Dynamic metabolism of endothelial triglycerides protects against atherosclerosis in mice

Figure 1. Loss of endothelial-specific ATGL leads to spontaneous vascular LD accumulation in vivo and ex vivo. (A) qRT-PCR analysis of Atgl mRNA in LECs from control and Atgl ECKO mice (n = 3/group).****P < 0.0001, unpaired, 2-tailed Student’s t test. (B) Representative Western blot analysis showing ATGL protein levels in LECs isolated from control and Atgl ECKO mice. Each replicate is from 3 independent experiments. (C) Representative confocal fluorescence images of LD detected with BODIPY 493/503 (green) in cultured LECs in EGM-2 media supplemented with either vehicle (–OA) or 1 mM OA overnight. Hoechst 33342 (blue) was used for nuclei staining. (D) Corresponding TG levels quantified in cell lysates (n = 3–4/group). *P < 0.01; **P = 0.003; ***P = 0.0001, 1-way ANOVA with Tukey’s post test. Scale bars: 10 μm. (E) qRT-PCR analysis of Atgl mRNA in FACS-purified aortic ECs (CD31+CD45–) from control and Atgl ECKO mice (n = 3/group). ***P < 0.001, unpaired, 2-tailed Student’s t test. (F) En face images of abdominal aorta from fasted control and Atgl ECKO mice exposed ex vivo to vehicle (–OA) or 1 mM BSA-complexed OA (+OA) in EGM-2 media for 4 hours (n = 5 mice/group). The aorta was…

Figure 2. ATGL deficiency leads to endothelial dysfunction. (A) Cumulative concentration-response curves of developed isometric tension in response to PE in aortic rings harvested from control and Atgl ECKO mice. (B) Cumulative concentration-response curves representing percentages of relaxation of precontracted vessels in response to ACh and (C) the NO˙ donor, SNP, in aortic rings harvested from control and Atgl ECKO mice. Data are represented as mean values ± SEM of 5 to 6 individual experiments (4 rings per mouse). *P < 0.05, 2-way ANOVA with Šidák’s multiple-comparison test. (D) Representative confocal images of en face immunostaining of eNOS protein levels (yellow) in thoracic aorta from control (far left), Atgl ECKO (middle), and eNOS–/– (far right) mice processed and stained identically. ECs were detected by immunostaining for VECAD (red), and nuclei were stained with DAPI (blue). (E) Quantification of aortic images (n = 3/group). **P < 0.01, unpaired, 2-tailed Student’s t test. Scale bars: 50 μm. (F) Representative Western blot analysis showing eNOS protein levels in aortic homogenates of control and Atgl ECKO mice and (G) quantification of n = 6/group. *P < 0.05, unpaired,…

Figure 3. Loss of ATGL in the endothelium upregulates proinflammatory gene expression and VCAM1 surface levels. (A) Volcano plot for differential expression genes (DEGs) from bulk RNA-Seq in LECs that fall above the threshold values ([log2(fold change [FC]) –1 or 1 and –log10(P) > 1.3], red lines) are pictured. Loss of ATGL upregulated 372 genes and downregulated 370 genes in LECs. Red colored dot represents Pnpla2 (ATGL gene name) for reference. (B) Canonical pathway analysis and URA of signaling pathways and gene regulators, respectively, that were significantly higher in Atgl ECKO compared with control LECs. (C) Clustered heatmap from RNA-Seq data showing significantly changed DEGs involved in inflammation between control and Atgl ECKO LEC (n = 3 replicates/group). (D) qRT-PCR analysis of Vcam1 and Ptgs2 mRNA in LECs at baseline and treated with mouse TNF-α overnight (10 ng/mL, 16 hours) from control and Atgl ECKO mice. n = 3/group. *P < 0.05; **P < 0.01; ****P < 0.0001, 2-way ANOVA with Šidák’s multiple-comparison test. (E) Basal and (F) TNF-α–stimulated (10 ng/mL,16 hours) surface VCAM1 levels between control and Atgl ECKO LECs (n = 4 replicates/group) determined by FACS and PE-VCAM1…

