PMC

Data are mean ± SD (n = 6) and normalized to the initial cell seeding number (indicated by dashed line). An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively.

OEC motility was assessed in response to 1.0 μM S1P and/or 50 ng/mL VEGF for 12h under normoxia and hypoxia (A). S1P and VEGF combined resulted in accelerated wound closure compared to either stimuli alone under both normoxia and hypoxia (B). Data are mean ± SD (n = 5) and normalized to the average untreated control values for either normoxia or hypoxia (indicated by the dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

A modified transwell assay was used to quantify directed migration/3D matrix invasion of OECs towards S1P in a fibrin gel (A). Gradients of S1P and VEGF were established by sustained delivery from an alginate hydrogel (B). While combined release of S1P and VEGF resulted in greater migration after 48h in both normoxia and hypoxia, S1P alone was equally sufficient under hypoxia (C). Data are mean ± SD (n = 4). An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

S1P and/or VEGF induced OEC sprouting in a fibrin gel after 1 day of culture in normoxia and hypoxia (A). S1P alone or in combination with VEGF led to significantly more sprouts per bead than VEGF alone under hypoxia (B). Scale bars represent 100 μm and 200 μm (DIC inlay). Data are mean ± SD (n = 4) and normalized to the average untreated control values for either normoxia or hypoxia (indicated by the dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

OEC expression of S1PR1 under normoxia and hypoxia was qualitatively confirmed by immunocytochemistry (A). The percentage of cells expressing S1PR1 on the cell surface, as confirmed with flow cytometry, was enhanced under hypoxia as compared to the normoxic control (B). Combined stimuli of S1P and VEGF resulted in greater S1PR1 mRNA production after 24h in normoxia (C). Combined stimuli also resulted in a greater proportion of S1PR1+ OECs under both normoxia and hypoxia (D and E). Scale bars represent 40 μm. Data are mean ± SD (n = 4) and normalized to the average untreated control values for either normoxia or hypoxia (C, E; indicated by dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.