A Comparative Genome-wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells

The genome-wide gene expression analysis of primary human trabecular meshwork (HTM) cells with known glucocorticoid (GC) responsiveness was not reported earlier. Therefore, the purpose of this study was to investigate genes and pathways involved in the GC responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. A perfusion cultured human anterior segment ex vivo model was utilized to identify the induction of GC-induced ocular hypertension in one eye of a paired eyes after dexamethasone treatment based on the maximum intraocular pressure response and in the contralateral eye, HTM cells were isolated to classify GC-responder and non-responder cells. Some previously reported and unique genes and their associated pathways were identified in HTM cells in response to dexamethasone treatment versus vehicle control and more significantly in GC-responder and non-responder cells. This study will open up the possibility of identifying suitable molecular targets which have the potential to treat GC-induced ocular hypertension/glaucoma.

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A Porcine Ex Vivo Model of Pigmentary Glaucoma

10.20944/preprints201803.0219.v1 ◽

2018 ◽

Author(s):

Yalong Dang ◽

Susannah Waxman ◽

Chao Wang ◽

Ralitsa T. Loewen ◽

Ming Sun ◽

...

Keyword(s):

Trabecular Meshwork ◽

Ex Vivo ◽

Anterior Segment ◽

Pigment Dispersion ◽

Pigmentary Glaucoma ◽

Ex Vivo Model ◽

Trabecular Meshwork Cells ◽

Gene Expression Microarrays ◽

Expression Microarrays ◽

And Behavior

Pigment dispersion can lead to pigmentary glaucoma, a poorly understood condition of younger myopic eyes with fluctuating high intraocular pressure. It has been difficult to investigate its pathogenesis without a model similar to human eyes in size and behavior. Here we present a porcine ex vivo model that recreates several features of pigmentary glaucoma, including intraocular hypertension, accumulation of pigment in the trabecular meshwork, and declining phagocytosis. We found that trabecular meshwork cells regulate outflow, form actin stress fibers, and have a decreased phagocytic activity. Gene expression microarrays and a pathway analysis of TM monolayers as well as ex vivo anterior segment perfusion cultures indicated that RhoA plays a central role in regulating the cytoskeleton, motility, and phagocytosis in the trabecular meshwork, providing new insights and targets to investigate in pigmentary glaucoma.

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Replacement of the Trabecular Meshwork Cells—A Way Ahead in IOP Control?

Biomolecules ◽

10.3390/biom11091371 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1371

Author(s):

Xiaochen Fan ◽

Emine K. Bilir ◽

Olivia A. Kingston ◽

Rachel A. Oldershaw ◽

Victoria R. Kearns ◽

...

Keyword(s):

Stem Cells ◽

Trabecular Meshwork ◽

Vision Loss ◽

Ex Vivo ◽

Open Angle Glaucoma ◽

Model Systems ◽

Ex Vivo Model ◽

Cell Replacement ◽

Trabecular Meshwork Cells ◽

Stem Cell Source

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM’s structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

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Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

F1000Research ◽

10.12688/f1000research.53572.1 ◽

2021 ◽

Vol 10 ◽

pp. 525

Author(s):

Raoul Verma-Fuehring ◽

Mohamad Dakroub ◽

Alicja Strzalkowska ◽

Piotr Strzalkowski ◽

Hong Han ◽

...

Keyword(s):

Trabecular Meshwork ◽

Structural Changes ◽

Ex Vivo ◽

Anterior Segment ◽

Large Change ◽

Invasive Technique ◽

Freeze Thaw ◽

Before And After ◽

Human Eyes ◽

Porcine Eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

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Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

10.7287/peerj.preprints.2736 ◽

2017 ◽

Author(s):

Yalong Dang ◽

Susannah Waxman ◽

Chao Wang ◽

Adrianna Jensen ◽

Ralitsa T Loewen ◽

...

Keyword(s):

Extracellular Matrix ◽

Trabecular Meshwork ◽

Degrees Of Freedom ◽

Ex Vivo ◽

Fluorescent Microscopy ◽

Anterior Segment ◽

Perfusion Culture ◽

Suicide Gene ◽

Control Group ◽

Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

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Transduction pattern of adeno‐associated viruses in the trabecular meshwork and anterior‐segment structures in a rat model of ocular hypertension

Acta Ophthalmologica ◽

10.1111/j.1755-3768.2019.5490 ◽

2019 ◽

Vol 97 (S263) ◽

Author(s):

Si Hyung Lee ◽

Sang Earn Woo ◽

Tae Kwann Park

Keyword(s):

Ocular Hypertension ◽

Rat Model ◽

Trabecular Meshwork ◽

Anterior Segment

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Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021942118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2021942118

Author(s):

Myoung Sup Shim ◽

April Nettesheim ◽

Angela Dixon ◽

Paloma B. Liton

Keyword(s):

Trabecular Meshwork ◽

Primary Cilia ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Mechanical Stretch ◽

Cell Types ◽

Elevated Pressure ◽

Compensatory Response ◽

Protein Levels ◽

Trabecular Meshwork Cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

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Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PeerJ ◽

10.7717/peerj.3629 ◽

2017 ◽

Vol 5 ◽

pp. e3629 ◽

Cited By ~ 12

Author(s):

Yalong Dang ◽

Susannah Waxman ◽

Chao Wang ◽

Adrianna Jensen ◽

Ralitsa T. Loewen ◽

...

Keyword(s):

Extracellular Matrix ◽

Trabecular Meshwork ◽

Degrees Of Freedom ◽

Culture Media ◽

Ex Vivo ◽

Fluorescent Microscopy ◽

Anterior Segment ◽

Perfusion Culture ◽

Control Group ◽

Freeze Thaw

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.

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A porcine ex vivo model of pigmentary glaucoma

10.1101/118448 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yalong Dang ◽

Susannah Waxman ◽

Chao Wang ◽

Ralista T. Loewen ◽

Ming Sun ◽

...

Keyword(s):

Trabecular Meshwork ◽

Ex Vivo ◽

Pigment Dispersion ◽

Pigmentary Glaucoma ◽

Ex Vivo Model ◽

Pigment Dispersion Syndrome ◽

And Behavior ◽

Human Eyes ◽

Pigment Accumulation

AbstractPigment dispersion syndrome can lead to pigmentary glaucoma (PG), a poorly understood condition of younger, myopic eyes with fluctuating, high intraocular pressure (IOP). The absence of a model similar in size and behavior to human eyes has made it difficult to investigate its pathogenesis. Here, we present a porcine ex vivo model that recreates the features of PG including intraocular hypertension, pigment accumulation in the trabecular meshwork and relative failure of phagocytosis. Inin vitromonolayer cultures as well as inex vivoeye perfusion cultures, we found that the trabecular meshwork (TM) cells that regulate outflow, form actin stress fibers and have a decreased phagocytosis. Gene expression microarray and pathway analysis indicated key roles of RhoA in regulating the TM cytoskeleton, motility, and phagocytosis thereby providing new targets for PG therapy.

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Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells

Scientific Reports ◽

10.1038/s41598-021-98766-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Megumi Watanabe ◽

Yosuke Ida ◽

Hiroshi Ohguro ◽

Chiaki Ota ◽

Fumihito Hikage

Keyword(s):

Trabecular Meshwork ◽

Ex Vivo ◽

Open Angle Glaucoma ◽

Smooth Muscle Actin ◽

Three Dimensional ◽

Tissue Inhibitor Of Metalloproteinase ◽

Three Dimension ◽

Trabecular Meshwork Cells ◽

2D And 3D

AbstractTo establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

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