A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-ß2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-ß2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

The Role and Mechanism of Nicotinamide Riboside in Oxidative Damage and a Fibrosis Model of Trabecular Meshwork Cells.

Purpose: To investigate the potential effects and mechanism of nicotinamide riboside (NR) on the oxidative stress and fibrosis model of human trabecular meshwork (HTM) cell line cells. Methods: HTM cells were pretreated with NR, followed by the induction of oxidative injury and fibrosis by hydrogen peroxide (H2O2) and TGF-ß2, respectively. Cell viability was tested using Hoechst staining and MTT assays, cell proliferation was assessed by EdU assay, and cell apoptosis was detected by flow cytometry and western blotting. DCFH-DA and DHE probes were used to measure the level of reactive oxygen species (ROS), and MitoTracker staining was used to measure the mitochondrial membrane potential (MMP). Fibrotic responses, including cell migration and deposition of extracellular matrix (ECM) proteins, were detected via Transwell assays, qRT-PCR, and immunoblotting. Results: NR pretreatment improved the viability, proliferation, and MMP of H2O2-treated HTM cells. Compared to cells treated solely with H2O2, HTM cells treated with both NR and H2O2, exhibited a reduced rate of apoptosis and generation of ROS. Compared with H2O2 pretreatment, NR pretreatment upregulated expression of the JAK2/Stat3 pathway but inhibited mitogen-activated protein kinase (MAPK) pathway expression. Moreover, 10-ng/mL TGF-ß2 promoted cell proliferation and migration, which were inhibited by NR pretreatment. Both qRT-PCR and immunoblotting showed that NR inhibited the expression of fibronectin in a TGF-ß2-induced fibrosis model. Conclusions: NR has a protective effect on oxidative stress and fibrosis in HTM cells, which may be related to the JAK2/Stat3 pathway and MAPK pathway. Translational Relevance: Our research provides the ongoing data for potential therapy of NAD+ precursors in glaucoma.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Genistein stimulates the viability and prevents myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-ß.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma leading to blindness. The search for ways to prevent/treat this entity is one of the main challenges of today's ophthalmology. One of such solution seems to be biologically active substances of natural origin, such as genistein (GEN), which can affect the function of isolated trabecular meshwork by the inhibition of protein tyrosine kinase. However, the role of GEN in viability as well as myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-ß is unknown. Using human trabecular meshwork cells (HTMCs) we investigated the effect of genistein on cell viability and myofibroblastic transformation stimulated by TGF-ß1 and TGF-ß2. Using Real-Time PCR, western blot and immunofluorescence we determined the effect on the expression changes of αSMA, TIMP1, collagen 1 and 3 at mRNA and protein level. We found that genistein increases the viability of HTMCs (1, 2, 3 µg/ml; P < 0.05 and 4, 5, 10, 15, 20 µg/ml; P < 0.01). Moreover, we found that addition of 10, 15 and 20 µg/ml is able to prevent myofibroblastic transformation of HTMCs by decreasing αSMA, TIMP1, collagen 1 and 3 mRNA and protein expression (P < 0.01). Based on the obtained results, we can conclude that genistein is a potential factor that can prevent the myofibroblastic transformation of HTMCs accompanying glaucoma. Describing GEN influence on myofibroblastic transformation processes in HTMC allows us to conclude that it can be considered a potential therapeutic agent or a substance supporting treatment in patients with 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.

Inhibition of TGF-ß2-Induced Trabecular Meshwork Fibrosis by Pirfenidone.

Purpose: Trabecular meshwork (TM) fibrosis is a crucial pathophysiological process in the development of primary open-angle glaucoma. Pirfenidone (PFD) is a new, broad-spectrum antifibrotic agent approved for the treatment of idiopathic pulmonary fibrosis. This study investigated the inhibitory effect of PFD on TM fibrosis and evaluated its efficacy in lowering intraocular pressure (IOP). Methods: Human TM cells were isolated, cultured, and characterized. Cell Counting Kit-8 was used to evaluate the proliferation and toxicity of different concentrations of PFD on normal or fibrotic TM cells. TM cells were treated with transforming growth factor beta-2 (TGF-ß2) in the absence or presence of PFD. Western blotting and immunofluorescence analyses were used to analyze changes in the TM cell cytoskeleton and extracellular matrix (ECM) proteins, including alpha-smooth muscle actin (α-SMA), F-actin, collagen IV (COL IV), and fibronectin (FN). An ocular hypertension (OHT) mouse model was induced with Ad-TGF-ß2C226/228S and then treated with PFD or latanoprost (LT) eye drops to confirm the efficacy of PFD in lowering IOP. Results: PFD inhibited the proliferation of fibrotic TM cells in a dose-dependent manner and inhibited TGF-ß2-induced overexpression of α-SMA, COL IV, and FN in TM cells. PFD stabilized F-actin. In vivo, PFD eye drops reduced the IOP of the OHT models and showed no significant difference compared with LT eye drops. Conclusions: PFD inhibited TGF-ß2-induced TM cell fibrosis by rearranging the disordered cytoskeleton and decreasing ECM deposition, thereby enhancing the aqueous outflow from the TM outflow pathway and lowering IOP, which provides a potential new approach to treating glaucoma. Translational Relevance: Our work with pirfenidone provides a new approach to treat glaucoma.

