Browsing by Subject "Ocular Hypertension / metabolism"
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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 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 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 Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(ARVO Journals, 2022-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Luan, Tomi; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFbeta2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFbeta2-TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods: Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFbeta2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 muM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFbeta2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results: Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFbeta2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFbeta2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFbeta2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions: These studies identify TGFbeta2-TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.Item Destabilizing COXIV in Muller Glia Increases Retinal Glycolysis and Alters Scotopic Electroretinogram(MDPI, 2022-12-12) Nsiah, Nana Yaa; Inman, Denise M.Muller glia (MG), the principal glial cell of the retina, have a metabolism that defies categorization into glycolytic versus oxidative. We showed that MG mount a strong hypoxia response to ocular hypertension, raising the question of their relative reliance on mitochondria for function. To explore the role of oxidative phosphorylation (OXPHOS) in MG energy production in vivo, we generated and characterized adult mice in which MG have impaired cytochrome c oxidase (COXIV) activity through knockout of the COXIV constituent COX10. Histochemistry and protein analysis showed that COXIV protein levels were significantly lower in knockout mouse retina compared to control. Loss of COXIV activity in MG did not induce structural abnormalities, though oxidative stress was increased. Electroretinography assessment showed that knocking out COX10 significantly impaired scotopic a- and b-wave responses. Inhibiting mitochondrial respiration in MG also altered the retinal glycolytic profile. However, blocking OXPHOS in MG did not significantly exacerbate retinal ganglion cell (RGC) loss or photopic negative response after ocular hypertension (OHT). These results suggest that MG were able to compensate for reduced COXIV stability by maintaining fundamental processes, but changes in retinal physiology and metabolism-associated proteins indicate subtle changes in MG function.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 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 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 Role of Glucocorticoids and Glucocorticoid Receptors in Glaucoma Pathogenesis(MDPI, 2023-10-27) Patel, Pinkal D.; Kodati, Bindu; Clark, Abbot F.The glucocorticoid receptor (GR), including both alternative spliced isoforms (GRalpha and GRbeta), has been implicated in the development of primary open-angle glaucoma (POAG) and iatrogenic glucocorticoid-induced glaucoma (GIG). POAG is the most common form of glaucoma, which is the leading cause of irreversible vision loss and blindness in the world. Glucocorticoids (GCs) are commonly used therapeutically for ocular and numerous other diseases/conditions. One serious side effect of prolonged GC therapy is the development of iatrogenic secondary ocular hypertension (OHT) and OAG (i.e., GC-induced glaucoma (GIG)) that clinically and pathologically mimics POAG. GC-induced OHT is caused by pathogenic damage to the trabecular meshwork (TM), a tissue involved in regulating aqueous humor outflow and intraocular pressure. TM cells derived from POAG eyes (GTM cells) have a lower expression of GRbeta, a dominant negative regulator of GC activity, compared to TM cells from age-matched control eyes. Therefore, GTM cells have a greater pathogenic response to GCs. Almost all POAG patients develop GC-OHT when treated with GCs, in contrast to a GC responder rate of 40% in the normal population. An increased expression of GRbeta can block GC-induced pathogenic changes in TM cells and reverse GC-OHT in mice. The endogenous expression of GRbeta in the TM may relate to differences in the development of GC-OHT in the normal population. A number of studies have suggested increased levels of endogenous cortisol in POAG patients as well as differences in cortisol metabolism, suggesting that GCs may be involved in the development of POAG. Additional studies are warranted to better understand the molecular mechanisms involved in POAG and GIG in order to develop new disease-modifying therapies to better treat these two sight threatening forms of glaucoma. The purpose of this timely review is to highlight the pathological and clinical features of GC-OHT and GIG, mechanisms responsible for GC responsiveness, potential therapeutic options, as well as to compare the similar features of GIG with POAG.