

To evaluate morphological abnormalities of Schlemm's canal (SC) among primary open-angle glaucoma (POAG) patients with a family history of POAG (group A), those without a family history of POAG (group B), and patients with normal-tension glaucoma (group C) from the aspect of aging. A total of 160 trabeculectomy specimens from 133 POAG patients were processed for light microscopy using immunohistochemical staining of thrombomodulin and transmission electron microscopy. The following parameters were statistically evaluated: SC length, the percentage of the thrombomodulin-negative area (PTNA) of SC, and the inner-wall SC endothelial cell density (SC-ECD/100 μm). No significant differences in age were observed among the three groups (group A: 56.71 ± 14.83; group B: 58.13 ± 18.13; group C: 56.61 ± 9.78). Length of SC in the group A patients (198.70 ± 81.65 μm, n = 70 eyes) was significantly shorter than in group B (250.30 ± 70.83 μm, n = 67 eyes), and group C (277.70 ± 65.52 μm, n = 23 eyes) patients. A positive correlation of patient age and SC length was observed in group B (r = 0.45, P = 0.0013), but SC length in group A tended to decrease with aging (r = -0.22, P = 0.07). No significant difference in SC was found between group A and B patients before age 50 years (P = 0.30). Correlations between patient age and increase of PTNA (r = 0.38, P = 0.0013) and patient age and decrease of SC-ECD (r = -0.53, P = 3.95 × 10(-6)) were observed only in the group B cases. Our findings suggest that SC in group A may easily collapse during middle age, while SC in group B remains open and SC endothelial cells drop out at around middle age.