Dr. Judith West-Mays is a Professor in the Department of Pathology and Molecular Medicine and Assistant Dean of the Medical Sciences Graduate Program at McMaster University, Ontario, Canada. She is an experienced vision scientist who obtained her Ph.D. from the School of Optometry at the University of Waterloo. She completed 3 years of Postdoctoral studies at Harvard Medical School and was recruited to McMaster in 2002 from the Tufts Center for Vision Research in Boston where she held a prestigious Career Award from Research to Prevent Blindness. Dr. West-Mays has extensive experience in investigating the molecular and genetic mechanisms of both eye development and disease. Specifically, she utilizes genetic mouse models and cell culture studies for determining the underlying causes of primary and secondary cataracts, glaucoma, and more recently, retinal degeneration.
Corneal development: Role of the periocular mesenchyme and Bi-directional signaling.
Exp Eye Res. 2020 Oct 08;:108231
Authors: Walker H, Akula M, West-Mays JA
The cornea is a highly specialized transparent tissue located at the anterior most surface of the eye. It consists of three main layers, the outer stratified squamous epithelium, the inner endothelium, and the intermediate stroma. Formation of these layers during development involves a complex interaction between ectodermal-derived structures, such as the overlying head ectoderm, and the periocular mesenchyme (POM), the latter of which is comprised of neural crest cells (NCC) and mesoderm derived progenitor cells. Regulation of corneal epithelial development, including both epithelial cell fate and stratification, has been shown to depend on numerous bi-directional mesenchymal-epithelial signaling pathways. In this review we pay particular attention to the genes and signaling pathways that involve the POM.
PMID: 33039457 [PubMed - as supplied by publisher]
AP-2β is required for formation of the murine trabecular meshwork and Schlemm's canal.
Exp Eye Res. 2020 Apr 27;:108042
Authors: Akula M, Taiyab A, Deschamps P, Yee S, Ball AK, Williams T, West-Mays JA
Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2β, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2β in the NCCs, particularly in the structures that control aqueous outflow using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2β neural crest cell knockout or AP-2β NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2β in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2β is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2β NCC KO mice.
PMID: 32353428 [PubMed - as supplied by publisher]
β-Catenin/Smad3 Interaction Regulates Transforming Growth Factor-β-Induced Epithelial to Mesenchymal Transition in the Lens.
Int J Mol Sci. 2019 Apr 27;20(9):
Authors: Taiyab A, Holms J, West-Mays JA
Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-β-induced signaling through both canonical, TGF-β/Smad, and non-canonical, β-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-β-mediated pathways using α-smooth muscle actin (α-SMA) as a molecular marker for EMT. We show that Smad3 inhibition via SIS3 prevents nuclear translocation of β-catenin and MRTF-A, and α-SMA expression, suggesting a key role of Smad3 in regulation of MRTF-A and β-catenin nuclear transport in LECs. Further, we demonstrate that inhibition of β-catenin/CBP interaction by ICG-001 decreased the amount of phosphorylated Smad3 upon TGF-β stimulation in addition to significantly decreasing the expression levels of TGF-β receptors, TBRII and TBRI. Overall, our findings demonstrate interdependence between the canonical and non-canonical TGF-β-mediated signaling pathways controlling EMT in the lens.
PMID: 31035577 [PubMed - in process]
Conditional Deletion of AP-2α and AP-2β in the Developing Murine Retina Leads to Altered Amacrine Cell Mosaics and Disrupted Visual Function.
Invest Ophthalmol Vis Sci. 2018 May 01;59(6):2229-2239
Authors: Hicks EA, Zaveri M, Deschamps PA, Noseworthy MD, Ball A, Williams T, West-Mays JA
Purpose: The combined action of the activating protein-2 (AP-2) transcription factors, AP-2α and AP-2β, is important in early retinal development, specifically in the formation of horizontal cells. However, in previous studies, it was not possible to analyze postnatal development and function of additional retinal subtypes.
Methods: We used a double conditional deletion of AP-2α and AP-2β from the retina to further examine the combinatory role of these genes in retinal cell patterning and function in postnatal adult mice as measured by Voronoi domain area and nearest-neighbor distance spatial analyses and ERGs, respectively.
