SIRT1 Activation Using CRISPR/dCas9 Promotes Regeneration of Human Corneal Endothelial Cells through Inhibiting Senescence

Human corneal endothelial cells (hCECs) are restricted in proliferative capacity in vivo. Reduction in the number of hCEC leads to persistent corneal edema requiring corneal transplantation. This study demonstrates the functions of SIRT1 in hCECs and its potential for corneal endothelial regeneration. Cell morphology, cell growth rates and proliferation-associated proteins were compared in normal and senescent hCECs. SIRT1 was activated using the CRISPR/dCas9 activation system (SIRT1a). The plasmids were transfected into CECs of six-week-old Sprague–Dawley rats using electroporation and cryoinjury was performed. Senescent cells were larger, elongated and showed lower proliferation rates and lower SIRT1 levels. SIRT1 activation promoted the wound healing of CECs. In vivo transfection of SIRT1a promoted the regeneration of CECs. The proportion of the S-phase cells was lower in senescent cells and elevated upon SIRT1a activation. SIRT1 regulated cell proliferation, proliferation-associated proteins, mitochondrial membrane potential, and oxidative stress levels. In conclusion, corneal endothelial senescence is related with a decreased SIRT1 level. SIRT1a promotes the regeneration of CECs by inhibiting cytokine-induced cell death and senescence. Gene function activation therapy using SIRT1a may serve as a novel treatment strategy for hCEC diseases.

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Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

10.21203/rs.3.rs-691242/v1 ◽

2021 ◽

Author(s):

Mohit Parekh ◽

Hefin Rhys ◽

Tiago Ramos ◽

Stefano Ferrari ◽

Sajjad Ahmad

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Ex Vivo ◽

Treatment Options ◽

Proliferative Capacity ◽

Corneal Endothelial Cells ◽

Human Corneal Endothelial Cells ◽

The Media

Abstract Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs), are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line – HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 hours. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labelling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

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Persimmon Leaves (Diospyros kaki) Extract Enhances the Viability of Human Corneal Endothelial Cells by Improving Na+-K+-ATPase Activity

Pharmaceuticals ◽

10.3390/ph15010072 ◽

2022 ◽

Vol 15 (1) ◽

pp. 72

Author(s):

Ramsha Afzal ◽

Hyung Bin Hwang

Keyword(s):

Endothelial Cells ◽

Atpase Activity ◽

Basolateral Membrane ◽

Corneal Edema ◽

Ethanol Extract ◽

Diospyros Kaki ◽

Corneal Endothelial Cells ◽

Human Corneal Endothelial Cells ◽

Corneal Decompensation

The Na+/K+-ATPase, present in the basolateral membrane of human corneal endothelial cells (HCECs), is known to play an important role for corneal transparency. Na+/K+-ATPase dysfunction is one of the major causes of corneal decompensation. The ethanol extract of Diospyros kaki (EEDK) has been reported to increase corneal cell viability. Thus, we treated HCECs with EEDK and studied its effects on HCECs survival and Na+/K+-ATPase against cytotoxic drugs like staurosporine (ST) and ouabain (OU). Firstly, survival assays, (MTT assay and live dead-imaging) showed that decreased HCECs viability by ST and OU was significantly recovered by EEDK co-treatment. Secondly, Na+/K+-ATPase activity assays revealed that EEDK enhanced Na+/K+-ATPase enzymatic activity (* p < 0.01) with/without ST and OU. Finally, Na+/K+-ATPase expression analysis (Western Blot and confocal microscopy) demonstrated that EEDK treatment with/without ST and OU facilitates Na+/K+-ATPase expression in HCECs. Taken together, our findings led us to the conclusion that EEDK might aid HCECs survival in vitro by increasing the activity and expression of Na+/K+-ATPase enzyme. Since Na+/K+-ATPase activity is important to maintain cellular function of HCECs, we suggest that EEDK can be a potential effective agent against corneal edema and related corneal disorders.

