Obesity accelerates endothelial-to-mesenchymal transition in adipose tissues of mice and humans

Figures

• 

  Figure 1. Adipose tissue endoMT in obesity-induced endothelial cell lineage tracing mice. (A) Overview schematic of the experimental strategy. (B) Representative flow cytometry gating strategy describes the identification of CD31-Acta2 + cells, then quantification of eYFP% of this population. (C) EndoMT rate defined as eYFP% of CD45-CD31-Acta2 + cells in subcutaneous and visceral adipose tissue from mice on Control or HF/HS diet for 13, 26, or 52 weeks (student's t-test, p-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001). (D) Linear regression between rate of EndoMT and age in individual mice in subcutaneous or visceral adipose tissue between Control diet and HF/HS diet (linear correlation analysis, p-values: * <0.05, ** <0.01).

• 

  Figure 2. Associations of EndoMT to time on diet, body weight, and adipose tissue location. Multivariate regression analysis quantified relative association of EndoMT to weeks on diet or weight of mice in (A) subcutaneous or (B) visceral adipose tissue (slope and p-value presented from multivariate regression analysis). (C) Pairwise analysis of EndoMT in subcutaneous and visceral adipose tissues from the same mice are connected by lines in 13, 26, and 15 weeks on HF/HS diet, with paired differences quantified and presented as EndoMT in visceral minus EndoMT in subcutaneous adipose tissue (paired student's t-test, p-values: * <0.05, ** <0.01). (D) Rate of EndoMT was quantified in lung, kidney, liver, subcutaneous adipose tissue, and visceral adipose tissue in negative control mice (Cre- with no lineage tracing) and in reporter mice (Cre + with lineage tracing) at 13 weeks on HF/HS diet (one-way ANOVA with post-hoc Tukey, p-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001).

• 

  Figure 3. Immunofluorescence imaging of endoMT cells in obese visceral adipose tissue of mice. Visceral adipose tissue from Cdh5CreERT2; Rosa26eYFP lineage tracing mice on HF/HS diet was immunofluorescently labeled for cell nuclei by DAPI, the eYFP reporter, Acta2, and CD31 overlaid with brightfield imaging to visualize EndoMT cells as DAPI + eYFP + Acta2 + CD31-. (A) A representative large vessel is shown, with a magnified section to highlight EndoMT cells by white arrows in (1) all channels, (2) brightfield, (3) DAPI, (4) eYFP, (5) Acta2, and (6) CD31. (B) A representative image of the stroma with small vessels is shown, with a magnified section to highlight an EndoMT cell by a white arrow in (1) all channels, (2) brightfield, (3) DAPI, (4) eYFP, (5) Acta2, and (6) CD31.

• 

  Figure 4. Transcriptomic analysis of murine endoMT cells in visceral adipose tissue of obese mice. (A) Differential expression of all genes identified through bulk RNA sequencing of CD31 + eYFP + endothelial cells vs. CD31-eYFP + EndoMT cells (n = 3 in each condition) was visualized by volcano plot showing the log2 (fold change) vs. the -log10 (p-value) of the change in gene expression, with genes significantly upregulated in endothelial cells in blue, EndoMT cells in red, and specific genes involved in endothelial cell function or EndoMT labeled. (B) Enrichment of GSEA Hallmark Pathways in all differentially expressed genes were visualized by log2 (fold change) and p-values listed, with pathways upregulated by 10% in red and downregulated by 10% in blue. (C) Enrichment profiles presented for terms enriched in endothelial cells (reactive oxygen species, fatty acid metabolism, peroxisome) and in EndoMT cells (epithelial mesenchymal transition, inflammatory response, TGFβ signaling).

• 

  Figure 5. Endomt in cultured human adipose-derived microvascular endothelial cells. (A) Overview schematic of experimental strategy. (B) Representative immunofluorescence image of HAMEC induced with pro-inflammatory cytokines, visualizing DAPI, CD31, and ACTA2. (C) Differential expression of all genes identified through bulk RNA sequencing of Control vs. EndoMT cells (n = 3 in each condition) was visualized by volcano plot showing the log2 (fold change) vs. the -log10 (p-value) of the change in gene expression, with genes significantly upregulated in Control cells in blue, EndoMT cells in red, and specific genes involved in endothelial cell function or EndoMT labeled. (D) Enrichment plots of GSEA Hallmark Pathway analysis of inflammatory response, TGFβ signaling, IL6/JAK/STAT3 signaling, and P53 pathway. (E) Genes that are significantly upregulated in both EndoMT conditions (HAMEC and Cdh5CreERT2; Rosa26eYFP HF/HS datasets), presented as relative log2 (fold change) and -log10 (p-value) quantifications, and genes with a 2-fold increase in both conditions labeled and highlighted in red.

• 

  Figure 6. Analysis of endothelial cells from single cell RNA sequencing datasets of visceral adipose tissue from obese patients. (A) UMAP of endothelial cells from datasets published in Vijay 2020, Hildreth 2021, and Emont 2022, with an EndoMT cell cluster highlighted by a red circle, identified by reduced relative PECAM1 expression and increased relative ACTA2 expression plotted by UMAP. (B) Log2 (fold change) and -log10 (p-value) of genes upregulated in the EndoMT cell cluster compared to other endothelial cells. (C) Relative KLF4 expression in endothelial cells plotted by UMAP. (D) Linear correlation between expression of KLF4 and combined expression of genes that are co-regulated in bulk RNA sequencing datasets from HAMEC and Cdh5CreERT2;Rosa26eYFP (r2 = 0.456, p-value <0.0001, left) and genes in the inflammatory response GSEA (r2 = 0.527, p-value <0.0001, right).