Browsing by Author "Millar, J. Cameron"
Now showing 1 - 20 of 34
- Results Per Page
- Sort Options
Item A Novel Mouse Model of TGFbeta2-Induced Ocular Hypertension Using Lentiviral Gene Delivery(MDPI, 2022-06-21) Patil, Shruti V.; Kasetti, Ramesh B.; Millar, J. Cameron; Zode, Gulab S.Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) beta2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFbeta2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFbeta2 in the TM. We developed an LV vector-encoding active hTGFbeta2(C226,228S) under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFbeta2(C226,228S). We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFbeta2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFbeta2(C226,228S) exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFbeta2(C226,228S) that induces TM dysfunction and outflow resistance.Item Activation of TRPV4 channels reduces IOP and improves outflow facility by regulating eNOS dependent nitric oxide release from the trabecular meshwork(2019-03-05) Kasetti, Ramesh; Maddineni, Prabhavathi; Millar, J. Cameron; Zode, Gulab; Patel, PinkalPurpose: Nitric oxide (NO) is known to reduce intraocular pressure (IOP) by relaxation of the trabecular meshwork (TM) and distal vessels of the conventional outflow pathway. However, the intrinsic mechanisms by which outflow pathway tissues regulate NO production is yet to be elucidated. In vascular endothelium, activation of mechanosensory transient receptor potential vanilloid 4 (TRPV4) channels results in endothelial nitic oxide synthase (eNOS) mediated NO release, which in turn promotes vasodilation. Here, we determined whether activation of TRPV4 regulates IOP and conventional outflow via NO release in the TM. Methods: In wildtype (WT) and glucocorticoid-induced ocular hypertensive (OHT) C57BL/6J mice, the effect of TRPV4 agonist GSK1016790A on IOP and outflow facility was determined using rebound tonometry and constant-flow infusion method respectively. Effect of TRPV4 agonist on eNOS activation and NO production was determined using Western blot and fluorometric DAF-FM assay in primary human TM cells and ex vivo cultured human TM donor tissues. We report for the first time a method for electrochemical measurement of NO in human anterior segment donor tissues using NO microsensors. Results: Topical administration of TRPV4 agonist GSK1016790A significantly reduced IOP (Pin WT and OHT mice compared to contralateral control eyes. In OHT mice, treatment with GSK1016790A resulted in increased outflow facility (P=0.02)compared to contralateral vehicle treated eyes. We further demonstrate that TRPV4 activation by GSK1016790A resulted in increased eNOS phosphorylation in GTM3 cells, primary human TM cells, and cultured human TM donor tissues. Activation of TRPV4 in primary TM cells and ex vivocultured human TM donor tissues resulted in increased DAF-FM fluorescence, which signifies increase in TRPV4-mediated NO production. Treatment of human anterior segments with TRPV4 agonist resulted in increased production of NO as detected electrochemically using NO microsensors. Nonselective inhibition of NOS by L-NAME abrogated the IOP lowering effect of TRPV4 agonist in mice and reduced TRPV4-mediated NO production in outflow pathway cells and donor tissues. Conclusion: TRPV4 activation improves IOP and outflow facility, perhaps by regulation of eNOS dependent NO release.Item ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load(Springer Nature, 2020-11-05) Kasetti, Ramesh B.; Patel, Pinkal D.; Maddineni, Prabhavathi; Patil, Shruti; Kiehlbauch, Charles; Millar, J. Cameron; Searby, Charles C.; Raghunathan, Vijaykrishna; Sheffield, Val C.; Zode, Gulab S.The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.Item BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork(ARVO Journals, 2018-04) Hernandez, Humberto; Millar, J. Cameron; Curry, Stacy M.; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFbeta2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFbeta2 signaling. We investigate the role of TGFbeta2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods: Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFbeta2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, alpha-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFbeta2, or