In vivo quantification of anterior and posterior chamber volumes in mice: implications for aqueous humor dynamics.

Purpose: Aqueous humor inflow rate, a key parameter influencing aqueous humor dynamics, is typically measured by fluorophotometery. Analyzing fluorophotometric data depends, inter alia, on the volume of aqueous humor in the anterior, but not the posterior, chamber. Previous fluorophotometric studies of aqueous inflow rate in mice have assumed the ratio of anterior:posterior volumes in mice to be similar to those in humans. Our goal was to measure anterior and posterior chamber volumes in mice to facilitate better estimates of aqueous inflow rates. Methods: We used standard near-infrared optical coherence tomography (OCT) and robotic visible-light OCT (vis-OCT) to visualize, reconstruct and quantify the volumes of the anterior and posterior chambers of the mouse eye in vivo. We used histology and micro-CT scans to validate relevant landmarks from ex vivo tissues to facilitate in vivo measurement. Results: Posterior chamber volume is 1.1 times the anterior chamber volume in BALB/cAnNCrl mice, i.e. the anterior chamber constitutes about 47% of the total aqueous humor volume, which is very dissimilar to the situation in humans. Anterior chamber volumes in 2-month-old BALB/cAnNCrl and 7-month-old C57BL6/J mice were 1.55 ± 0.36 µL (n=10) and 2.41 ± 0.29 µL (n=8), respectively. This implies that previous studies likely over-estimated aqueous inflow rate by approximately two-fold. Conclusions: It is necessary to reassess previously reported estimates of aqueous inflow rates, and thus aqueous humor dynamics in the mouse. For example, we now estimate that only 0-15% of aqueous humor drains via the pressure-independent (unconventional) route, similar to that seen in humans and monkeys.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

The Factors Affecting the Stability of IOP Homeostasis.

Purpose: 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). Methods: 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. Results: 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. Conclusions: 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.

Magnetically Steered Cell Therapy For Functional Restoration Of Intraocular Pressure Control In Open-Angle Glaucoma.

Trabecular meshwork (TM) cell therapy has been proposed as a next-generation treatment for elevated intraocular pressure (IOP) in glaucoma, the most common cause of irreversible blindness. Using a magnetic cell steering technique with excellent efficiency and tissue-specific targeting, we delivered two types of cells into a mouse model of glaucoma: either human adipose-derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells). We observed a 4.5 [3.1, 6.0] mmHg or 27% reduction in intraocular pressure (IOP) for nine months after a single dose of only 1500 magnetically-steered hAMSCs, associated with restoration of function to the conventional outflow pathway, as judged by increased outflow facility and TM cellularity. iPSC-TM cells were also effective, but less so, showing only a 1.9 [0.4, 3.3] mmHg or 13% IOP reduction and increased risk of tumorigenicity. In both cases, injected cells remained detectable in the iridocorneal angle three weeks post-transplantation. Based on the locations of the delivered cells, the mechanism of IOP lowering is most likely paracrine signaling. We conclude that magnetically-steered hAMSC cell therapy has potential for long-term treatment of ocular hypertension in glaucoma. One Sentence Summary: A novel magnetic cell therapy provided effective intraocular pressure control in a mouse model of glaucoma, motivating future translational studies.

Aging and intraocular pressure homeostasis in mice.

Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.

ANGPTL7 and Its Role in IOP and Glaucoma.

Purpose: Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics. Methods: IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured. Results: IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies. Conclusions: Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.

A Histomorphometric and Computational Investigation of the Stabilizing Role of Pectinate Ligaments in the Aqueous Outflow Pathway.

Murine models are commonly used to study glaucoma, the leading cause of irreversible blindness. Glaucoma is associated with elevated intra-ocular pressure (IOP), which is regulated by the tissues of the aqueous outflow pathway. In particular, pectinate ligaments (PLs) connect the iris and trabecular meshwork (TM) at the anterior chamber angle, with an unknown role in maintenance of the biomechanical stability of the aqueous outflow pathway, thus motivating this study. We conducted histomorphometric analysis and optical coherence tomography-based finite element (FE) modeling on three cohorts of C57BL/6 mice: "young" (2-6 months), "middle-aged" (11-16 months), and "elderly" (25-32 months). We evaluated the age-specific morphology of the outflow pathway tissues. Further, because of the known pressure-dependent Schlemm's canal (SC) narrowing, we assessed the dependence of the SC lumen area on varying IOPs in age-specific FE models over a physiological range of TM/PL stiffness values. We found age-dependent changes in morphology of outflow tissues; notably, the PLs were more developed in older mice compared to younger ones. In addition, FE modeling demonstrated that murine SC patency is highly dependent on the presence of PLs and that increased IOP caused SC collapse only with sufficiently low TM/PL stiffness values. Moreover, the elderly model showed more susceptibility to SC collapse compared to the younger models. In conclusion, our study elucidated the previously unexplored role of PLs in the aqueous outflow pathway, indicating their function in supporting TM and SC under elevated IOP.