Viral Vector-Induced Ocular Hypertension in Mice.

Viral transduction of the mouse trabecular meshwork using a variety of transgenes associated with glaucoma generates an inducible and reproducible method for generating ocular hypertension due to increased aqueous humor outflow resistance of the conventional outflow pathway. Both adenovirus serotype 5 (Ad5) and lentiviruses have selective tropism for the mouse trabecular meshwork with intraocular injections. Accurate intraocular pressures are easily measured using a rebound tonometer, and aqueous humor outflow facilities can be measured in anesthetized live mice.

High VEGF Concentrations Accelerate Human Trabecular Meshwork Fibrosis in a TAZ-Dependent Manner.

We aimed to investigate the effects of different concentrations of vascular endothelial growth factor (VEGF) on the extracellular matrix (ECM) and fibrotic proteins in human trabecular meshwork (TM) cells. We also explored how the Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) signaling pathway modulates VEGF-induced fibrosis. We determined cross-linked actin network (CLAN) formation using TM cells. Changes in fibrotic and ECM protein expression were determined. High VEGF concentrations (10 and 30 ng/mL) increased TAZ and decreased p-TAZ/TAZ expression in TM cells. Western blotting and real-time PCR revealed no YAP expression changes. Fibrotic and ECM protein expression decreased at low VEGF concentrations (1 and 10 ρg/mL) and significantly increased at high VEGF concentrations (10 and 30 ng/mL). CLAN formation increased in TM cells treated with high VEGF concentrations. Moreover, TAZ inhibition by verteporfin (1 µM) rescued TM cells from high-VEGF-concentration-induced fibrosis. Low VEGF concentrations reduced fibrotic changes, whereas high VEGF concentrations accelerated fibrosis and CLAN formations in TM cells in a TAZ-dependent manner. These findings reflect the dose-dependent influences of VEGF on TM cells. Moreover, TAZ inhibition might be a therapeutic target for VEGF-induced TM dysfunction.

Activation of Sonic Hedgehog Signaling Pathway Regulates Human Trabecular Meshwork Cell Function.

Purpose: To investigate the effects of Sonic hedgehog (Shh) signaling on primary human trabecular meshwork (HTM) cells. Methods: Primary HTM cells were isolated from healthy donors and cultured. Recombinant Shh (rShh) protein and cyclopamine were used to activate and inhibit the Shh signaling pathway, respectively. A cell viability assay was performed to assess the effects of rShh on the activity of primary HTM cells. Functional assessment of cell adhesion and phagocytosis was also performed. The proportion of apoptotic cells was examined using flow cytometry. Fibronectin (FN) and transforming growth factor beta2 (TGF-ß2) protein were detected to assess the influence of rShh on the metabolism of the extracellular matrix (ECM). Real-time polymerase chain reaction (RT-PCR) and western blot analyses were used to examine mRNA and protein expression of Shh signaling pathway-associated factors GLI Family Zinc Finger 1 (GLI1) and Suppressor of Fused (SUFU). Results: rShh significantly enhanced primary HTM cell viability at a concentration of 0.5 µg/mL. rShh increased the adhesion and phagocytic abilities of primary HTM cells, and decreased cell apoptosis. FN and TGF-ß2 protein expression increased in primary HTM cells treated with rShh. rShh upregulated the transcriptional activity and protein levels of GLI1, and downregulated those of SUFU. Correspondingly, the rShh-induced GLI1 upexpression was partially blocked by pretreatment with the Shh pathway inhibitor cyclopamine at a concentration of 10 µM. Conclusions: Activation of Shh signaling can regulate the function of primary HTM cells through GLI1. Regulation of Shh signaling may be a potential target for attenuating cell damage in glaucoma.