Results: Conditional deletion of both AP-2α and AP-2β from the retina resulted in a variety of abnormalities, including the absence of horizontal cells, defects in the photoreceptor ribbons in which synapses failed to form, along with evidence of aberrant amacrine cell arrangement. Although no significant changes in amacrine cell population numbers were observed in the double mutants, significant irregularities in the mosaic patterning of amacrine cells was observed as demonstrated by both Voronoi domain areas and nearest-neighbor distances analyses. These changes were further accompanied by an alteration in the retinal response to light as recorded by ERGs. In particular, in the double-mutant mice lacking AP-2α and AP-2β, the b-wave amplitude, representative of interneuron signal processing, was significantly reduced compared with control littermates.
Conclusions: Together these findings demonstrate the requirement for both AP-2α and AP-2β in proper amacrine mosaic patterning and a normal functional light response in the retina.
PMID: 29715367 [PubMed - in process]
Relationship between neural crest cell specification and rare ocular diseases.
J Neurosci Res. 2018 Apr 16;:
Authors: Akula M, Park JW, West-Mays JA
Development of the eye is closely associated with neural crest cell migration and specification. Eye development is extremely complex, as it requires the working of a combination of local factors, receptors, inductors, and signaling interactions between tissues such as the optic cup and periocular mesenchyme (POM). The POM is comprised of neural crest-derived mesenchymal progenitor cells that give rise to numerous important ocular structures including those tissues that form the optic cup and anterior segment of the eye. A number of genes are involved in the migration and specification of the POM such as PITX2, PITX3, FOXC1, FOXE3, PAX6, LMX1B, GPR48, TFAP2A, and TFAP2B. In this review, we will discuss the relevance of these genes in the development of the POM and how mutations and defects result in rare ocular diseases.
PMID: 29660784 [PubMed - as supplied by publisher]
Dual function of TGFβ in lens epithelial cell fate: implications for secondary cataract.
Mol Biol Cell. 2017 Apr 01;28(7):907-921
Authors: Boswell BA, Korol A, West-Mays JA, Musil LS
The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFβ has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified. We show here for the first time that TGFβ can induce purified primary lens epithelial cells within the same culture to undergo differentiation into either lens fiber cells or myofibroblasts. Marker analysis confirmed that the two cell phenotypes were mutually exclusive. Blocking the p38 kinase pathway, either with direct inhibitors of the p38 MAP kinase or a small-molecule therapeutic that also inhibits the activation of p38, prevented TGFβ from inducing epithelial-myofibroblast transition and cell migration but did not prevent fiber cell differentiation. Rapamycin had the converse effect, linking MTOR signaling to induction of fiber cell differentiation by TGFβ. In addition to providing novel potential therapeutic strategies for PCO, our findings extend the so-called TGFβ paradox, in which TGFβ can induce two disparate cell fates, to a new epithelial disease state.
PMID: 28209733 [PubMed - indexed for MEDLINE]
Matrix metalloproteinase 9 is associated with peritoneal membrane solute transport and induces angiogenesis through β-catenin signaling.
Nephrol Dial Transplant. 2017 01 01;32(1):50-61
Authors: Padwal M, Siddique I, Wu L, Tang K, Boivin F, Liu L, Robertson J, Bridgewater D, West-Mays J, Gangji A, Brimble KS, Margetts PJ
Background: For patients using peritoneal dialysis (PD), the peritoneal membrane can develop fibrosis and angiogenesis, leading to ultrafiltration failure, chronic hypervolemia and increased risk of technique failure and mortality. Matrix metalloproteinases (MMPs), and specifically the gelatinases (MMP2 and MMP9), may be involved in peritoneal membrane injury.
Methods: From stable PD patients, mesothelial cells were assayed for MMP gene expression. MMP9 was overexpressed in mouse peritoneum by adenovirus, and MMP9 -/- mice were subjected to transforming growth factor β (TGF-β)-induced peritoneal fibrosis.
Results: MMP9 mRNA expression correlated with peritoneal membrane solute transport properties. Overexpression of MMP9 in the mouse peritoneum induced submesothelial thickening and angiogenesis. MMP9 induced mesothelial cell transition to a myofibroblast phenotype measured by increased alpha smooth muscle actin and decreased E-cadherin expression. Angiogenesis was markedly reduced in MMP9 -/- mice treated with an adenovirus expressing active TGF-β compared with wild-type mice. TGF-β-mediated E-cadherin cleavage was MMP9 dependent, and E-cadherin cleavage led to β-catenin-mediated signaling. A β-catenin inhibitor blocked the angiogenic response induced by AdMMP9.
