Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

Abstract

Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

Inhibition of mTOR by Rapamycin Aggravates Corneal Epithelial Stem Cell Deficiency by Upregulating Inflammatory Response

“…A recent study by Gidfar et al showed that rapamycin increased survival of CESCs and reduced apoptosis in cultures of human corneal epithelial cells, as opposed to our in vivo findings. These findings by Gidfar et al might provide the rationale of using rapamycin for an ex vivo expansion of CESC before cell transplantation, but do not serve as a rationale for direct injection of rapamycin into the eye for the treatment of a disease. Because CESCs are vulnerable to collateral damage exerted by inflammatory cells, the effects of rapamycin on CESCs should be investigated in the context of inflammatory microenvironment, apart from its direct effects on the cells.…”

“…Previous studies demonstrated that mTOR inhibition with rapamycin increased the survival of human oral mucosal epithelial stem cells and CESCs in culture and prevented stem cell senescence . Similarly, other studies showed that intestinal and oral mucosal epithelial stem cells were protected against apoptosis upon ionizing radiation in mice by rapamycin .…”

Corneal Wound Healing Effects of Mesenchymal Stem Cell Secretome Delivered Within a Viscoelastic Gel Carrier

“…Primary human corneal epithelial cells (HCECs) were harvested from human cadaver corneas kindly provided by Eversight (Chicago, IL) as described elsewhere . Briefly, the iris, conjunctiva, and sclera were removed and the cornea was washed with phosphate‐buffered saline (PBS) at least three times.…”

Current and emerging therapies for corneal neovascularization

“…In addition, rapamycin can promote T-cell anergy independently of the inhibition of proliferation even in the presence of T-cell receptor activation and co-stimulation by CD28 and IL-2. 183, 184 Systemic and topical administration of sirolimus has been associated with a significant decrease of corneal opacity and vascularization as well as decreased corneal IL-6 and TGF-β 1 levels after alkali ocular injury in mice. 185 Moreover, topical everolimus significantly decreased CNV induced with silver nitrate in rats, by decreasing the expression of VEGFR-2 and ERK 1/2.…”