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.

Microchip encapsulation and microRNA-7 overexpression of trabecular meshwork mesenchymal stem/stromal cells improve motor function after spinal cord injury.

Manipulation of stem cells and microencapsulation through microfluidic chips has shown more promising results in treating complex conditions, such as spinal cord injury (SCI), than traditional treatments. This study aimed to investigate the potency of neural differentiation and its therapeutic role in SCI animal model of trabecular meshwork mesenchymal stem/stromal cells (TMMSCs) via miR-7 overexpression and microchip-encapsulated. TMMSCs are transduced with miR-7 via a lentiviral vector (TMMSCs-miR-7[+]) and encapsulated in alginate-reduced graphene oxide (alginate-rGO) hydrogel via a microfluidic chip. Neuronal differentiation of transduced cells in hydrogel (3D) and tissue cultures plate (2D) was assessed by expressing specific mRNAs and proteins. Further evaluation is being carried out through 3D and 2D TMMSCs-miR-7(+ and -) transplantation into the rat contusion SCI model. TMMSCs-miR-7(+) encapsulated in the microfluidic chip (miR-7-3D) increased nestin, ß-tubulin III, and MAP-2 expression compared with 2D culture. Moreover, miR-7-3D could improve locomotor behavior in contusion SCI rats, decrease cavity size, and increase myelination. Our results revealed that miR-7 and alginate-rGO hydrogel were involved in the neuronal differentiation of TMMSCs in a time-dependent manner. In addition, the microfluidic-encapsulated miR-7 overexpression TMMSCs represented a better survival and integration of the transplanted cells and the repair of SCI. Collectively, the combination of miR-7 overexpression and encapsulation of TMMSCs in hydrogels may represent a promising new treatment for SCI.

Long non-coding RNA SNHG11 regulates the Wnt/ß-catenin signaling pathway through rho/ROCK in trabecular meshwork cells.

Trabecular meshwork (TM) cell dysfunction is the leading cause of elevated intraocular pressure (IOP) and glaucoma. The long non-coding RNA (lncRNA) small nucleolar RNA host gene 11 (SNHG11) is associated with cell proliferation and apoptosis, but its biological functions and role in glaucoma pathogenesis remain unclear. In the present study, we investigated the role of SNHG11 in TM cells using immortalized human TM and glaucomatous human TM (GTM3 ) cells and an acute ocular hypertension mouse model. SNHG11 expression was depleted using siRNA targeting SNHG11. Transwell assays, quantitative real-time PCR analysis (qRT-PCR), western blotting, and CCK-8 assay were used to evaluate cell migration, apoptosis, autophagy, and proliferation. Wnt/ß-catenin pathway activity was inferred from qRT-PCR, western blotting, immunofluorescence, and luciferase reporter and TOPFlash reporter assays. The expression of Rho kinases (ROCKs) was detected using qRT-PCR and western blotting. SNHG11 was downregulated in GTM3 cells and mice with acute ocular hypertension. In TM cells, SNHG11 knockdown inhibited cell proliferation and migration, activated autophagy, and apoptosis, repressing the Wnt/ß-catenin signaling pathway, and activated Rho/ROCK. Wnt/ß-catenin signaling pathway activity increased in TM cells treated with ROCK inhibitor. SNHG11 regulated Wnt/ß-catenin signaling through Rho/ROCK by increasing GSK-3ß expression and ß-catenin phosphorylation at Ser33/37/Thr41 while decreasing ß-catenin phosphorylation at Ser675. We demonstrate that the lncRNA SNHG11 regulates Wnt/ß-catenin signaling through Rho/ROCK via ß-catenin phosphorylation at Ser675 or GSK-3ß-mediated phosphorylation at Ser33/37/Thr41, affecting cell proliferation, migration, apoptosis, and autophagy. Through its effects on Wnt/ß-catenin signaling, SNHG11 is implicated in glaucoma pathogenesis and is a potential therapeutic target.

NFATc1 Regulation of Dexamethasone-Induced TGFB2 Expression Is Cell Cycle Dependent in Trabecular Meshwork Cells.

