Microvascular endothelial scavenger receptor class B type I protects against heart failure with preserved ejection fraction by inhibiting T-cell cardiotropism

Figures

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  Figure 1. SR-B1 is predominantly present in cardiac microvascular endothelial cells and significantly decreased in the HFpEF heart. (A) Immunofluorescence staining of SR-B1 in the mouse cardiac left ventricle co-stained with fluorescence-conjugated isolectin B4 (IsoB4) to label endothelial cells and DAPI to label nuclei. Scale bar, 25 μm. Arrow, SR-B1/IsoB4 co-localization. Dashed ellipse: vascular lumen. (B) Double-staining of SR-B1 and CD31 in the cardiac left ventricle of a healthy human. Scale bar, 100 μm. Arrow, SR-B1/CD31 co-localization. Dashed ellipse: vascular lumen. (C) Scarb1 (SR-B1) and Pecam1 (CD31) mRNA abundance was determined in adult mouse cardiac endothelial cells (MCECs) and non-MCECs by RT-qPCR; Pecam1 transcript level served as quality control of purified MCECs (n = 3). Statistical significance was evaluated by Welch’s t test. (D) Upper panel, SR-B1 protein abundance was determined in adult mouse cardiac endothelial cells (MCECs) and non-MCECs by immunoblotting; CD31 abundance served as quality control of purified MCECs; GAPDH as a loading control. Lower panel, Quantification of SR-B1 protein abundance in MCECs and non-MCECs (n = 5). Statistical significance was…

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  Figure 2. Endothelial-restricted SR-B1 deletion drives cardiac diastolic dysfunction in HFpEF. (A) A schematic overview of the CRISPR-based genome-editing strategy used to generate the SR-B1 deficiency mice. (B) Scarb1 (SR-B1) mRNA abundance was assessed in the cardiac ventricle of Sfl/fl and SΔEC mice 2 weeks post-tamoxifen administration (n = 11, Mann–Whitney test). (C) SR-B1 protein abundance was evaluated in mouse cardiac endothelial cells (MCECs) isolated from Sfl/fl mice and SΔEC mice 2 weeks after tamoxifen administration by immunoblotting and statistically evaluated via Student’s t test (n = 5, 4). (D–L) Sfl/fl mice and SΔEC mice were fed with a normal diet or HFD plus L-NAME regimens for 20 weeks to investigate the effect of SR-B1 deletion on cardiac function of HFpEF mice (n = 9, 9, 10, 10; statistical differences were evaluated by two-way ANOVA followed by Bonferroni post-hoc test). (D) Workflow of in vivo experiments. (E) Representative M-mode images in the parasternal short-axis view. (F) FS, left ventricular fractional shortening. (G) LVPWd, left ventricular diastolic posterior wall thickness. (H) LV mass, left ventricular mass. (I) LVRI, left ventricular remodeling index.…

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  Figure 3. Endothelial-specific SR-B1 deficiency aggravates cardiac pathological remodeling, and AAV-mediated restoration of SR-B1 expression in SΔEC mice effectively ameliorates cardiac dysfunction and remodeling induced by SR-B1 deletion. (A–F) Cardiac pathological examinations were conducted in Sfl/fl and SΔEC mice euthanized after 20 weeks of either a normal diet or HFD plus L-NAME (A two-way ANOVA followed by Bonferroni post-hoc test was applied to assess the significant difference). (A) Ratio between cardiac ventricle weight and tibia length (TL) in Sfl/fl and SΔEC mice euthanized after 20 weeks of normal diet or HFD plus L-NAME (n = 9, 9, 10, 10). (B) Ratio between wet lung weight and TL (n = 9, 9, 10, 10). (C) Representative images of hematoxylin & eosin (H&E; scale bar, 2.5 mm) and Masson’s trichrome (MT; scale bar, 250 μm) staining in transversal sections of heart. (D) Fibrosis area in Masson’s trichrome-stained transversal sections was analyzed (n = 9, 9, 10, 10). (E) Col3a1, Col1a1, and Bnp mRNA abundance in the left cardiac ventricle were measured via RT-qPCR (n = 9, 9, 9, 8). (F) Representative images of immunofluorescence staining of αSMA to label myofibroblast co-stained…

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  Figure 4. Genetic lineage tracing of endothelial cells in vivo reveals that cardiac endothelial cells cannot transdifferentiate into myofibroblast in HFpEF. (A) Representative bright-field images of MCECs incubated with vehicle or 10 ng/mL TGF-β1, captured on day 4, are presented. Scale bar, 100 μm. (B) EnMT-related genes and Scarb1 (SR-B1) mRNA abundance were measured in MCECs isolated from wild-type mice following stimulation with different factors via RT-qPCR (n = 7). Statistical analyses were performed using distinct approaches based on data distribution characteristics: Col1a1 and Col3a1 expression data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test; Acta2 (αSMA) expression was evaluated using Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons; and the remaining genes were assessed using ordinary one-way ANOVA with Bonferroni’s multiple comparisons correction. (C) Immunofluorescent staining of CD31 and αSMA to investigate the potential ability of MCECs in the cardiac fibrosis area of HFpEF mice. Scale bar, 50 μm. (D) Workflow of experiment to generate endothelial-specific SR-B1-deficient and RFP overexpression mice (a…

