Figure 3: Lung vascular injury expands the proliferative S1PR1⁺ endothelial cell population.

A and B, GFP-gated EC (GFP⁺CD31⁺CD45⁻) from LPS (10 mg/kg, i.p) exposed lungs of S1PR1-GFP reporter mice. A, representative FACS plot at indicated time points. B, quantification of GFP⁺CD31⁺CD45⁻ (S1PR1⁺ EC) as a percentage of total lung EC (CD31⁺CD45⁻) post injury (n=7 mice/group). C and D, lung sections were immunostained with endothelial-specific marker, von-Willebrand Factor (vWF) and S1PR1⁺ EC were determined using confocal analysis. C, a representative micrograph. The inset shows x5 magnified vessel. Scale bar 50 μm. D, number of S1PR1⁺ EC over vessel area in lungs of S1PR1-GFP reporter mice (n=4 mice/group). E, a representative micrograph showing BrdU⁺S1PR1⁺ EC in unexposed or LPS-exposed S1PR1-GFP reporter mice lungs. Mice received LPS (10 mg/kg, i.p) followed by BrdU (80mg/kg, i.p.) injection 4h before harvesting lungs. Lungs sections were immunostained with anti-BrdU antibody (n=3 mice/group). Scale bar 50 μm. F, a representative scatter plot of showing S1PR1⁺ EC proliferation using CD31⁺CD45⁻Ki-67⁺ antibodies after without or with LPS challenge (10 mg/kg, i.p). B and D individual data with mean ±SD. Data in B were analyzed using one-way ANOVA followed by Post hoc Tukey’s multiple comparisons test, while in D unpaired t test was used (See also Online Table II). B, p=0.0211, p=2.56E-15, p=4.23E-15, p=5.48E-14; and D, p=3.14E-05 indicate significance relative to time “0h LPS or No LPS”. ns=not significant.