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.

Targeting YAP/TAZ mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology.

Pathological alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared with that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell mechanosignaling via YAP and transcriptional coactivator with PDZ-binding motif (TAZ) in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP/TAZ activity in primary human SC cells, and whether disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP/TAZ activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP/TAZ mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Finally, we found that perfusion of the clinically used, small molecule YAP/TAZ inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP/TAZ mechanosignaling in SC cell dysfunction and suggest that YAP/TAZ inhibition has therapeutic value for treating ocular hypertension in glaucoma.NEW & NOTEWORTHY Pathologically altered biomechanical properties of the Schlemm's canal (SC) inner wall microenvironment were recently validated as the cause for increased outflow resistance in ocular hypertensive glaucoma. However, the involvement of specific mechanotransduction pathways in these disease processes is largely unclear. Here, we demonstrate that Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are central regulators of glaucoma-like SC cell dysfunction in response to extracellular matrix stiffening and that targeted disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and enhances outflow function.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD).

Aging and intraocular pressure homeostasis in mice.

Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.

Estrogen dysregulation, intraocular pressure, and glaucoma risk.

Characterized by optic nerve atrophy due to retinal ganglion cell (RGC) death, glaucoma is the leading cause of irreversible blindness worldwide. Of the major risk factors for glaucoma (age, ocular hypertension, and genetics), only elevated intraocular pressure (IOP) is modifiable, which is largely regulated by aqueous humor outflow through the trabecular meshwork. Glucocorticoids such as dexamethasone have long been known to elevate IOP and lead to glaucoma. However, several recent studies have reported that steroid hormone estrogen levels inversely correlate with glaucoma risk, and that variants in estrogen signaling genes have been associated with glaucoma. As a result, estrogen dysregulation may contribute to glaucoma pathogenesis, and estrogen signaling may protect against glaucoma. The mechanism for estrogen-related protection against glaucoma is not completely understood but likely involves both regulation of IOP homeostasis and neuroprotection of RGCs. Based upon its known activities, estrogen signaling may promote IOP homeostasis by affecting extracellular matrix turnover, focal adhesion assembly, actin stress fiber formation, mechanosensation, and nitric oxide production. In addition, estrogen receptors in the RGCs may mediate neuroprotective functions. As a result, the estrogen signaling pathway may offer a therapeutic target for both IOP control and neuroprotection. This review examines the evidence for a relationship between estrogen and IOP and explores the possible mechanisms by which estrogen maintains IOP homeostasis.

Targeting YAP mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology.

Pathologic alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared to that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell YAP mechanosignaling in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP activity in primary human SC cells, and whether disruption of YAP mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Lastly, we found that perfusion of the clinically-used, small molecule YAP inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP mechanosignaling in SC cell dysfunction and suggest that YAP inhibition has therapeutic value for treating ocular hypertension in glaucoma.

Genetic background determines severity of Loxl1-mediated systemic and ocular elastosis in mice.

Pseudoexfoliation syndrome (PEX) is a systemic, age-related disorder characterized by elastosis and extracellular matrix deposits. Its most significant ocular manifestation is an aggressive form of glaucoma associated with variants in the gene encoding lysyl oxidase-like 1 (LOXL1). Depending upon the population, variants in LOXL1 can impart risk or protection for PEX, suggesting the importance of genetic context. As LOXL1 protein levels are lower and the degree of elastosis is higher in people with PEX, we studied Loxl1-deficient mice on three different genetic backgrounds: C57BL/6 (BL/6), 129S×C57BL/6 (50/50) and 129S. Early onset and high prevalence of spontaneous pelvic organ prolapse in BL/6 Loxl1-/- mice necessitated the study of mice that were <2 months old. Similar to pelvic organ prolapse, most elastosis endpoints were the most severe in BL/6 Loxl1-/- mice, including skin laxity, pulmonary tropoelastin accumulation, expansion of Schlemm's canal and dilation of intrascleral veins. Interestingly, intraocular pressure was elevated in 50/50 Loxl1-/- mice, depressed in BL/6 Loxl1-/- mice and unchanged in 129S Loxl1-/- mice compared to that of control littermates. Overall, the 129S background was protective against most elastosis phenotypes studied. Thus, repair of elastin-containing tissues is impacted by the abundance of LOXL1 and genetic context in young animals.

