Browsing by Subject "Trabecular meshwork"

Now showing 1 - 10 of 15
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    An ex vivo model of human corneal rim perfusion organ culture
    (Elsevier, 2022) Peng, Michael; Margetts, Tyler J.; Sugali, Chenna Kesavulu; Rayana, Naga Pradeep; Dai, Jiannong; Sharma, Tasneem P.; Raghunathan, Vijay Krishna; Mao, Weiming; Ophthalmology, School of Medicine
    The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 μl/min, the outflow facility was 0.359 ± 0.216 μl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.
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    Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway
    (MDPI, 2021-10-24) Soundararajan, Avinash; Ghag, Sachin Anil; Vuda, Sai Supriya; Wang, Ting; Pattabiraman, Padmanabhan Paranji; Ophthalmology, School of Medicine
    The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFβ2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.
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    Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms
    (Association for Research in Vision and Ophthalmology, 2022) McDowell, Colleen M.; Kizhatil, Krishnakumar; Elliott, Michael H.; Overby, Darryl R.; van Batenburg-Sherwood, Joseph; Millar, J. Cameron; Kuehn, Markus H.; Zode, Gulab; Acott, Ted S.; Anderson, Michael G.; Bhattacharya, Sanjoy K.; Bertrand, Jacques A.; Borras, Terete; Bovenkamp, Diane E.; Cheng, Lin; Danias, John; De Ieso, Michael Lucio; Du, Yiqin; Faralli, Jennifer A.; Fuchshofer, Rudolf; Ganapathy, Preethi S.; Gong, Haiyan; Herberg, Samuel; Hernandez, Humberto; Humphries, Peter; John, Simon W.M.; Kaufman, Paul L.; Keller, Kate E.; Kelley, Mary J.; Kelly, Ruth A.; Krizaj, David; Kumar, Ajay; Leonard, Brian C.; Lieberman, Raquel L.; Liton, Paloma; Liu, Yutao; Liu, Katy C.; Lopez, Navita N.; Mao, Weiming; Mavlyutov, Timur; McDonnell, Fiona; McLellan, Gillian J.; Mzyk, Philip; Nartey, Andrews; Pasquale, Louis R.; Patel, Gaurang C.; Pattabiraman, Padmanabhan P.; Peters, Donna M.; Raghunathan, Vijaykrishna; Rao, Ponugoti Vasantha; Rayana, Naga; Raychaudhuri, Urmimala; Reina-Torres, Ester; Ren, Ruiyi; Rhee, Douglas; Chowdhury, Uttio Roy; Samples, John R.; Samples, E. Griffen; Sharif, Najam; Schuman, Joel S.; Sheffield, Val C.; Stevenson, Cooper H.; Soundararajan, Avinash; Subramanian, Preeti; Sugali, Chenna Kesavulu; Sun, Yang; Toris, Carol B.; Torrejon, Karen Y.; Vahabikashi, Amir; Vranka, Janice A.; Wang, Ting; Willoughby, Colin E.; Xin, Chen; Yun, Hongmin; Zhang, Hao F.; Fautsch, Michael P.; Tamm, Ernst R.; Clark, Abbot F.; Ethier, C. Ross; Stamer, W. Daniel; Ophthalmology, School of Medicine
    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.
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    Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells
    (Elsevier, 2022) Peng, Michael; Rayana, Naga Pradeep; Dai, Jiannong; Sugali, Chenna Kesavulu; Baidouri, Hasna; Suresh, Ayush; Raghunathan, Vijay Krishna; Mao, Weiming; Ophthalmology, School of Medicine
    Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labeled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blastcidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.
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    Glucocorticoid-Induced Ocular Hypertension and Glaucoma
    (Dove Press, 2024-02-16) Harvey, Devon Hori; Sugali, Chenna Kesavulu; Mao, Weiming; Ophthalmology, School of Medicine
    Glucocorticoid (GC) therapy is indicated in many diseases, including ocular diseases. An important side-effect of GC therapy is GC-induced ocular hypertension (GIOHT), which may cause irreversible blindness known as GC-induced glaucoma (GIG). Here, we reviewed the pathological changes that contribute to GIOHT including in the trabecular meshwork and Schlemm’s canal at cellular and molecular levels. We also discussed the clinical aspects of GIOHT/GIG including disease prevalence, risk factors, the type of GCs, the route of GC administration, and management strategies.
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    GSK3β Inhibitors Inhibit TGFβ Signaling in the Human Trabecular Meshwork
    (Association for Research in Vision and Ophthalmology, 2024) Sugali, Chenna Kesavulu; Rayana, Naga Pradeep; Dai, Jiannong; Harvey, Devon H.; Dhamodaran, Kamesh; Mao, Weiming; Ophthalmology, School of Medicine
    Purpose: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT). Methods: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. Results: We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes. Conclusions: Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.