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  Figure 5. Single-cell RNA sequencing reveals that endothelial SR-B1 deletion promotes inflammatory activation of the cardiac microvascular endothelial cell, which is confirmed by increasing cardiac abundance of CXCL10 and CXCR3-positive T cell infiltration in the left cardiac ventricle. (A) A schematic diagram of a single-cell transcriptomic sequencing experiment. (B) Projection of cardiac ECs onto UMAP. (C) UMAP visualization of cardiac capillary ECs. (D) The comparison of the percentage of cardiac capillary EC subclusters from left cardiac ventricles of different groups. (E) Heatmap depicts expression levels of differentially expressed marker genes in subclusters of cardiac microvascular ECs. Yellow represents high gene expression, and purple represents low gene expression. (F) Scarb1 (SR-B1), Cxcl1, Cxcl2, Cxcl10, and Cxcr3 mRNA abundance were measured in left ventricle of Sfl/fl and SΔEC HFpEF mice (n = 9, 8, the fold change differences between Cxcl10 and Cxcl1 or Cxcl2 were analyzed using a two-way ANOVA followed by Bonferroni post-hoc test, while the remaining comparisons were assessed using Student’s t test or Mann–Whitney test). (G) Immunoblotting of SR-B1, CXCL10, and CXCR3 in the…

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  Figure 6. The transcriptional factor IRF1 is identified as a direct mediator responsible for CXCL10 transcript upregulated by microvascular endothelial SR-B1 deletion. (A) SCARB1 (SR-B1), CXCL1, CXCL2, and CXCL10 mRNA abundances were measured in HCMECs transfected with SCARB1 siRNA (siSR-B1) or a normal control siRNA (siNC). Significant differences were analyzed by Student’s t test (n = 7). (B) CXCL10 mRNA abundance was determined in different groups via RT-qPCR (n = 5; statistical differences were evaluated by two-way ANOVA followed by Bonferroni post-hoc test). HCMECs transfected with siNC or siSR-B1 were incubated with lipopolysaccharide (LPS, 20 ng/mL) for 8 h. (C) CXCL10 protein abundance was evaluated in the four groups by immunoblotting (n = 9; two-way ANOVA followed by Bonferroni post-hoc test). (D) SR-B1 and CXCL10 protein abundance was assessed by immunoblotting in HCMECs co-transfected with siRNA (siNC or siSR-B1) and lentivirus (overexpression of homoSR-B1 [oeSR-B1] or control lentivirus [lvNC]), and analyzed via one-way ANOVA followed by Bonferroni post-hoc test (n = 5). (E) IRF1-binding motif obtained from JASPAR website (Matrix ID Ma0050.2). Predicted site was located in…

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  Figure 7. Endothelial-restricted CXCL10 overexpression deteriorates cardiac diastolic dysfunction and pathological remodeling in HFpEF, correlating with enhanced infiltration of CXCR3 + T cells in the heart. (A) A schematic diagram of the experiment workflow to investigate the role of endothelial CXCL10 in HFpEF mice. Echocardiographic evaluation of cardiac functional changes of HFpEF mice infected with AAV1-mediated endothelial-restricted CXCL10 (HFpEFCXCL10) or control ZsGreen (HFpEFZsGreen) delivery ((B–I), n = 7, 8; statistical differences were evaluated by Student’s t test). (B) FS. (C) LVPWd. (D) LV mass, left ventricular mass. (E) LVRI. (F) Heart rate in parasternal short-axis view. (G) MPI. (H) E/e′ ratio. (I) Running distance during the exercise exhaustion test. (J) Ratio between cardiac ventricle weight and tibia length (TL) (n = 7, 8; Mann–Whitney test). (K) Ratio between wet lung weight and TL (n = 7, 8; Mann–Whitney test). (L) Representative images of H&E (scale bar, 2.5 mm) and MT (scale bar, 250 μm) staining. (M) Fibrosis area in MT-stained transversal sections relative to HFpEFZsGreen mice (n = 7, 8; Student’s t test). (N) Col3a1, Col1a1, Bnp, Cxcl10, and Cxcr3 mRNA…

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  Figure 8. IRF1-CXCL10/CXCR3 axis implicates in the pathogenesis of clinical HFpEF patients. (A) BNP and COL3A1 mRNA abundances were assessed in healthy and HFpEF individuals by RT-qPCR (n = 5, 12; Mann–Whitney test). Data were presented as mean ± SEM. (B) SCARB1 (SR-B1) mRNA abundance was evaluated in healthy and HFpEF individuals (n = 5, 12; Student’s t test). Data were presented as mean ± SEM. (C) IRF1, CXCL10, and CXCR3 mRNA abundances in healthy and HFpEF individuals (n = 5,12; Welch’s or Student’s t test). Data were presented as mean ± SEM. (D) Plasma CXCL10 concentrations were significantly elevated in HFpEF patients (respectively, n = 67, 109; Mann–Whitney test). The box plots are defined as follows: Center of box, Median (healthy: 114.75 pg/mL; HFpEF: 159.95 pg/mL). Bounds, Interquartile range (IQR; 25th to 75th percentiles) (healthy: 68.92–145.20 pg/mL; HFpEF: 91.46–296.91 pg/mL); Bounds of whiskers, Lowest and highest values within 1.5 × IQR (lower whisker: healthy 26.91 pg/mL, HFpEF 23.27 pg/mL; upper whisker: healthy 249.27 pg/mL, HFpEF 593.29 pg/mL); Minima and Maxima, Full data range (healthy: 26.91–482.91 pg/mL; HFpEF: 23.27–1039.95 pg/mL). (E) Bivariate linear correlation…