Occurrence of distinctive cells and effects of irradiance on vascular formation in leaves of shade-tolerant C4 grass Paspalum conjugatum.

The denser leaf vasculature of C4 plants than of C3 plants may suit rapid export of assimilates associated with their higher photosynthetic rate. However, some C4 grasses have partially reduced leaf vasculature with vascular bundle (VB)-free bundle-sheath cells called distinctive cells (DCs). The shade-tolerant C4 grass Paspalum conjugatum has such a reduced leaf vascular system with DCs. We examined whether irradiance during growth affects vascular formation in leaves of P. conjugatum grown under 100%, 30%, or 14% sunlight for 1 month alongside the C4 grass maize. Under all conditions, P. conjugatum leaves had partially reduced vasculature: DCs and incomplete small VBs without phloem occurred between VBs with a normal structure consisting of both xylem and phloem. Shaded plants had less phloem in the small VBs than the full-sunlit plants. In maize, however, all VBs always had both xylem and phloem under all light conditions. The net photosynthetic rate of both grasses was reduced under shade; that of P. conjugatum was always lower than that of maize under all light conditions, but was reduced less by shade than that of maize. The light compensation point was lower in P. conjugatum than in maize, indicating that P. conjugatum acclimatizes better to low light. The reduction of phloem in VBs of P. conjugatum may be an acclimatization to shade, because dense vasculature may be expensive for C4 plants growing in environments where the higher photosynthetic rate is not realized.

Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.

PURPOSE: To develop analysis software for cultured human corneal endothelial cells (HCECs). METHODS: Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). RESULTS: Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). CONCLUSIONS: The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.

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

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-ß-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 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-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-ß-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.