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1.
Invest Ophthalmol Vis Sci ; 65(6): 4, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38833261

RESUMO

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.


Assuntos
Humor Aquoso , Homeostase , Pressão Intraocular , Malha Trabecular , Pressão Intraocular/fisiologia , Homeostase/fisiologia , Humor Aquoso/fisiologia , Humor Aquoso/metabolismo , Humanos , Malha Trabecular/metabolismo , Malha Trabecular/fisiologia , Óxido Nítrico/metabolismo , Modelos Teóricos
2.
bioRxiv ; 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-37886472

RESUMO

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.

3.
Am J Physiol Cell Physiol ; 326(2): C513-C528, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38105758

RESUMO

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.


Assuntos
Glaucoma , Proteínas de Sinalização YAP , Animais , Humanos , Camundongos , Mecanotransdução Celular , Canal de Schlemm , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional
4.
bioRxiv ; 2023 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-37781615

RESUMO

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.

5.
Invest Ophthalmol Vis Sci ; 64(7): 36, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37358489

RESUMO

Purpose: The aim of this study was to test the hypothesis that nitric oxide (NO) mediates a pressure-dependent, negative feedback loop that maintains conventional outflow homeostasis and thus IOP. If true, holding pressure during ocular perfusions will result in uncontrolled production of NO, hyper-relaxation of the trabecular meshwork, and washout. Methods: Paired porcine eyes were perfused at constant pressure of 15 mm Hg. After 1 hour acclimatization, one eye was exchanged with N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME) (50 µm) and the contralateral eye with DBG, and perfused for 3 hours. In a separate group, one eye was exchanged with DETA-NO (100 nM) and the other with DBG and perfused for 30 minutes. Changes in conventional outflow tissue function and morphology were monitored. Results: Control eyes exhibited a washout rate of 15% (P = 0.0026), whereas eyes perfused with L-NAME showed a 10% decrease in outflow facility from baseline over 3 hours (P < 0.01); with nitrite levels in effluent positively correlating with time and facility. Compared with L-NAME-treated eyes, significant morphological changes in control eyes included increased distal vessel size, number of giant vacuoles, and juxtacanalicular tissue separation from the angular aqueous plexi (P < 0.05). For 30-minute perfusions, control eyes showed a washout rate of 11% (P = 0.075), whereas DETA-NO-treated eyes showed an increased washout rate of 33% from baseline (P < 0.005). Compared with control eyes, significant morphological changes in DETA-NO-treated eyes also included increased distal vessel size, number of giant vacuoles and juxtacanalicular tissue separation (P < 0.05). Conclusions: Uncontrolled NO production is responsible for washout during perfusions of nonhuman eyes where pressure is clamped.


Assuntos
Humor Aquoso , Pressão Intraocular , Óxido Nítrico , Perfusão , Animais , Constrição , NG-Nitroarginina Metil Éster/farmacologia , Suínos , Malha Trabecular
6.
Invest Ophthalmol Vis Sci ; 63(2): 12, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35129590

RESUMO

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.


Assuntos
Humor Aquoso/fisiologia , Consenso , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Hipertensão Ocular/metabolismo , Malha Trabecular/metabolismo , Animais , Modelos Animais de Doenças , Glaucoma/fisiopatologia , Camundongos , Hipertensão Ocular/fisiopatologia , Tonometria Ocular
7.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502356

RESUMO

Previous studies have shown that glaucomatous Schlemm's canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm's canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes. RNA and protein quantification of both fibrotic and endothelial cell markers was carried out on both healthy and gSCECs. Functional assays to assess cell density, size, migration, proliferation, and mitochondrial function of these cells were also carried out. Indeed, we found that gSCECs deviate from typical endothelial cell characteristics and exhibit a more fibrotic phenotype. For example, gSCECs expressed significantly higher protein levels of the fibrotic markers α-SMA, collagen I-α1, and fibronectin, as well as significantly increased protein expression of TGFß-2, the main driver of fibrosis, compared to healthy SCECs. Interestingly, we observed a significant increase in protein expression of endothelial marker VE-cadherin in gSCECs, compared to healthy SCECs. gSCECs also appeared to be significantly larger, and surprisingly proliferate and migrate at a significantly higher rate, as well as showing significantly reduced mitochondrial activity, compared to healthy SCECs.


Assuntos
Fibrose/fisiopatologia , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Antígenos CD/metabolismo , Humor Aquoso/metabolismo , Caderinas/metabolismo , Contagem de Células , Movimento Celular , Proliferação de Células , Células Endoteliais/metabolismo , Endotélio , Matriz Extracelular , Olho/metabolismo , Humanos , Mitocôndrias , Porosidade , Cultura Primária de Células , Esclera , Malha Trabecular , Fator de Crescimento Transformador beta2/metabolismo
8.
Mol Ther Methods Clin Dev ; 20: 86-94, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33376757

RESUMO

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm's canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

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