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Long noncoding RNA KCNQ1OT1 induces pyroptosis in diabetic corneal endothelial keratopathy

AJP Cell Physiology ◽

10.1152/ajpcell.00053.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. C346-C359 ◽

Cited By ~ 4

Author(s):

Yanyan Zhang ◽

Zhen Song ◽

Xuran Li ◽

Shuo Xu ◽

Sujun Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Long Noncoding Rna ◽

Noncoding Rna ◽

Rna Binding ◽

Cultured Cells ◽

Corneal Edema ◽

Corneal Endothelial Cells ◽

Caspase 1

Diabetic corneal endothelial keratopathy is an intractable ocular complication characterized by corneal edema and endothelial decompensation, which seriously threaten vision. It has been suggested that diabetes is associated with pyroptosis, a type of programmed cell death via the activation of inflammation. Long noncoding RNA KCNQ1OT1 is commonly associated with various pathophysiological mechanisms of diabetic complications, including diabetic cardiomyopathy and diabetic retinopathy. However, whether KCNQ1OT1 is capable of regulating pyroptosis and participates in the pathogenesis of diabetic corneal endothelial keratopathy remains unknown. The aim of this study was to investigate the mechanisms of KCNQ1OT1 in diabetic corneal endothelial keratopathy. Here, we reveal that KCNQ1OT1 and pyroptosis can be triggered in diabetic human and rat corneal endothelium, along with the high glucose-treated corneal endothelial cells. However, miR-214 expression was substantially decreased in vivo and in experiments with cultured cells. LDH assay was also used to verify the existence of pyroptosis in high glucose-treated cells. Bioinformatics prediction and luciferase assays showed that KCNQ1OT1 may function as a competing endogenous RNA binding miR-214 to regulate the expression of caspase-1. To further analyze the KCNQ1OT1-mediated mechanism, miR-214 mimic and inhibitor were introduced into the high glucose-treated corneal endothelial cells. The results showed that upregulation of miR-214 attenuated pyroptosis; conversely, knockdown of miR-214 promoted it. In addition, KCNQ1OT1 knockdown by a small interfering RNA decreased pyroptosis factors expressions but enhanced miR-214 expression in corneal endothelial cells. To understand the signaling mechanisms underlying the prepyroptotic properties of KCNQ1OT1, si-KCNQ1OT1 was cotransfected with or without miR-214 inhibitor. The results showed that pyroptosis was repressed after silencing KCNQ1OT1 but was reversed by cotransfection with miR-214 inhibitor, suggesting that KCNQ1OT1 mediated pyroptosis induced by high glucose via targeting miR-214. Therefore, the KCNQ1OT1/miR-214/caspase-1 signaling pathway represents a new mechanism of diabetic corneal endothelial keratopathy progression, and KCNQ1OT1 could potentially be a novel therapeutic target.

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In Vivo Fluorescence Visualization of Anterior Chamber Injected Human Corneal Endothelial Cells Labeled With Quantum Dots

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.19-27788 ◽

2019 ◽

Vol 60 (12) ◽

pp. 4008 ◽

Cited By ~ 1

Author(s):

Munetoyo Toda ◽

Hiroshi Yukawa ◽

Jun Yamada ◽

Morio Ueno ◽

Shigeru Kinoshita ◽

...

Keyword(s):

Quantum Dots ◽

Endothelial Cells ◽

Anterior Chamber ◽

Corneal Endothelial Cells ◽

Human Corneal Endothelial Cells ◽

In Vivo Fluorescence

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Balance of S1P1and S1P2signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00097.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. H33-H42 ◽

Cited By ~ 55

Author(s):

Jen-Fu Lee ◽

Sharon Gordon ◽

Rosendo Estrada ◽

Lichun Wang ◽

Deanna L. Siow ◽

...

Keyword(s):

Endothelial Cells ◽

Peripheral Circulation ◽

Microvascular Permeability ◽

Cremaster Muscle ◽

Sprague Dawley ◽

Vascular Bed ◽

Cultured Endothelial Cells ◽

Sprague Dawley Rats ◽

Inhibitory Signaling

Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P1-5receptors). S1P1and S1P2receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P1-mediated signaling by SEW2871 and FTY720, two agonists of S1P1, significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P1-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P2signaling by JTE-013, a specific antagonist of S1P2, S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P1- and S1P2-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P1and S1P2signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.

