Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine

Okumura, Naoki; Kay, EunDuck P.; Nakahara, Makiko; Hamuro, Junji; Kinoshita, Shigeru; Koizumi, Noriko

doi:10.1371/journal.pone.0058000

Cited by 146 publications

(144 citation statements)

References 41 publications

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“…The Descemet's membrane and endothelium complex was stripped from corneas and treated with 0.2% collagenase type II and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 3 hours at 37°C. After centrifugation, the primary cells were resuspended in culture medium: OptiMEM-I supplemented with 5% fetal bovine serum, 10 µM Y-27632, 0.5 µg/ml R-spondin and 10 µM SB431542 (TGF-β RI Kinase inhibitor VI) (16) with 0.05% trypsin and 5 mM EDTA in phosphate-buffered saline (PBS) for 10 minutes. Second or third passage human CEC were used for all experiments.…”

Section: Methodsmentioning

confidence: 99%

“…A major hallmark of the mesenchymal transition is down-regulation of the junctional protein E-cadherin and upregulation of cytoskeletal proteins such as fibronectin and vimentin (19)(20)(21). Moreover, there is also increased expression of α1 (COL1A1) and α2 (COL1A2) chains of type I collagen (21,22), and this is also observed in CEC (12, 16,23). It has been reported that overexpression of transcription factors such as members of SNAI family, SNAI1 and SNAI2, and members of ZEB family, ZEB1 and ZEB2, regulate suppression of E-cadherin expression and overexpression of fibronectin, vimentin, and α-smooth muscle actin (αSMA) (24)(25)(26).…”

mentioning

confidence: 99%

“…Moreover, severely injured CEC can undergo mesenchymal transition through which they lose their polarity and assume a fibroblastic phenotype. These CEC also exhibit enhanced migration, proliferation and secrete type I collagen leading to retrocorneal membrane formation (11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

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Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

Lee

Jung

Heur

2018

Journal of Biological Chemistry

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Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness due to endothelial dysfunction. We previously reported that IL-1β stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-boxbinding homeobox 1 (ZEB1), and ZEB2. This, in turn, induced expression of fibronectin, vimentin and, type I collagen and suppressed E-cadherin in CECs in vitro and ex vivo. siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.The cornea is the anterior, transparent tissue of the human eye and consists of epithelium, stroma and endothelium. The corneal endothelium is composed of a monolayer of cells and plays a critical role in maintaining corneal transparency that is critical for sharp vision through its pump function (1,2). Adult human corneal endothelial cells (CEC) are usually mitotically inactive and arrested at the G 1 phase of the cell cycle (3,4). Because of the cell cycle arrest, there is a progressive decline in CEC density that can be further accelerated by injury, such as intraocular surgery or infection. When the density decreases below a critical threshold, corneal edema ensues, leading to loss of transparency and vision. Vision loss secondary to endothelial dysfunction is a common indication for corneal transplantation in developed nations.There have been many attempts to overcome the cell cycle arrest using various approaches including the use of growth factors such as FGF, EGF, and TGF-β, the use of endothelial cell growth supplements (5-9), and disruption of contact inhibition using EDTA (10). Moreover, severely injured CEC can undergo mesenchymal transition through which they lose their polarity and assume a fibroblastic phenotype. These CEC a...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

Lee

Jung

Heur

2018

Journal of Biological Chemistry

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“…The Descemet membrane and endothelium complex was stripped from corneas and treated with 0.2% collagenase type II and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 3 h at 37°C. After centrifugation, the primary cells were resuspended in culture medium (OptiMEM I supplemented with 5% fetal bovine serum, 10 M Y27632, 0.5 g/ml R-spondin, and 10 M SB431542 (TGF-␤ receptor inhibitor kinase inhibitor VI) (48)) and plated on 24-well tissue culture plates precoated with FNC Coating Mix (Biological Research Faculty and Facility, Inc., Ijamsville, MD) and laminin (Sigma). For subculture, confluent primary cultures were treated with 0.05% trypsin and 5 mM EDTA in PBS for 10 min.…”

Section: Methodsmentioning

confidence: 99%

WNT10B Enhances Proliferation through β-Catenin and RAC1 GTPase in Human Corneal Endothelial Cells

Lee

Heur

2015

Journal of Biological Chemistry

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Background: Adult human corneal endothelial cells are G 1 -arrested, often necessitating transplantation in patients with endothelial dysfunction. Results: WNT10B enhances proliferation in human corneal endothelial cells. Conclusion: Modulation of the WNT10B pathway could be used to treat vision loss because of corneal endothelial dysfunction. Significance: Modulation of the WNT10B pathway may provide a means of addressing the impending increase in donor cornea demand.

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“…12,13 After culturing for 14 days, the FECD-derived CECs and the control CECs were immortalized using SV40 and hTERT to produce iFECD and iHCEC cell lines, respectively. The coding sequences of the SV40 large T antigen and hTERT genes were amplified by PCR and were TA-cloned into a commercial lentiviral vector.…”

Section: Immortalization Of Cecsmentioning

confidence: 99%

Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy

Okumura

Minamiyama

et al. 2015

Laboratory Investigation

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Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is a major cause of corneal transplantation. It is characterized by abnormal deposition of extracellular matrix (ECM), such as corneal guttae, accompanied by a loss of endothelial cells. Although recent studies have revealed several genomic factors, the molecular pathophysiology of FECD has not yet been revealed. In this study, we establish a cellular in vitro model by using immortalized corneal endothelial cells obtained from late-onset FECD and control patients and examined the involvement of epithelial mesenchymal transition (EMT) on excessive ECM production. We demonstrate that the EMT-inducing genes ZEB1 and SNAI1 were highly expressed in corneal endothelial cells in FECD and were involved in excessive production of ECM proteins, such as type I collagen and fibronectin through the transforming growth factor (TGF)-β signaling pathway. Furthermore, we found that SB431542, a specific inhibitor of TGF-β type I ALK receptors, suppressed the expression of ZEB1 and Snail1 followed by reduced production of ECM. These findings suggest that increased expression levels of ZEB1 and Snail1 in FECD cells were responsible for an increased responsiveness to TGF-β present in the aqueous humor and excessive production of ECM. In addition, these results suggest that the regulation of EMT-related genes by blocking the TGF-β signaling pathway may be a feasible therapeutic strategy for FECD.

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Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine

Cited by 146 publications

References 41 publications

Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

WNT10B Enhances Proliferation through β-Catenin and RAC1 GTPase in Human Corneal Endothelial Cells

Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy

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