Conclusions: Our data suggest that MMP9 is involved in peritoneal membrane injury possibly through cleavage of E-cadherin and induction of β-catenin signaling. MMP9 is a potential biomarker for peritoneal membrane injury and is a therapeutic target to protect the peritoneal membrane in PD patients.
PMID: 27190383 [PubMed - indexed for MEDLINE]
Aberrant Collagen Composition of the Trabecular Meshwork Results in Reduced Aqueous Humor Drainage and Elevated IOP in MMP-9 Null Mice.
Invest Ophthalmol Vis Sci. 2016 Nov 01;57(14):5984-5995
Authors: De Groef L, Andries L, Siwakoti A, Geeraerts E, Bollaerts I, Noterdaeme L, Etienne I, Papageorgiou AP, Stalmans I, Billen J, West-Mays JA, Moons L
Purpose: Homeostatic turnover of the trabecular meshwork extracellular matrix (ECM) is essential to regulate aqueous humor outflow and to maintain intraocular pressure homeostasis. In this study, we evaluated aqueous humor turnover, intraocular pressure, and trabecular meshwork organization in MMP-9 null mice.
Methods: Intraocular pressure and aqueous humor turnover were measured in MMP-9 null versus wild-type mice. Morphology of the anterior segment of the eye, with special attention to the structural organization of the trabecular meshwork, was investigated by means of optical coherence tomography, light microscopy, and transmission electron microscopy. Furthermore, using quantitative real-time polymerase chain reaction and immunostainings, we evaluated the ECM composition of the trabecular meshwork. Finally, the integrity and function of the retina and optic nerve were assessed, via optical coherence tomography, histologic techniques, and optomotor testing.
Results: MMP-9 null mice displayed early-onset ocular hypertension and reduced aqueous humor turnover. While transmission electron microscopic analysis did not reveal any abnormalities in the cellular organization of the trabecular meshwork, detailed investigation of collagen expression indicated that there is an aberrant trabecular meshwork ECM composition in MMP-9 null mice. Notably, at the age of 13 months, no glaucomatous neurodegeneration was seen in MMP-9 null mice.
Conclusions: Our observations corroborate MMP-9 as an important remodeler of the collagenous composition of the trabecular meshwork and provide evidence for a causal link between MMP-9 deficiency, trabecular meshwork ultrastructure, and ocular hypertension.
PMID: 27820954 [PubMed - indexed for MEDLINE]
β-Catenin/CBP-Dependent Signaling Regulates TGF-β-Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells.
Invest Ophthalmol Vis Sci. 2016 Oct 01;57(13):5736-5747
Authors: Taiyab A, Korol A, Deschamps PA, West-Mays JA
Purpose: Transforming growth factor-β-induced epithelial-mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-β-induced PCO have not been fully characterized. Here, we focus on examining the role of β-catenin/cyclic AMP response element-binding protein (CREB)-binding protein (CBP) and β-catenin/T-cell factor (TCF)-dependent signaling in regulating cytoskeletal dynamics during TGF-β-induced EMT in lens epithelial explants.
Methods: Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-β2 in the presence or absence of the β-catenin/CBP interaction inhibitor, ICG-001, or the β-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed.
Results: An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-β, and colocalized with F-actin filaments. Inhibition of β-catenin/CBP interactions, but not β-catenin/TCF interactions, led to a decrease in TGF-β-induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, α-smooth muscle actin (α-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of β-catenin/CBP-dependent signaling also prevented TGF-β-induced downregulation of epithelial cadherin (E-cadherin) in lens explants.
Conclusions: We show that β-catenin/CBP-dependent signaling regulates fascin, MMP9, and α-SMA expression during TGF-β-induced EMT. We demonstrate that β-catenin/CBP-dependent signaling is crucial for TGF-β-induced EMT in the lens.
PMID: 27787561 [PubMed - indexed for MEDLINE]
RhoA/ROCK signaling regulates TGFβ-induced epithelial-mesenchymal transition of lens epithelial cells through MRTF-A.
