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

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

Antioxidants ◽

10.3390/antiox9111085 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1085

Author(s):

Hye Jun Joo ◽

Dae Joong Ma ◽

Jin Sun Hwang ◽

Young Joo Shin

Keyword(s):

Endothelial Cells ◽

Corneal Transplantation ◽

Corneal Edema ◽

Corneal Endothelial Cells ◽

Sprague Dawley ◽

Human Corneal Endothelial Cells ◽

Sprague Dawley Rats ◽

Associated Proteins ◽

Activation Therapy

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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