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TME5C exerts cytoprotective and angiogenic functions
between two groups under multiple conditions using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison tests using GraphPad Software (La Jolla, CA, USA). Differences in animal survival (Kaplan-Meier survival curves) were analyzed by log-rank test. A P-value < 0.05 was considered statistically significant.
Results
TME5C, but not TME5A or TME5B, stimulates proliferation of endothelial cells
Figure 1A shows the amino acid sequences of TM mutants TME5A, TME5B, and TME5C. We first exam- ined the effects of each TM mutant on the proliferation of
Figure 4. TME5C increases the levels of p-ERK, p-AKT, p-p38, and Mcl-1 in endothelial cells. (A and C). HUVECs or HHSECs were exposed to control diluents (PBS as control) or TM mutants (500 nM). After 48 h, proteins were extracted and subjected to western blot analyses. The membrane was sequentially probed with the indicated antibodies. (B and D). Relative quantifications of p-ERK, p-AKT, p-p38, and Mcl-1. ImageJ software was used to measure the band intensities after western blotting. All experiments were performed three times. Results represent the mean ± SD. *P<0.05. TM: thrombomodulin; HUVECs: human umbilical vein endothelial cells; HHSECs: human hepatic sinusoidal endothelial cells.
HUVECs or HHSECs. Exposure to TME5C (25-1000 nM) but not molar equivalents of TME5A or TME5B stimulat- ed their proliferation in a dose-dependent manner, as assessed by BrdU incorporation assays. For example, 500 nM TME5C stimulated proliferation of HUVEC and HHSECs by nearly 1.5-fold. The highest dose of TME5C (1000 nM) did not further stimulate the proliferation of HUVECs and HHSECs (Figure 1B). TME5 also stimulated the proliferation of endothelial cells in a dose-dependent manner, which was consistent with our previous study.25 TME5 produced the maximum pro-proliferative effect at a concentration of 30 nM (Online Supplementary Figure S1).
We conducted further experiments with a TME5C mutant with a single amino acid substitution (G→A, Table 1). This mutant form of TME5C lost the ability to
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