Endothelial-immune crosstalk contributes to vasculopathy in non-alcoholic fatty liver disease (2022)
Figure 3

Figure 3. Validation of chemokine hallmarks in human endothelial cells and mouse disease models.

(a) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. Results are indicated by mean ± SD. *p<0.05; **p<0.01; ns, not significant (Mann-Whitney test).

(b) Left: Representative images showing non-treated and LPS+FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585nm, and readings were normalized with blanks (n = 3 donors/ group). Results are indicated by mean ± SD. ns, not significant (Two-way ANOVA comparing NAFLD and healthy groups).

(c) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in LPS+FFA-treated or non-treated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to non-treated healthy BOECs, with 3 technical replicates per donor. Results are indicated by mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Two-way ANOVA).

(d) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs. *p<0.05; ns, not significant (Mann-Whitney test). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers).

(e) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander's Coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analysed, with 10-20 independent regions of interest per mouse were analysed for image quantification. ****p<0.0001 (Mann-Whitney test).

(f) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW: Aortic wall; L: lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analysed, with 10-20 delimitated endothelial regions per mouse analysed for image quantification. ****p<0.0001 (Mann-Whitney test).

(g) Left: Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet. Sections were stained for leukocyte markers CD45 (red) to determine the extent of leukocyte infiltration. Dotted lines demarcate the aortic walls (AW). L: lumen. Scale bar: 20 µm. Right: Box-whisker plot of mean fluorescence intensity (MFI) of CD45 signals as a readout for the amount of infiltrated CD45+ leukocytes within the aortic walls. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). n = 3 mice per group, with 2 tissue sections analyzed per animal. **p<0.01 (Mann-Whitney).