Figure 4. Loss of endothelial-specific ATGL leads to ER stress–induced inflammation. (A) Clustered heatmap from RNA-Seq data showing significantly different DEGs involved in ER stress between control and Atgl ECKO LECs. n = 3 replicates/group. (B) Representative Western blot analysis showing higher baseline ER stress marker (ATF4, CHOP) protein levels as well as heightened ER stress and inflammatory (VCAM1, COX2) responsiveness to palmitate (0–0.25 mM, 16 hours) dosing between control and Atgl ECKO LECs. Quantification of VCAM1 (C), COX2 (D), CHOP (E), and ATF4 (F) from palmitate dosing. n = 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.0001; ****P < 0.001, 2-way ANOVA, Šidák’s multiple-comparison test. (G) Representative Western blot analysis showing rescue of baseline VCAM1 and ER stress marker (ATF4, CHOP) levels as well as rescue of heightened VCAM1, COX2, ATF4, and CHOP levels in response to palmitate (100 μM, 16 hours) in the presence or absence of the global ER stress inhibitor 4-PBA (2.5 mM, 8-hour pretreatment) in Atgl ECKO LECs. Quantification of VCAM1 (H), COX2 (I), ATF4 (J), and CHOP (K) from G (n = 3 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001;…

Figure 5. Endothelial deficiency of ATGL accelerates atherosclerosis. (A) Representative en face images of the luminal surface of mouse aorta (aortic root to common iliac bifurcation) stained for Oil Red O to delineate lipid-rich lesions between control and Atgl ECKO on an ApoE–/– background following 12 weeks of atherogenic diet feeding. (B) Corresponding quantification of plaque area as a percentage of total aortic luminal area (n = 10–12/group). ****P < 0.0001, unpaired, 2-tailed Student’s t test. (C) Representative histological staining of aortic sinus stained with Oil Red O and (D) BCA cross sections stained with Oil Red O and hematoxylin for plaque lesions. Scale bars: 500 μm. (E) Quantification of the absolute plaque area (n = 8–9/group) of lesions present in the aortic root and (F) BCA (n = 8–9/group). ****P < 0.0001, unpaired, 2-tailed Student’s t test. (G) Representative immunofluorescence images of aortic sinus cross sections staining of CD68+ macrophages (red) and smooth muscle α-actin+ (SMC) (green) cells. Nuclei were DAPI counterstained (blue) (n = 5/group). Scale bars: 750 μm. (H) Bar graph showing quantification of CD68-positive area as a percentage of plaque lesion area.…

Figure 6. Endothelial deficiency of ATGL upregulates ER stress and proinflammatory gene expression in aortic ECs following a short-term atherogenic diet. (A) Uniform manifold approximation and projection (UMAP) representation of aligned gene expression data in single cells extracted from aortas of control and Atgl ECKO mice injected with rAAV8-mPcsk9 and fed an atherogenic diet for 4 weeks. (B) Heatmap depicting gene-expression patterns of known markers of fibroblasts, SMCs, RBCs, ECs, and CD45+ immune cells. (C) Volcano plot depicting DGE patterns in the EC cluster between Atgl ECKO plus mPcsk9 compared with control plus mPcsk9 mice and expressed as log2 fold change along the y axis versus the percentage of cell expression of individual genes along the x axis. (D) Pathway enrichment of upregulated differentially expressed genes in the EC cluster between Atgl ECKO plus mPcsk9 compared with control plus mPcsk9 mice, expressed as log[–P], and analyzed by IPA. (E) Expression profiles in the EC cluster showing relative expression of the ER stress genes Hspa5, Ddit3, and Atf4 and the proinflammatory gene Vcam1 between control plus mPcsk9 and Atgl ECKO plus mPcsk9 mice. (F) Quantification of MFI…