Mol Med. 2016 Sep 29;22:
Authors: Korol A, Taiyab A, West-Mays JA
Transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) leads to the formation of ocular fibrotic pathologies, such as anterior subcapsular cataract and posterior capsule opacification. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT, however, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin related transcription factor A (MRTF-A), an actin-binding protein, might be an important mediator of TGFβ-induced EMT in lens epithelial cells. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFβ-induced nuclear accumulation of MRTF-A, and ultimate expression of α-smooth muscle actin (αSMA), a marker of a contractile, myofibroblast phenotype. Using rat lens epithelial explants, we demonstrate that ROCK inhibition using Y-27632 prevents TGFβ-induced nuclear accumulation of MRTF-A, E-cadherin/β-catenin complex disassembly, and αSMA expression. Using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, we further demonstrate the requirement of MRTF-A nuclear localization and activity in the induction of αSMA expression. Overall, our findings suggest that TGFβ-induced cytoskeletal reorganization through RhoA/ROCK/MRTF-A signaling is critical to EMT of lens epithelial cells.
PMID: 27704140 [PubMed - as supplied by publisher]
Conditional deletion of AP-2β in mouse cranial neural crest results in anterior segment dysgenesis and early-onset glaucoma.
Dis Model Mech. 2016 Aug 01;9(8):849-61
Authors: Martino VB, Sabljic T, Deschamps P, Green RM, Akula M, Peacock E, Ball A, Williams T, West-Mays JA
Anterior segment dysgenesis (ASD) encompasses a group of developmental disorders in which a closed angle phenotype in the anterior chamber of the eye can occur and 50% of patients develop glaucoma. Many ASDs are thought to involve an inappropriate patterning and migration of the periocular mesenchyme (POM), which is derived from cranial neural crest cells (NCCs) and mesoderm. Although, the mechanism of this disruption is not well understood, a number of transcriptional regulatory molecules have previously been implicated in ASDs. Here, we investigate the function of the transcription factor AP-2β, encoded by Tfap2b, which is expressed in NCCs and their derivatives. Wnt1-Cre-mediated conditional deletion of Tfap2b in NCCs resulted in post-natal ocular defects typified by opacity. Histological data revealed that the conditional AP-2β NCC knockout (KO) mutants exhibited dysgenesis of multiple structures in the anterior segment of the eye including defects in the corneal endothelium, corneal stroma, ciliary body and disruption in the iridocorneal angle with adherence of the iris to the cornea. We further show that this phenotype leads to a significant increase in intraocular pressure and a subsequent loss of retinal ganglion cells and optic nerve degeneration, features indicative of glaucoma. Overall, our findings demonstrate that AP-2β is required in the POM for normal development of the anterior segment of the eye and that the AP-2β NCC KO mice might serve as a new and exciting model of ASD and glaucoma that is fully penetrant and with early post-natal onset.
PMID: 27483349 [PubMed - in process]
AP-2β Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development.
Invest Ophthalmol Vis Sci. 2016 Mar;57(3):1072-81
Authors: Chen L, Martino V, Dombkowski A, Williams T, West-Mays J, Gage PJ
PURPOSE: The homeodomain transcription factor, PITX2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the PITX2 remain unknown. The purpose of this study was to discover and validate PITX2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea.
METHODS: Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium.
RESULTS: We show that PITX2 is required for the expression of Tfap2b, encoding the AP-2β transcription factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2β. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2β. Endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of AP-2β, whereas LYVE1+ lymphatic vessels are not found.
CONCLUSIONS: The AP-2β transcription factor is an important effector of PITX2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike PITX2, AP-2β is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional PITX2-dependent factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development.
PMID: 26968737 [PubMed - indexed for MEDLINE]
AP-2α is required after lens vesicle formation to maintain lens integrity.
Dev Dyn. 2014 Oct;243(10):1298-309
Authors: Kerr CL, Zaveri MA, Robinson ML, Williams T, West-Mays JA
BACKGROUND: Transcription factors are critical in regulating lens development. The AP-2 family of transcription factors functions in differentiation, cell growth and apoptosis, and in lens and eye development. AP-2α, in particular, is important in early lens development, and when conditionally deleted at the placode stage defective separation of the lens vesicle from the surface ectoderm results. AP-2α's role during later stages of lens development is unknown. To address this, the MLR10-Cre transgene was used to delete AP-2α from the lens epithelium beginning at embryonic day (E) 10.5.
RESULTS: The loss of AP-2α after lens vesicle separation resulted in morphological defects beginning at E18.5. By P4, a small highly vacuolated lens with a multilayered epithelium was evident in the MLR10-AP-2α mutants. Epithelial cells appeared elongated and expressed fiber cell specific βB1 and γ-crystallins. Epithelial cell polarity and lens cell adhesion was disrupted and accompanied by the misexpression of ZO-1, N-Cadherin, and β-catenin. Cell death was observed in the mutant lens epithelium between postnatal day (P) 14 and P30, and correlated with altered arrangements of cells within the epithelium.