Ad5.TGFbeta2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results: MTM cells express TM markers and respond to DEX and TGFbeta2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFbeta2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFbeta2, and Ad5.TGFbeta2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions: We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.Item CNS axonal degeneration and transport deficits at the optic nerve head precede structural and functional loss of retinal ganglion cells in a mouse model of glaucoma(BioMed Central Ltd., 2020-08-27) Maddineni, Prabhavathi; Kasetti, Ramesh B.; Patel, Pinkal D.; Millar, J. Cameron; Kiehlbauch, Charles; Clark, Abbot F.; Zode, Gulab S.BACKGROUND: Glaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP. METHODS: C57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT. RESULTS: Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). CONCLUSIONS: These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.Item Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms(ARVO Journals, 2022-02) McDowell, Colleen M.; Kizhatil, Krishnakumar; Elliott, Michael H.; Overby, Darryl R.; van Batenburg-Sherwood, Joseph; Millar, J. Cameron; Kuehn, Markus H.; Zode, Gulab S.; 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, Rudolph; 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 A.; 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. DanielDue 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.Item Cost Analysis of Robotic Exoskeletons for Use in Inpatient Rehabilitation Post-Spinal Cord Injury (CARE4U In Rehab Post-SCI)(2023-05) Baltz, Sara A.; Millar, J. CameronRecent healthcare technology innovations have dramatically reshaped spinal cord injury (SCI) recovery outcomes. One new type of therapy, robotic gait training (RGT), utilizes a device consisting of a motorized "exoskeleton" frame to facilitate various ambulatory activities. However, barriers such as upfront costs and stakeholder buy-in hinder further research into whether RGT surpasses usual care gait training (UC) (e.g., body weight-supported treadmill training, conventional overground walking) for treating acute SCI. Additional contributions to RGT and UC cost data via cost-effectiveness and cost-benefit analyses would aid stakeholders in their determinations, and any significant findings could lead to the financial benefit of healthcare institutions as well as to the therapeutic benefit of patients. Recent healthcare technology innovations have dramatically reshaped spinal cord injury (SCI) recovery outcomes. One new type of therapy, robotic gait training (RGT), utilizes a device consisting of a motorized "exoskeleton" frame to facilitate various ambulatory activities. However, barriers such as upfront costs and stakeholder buy-in hinder further research into whether RGT surpasses usual care gait training (UC) (e.g., body weight-supported treadmill training, conventional overground walking) for treating acute SCI. Additional contributions to RGT and UC cost data via cost-effectiveness and cost-benefit analyses would aid stakeholders in their determinations, and any significant findings could lead to the financial benefit of healthcare institutions as well as to the therapeutic benefit of patients.Item Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation(MDPI, 2022-02-22) Kodati, Bindu; Merchant, Shawn A.; Millar, J. Cameron; Liu, YangMutations in PITX2 cause Axenfeld-Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.Item Effect of ATP-sensitive Potassium Channel Openers on Intraocular Pressure in Ocular Hypertensive Animal Models(ARVO Journals, 2022-02-01) Roy Chowdhury, Uttio; Millar, J. Cameron; Holman, Bradley H.; Anderson, Kjerston J.; Dosa, Peter I.; Roddy, Gavin W.; Fautsch, Michael P.Purpose: To evaluate the effect of ATP-sensitive potassium channel openers cromakalim prodrug 1 (CKLP1) and diazoxide on IOP in three independent mouse models of ocular hypertension. Methods: Baseline IOP was measured in TGFbeta2 overexpression, steroid-induced, and iris dispersion (DBA/2J) ocular hypertension mouse models, followed by once daily eyedrop administration with CKLP1 (5 