Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation

Abstract

Citation: Nakahara M, Okumura N, Nakano S, Koizumi N. Effect of a p38 mitogen-activated protein kinase inhibitor on corneal endothelial cell proliferation. Invest Ophthalmol Vis Sci. 2018;59:4218-4227. https:// doi.org/10.1167/iovs.18-24394 PURPOSE. We have performed clinical research on cell-based therapy for corneal endothelial decompensation since 2013. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for promoting proliferation of human corneal endothelial cells (HCECs).METHODS. HCECs were cultured in media supplemented with various low-molecular-weight compounds to screen for the effect of those compounds on cell proliferation. Activation of substrates of p38 MAPK and cell cycle regulatory proteins were evaluated by western blotting. Corneal endothelial wounds were created in a rabbit model, and p38 MAPK was applied in eye drop form, followed by evaluation of cell proliferation in the corneal endothelium by Ki67-immunostaining. RESULTS.HCECs cultured with SB203580 exhibited hexagonal morphology and similar size and morphology, whereas control HCECs cultured without inhibitor exhibited monolayer morphology and varied in size and morphology. Flow cytometry demonstrated that cell proliferation was significantly increased by SB203580. Western blotting showed activation of ATF2 and HSP27 (substrates of p38 MAPK), and upregulation of cyclin D and downregulation of p27 were induced by inhibiting p38 MAPK. In the rabbit model, promotion of wound healing of the corneal endothelium was associated with significant upregulation of Ki67-positive proliferating cells following topical administration of SB203580 when compared with untreated endothelium (50.9% and 36.1%, respectively).CONCLUSIONS. Activation of p38 MAPK signaling due to culture stress might suppress the proliferation of HCECs, whereas a p38 MAPK inhibitor can counteract this activation and enable efficient in vitro HCEC expansion.

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion

“…The progression to senescence in vitro remains a significant barrier to the expansion of cultured CEnC. Identification of oxidative stress as a component of CEnC senescence has prompted investigators to add ascorbic acid as a supplement to early media formulations (added to F99 and M4) to reduce/ inhibit oxidant-induced stress/apoptosis 54,55 and to use p38MAPK inhibitors to delay the onset of senescence, but this approach has shown mixed results 18,56,57 . In addition, senescent CEnC in vitro are characterized by the Senescence-Associated Secretory Phenotype (SASP), which includes the secretion of immune-regulating factors (cytokines, chemokines and growth factors) that have been demonstrated to support tumorigenesis in adjacent epithelial tissues [58][59][60][61] .…”

P38α Deficiency in Macrophages Ameliorates Murine Experimental Colitis by Regulating Inflammation and Immune Process

“…MAPKs are involved in multiple physiological processes, including cell growth, metabolism, differentiation and cell death (Huang, et al 2009;Roux and Blenis 2004;Zhang and Liu 2002). Activation of p38 MAPK suppresses the proliferation of corneal endothelial cells, whereas the p38 MAPK inhibitor counteracts this effect (Nakahara, et al 2018). P38α de ciency in neonatal muscle regulates cellular hyperproliferation and maturation (Perdiguero, et al 2007).…”

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion

“…The progression to senescence in vitro remains a significant barrier to the expansion of cultured CEnC. Identification of oxidative stress as a component of CEnC senescence has prompted investigators to add ascorbic acid as a supplement to early media formulations (added to F99 and M4) to reduce/inhibit oxidant-induced stress/apoptosis (Serbecic and Beutelspacher, 2005; Shima et al, 2011) and to use p38MAPK inhibitors to delay the onset of senescence, but this approach has shown mixed results (Hongo et al, 2017; Nakahara et al, 2018; Sheerin et al, 2012). In addition, senescent CEnC in vitro are characterized by the Senescence-Associated Secretory Phenotype (SASP), which includes the secretion of immune-regulating factors (cytokines, chemokines and growth factors) that have been demonstrated to support tumorigenesis in adjacent epithelial tissues (Georgilis et al, 2018; Laberge et al, 2015; Lau and David, 2019; Lopes-Paciencia et al, 2019).…”