CONCLUSIONS: Our findings demonstrate that AP-2α continues to be required after lens vesicle separation to maintain a normal lens epithelial cell phenotype and overall lens integrity and to ensure correct fiber cell differentiation.
PMID: 24753151 [PubMed - indexed for MEDLINE]
Matrix metalloproteinase-9-null mice are resistant to TGF-β-induced anterior subcapsular cataract formation.
Am J Pathol. 2014 Jul;184(7):2001-12
Authors: Korol A, Pino G, Dwivedi D, Robertson JV, Deschamps PA, West-Mays JA
Epithelial-mesenchymal transition (EMT) is associated with fibrotic diseases in the lens, such as anterior subcapsular cataract (ASC) formation. Often mediated by transforming growth factor (TGF)-β, EMT in the lens involves the transformation of lens epithelial cells into a multilayering of myofibroblasts, which manifest as plaques beneath the lens capsule. TGF-β-induced EMT and ASC have been associated with the up-regulation of two matrix metalloproteinases (MMPs): MMP-2 and MMP-9. The current study used MMP-2 and MMP-9 knockout (KO) mice to further determine their unique roles in TGF-β-induced ASC formation. Adenoviral injection of active TGF-β1 into the anterior chamber of all wild-type and MMP-2 KO mice led to the formation of distinct ASC plaques that were positive for α-smooth muscle actin, a marker of EMT. In contrast, only a small proportion of the MMP-9 KO eyes injected with adenovirus-expressing TGF-β1 exhibited ASC plaques. Isolated lens epithelial explants from wild-type and MMP-2 KO mice that were treated with TGF-β exhibited features indicative of EMT, whereas those from MMP-9 KO mice did not acquire a mesenchymal phenotype. MMP-9 KO mice were further bred onto a TGF-β1 transgenic mouse line that exhibits severe ASC formation, but shows a resistance to ASC formation in the absence of MMP-9. These findings suggest that MMP-9 expression is more critical than MMP-2 in mediating TGF-β-induced ASC formation.
PMID: 24814605 [PubMed - indexed for MEDLINE]
Nuclear translocation of myocardin-related transcription factor-A during transforming growth factor beta-induced epithelial to mesenchymal transition of lens epithelial cells.
Mol Vis. 2013;19:1017-28
Authors: Gupta M, Korol A, West-Mays JA
PURPOSE: Transforming growth factor beta (TGFβ) is a known inducer of epithelial to mesenchymal transition (EMT), and studies in other systems have shown that nuclear localization of the myocardin-related transcription factor (MRTF) is downstream of TGFβ. In the following study, we investigated whether nuclear translocation of MRTF-A or MRTF-B is involved in TGFβ-induced EMT of lens epithelial cells (LECs). We further investigated the relationship between matrix metalloproteinase-2 and -9 (MMP-2/9) and MRTF in the EMT of LECs.
METHODS: Rat lens explant cultures were used as the model system. Explants were treated with TGFβ, an MMP-2/9 inhibitor, or actin binding drugs and immunostained for alpha smooth muscle actin (αSMA), MRTF-A, and MRTF-B. Cytoplasmic and nuclear intensities of cells were measured using ImageJ. Production of αSMA was measured using western blot analysis and ImageJ.
RESULTS: Untreated explant cells exhibited little αSMA expression, and MRTF-A and B were found to reside primarily in the cytosol. However, when stimulated with TGFβ, a significantly greater number of cells exhibited nuclear expression of MRTF-A, accompanied by an increase in αSMA expression. However, MRTF-B remained in the cytoplasm following TGFβ treatment. Cotreatment with an MMP-2/9 inhibitor and TGFβ resulted in reduced MRTF-A nuclear localization and αSMA expression compared to cells treated with TGFβ alone.
CONCLUSIONS: Our results are the first to demonstrate the expression of MRTF-A in LECs and that its nuclear translocation can be stimulated by TGFβ. Our data further suggest that MMP-2 and -9 are involved in the translocation of MRTF-A in LECs during TGFβ-induced EMT.
