TSC22D1 promotes liver sinusoidal endothelial cell dysfunction and induces macrophage M1 polarization in non-alcoholic fatty liver disease

Figure 1 Single-cell atlas of liver tissue from two groups of mice. A: T-distributed stochastic neighbor embedding plot of single cells from liver tissue of two groups of mice; B: Uniform manifold approximation plot of single cells from liver tissue of two groups of mice; C: Heatmap of characteristic genes of various cell types in liver tissue; D: Cell proportion plot in each sample. t-SNE: T-distributed stochastic neighbor embedding; UMAP: Uniform manifold approximation; NK: Nature killer.

Figure 2 Cell communication analysis in nonalcoholic fatty liver disease. A: Differential gene expression analysis showing up- and down-regulated genes across all eight clusters. An adjusted P value < 0.01 is indicated in red, while an adjusted P value ≥ 0.01 is indicated in black; B: Kyoto Encyclopedia of Genes and Genomes enrichment analysis of genetic variations in hepatic sinusoidal endothelial cells; C: Gene Ontology enrichment analysis of genetic variations in hepatic sinusoidal endothelial cells; D: Analysis of TSC22D1 expression levels in hepatic sinusoidal endothelial cells and the strength of communication with other cell types; E: Different cells interact via the tumor necrosis factor-like weak inducer of apoptosis fibroblast growth factor-inducible 14 signaling pathway. NK: Nature killer; FC: Fold change; COVID-19: Corona virus disease 2019; MAPK: Mitogen-activated protein kinase; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology; BP: Biological process; CC: Cellular component; MF: Molecular function; 5’-UTR: 5’ untranslated region; mRNA: Message RNA.

Figure 3 High-fat diet upregulates TSC22D1 expression and promotes liver sinusoidal endothelial cell dysfunction. A: Hematoxylin eosin, Sirius red, and Oil red O staining of liver tissues from three groups of mice; B: Biochemical indices of the three groups of mice; C: Electron microscopy showing fenestrae in liver tissues with orange arrows; D: Immunohistochemical staining for cluster of differentiation (CD) 34, laminin, and angiotensin-2; E: Immunohistochemical staining for α-smooth muscle actin, E-cadherin, and N-cadherin; F: Western blot analysis for TSC22D1 expression; G: Immunofluorescence staining for CD31 and TSC22D1. Scale bar 100 μm, 100 × magnification. Scale bar 50 μm, 200 × magnification. Scale bar 2 μm, 5000 × magnification. n = 6 mice each group. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. One-way analysis of variance. HE: Hematoxylin eosin; HFD: High-fat diet; CD: Cluster of differentiation; Ang-2: Angiotensin-2; NS: No significant; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TG: Triglyceride; SEM: Scanning electron microscopy; SMA: Smooth muscle actin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 4 Oxidized low-density lipoprotein upregulates TSC22D1 expression and promotes microvascular and endothelial-mesenchymal transition of liver sinusoidal endothelial cells. A: Oil red O staining for the verification of the oxidized low-density lipoprotein (oxLDL) intervention model; B: Western blot analysis of TSC22D1 expression in normal and oxLDL-treated liver sinusoidal endothelial cells; C: Tube formation assay; D: Immunofluorescence staining for cluster of differentiation 34, laminin, and angiotensin-2, α-smooth muscle actin, E-cadherin, and N-cadherin; E: Fluorescence double staining for TSC22D1 and tumor necrosis factor-like weak inducer of apoptosis. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Student’s t test. Scale bar 50 μm, 200 × magnification. NC: Negative control; oxLDL: Oxidized low-density lipoprotein; CD: Cluster of differentiation; DAPI: 4’,6-diamidino-2-phenylindole; Ang-2: Angiotensin-2; SMA: Smooth muscle actin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TWEAK: Tumor necrosis factor-like weak inducer of apoptosis.