Development and testing of a metabolic chamber for effluent collection during whole eye perfusions.

Ocular hypertension is caused by dysregulated outflow resistance regulation by the conventional outflow (CO) pathway. The physiology of the CO pathway can be directly studied during ex vivo ocular perfusions. In addition to measuring outflow resistance generation by the CO tissues, perfusion media that is conditioned by CO pathway cells can be collected upon exiting the eye as effluent. Thus, contents of effluent include factors contributed by upstream cells that report on the (dys)functionality of the outflow tissues. Two methods have been used in the past to monitor effluent contents from perfused eyes, each with their limitations. To overcome these limitations, we designed and printed a metabolic chamber to accommodate eyes of different sizes during perfusions. To test this new chamber, human eyes were perfused for 4 h at constant flow rate of 2.5 µl/min, while pressure was continuously monitored and effluent was collected every hour. Facility was 0.28 ± 0.16 µl/min/mmHg for OD eyes and 0.33 ± 0.11 µl/min/mmHg for OS eyes (n = 3). Effluent samples were protein rich, with protein concentration ranging from 2700 to 10,000 µg/ml for all eyes and timepoints (N = 3). Effluent samples expressed proteins that were actively secreted by the TM and easily detectible including MYOC and MMP2. Taken together, our model provides a reliable method to collect effluent from ex vivo human eyes, while maintaining whole globe integrity.

Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells.

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.

Shear stress induces autophagy in Schlemm's canal cells via primary cilia-mediated SMAD2/3 signaling pathway.

The Schlemm's canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibitor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.

Differential roles of FOXC2 in the trabecular meshwork and Schlemm's canal in glaucomatous pathology.

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.

Greater Outflow Facility Increase After Targeted Trabecular Bypass in Angiographically Determined Low-low Regions.

PURPOSE: To investigate the impact of trabecular bypass surgery targeted to angiographically determined high- vs. low-aqueous humor outflow areas on outflow facility (C) and intraocular pressure (IOP). DESIGN: Ex vivo comparative study. SUBJECTS: Postmortem ex vivo porcine and human eyes. METHODS: Porcine (n = 14) and human (n = 13) whole globes were acquired. In both species, anterior segments were dissected, mounted onto a perfusion chamber, and perfused using Dulbecco's phosphate buffered solution containing glucose in a constant flow paradigm to achieve a stable baseline. Fluorescein was perfused into the anterior chamber and used to identify baseline segmental high- and low-flow regions of the conventional outflow pathways. The anterior segments were divided into 2 groups, and a 5 mm needle goniotomy was performed in either a high- or low-flow area. Subsequently, C and IOP were quantitatively reassessed and compared between surgery in baseline "high-flow" and "low-flow" region eyes followed by indocyanine green angiography. MAIN OUTCOME MEASURES: Outflow facility. RESULTS: In all eyes, high- and low-flow segments could be identified. Performing a 5-mm goniotomy increased outflow facility to a variable extent depending on baseline flow status. In the porcine high-flow group, C increased from 0.31 ± 0.09 to 0.39 ± 0.09 µL/mmHg/min (P = 0.12). In the porcine low-flow group, C increased from 0.29 ± 0.03 to 0.56 ± 0.10 µL/mmHg/min (P < 0.001). In the human high-flow group, C increased from 0.38 ± 0.20 to 0.41 ± 0.20 µL/mmHg/min (P = 0.02). In the human low-flow group, C increased from 0.25 ± 0.11 to 0.32 ± 0.11 µL/mmHg/min (<0.001). There was statistically significant greater increase in C for eyes where surgery was targeted to baseline low-flow regions in both porcine (0.07 ± 0.09 vs. 0.27 ± 0.13, P = 0.007 µL/mmHg/min, high vs low flow) and human eyes (0.03 ± 0.03 vs. 0.07 ± 0.02, P = 0.03 µL/mmHg/min, high vs. low flow). CONCLUSIONS: Targeting surgery to low-flow areas of the trabecular meshwork yields higher overall facility increase and IOP reduction compared to surgery in high-flow areas. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.