PMID: 23687438 [PubMed - indexed for MEDLINE]
Altered expression of transforming growth factor beta 1 and matrix metalloproteinase-9 results in elevated intraocular pressure in mice.
Mol Vis. 2013;19:684-95
Authors: Robertson JV, Siwakoti A, West-Mays JA
PURPOSE: Extracellular matrix remodeling is thought to have profound effects on tissue architecture and associated function. We have shown previously that overexpression of transforming growth factor beta (TGFβ), which stimulates matrix accumulation, results in altered morphology, cataract, and ocular hypertension in rodents. We have further shown that TGFβ-induced cataracts can be mitigated through inhibition of the matrix metalloproteinases (MMP) MMP-2 and MMP-9. We therefore sought to determine whether loss of MMP expression also altered TGFβ-induced changes in intraocular pressure (IOP).
METHODS: To carry out this study, TGFβ1 transgenic mice were bred onto a MMP-9 null background. IOP measurements were made at 1- to 2-, 2- to 3-, and 3- to 4-month time points using a TonoLab rebound tonometer. Histological and immunofluorescence findings were obtained at the same time points.
RESULTS: Our results demonstrate that lens-specific expression of TGFβ1 in mice results in altered morphology of the anterior segment and an accompanying significant increase in IOP. TGFβ1 transgenic mice bred onto the MMP-9 null background exhibited a further increase in IOP. Interestingly, the MMP-9-deficient animals (without the TGFβ transgene), which exhibited normal angle morphology, had increased IOP levels compared to their wild-type littermates.
CONCLUSION: These results indicate that TGFβ and MMP-9 likely act independently in regulating IOP. Additionally, MMP-9 plays an important role in maintaining IOP, and further investigation into the mechanisms of MMP-9 activity in the anterior angle may give clues to how extracellular matrix remodeling participates in ocular hypertension and glaucoma.
PMID: 23559862 [PubMed - indexed for MEDLINE]
Activation of the hedgehog signaling pathway in the developing lens stimulates ectopic FoxE3 expression and disruption in fiber cell differentiation.
Invest Ophthalmol Vis Sci. 2012 Jun 05;53(7):3316-30
Authors: Kerr CL, Huang J, Williams T, West-Mays JA
PURPOSE: The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens.
METHODS: We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm.
RESULTS: Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death.
CONCLUSIONS: Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.
PMID: 22491411 [PubMed - indexed for MEDLINE]
Overlapping expression patterns and redundant roles for AP-2 transcription factors in the developing mammalian retina.
Dev Dyn. 2012 Apr;241(4):814-29
Authors: Bassett EA, Korol A, Deschamps PA, Buettner R, Wallace VA, Williams T, West-Mays JA
BACKGROUND: We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2β and AP-2γ, showed expression patterns similar to AP-2α.
RESULTS: Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2β are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/β-immunopositive population. To address possible redundant roles for AP-2α and AP-2β during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone.
CONCLUSIONS: These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.
PMID: 22411557 [PubMed - indexed for MEDLINE]
Corneal epithelial cell adhesion and growth on EGF-modified aminated PDMS.
J Biomed Mater Res A. 2010 Jun 01;93(3):1043-9
Authors: Klenkler BJ, Dwivedi D, West-Mays JA, Sheardown H
Growth factor tethering has significant potential to mediate cellular responses in biomaterials and tissue engineering. We have previously demonstrated that epidermal growth factor (EGF) can be tethered to polydimethylsiloxane (PDMS) substrates and that these surfaces promoted interactions with human corneal epithelial cells in vitro. The goal of the current work was to better understand the specific effects of the tethered growth factor on the cells. The EGF was reacted with a homobifunctional N-hydroxysuccinimide (NHS) polyethylene glycol (PEG) derivative, and then bound to allyamine plasma-modified PDMS. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium for periods of up to 5 days. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Expression of matrix proteins and alpha(6)-integrins was assessed by immunostaining and confocal microscopy. A centrifugation assay was used to determine cell adhesion under an applied detachment force. Binding of EGF was found to significantly increase cell numbers and coverage across the surfaces at 5 days of culture in vitro. Immunofluorescence experiments indicate increased expression of fibronectin, laminin, and alpha(6)-integrins on the EGF-modified surfaces, and expression is localized at the cell-material interface as observed by confocal microscopy. In accordance with these results, the highest quantity of adherent cells is found on the EGF-modified subtrates at 5 days of culture. The results provide initial evidence that binding of EGF may be used to improve the epithelialization of and the adhesion of the cells on a polymeric artificial cornea device.