mM) or diazoxide (5 mM). The IOP was measured in conscious animals with a handheld rebound tonometer. Aqueous humor dynamics were assessed by a constant perfusion method. Effect of treatment on ocular tissues was evaluated by transmission electron microscopy. Results: CKLP1 decreased the IOP by 20% in TGFbeta2 overexpressing mice (n = 6; P < 0.0001), 24% in steroid-induced ocular hypertensive mice (n = 8; P < 0.0001), and 43% in DBA/2J mice (n = 15; P < 0.0001). Diazoxide decreased the IOP by 32% in mice with steroid-induced ocular hypertension (n = 13; P < 0.0001) and by 41% in DBA/2J mice (n = 4; P = 0.005). An analysis of the aqueous humor dynamics revealed that CKLP1 decreased the episcleral venous pressure by 29% in TGFbeta2 overexpressing mice (n = 13; P < 0.0001) and by 72% in DBA/2J mice (n = 4 control, 3 treated; P = 0.0002). Diazoxide lowered episcleral venous pressure by 35% in steroid-induced ocular hypertensive mice (n = 3; P = 0.03). Tissue histology and cell morphology appeared normal when compared with controls. Accumulation of extracellular matrix was reduced in CKLP1- and diazoxide-treated eyes in the steroid-induced ocular hypertension model. Conclusions: ATP-sensitive potassium channel openers CKLP1 and diazoxide effectively decreased the IOP in ocular hypertensive animal models by decreasing the episcleral venous pressure, supporting a potential therapeutic application of these agents in ocular hypertension and glaucoma.Item Effect of Cromakalim Prodrug 1 (CKLP1) on Aqueous Humor Dynamics and Feasibility of Combination Therapy With Existing Ocular Hypotensive Agents(ARVO Journals, 2017-11-01) Roy Chowdhury, Uttio; Rinkoski, Tommy A.; Bahler, Cindy K.; Millar, J. Cameron; Bertrand, Jacques A.; Holman, Bradley H.; Sherwood, Joseph M.; Overby, Darryl R.; Stoltz, Kristen L.; Dosa, Peter I.; Fautsch, Michael P.Purpose: Cromakalim prodrug 1 (CKLP1) is a water-soluble ATP-sensitive potassium channel opener that has shown ocular hypotensive properties in ex vivo and in vivo experimental models. To determine its mechanism of action, we assessed the effect of CKLP1 on aqueous humor dynamics and in combination therapy with existing ocular hypotensive agents. Methods: Outflow facility was assessed in C57BL/6 mice by ex vivo eye perfusions and by in vivo constant flow infusion following CKLP1 treatment. Human anterior segments with no trabecular meshwork were evaluated for effect on pressure following CKLP1 treatment. CKLP1 alone and in combination with latanoprost, timolol, and Rho kinase inhibitor Y27632 were evaluated for effect on intraocular pressure in C57BL/6 mice and Dutch-belted pigmented rabbits. Results: CKLP1 lowered episcleral venous pressure (control: 8.9 +/- 0.1 mm Hg versus treated: 6.2 +/- 0.1 mm Hg, P < 0.0001) but had no detectable effect on outflow facility, aqueous humor flow rate, or uveoscleral outflow. Treatment with CKLP1 in human anterior segments without the trabecular meshwork resulted in a 50% +/- 9% decrease in pressure, suggesting an effect on the distal portion of the conventional outflow pathway. CKLP1 worked additively with latanoprost, timolol, and Y27632 to lower IOP, presumably owing to combined effects on different aspects of aqueous humor dynamics. Conclusions: CKLP1 lowered intraocular pressure by reducing episcleral venous pressure and lowering distal outflow resistance in the conventional outflow pathway. Owing to this unique mechanism of action, CKLP1 works in an additive manner to lower intraocular pressure with latanoprost, timolol, and Rho kinase inhibitor Y27632.Item Effects of TGFβ2 and BMP4 Downstream Targets ID1 and ID3 in Trabecular Meshwork: Implications In Lowering IOP(2017-12-01) Mody, Avani A.; Clark, Abbot F.; Millar, J. Cameron; Krishnamoorthy, Raghu R.Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma, which is a major cause of irreversible vision loss. The major risk factor associated with POAG is increased intra ocular pressure (IOP). Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) increases the deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor I (PAI-1) and ECM cross-linking enzymes. These disruptions in ECM physiology in the TM elevate IOP and decrease aqueous humor outflow facility. Bone morphogenetic proteins (BMPs) regulate TGFβ2 induced profibrotic ECM production. The underlying mechanism for BMP4 inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 induces inhibitor of DNA binding proteins (ID1, ID3), which are negative transcription regulators that suppress fibrosis by regulating ECM component expression. Our study sought to determine whether ID1and ID3 proteins are downstream targets of BMP4 in TM and thereby attenuate TGFβ2 induction of ECM proteins in TM cells, elevated IOP and decreased outflow facility. Methods: Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. GTM3 cells were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression and promoter activities. IOP and AH outflow facility changes were studied in female BALB/cJ mice. Ad5-CMV-ID1 and Ad5-CMV-ID3 viral vectors along with Ad5-CMV-TGFβ2C226S/C288S were injected intravitreally. Ad5-CMV-TGFβ2C226S/C288S was injected along with Ad5- null vector as positive control, while Ad5-null injected mice were included as a negative control. IOP was measured using a Tonolab impact tonometer and AH outflow facility was measured using a constant flow infusion method. In addition we also performed a luciferase reporter activity array assay to study the effect of ID1, ID3 and TGFβ2 on various transcription factor activity in TM. Results: Basal expression of ID1-3 was detected in primary human TM cells. BMP4 significantly induced early expression of ID1 and ID3 mRNA (p [less than] < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells (p< 0.01). Transduction of mouse eyes with ID1 or ID3 significantly blocked TGFβ2 induced ocular hypertension (P [less than] 0.0001) and AH outflow facility changes in living mice. Further, ID1 (P [less than] 0.01) and ID3 increase NFκB, Notch and cAMP/PKA activity, even in presence TGFβ2. Conclusions: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 profibrotic activity in human TM cells. In addition ID1 and ID3 suppresses elevated IOP and decreases AH outflow facility induced by TGFβ2. Further, increased activity of NFκB, Notch and cAMP/PKA suggest that ID1 and ID3 may regulate TGFβ2 effects in TM via activation of the MMP pathway, inhibition of PAI-1 and Rho kinase activity, resisting cytoskeleton changes and lead to increased TM cellularity. This strongly supports ID1 and/or ID3 as robust candidates as a basis for developing disease-modifying IOP lowering therapies in POAG.Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Functional impairment of TRPV4-eNOS signaling in glaucomatous trabecular meshwork contributes to elevation of intraocular pressure.(2020) Kasetti, Ramesh; Zode, Gulab S.; Maddineni, Prabhavathi; Sonkusare, Swapnil; Millar, J. Cameron; Chen, Yen-Lin; Patel, PinkalPurpose: Nitric oxide (NO) is known to reduce intraocular pressure (IOP) by relaxation of the trabecular meshwork (TM). However, the intrinsic mechanism regulating NO production in the TM is yet to be elucidated. Here, we examined whether transient receptor potential vanilloid 4 (TRPV4) channels regulate IOP via endothelial nitic oxide synthase (eNOS) signaling. We also assess whether TRPV4-eNOS signaling is impaired in glaucoma. Methods: In WT C57BL/6J mice, the effect of the TRPV4 agonist GSK1016790A (GSK101) on IOP and outflow facility was determined using rebound tonometry and constant-flow infusion method, respectively. The effect of GSK101 on eNOS activation and NO production was determined using Western blot and a fluorometric DAF-FM assay in normal and glaucomatous primary human TM cells. We further examined whether TRPV4 channel function is impaired in glaucoma using high-speed Calcium imaging in normal and glaucomatous human primary TM cells. Results: Topical administration of GSK101 (20 µM) significantly improved outflow facility (P=0.05) and reduced IOP (P< 0.0001) in WT mice. GSK101 (20 nM) treatment resulted in increased eNOS phosphorylation in primary human TM cells and donor tissues. GSK101 treatment also resulted in increased DAF-FM fluorescence, which signifies an increase in NO production. Importantly, human glaucomatous primary TM cells exhibited significantly attenuated Calcium influx, eNOS phosphorylation, and NO production in response to GSK101 when compared to normal