Restoration of Mitochondrial Integrity, Telomere Length, and Sensitivity to Oxidation by In Vitro Culture of Fuchs' Endothelial Corneal Dystrophy Cells

Gendron, Sébastien P.; Thériault, Marius; Proulx, Stéphanie; Brunette, Isabelle; Rochette, Patrick J.

doi:10.1167/iovs.16-20551

Cited by 25 publications

(16 citation statements)

References 49 publications

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“…The goal of these experiments was to ascertain whether CECs from normal human corneas can be stimulated to proliferate in organ culture where the low basal proliferation may better reflect the in vivo situation 34 compared with dissociated culture systems that produce high levels of proliferation of normal and dystrophic/FECD CECs. 7,8,[44][45][46] In this human corneal organ culture model, unstimulated CECs in the mid zone had very few cells incorporating EdU, indicating that this model is more reflective of the in vivo situation where the number of cells changes only slowly.…”

Section: Discussionmentioning

confidence: 84%

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

Eveleth

Pizzuto

Weant

et al. 2020

Journal of Ocular Pharmacology and Therapeutics

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Purpose: Corneal endothelial dystrophies are characterized by endothelial cell loss and dysfunction. Recent evidence suggests that corneal endothelial cells (CECs) can regenerate although they do not do so under normal conditions. This work sought to test whether CECs can be stimulated to proliferate in organ culture by wounding and/or by treatment with the engineered human fibroblast growth factor 1 (FGF1) derivative TTHX1114. Methods: Human donor corneas obtained from eye banks were maintained in organ culture in the presence or absence of TTHX1114. Wounds in the corneas were created by quartering the corneas. The CEC monolayer was identified as a regular layer by Hoechst staining of the nuclear DNA with cell outlines delineated by immunohistochemical identification of ZO-1. Nuclei and nuclei incorporating 5-ethynyl-2¢-deoxyuridine (EdU) were counted using ImageJ. Results: CECs in normal corneas in undisturbed monolayers had low, but measurable, rates of proliferation. CECs at the edge of a wound had higher rates of proliferation, probably due to the release of contact inhibition. TTHX1114 increased proliferation at wound edges. After 7 days of culture, proliferating CECs formed contiguous groups of labeled cells that did not migrate away from one another. TTHX1114-treated cells, including the EdU labeled proliferating cells, retained normal morphology, including cell/cell junction ZO-1 staining. Conclusions: Proliferation of CECs in organ-cultured corneas is low, but can be stimulated by wounding or by the administration of TTHX1114 with the effects of each being additive. The CEC monolayer appears to have a population of progenitor cells that are susceptible to stimulation.

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Section: Discussionmentioning

confidence: 84%

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

Eveleth

Pizzuto

Weant

et al. 2020

Journal of Ocular Pharmacology and Therapeutics

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“…Total RNA was isolated from postconfluent CEC cultures, as described. 39 Passage 2 or 3 cells were grown and cultured for 24–38 days, with the addition of hydrocortisone (0.4 μg/mL) during the last 7 days of culture. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Toronto, ON, CA) and RNA quality was analyzed using the 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada).…”

Section: Methodsmentioning

confidence: 99%

Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model

Goyer

Thériault

Gendron

et al. 2018

Tissue Engineering Part A

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Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1–3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and β1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor β-induced protein (TGFβIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin β5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and β1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFβIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

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“…Descemet's membrane were kept overnight at 37 °C in growth medium, as previously described 10 . Explants were then washed with Opti-Mem-I (for all markers but CM-H 2 DCFDA; Invitrogen, Burlington, ON, Canada) or PBS (for CM-H 2 DCFDA).…”

Section: Markers Used On Fecd and Healthy Endothelium Explants Cornementioning

confidence: 99%

“…In this hypothetic vicious circle, mitochondria allow for a compensation mechanism for cell depletion, but this eventually leads to an energetic exhaustion that we call "mitochondrial burnout". We previously showed that, in the same FECD explant, individual CECs present different levels of mitochondrial mass 10 , suggesting that each CEC is not at the same stage of the vicious circle. In an attempt to decipher the chronology of events that leads to cell death in FECD, we analyzed the different elements of the vicious circle using human FECD corneal endothelial explants.…”

mentioning

confidence: 99%

Chronology of cellular events related to mitochondrial burnout leading to cell death in Fuchs endothelial corneal dystrophy

Méthot

Proulx

Brunette

et al. 2020

Sci Rep

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fuchs endothelial corneal dystrophy (fecD) is a degenerative eye disease characterized by corneal endothelial cell (CEC) death and the formation of guttae, an abnormal thickening of CEC's basement membrane. At the tissue level, an oxidative stress causing mitochondrial damage and CEC death have been described to explain FECD pathogenesis. At the cellular level, our group has previously observed significant variability in the mitochondrial mass of FECD CECs. This led us to hypothesize that mitochondrial mass variability might play a key role in the chronology of events eventually leading to CEC death in FECD. We thus used different fluorescent markers to assess mitochondrial health and functionality as a function of mitochondrial mass in FECD corneal endothelial explants, namely, intramitochondrial calcium, mitochondrial membrane potential, oxidation level and apoptosis. This has led us to describe for the first time a sequence of events leading to what we referred to as a mitochondrial burnout, and which goes as follow. FECD CECs initially compensate for endothelial cell losses by incorporating mitochondrial calcium to help generating more ATP, but this leads to increased oxidation. CECs then resist the sustained need for more ATP by increasing their mitochondrial mass, mitochondrial calcium and mitochondrial membrane potential. At this stage, CECs reach their maximum capacity and start to cope with irreversible oxidative damage, which leads to mitochondrial burnout. This burnout is accompanied by a dissipation of the membrane potential and a release of mitochondrial calcium, which in turn leads to cell death by apoptosis. The cornea is composed of three cellular layers, i.e. the epithelium, the stroma and the endothelium 1. The stroma needs to be maintained partially dehydrated to allow for the optimal arrangement of its collagen fibrils necessary to ensure its transparency 2. This ATP-demanding function is achieved mainly by the corneal endothelium, a leaky cell monolayer that pumps fluid out of the stroma, into the anterior chamber, maintaining the proper hydration of the corneal stroma 2,3. Fuchs endothelial corneal dystrophy (FECD) is characterized by a loss of corneal endothelial cells (CECs), abnormal extracellular matrix deposition on its basal membrane (Descemet's membrane) leading to the formation of excrescences called guttae, and changes in cellular morphology 4. The loss of CECs ultimately compromises the role of the endothelium to preserve corneal deturgescence. This leads to corneal edema and vision loss 4. FECD affects 4 percent of the US populations over 40 years old 5 and the only treatment currently available for FECD is corneal transplantation 6,7. Although the exact cause of FECD is not yet determined, there is growing evidence that oxidative stress and mitochondria are major contributors 8,9. The mechanism and the sequence of events leading to mitochondrial dysfunction and cell loss still remain to be elucidated. Manifestations of cumulative oxidative damage and mitochondrial dysfunction in ...

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Restoration of Mitochondrial Integrity, Telomere Length, and Sensitivity to Oxidation by In Vitro Culture of Fuchs' Endothelial Corneal Dystrophy Cells

Cited by 25 publications

References 49 publications

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

Proliferation of Human Corneal Endothelia in Organ Culture Stimulated by Wounding and the Engineered Human Fibroblast Growth Factor 1 Derivative TTHX1114

Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model

Chronology of cellular events related to mitochondrial burnout leading to cell death in Fuchs endothelial corneal dystrophy

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