PMID: 19753622 [PubMed - indexed for MEDLINE]
AP-2alpha knockout mice exhibit optic cup patterning defects and failure of optic stalk morphogenesis.
Hum Mol Genet. 2010 May 01;19(9):1791-804
Authors: Bassett EA, Williams T, Zacharias AL, Gage PJ, Fuhrmann S, West-Mays JA
Appropriate development of the retina and optic nerve requires that the forebrain-derived optic neuroepithelium undergoes a precisely coordinated sequence of patterning and morphogenetic events, processes which are highly influenced by signals from adjacent tissues. Our previous work has suggested that transcription factor activating protein-2 alpha (AP-2alpha; Tcfap2a) has a non-cell autonomous role in optic cup (OC) development; however, it remained unclear how OC abnormalities in AP-2alpha knockout (KO) mice arise at the morphological and molecular level. In this study, we show that patterning and morphogenetic defects in the AP-2alpha KO optic neuroepithelium begin at the optic vesicle stage. During subsequent OC formation, ectopic neural retina and optic stalk-like tissue replaced regions of retinal pigment epithelium. AP-2alpha KO eyes also displayed coloboma in the ventral retina, and a rare phenotype in which the optic stalk completely failed to extend, causing the OCs to be drawn inward to the midline. We detected evidence of increased sonic hedgehog signaling in the AP-2alpha KO forebrain neuroepithelium, which likely contributed to multiple aspects of the ocular phenotype, including expansion of PAX2-positive optic stalk-like tissue into the OC. Our data suggest that loss of AP-2alpha in multiple tissues in the craniofacial region leads to severe OC and optic stalk abnormalities by disturbing the tissue-tissue interactions required for ocular development. In view of recent data showing that mutations in human TFAP2A result in similar eye defects, the current findings demonstrate that AP-2alpha KO mice provide a valuable model for human ocular disease.
PMID: 20150232 [PubMed - indexed for MEDLINE]
Development and use of the lens epithelial explant system to study lens differentiation and cataractogenesis.
Prog Retin Eye Res. 2010 Mar;29(2):135-43
Authors: West-Mays JA, Pino G, Lovicu FJ
Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal lens biology and its pathology. Much of this has been made possible through the establishment and use of the lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of lens epithelium on its native substratum, has been used effectively to study many cellular processes, including lens epithelial cell proliferation, fiber cell differentiation, cell apoptosis as well as epithelial-to-mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFbeta family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in lens cells. There is no question that the lens epithelial explant system has served as a powerful tool to further our understanding of lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant lens cell behavior are to be trialed.
PMID: 20006728 [PubMed - indexed for MEDLINE]
Ocular gene transfer of active TGF-beta induces changes in anterior segment morphology and elevated IOP in rats.
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):308-18
Authors: Robertson JV, Golesic E, Gauldie J, West-Mays JA
Purpose. Transforming growth factor beta (TGF-beta) is known to play a crucial role in wound healing and fibrotic tissue remodeling. A large body of evidence suggests a role for this cytokine in the pathogenesis of glaucoma; however, the mechanisms by which it affects anterior segment morphology are not well understood. Therefore, the purpose of this study was to examine the effects of TGF-beta overexpression on anterior segment morphology and subsequent effects on intraocular pressure. Methods. Adenoviral gene transfer was used to deliver active TGF-beta1 to the rat eye. Measurements of intraocular pressure were taken with a tonometer on days 0, 14, 21, and 29. Histologic analysis was undertaken to examine anterior segment morphology, and markers of matrix deposition and fibrosis were used. Results. Gene transfer of TGF-beta in the anterior segment resulted in the formation of peripheral anterior synechiae (PAS), which consisted of a fibroproliferative region of corneal endothelial cells, matrix accumulation, and decrease in trabecular meshwork expression of alpha-smooth muscle actin. These features were accompanied by ocular hypertension. Conclusions. Gene transfer of TGF-beta into the anterior segment induces aberrant PAS associated with the transition of corneal endothelial cells and subsequent matrix deposition. These features are highly reminiscent of human iridocorneal endothelial (ICE) syndrome. Gene transfer of TGF-beta can, therefore, be used to induce anatomic changes in the anterior segment in a rodent model that result in ocular hypertension.
PMID: 19696167 [PubMed - indexed for MEDLINE]