Figure 5 TSC22D1 promotes the microvascular and endothelial-mesenchymal transition of liver sinusoidal endothelial cells via the tumor necrosis factor-like weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway. A: Western blot analysis for the verification of TSC22D1 overexpression plasmid; B: Immunofluorescence staining for cluster of differentiation 34, laminin, angiotensin-2, α-smooth muscle actin, E-cadherin, and N-cadherin; C: Tube formation assay; D: Electron microscopy showing fenestrae density in liver sinusoidal endothelial cells; E: Fluorescence double staining for TSC22D1 and tumor necrosis factor-like weak inducer of apoptosis (TWEAK); F: Western blot analysis for TSC22D1 and TWEAK/fibroblast growth factor-inducible 14 pathway. Scale bar 100 μm, 100 × magnification. Scale bar 50 μm, 200 × magnification. Scale bar 10 μm, 1000 × magnification. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. One-way analysis of variance. NS: No significant; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; CD: Cluster of differentiation; DAPI: 4’,6-diamidino-2-phenylindole; Ang-2: Angiotensin-2; SMA: Smooth muscle actin; TWEAK: Tumor necrosis factor-like weak inducer of apoptosis; FN14: Fibroblast growth factor-inducible 14.

Figure 6 Tumor necrosis factor-like weak inducer of apoptosis promotes macrophage M1 polarization secreted by liver sinusoidal endothelial cells overexpressing TSC22D1 via the tumor necrosis factor-like weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway. A: Enzyme-linked immunosorbent assay (ELISA) analysis of tumor necrosis factor-like weak inducer of apoptosis in the supernatants of liver sinusoidal endothelial cells (LSECs); B: Flow cytometry analysis detecting the expression levels of M1 and M2 macrophages; C: ELISA analysis of interleukin (IL)-6, tumor necrosis factor-α, IL-10, and transforming growth factor-β in the supernatants of macrophages cultured in conditioned media from LSECs; D: Quantitative real-time polymerase chain reaction analysis of inducible nitric oxide synthase, monocyte chemoattractant protein-1, arginase-1 and cluster of differentiation 206 in macrophages. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. One-way analysis of variance. TWEAK: Tumor necrosis factor-like weak inducer of apoptosis; CD: Cluster of differentiation; FITC: Fluorescein Isothiocyanate; APC: Antigen-presenting cell; NS: No significant; IL: Interleukin; TNF: Tumor necrosis factor; TGF: Transforming growth factor; iNOS: Inducible nitric oxide synthase; MCP: Monocyte chemoattractant protein; Arg-1: Arginase-1.

Figure 7 Targeting TSC22D1 alleviates liver sinusoidal endothelial cells dysfunction and inhibits macrophage M1 polarization via the tumor necrosis factor-like weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway in vivo. A: The interference efficiency of different short hairpin RNAs against TSC22D1 via quantitative real-time polymerase chain reaction analysis; B: Hematoxylin eosin, Sirius red, and Oil red O staining of liver tissues from the adeno-associated virus serotype 8-short hairpin RNA-negative control group and the recombinant adeno-associated virus serotype 8 carrying TSC22D1-targeting short hairpin RNA vector group; C: Fluorescence double staining for cluster of differentiation (CD) 31 and TSC22D1; D: Immunohistochemical staining for CD34, laminin, angiotensin-2, α-smooth muscle actin, E-cadherin, and N-cadherin in liver tissues; E: Immunohistochemical staining for mouse epidermal growth factor-like module-containing mucin-like hormone receptor-like 1, inducible nitric oxide synthase, and CD206; F: Western blot analysis for TSC22D1 and tumor necrosis factor-like weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway. Scale bar 100 μm, 100 × magnification. Scale bar 50 μm, 200 × magnification. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Student’s t test. n = 6 mice each group. sh-TSC22D1: The recombinant adeno-associated virus serotype 8 carrying TSC22D1-targeting short hairpin RNA vector; sh-NC: The recombinant adeno-associated virus serotype 8 carrying negative control short hairpin RNA vector; F4/80: Mouse epidermal growth factor-like module-containing mucin-like hormone receptor-like 1; HE: Hematoxylin eosin; CD: Cluster of differentiation; DAPI: 4’,6-diamidino-2-phenylindole; Ang-2: Angiotensin-2; SMA: Smooth muscle actin; iNOS: Inducible nitric oxide synthase; NS: No significant; TWEAK: Tumor necrosis factor-like weak inducer of apoptosis; FN14: Fibroblast growth factor-inducible 14; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.