human primary TM cells. Conclusion: TRPV4 channel activation lowers IOP by regulating NO in the TM. Functional impairment of these channels may contribute to glaucomatous pathophysiology.Item Glucocorticoid receptor GRbeta regulates glucocorticoid-induced ocular hypertension in mice(Springer Nature, 2018-01-16) Patel, Gaurang C.; Liu, Yang; Millar, J. Cameron; Clark, Abbot F.Prolonged glucocorticoid (GC) therapy can cause GC-induced ocular hypertension (OHT), which if left untreated progresses to iatrogenic glaucoma and permanent vision loss. The alternatively spliced isoform of glucocorticoid receptor GRbeta acts as dominant negative regulator of GR activity, and it has been shown that overexpressing GRbeta in trabecular meshwork (TM) cells inhibits GC-induced glaucomatous damage in TM cells. The purpose of this study was to use viral vectors to selectively overexpress the GRbeta isoform in the TM of mouse eyes treated with GCs, to precisely dissect the role of GRbeta in regulating steroid responsiveness. We show that overexpression of GRbeta inhibits GC effects on MTM cells in vitro and GC-induced OHT in mouse eyes in vivo. Ad5 mediated GRbeta overexpression reduced the GC induction of fibronectin, collagen 1, and myocilin in TM of mouse eyes both in vitro and in vivo. GRbeta also reversed DEX-Ac induced IOP elevation, which correlated with increased conventional aqueous humor outflow facility. Thus, GRbeta overexpression reduces effects caused by GCs and makes cells more resistant to GC treatment. In conclusion, our current work provides the first evidence of the in vivo physiological role of GRbeta in regulating GC-OHT and GC-mediated gene expression in the TM.Item Glucocorticoid receptor GRβ regulates glucocorticoid-induced ocular hypertension and glaucoma in mice(2017-03-14) Liu, Yang; Millar, J. Cameron; Clark, Abbot; Patel, GaurangPurpose: Glucocorticoid (GC) induced ocular hypertension (OHT) is a serious side effect of prolonged GC therapy and if left untreated it can lead to iatrogenic glaucoma and permanent vision loss. The Alternatively spliced isoform of glucocorticoid receptor GRβ acts as a dominant negative regulator of GC activity. Our previous studies have shown that GRβ regulates GC responsiveness and that overexpressing GRβ in trabecular meshwork (TM) cells inhibits GC-induced and glaucomatous damage in TM cells. The purpose of this study was to determine whether increased expression of GRβ can reverse GC-induced OHT in mice. Methods: Mouse trabecular meshwork cells (MTM) were transduced with Ad5.null or Ad5.hGRβ expression vectors at MOI-50. After 24 hours MTM cells were treated with dexamethasone (DEX) or vehicle control (0.1% ethanol). To generate GC-OHT, C57BL/6J mice received weekly bilateral periocular (administrated through conjunctival fornix) injections of dexamethasone acetate (DEX-Ac, 200ug/eye). Several weeks after DEX-Ac administration, mouse eyes were injected intravitreally with Ad5.null or Ad5.hGRβ expression vectors (3x107 pfu/eye) to transduce the TM. Nighttime intraocular pressure (IOP) was measured using a TonoLab rebound tonometer, and outflow facilities were measured in living mice using our constant flow infusion technique. Fibronectin and collagen I expression were evaluated using immunoblotting of mouse anterior segment tissues. The unpaired Student’s t-test (2-tailed) and One-way ANOVA were used for statistical analysis. Results: DEX treatment of MTM cells increased fibronection expression, whereas transduction of MTM cells with Ad5.hGRβ maintained fibronectin expression at control levels as shown by immunocytochemistry. DEX-Ac significantly increased IOP from days 3-44 (n=23, p Conclusion: Overexpression of GRβ in the TM of mouse eyes reversed GC-OHT. GRβ gene therapy may be a useful therapeutic approach to treat GC-OHT and glaucoma.Item Glucocorticoid Receptor Transactivation Is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma(ARVO Journals, 2019-05) Patel, Gaurang C.; Millar, J. Cameron; Clark, Abbot F.Purpose: Glucocorticoid (GC)-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and alpha-smooth muscle actin (alpha-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusions: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.Item