Although elevated TGFß2 levels appear to be a causative factor in glaucoma pathogenesis, little is known about how TGFß2 expression is regulated in the trabecular meshwork (TM). Here, we investigated if activation of the cytokine regulator NFATc1 controlled transcription of TGFß2 in human TM cells by using dexamethasone (DEX) to induce NFATc1 activity. The study used both proliferating and cell cycle arrested quiescent cells. Cell cycle arrest was achieved by either cell-cell contact inhibition or serum starvation. ß-catenin staining and p21 and Ki-67 nuclear labeling were used to verify the formation of cell-cell contacts and activity of the cell cycle. NFATc1 inhibitors cyclosporine A (CsA) or 11R-VIVIT were used to determine the role of NFATc1. mRNA levels were determined by RT-qPCR. DEX increased TGFß2 mRNA expression by 3.5-fold in proliferating cells but not in quiescent cells or serum-starved cells, and both CsA and 11R-VIVIT inhibited this increase. In contrast, the expression of other DEX/NFATc1-induced mRNAs (myocilin and ß3 integrin) occurred regardless of the proliferative state of the cells. These studies show that NAFTc1 regulates TGFß2 transcription in TM cells and reveals a previously unknown connection between the TM cell cycle and modulation of gene expression by NFATc1 and/or DEX in TM cells.

TGF-ß-3 Induces Different Effects from TGF-ß-1 and -2 on Cellular Metabolism and the Spatial Properties of the Human Trabecular Meshwork Cells.

To compare the effects among three TGF-ß isoforms (TGF-ß-1, TGF-ß-2, and TGF-ß-3) on the human trabecular meshwork (HTM), two-dimensional (2D) and three-dimensional (3D) cultures of commercially available certified immortalized HTM cells were used, and the following analyses were conducted: (1) trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D); (2) a real-time cellular metabolic analysis (2D); (3) analysis of the physical property of the 3D HTM spheroids; and (4) an assessment of the gene expression levels of extracellular matrix (ECM) components (2D and 3D). All three TGF-ß isoforms induced a significant increase in TEER values and a relative decrease in FITC dextran permeability in the 2D-cultured HTM cells, but these effects were the most potent in the case of TGF-ß-3. The findings indicated that solutions containing 10 ng/mL of TGF-ß-1, 5 ng/mL of TGF-ß-2, and 1 ng/mL of TGF-ß-3 had nearly comparable effects on TEER measurements. However, a real-time cellular metabolic analysis of the 2D-cultured HTM cells under these concentrations revealed that TGF-3-ß induced quite different effects on the metabolic phenotype, with a decreased ATP-linked respiration, increased proton leakage, and decreased glycolytic capacity compared with TGF-ß-1 and TGF-ß-2. In addition, the concentrations of the three TGF-ß isoforms also caused diverse effects on the physical properties of 3D HTM spheroids and the mRNA expression of ECMs and their modulators, in many of which, the effects of TGF-ß-3 were markedly different from TGF-ß-1 and TGF-ß-2. The findings presented herein suggest that these diverse efficacies among the TGF-ß isoforms, especially the unique action of TGF-ß-3 toward HTM, may induce different effects within the pathogenesis of glaucoma.

Isoprenylation of Monomeric GTPases in Human Trabecular Meshwork Cells.

Small monomeric GTPases, including those belonging to the Rho family, regulate a diverse array of intracellular signaling pathways which affect vesicle transport/trafficking, endocytosis, cell cycle progression, cell contractility, and formation of stress fibers or focal adhesions. Functional activation of newly synthesized small monomeric GTPases is facilitated by a multi-step posttranslational process involving transferase-catalyzed addition of farnesyl or geranylgeranyl isoprenoids to conserved cysteine residues within a unique carboxy terminal -CaaX motif. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semi-quantitatively evaluate the functional consequence of posttranslational isoprenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational isoprenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.

α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFß2.