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    High Glucose-Induced Transcriptomic Changes in Human Trabecular Meshwork Cells
    (Research Square, 2024-12-24) Singh, Shivendra; Patel, Niketa A.; Soundararajan, Avinash; Pattabiraman, Padmanabhan P.; Ophthalmology, School of Medicine
    Glaucoma is a leading cause of irreversible blindness, often associated with elevated intraocular pressure (IOP) due to trabecular meshwork (TM) dysfunction. Diabetes mellitus (DM) is recognized as a significant risk factor for glaucoma; however, the molecular mechanisms through which hyperglycemia affects TM function remain unclear. This study investigated the impact of high glucose on gene expression in human TM (HTM) cells to uncover pathways that contribute to TM dysfunction and glaucoma pathogenesis under diabetic conditions. Primary HTM cells were cultured under normoglycemic (5.5 mM) and hyperglycemic (30 mM) conditions for seven days, followed by mRNA sequencing (mRNA-seq) to identify differentially expressed genes, with quantitative PCR (qPCR) used for confirmatory analysis. STRING network analysis was performed to predict interactions among upregulated and downregulated proteins. mRNA-seq analysis revealed 25 significantly differentially expressed genes in high glucose conditions, including upregulated genes associated with oxidative stress, apoptosis, autophagy, immune response, and fibrosis. Notably, TXNIP was significantly upregulated, indicating increased oxidative stress and apoptosis in TM cells, while downregulation of autophagy-related genes, such as HSPA6 and LAMP3, suggests compromised protein quality control. Immune response genes, including CCL7 and CHI3L1, were upregulated, suggesting an inflammatory response to oxidative stress. Increased expression of fibrosis-related genes, such as SNAI1, FGF7, and KRT19, supports the hypothesis of ECM accumulation in diabetic conditions, potentially elevating IOP. Chronic hyperglycemia in diabetic patients could therefore lead to TM dysfunction, impair aqueous humor outflow, and elevate IOP, thereby increasing glaucoma risk. Targeting oxidative stress and fibrosis pathways offers therapeutic strategies to mitigate glaucoma progression in diabetic populations.
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    Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation
    (Wiley, 2023) Wang, Ting; Soundararajan, Avinash; Rabinowitz, Jeffrey; Jaiswal, Anant; Osborne, Timothy; Pattabiraman, Padmanabhan Paranji; Ophthalmology, School of Medicine
    Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.
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    Modulation of Intralocular Pressure by Tuning Transcriptional Control of Lipid Synthesis
    (2024-06) Wang, Ting; Morral, Nuria; Pattabiraman, Padmanabhan; Corson, Timothy W.; Landreth, Gary E.; Perrin, Benjamin J.
    Glaucoma is an age-related optic neuropathy and is one of the leading causes of irreversible blindness. Primary open-angle glaucoma (POAG) is the predominant subtype of glaucoma. Elevated intraocular pressure (IOP) is a major risk factor for POAG and lowering IOP is the most effective therapeutic strategy. IOP is maintained by the balance of aqueous humor (AH) generation by the ciliary body and drainage by conventional outflow pathway including trabecular meshwork (TM). TM is a highly contractile and mechanosensitive tissue, and its contractility regulated by the actin cytoskeleton and extracellular matrix (ECM) is directly related to IOP regulation. Using multiomics analysis in human TM (HTM) cells, I identified that mechanical stretch caused the activation of sterol regulatory element binding proteins (SREBPs) related-lipid biogenesis pathways. Further, using immunofluorescence, and constitutive activation of each SREBP isoform, I discovered the mechanosensing role of SREBPs in HTM cells and mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo resulted in significant IOP lowering. Conversely, significantly elevated IOP was observed after using the pharmacological activator of SREBPs by clozapine and constitutive activation of SREBPs ex vivo and in vivo, respectively. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways and phospholipids, cholesterol, and triglyceride levels. The increased lipid biogenesis was found after constitutive activation of SREBP isoforms in HTM cells but with slightly different effects between each isoform. Further, I showed that fatostatin mitigated actin polymerization machinery and stabilization, and identified that SREBPs activation is a critical regulator of ECM engagement to the matrix sites. Lastly, I identified that cholesterol levels play an important role in regulating actin polymerization, focal adhesion formation, cell-ECM interactions, and membrane tension in HTM cells. Therefore, we have established the direct connection between cholesterol and TM contractility. Overall, I postulate that lowering de novo lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.
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    Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork
    (Wiley, 2022) Soundararajan, Avinash; Wang, Ting; Ghag, Sachin A.; Kang, Min H.; Pattabirama, Padmanabhan P.; Ophthalmology, School of Medicine
    This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and β5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and β5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-β2 (TGFβ2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFβ2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.