The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells

Abstract

Citation: Hongo A, Okumura N, Nakahara M, Kay EP, Koizumi N. The effect of a p38 mitogen-activated protein kinase inhibitor on cellular senescence of cultivated human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2017;58:3325-3334. DOI:10.1167/iovs.16-21170 PURPOSE. We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs).METHODS. HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-b-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA.RESULTS. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 6 657 cells/mm 2 and 1752 6 628 cells/mm 2 , respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na þ /K þ -ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-b-gal staining, a low nuclear/ cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580.CONCLUSIONS. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

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

“…15 Even if cultured HCECs do not acquire these fibroblastic changes, they can undergo senescence, which decreases the cell density and again produces cells that are not applicable for clinical use. 16 An additional complication is that the proliferative ability of HCECs is limited even under ideal culture conditions, so multiple procedures have been developed to promote cell proliferation. [29][30][31][32] For instance, we reported that the use of a ROCK inhibitor, 13 conditioned medium derived from mesenchymal stem cells, 14 and laminin 511 fragment as a culture substrate 17 enhances cell proliferation.…”

“…Corneal endothelial growth medium was prepared by conditioning basal medium mix (OptiMEM-I, 8% fetal bovine serum [ 14,16 Briefly, confluent 3T3 fibroblasts were incubated with 4 lg/mL mitomycin C (MMC) (Kyowa Hakkko Kirin Co., Ltd., Tokyo, Japan) for 2 hours, seeded on plastic dishes at a cell density of 2 3 10 4 cells/cm 2 , and cultured with DMEM (Life Technologies Corp., Grand Island, NY, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin (Nacalai Tesque, Inc., Kyoto, Japan). The 3T3 fibroblasts were washed 3 times with PBS and cultured with basal medium mix for an additional 24 hours.…”

“…By contrast, HCECs cultured with SB203580, SB431542, and Y-27632 exhibited hexagonal morphology and similar cell size and morphology, which is consistent with our previous reports. 13,16 HCECs cultured with some inhibitors, such as Withaferin A, LY2904002, SAHA, and IBMX, grew as monolayers but varied in cell size and morphology like the control cells (Fig. 1A, Supplementary Fig.…”

“…1B). We further evaluated the effect of a combination of SB203580 16 and Y-27632 13 or SB431542, 15 which we previously reported to be beneficial for culturing HCECs. SB203580 and Y-27632 (but not SB431542) significantly increased the incorporation of BrdU.…”

“…10 Multiple problems arise when culturing HCECs, as these cells have limited proliferative ability, undergo transformation into a fibroblastic phenotype that will not regenerate corneal endothelium, and exhibit senescence that decreases the cell density. [12][13][14][15][16] These undesirable features of HCECs extend the duration required to obtain sufficient cell numbers, which will ultimately increase the final cost of a regenerative medical product.…”