α-Synuclein (α-Syn) is implicated in Parkinson's disease (PD), a neuromotor disorder with prominent visual symptoms. The underlying cause of motor dysfunction has been studied extensively, and is attributed to the death of dopaminergic neurons mediated in part by intracellular aggregation of α-Syn. The cause of visual symptoms, however, is less clear. Neuroretinal degeneration due to the presence of aggregated α-Syn has been reported, but the evidence is controversial. Other symptoms including those arising from primary open angle glaucoma (POAG) are believed to be the side-effects of medications prescribed for PD. Here, we explored the alternative hypothesis that dysfunction of α-Syn in the anterior eye alters the interaction between the actin cytoskeleton of trabecular meshwork (TM) cells with the extracellular matrix (ECM), impairing their ability to respond to physiological changes in intraocular pressure (IOP). A similar dysfunction in neurons is responsible for impaired neuritogenesis, a characteristic feature of PD. Using cadaveric human and bovine TM tissue and primary human TM cells as models, we report two main observations: 1) α-Syn is expressed in human and bovine TM cells, and significant amounts of monomeric and oligomeric α-Syn are present in the AH, and 2) primary human TM cells and human and bovine TM tissue endocytose extracellular recombinant monomeric and oligomeric α-Syn via the prion protein (PrPC), and upregulate fibronectin (FN) and α-smooth muscle actin (α-SMA), fibrogenic proteins implicated in POAG. Transforming growth factor ß2 (TGFß2), a fibrogenic cytokine implicated in ∼50% cases of POAG, is also increased, and so is RhoA-associated coiled-coil-containing protein kinase 1 (ROCK-1). However, silencing of α-Syn in primary human TM cells reduces FN, α-SMA, and ROCK-1 in the absence or presence of over-expressed active TGFß2, suggesting modulation of FN and ROCK-1 independent of, or upstream of TGFß2. These observations suggest that extracellular α-Syn modulates ECM proteins in the TM independently or via PrPC by activating the RhoA-ROCK pathway. These observations reveal a novel function of α-Syn in the anterior eye, and offer new therapeutic options.

MicroRNA-210-3p mediates trabecular meshwork extracellular matrix accumulation and ocular hypertension - Implication for novel glaucoma therapy.

Elevation of intraocular pressure (IOP) is a major, controllable risk factor of primary open-angle glaucoma (POAG). Transforming growth factor-ß2 (TGF-ß2)-induced excessive accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) has been demonstrated to contribute significantly to the development of high IOP. We previously showed that treatment with salidroside (Sal), a plant-derived glucoside, can ameliorate the TGF-ß2-induced ECM expression in cultured human TM cells and reduce TGF-ß2-induced ocular hypertension in mice. In the current study, its underlying molecular mechanism associated with microRNA-210-3p (miR-210-3p) was characterized. We discovered that, in TM tissues of POAG patients, there was an increase in miR-210-3p. And miR-210-3p mediated a portion of the pathological effects of TGF-ß2 in vitro (excessive accumulation of ECM in cultured human TM cells) and in vivo (mouse ocular hypertension and ECM accumulation in the TM). Most interestingly, miR-210-3p was down-regulated by Sal, which appeared to mediate a significant portion of its IOP-lowering effect. Thus, these results shed light on the probable molecular mechanisms of TGF-ß2 and Sal and indicate that manipulation of miR-210-3p level/activity represents a potential new therapeutic strategy for POAG.

Intracameral Injection of AAV-DJ.COMP-ANG1 Reduces the IOP of Mice by Reshaping the Trabecular Outflow Pathway.

Purpose: The angiopoietin-1 (ANG1)-TIE signaling pathway orchestrates the development and maintenance of the Schlemm's canal (SC). In this study, we investigated the impact of adeno-associated virus (AAV)-mediated gene therapy with cartilage oligomeric matrix protein-ANG1 (COMP-ANG1) on trabecular outflow pathway. Methods: Different serotypes of AAVs were compared for transduction specificity and efficiency in the anterior segment. The selected AAVs encoding COMP-ANG1 or ZsGreen1 (control) were delivered into the anterior chambers of wild-type C57BL/6J mice. The IOP and ocular surface were monitored regularly. Ocular perfusion was performed to measure the outflow facility and label flow patterns of the trabecular drainage pathway. Structural features of SC as well as limbal, retinal, and skin vessels were visualized by immunostaining. Ultrastructural changes in the SC and trabecular meshwork were observed under transmission electron microscopy. Results: AAV-DJ could effectively infect the anterior segment. Intracameral injection of AAV-DJ.COMP-ANG1 lowered IOP in wild-type C57BL/6J mice. No signs of inflammation or angiogenesis were noticed. Four weeks after AAV injection, the conventional outflow facility and effective filtration area were increased significantly (P = 0.005 and P = 0.04, respectively). Consistently, the area of the SC was enlarged (P < 0.001) with increased density of giant vacuoles in the inner wall (P = 0.006). In addition, the SC endothelia lay on a more discontinuous basement membrane (P = 0.046) and a more porous juxtacanalicular tissue (P = 0.005) in the COMP-ANG1 group. Conclusions: Intracamerally injected AAV-DJ.COMP-ANG1 offers a significant IOP-lowering effect by remodeling the trabecular outflow pathway of mouse eyes.