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In Vitro Evaluation and Transplantation of Human Corneal Endothelial Cells Cultured on Biocompatible Carriers

Cell Transplantation ◽

10.1177/0963689720923577 ◽

2020 ◽

Vol 29 ◽

pp. 096368972092357 ◽

Cited By ~ 1

Author(s):

Daniele Spinozzi ◽

Alina Miron ◽

Jessica T. Lie ◽

Mehrdad Rafat ◽

Neil Lagali ◽

...

Keyword(s):

Endothelial Cells ◽

Reference Model ◽

Corneal Transplantation ◽

Corneal Endothelial Cells ◽

Effective Treatment Option ◽

Human Donor ◽

Human Corneal Endothelial Cells ◽

Descemet Membrane ◽

Anterior Lens Capsule

Corneal transplantation is currently the only effective treatment option for dysfunctional corneal endothelial cells (CEC). In this study, we test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), LinkCell™ bioengineered collagen sheets of 20-µm thickness (LK20), and denuded Descemet membrane (dDM) as tissue-engineered grafts for Descemet membrane (DM) endothelial keratoplasty (DMEK) to bypass the problem of donor tissue availability. Primary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96% ± 4%). hCEC on HALC and LK20 showed unremarkable expression of zonula occludens-1 (ZO-1) and Na+/K+-adenosine triphosphatase (ATPase), while Na+/K+-ATPase expression of cells seeded on dDM was mainly cytoplasmic. All hCEC–carrier constructs were evaluated by simulating DMEK surgery in vitro using a human donor cornea without DM mounted on an artificial anterior chamber (AC) and a regular DMEK-graft used as a surgical reference model. During in vitro surgery, hCEC–HALC constructs behaved most similarly to a DMEK-graft during implantation and unfolding, showing good adhesion to the bare stroma. On the other hand, hCEC–LK20 and hCEC–dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC–dDM constructs showed similar graft adherence as hCEC–HALC constructs, while adherence of hCEC–LK20 constructs was less effective. After the in vitro surgery, the estimated area populated by viable cells on the hCEC–HALC and hCEC–LK20 constructs was ∼83% and ∼67%, respectively. Overall, hCEC–HALC constructs behaved most similarly to a DMEK-graft during in vitro DMEK surgery, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC–LK20 and hCEC–dDM constructs.

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Follow-up study of human corneal endothelial cells, photographed in vivo before enucleation and 20 years later in grafts

Acta Ophthalmologica Scandinavica ◽

10.1034/j.1600-0420.1999.770305.x ◽

1999 ◽

Vol 77 (3) ◽

pp. 273-276 ◽

Cited By ~ 7

Author(s):

Kristiina Vasara ◽

Kirsi Setälä ◽

Pekka Ruusuvaara

Keyword(s):

Endothelial Cells ◽

Corneal Endothelial Cells ◽

Human Corneal Endothelial Cells ◽

Follow Up Study

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Human corneal endothelial cells grafts to replace cadaveric donor corneas

10.31219/osf.io/p9x7e ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Endothelial Cells ◽

Culture Media ◽

Corneal Transplantation ◽

Ocular Tissue ◽

Corneal Endothelial Cells ◽

Cadaveric Donor ◽

Human Corneal Endothelial Cells ◽

The Future ◽

Limbal Stem Cell Transplantation ◽

Donor Corneas

Human corneal endothelial cells (HCEC) grafts to replace cadaveric donor corneas have made tremendous progress in recent years, and this article highlights the most recent discoveries and important achievements in this area. Cell injection therapy with cultured HCEC has just finished its first clinical research, which showed promising results. This follows on the heels of the success of autologous limbal stem cell transplantation in bioengineered ocular tissue transplantation. Transplantation techniques like bioengineered endothelium sheet transplantation and full-thickness corneal transplantation are likely to become more prevalent in the future decade. This goal will be achieved by a combination of current advancements in HCEC propagation, high-quality culture media, and 3D or 4D tissue culture, which will be the trend in the future.

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