ID1 and ID3 are Negative Regulators of TGFbeta2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice(ARVO Journals, 2021-05-03) Mody, Avani A.; Millar, J. Cameron; Clark, Abbot F.Purpose: In POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFbeta2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFbeta2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFbeta2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes. Methods: IOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFbeta2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice. Results: Over-expressing ID1 and ID3 significantly blocked TGFbeta2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFbeta2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFbeta2. Knockdown of ID1 or ID3 expression exacerbated TGFbeta2-induced ocular hypertension. Conclusions: Increased expression of ID1 and ID3 suppressed both TGFbeta2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.Item Increased synthesis and deposition of extracellular matrix proteins leads to endoplasmic reticulum stress in the trabecular meshwork(Springer Nature, 2017-11-02) Kasetti, Ramesh B.; Maddineni, Prabhavathi; Millar, J. Cameron; Clark, Abbot F.; Zode, Gulab S.Increased synthesis and deposition of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) is associated with TM dysfunction and intraocular pressure (IOP) elevation in glaucoma. However, it is not understood how ECM accumulation leads to TM dysfunction and IOP elevation. Using a mouse model of glucocorticoid (GC)-induced glaucoma, primary human TM cells and human post-mortem TM tissues, we show that increased ECM accumulation leads to endoplasmic reticulum (ER) stress in the TM. The potent GC, dexamethasone (Dex) increased the secretory protein load of ECM proteins in the ER of TM cells, inducing ER stress. Reduction of fibronectin, a major regulator of ECM structure, prevented ER stress in Dex-treated TM cells. Overexpression of fibronectin via treatment with cellular fibronectin also induced chronic ER stress in primary human TM cells. Primary human TM cells grown on ECM derived from Dex-treated TM cells induced ER stress markers. TM cells were more prone to ER stress from ECM accumulation compared to other ocular cell types. Moreover, increased co-localization of ECM proteins with ER stress markers was observed in human post-mortem glaucomatous TM tissues. These data indicate that ER stress is associated with increased ECM accumulation in mouse and human glaucomatous TM tissues.Item Intensity of Usual Care Therapeutic Interventions in Inpatient Rehabilitation - A Pilot Study(2019-12) Di Pasquale, Jake A.; Millar, J. Cameron; Ranjan, Amalendu P.Minimal evidence exists describing key dosing parameters of interventions used during the subacute phase of recovery after neurological injury. This prospective cohort study aims to assess cardiorespiratory strain resulting from novel and conventional therapeutic interventions. Gait training provided relatively more moderate to vigorous exercise, reaching the associated %HRR 25% and 42% of the time in patients post spinal cord injury (SCI) and stroke, respectively. Specifically, EksoGT overground robotic gait training appears more effective, evoking targeted %HRR for 48% and 52% of sessions. Rate of Perceived Exertion (RPE) was moderately correlated with very light intensity in patients with SCI but ultimately ineffective at gauging %HRR. EksoGT can administer moderate to vigorous intensity exercise to patients with severe disabilities. Inpatient rehabilitation is inherently variable in method and population but can provide minimally sufficient exercise intensity. Further research into the dose-response relationship and accurate estimation of intensity are needed.Item Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma(Springer Nature Limited, 2024-03-24) Patil, Shruti V.; Kaipa, Balasankara R.; Ranshing, Sujata; Sundaresan, Yogapriya; Millar, J. Cameron; Nagarajan, Bhavani; Kiehlbauch, Charles; Zhang, Qihong; Jain, Ankur; Searby, Charles C.; Scheetz, Todd E.; Clark, Abbot F.; Sheffield, Val C.; Zode, Gulab S.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-MYOC(Y437H) 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.