IFN-γ reprograms cardiac microvascular endothelial cells to mediate doxorubicin transport and influences the sensitivity of mice to doxorubicin-induced cardiotoxicity

Figures

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  Fig. 1. Different sensitivities to DIC are observed in mice. a, Kaplan–Meier survival curves of mice pretreated with saline (control) or 12.3 mg/ml (clinical dose) DOX (i.p.) on days 0 and 3 (n = 50 mice per group). b, Representative echocardiographic photographs showing the cardiac function of the control, insDIC and senDIC mice. ***P < 0.001. n = 15. c, The proportions of mice in the insDIC group and senDIC group were analyzed. d, Representative images of TUNEL-stained sections of heart tissues from control mice and insDIC or senDIC mice. ***P < 0.001. Scale bars, 10 µm. n = 6. e, Representative TEM images of heart tissues from control and insDIC or senDIC mice. Scale bars, 1 µm. n = 3. f, The expression of DIC markers (Atp5a1, Esrra, Sdha, miR-330-5p, Atp2a2, Qki5 and lnc-Mhrt) in control mice and insDIC or senDIC mice was detected via qRT‒PCR. ***P < 0.001; n.s., not significant. n = 6. All the data are presented as the mean ± s.e.m.

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  Fig. 2. A lower myocardial distribution of DOX is associated with the insensitivity of DIC. a, Left: SERS spectra of heart tissues from mice treated with different concentrations of DOX. Right: changes in the heart DOX I1242/I723 ratio with the corresponding concentration in mice.b, SERS spectra of the DOX concentration in heart tissues from mice in the control (blue line), insDIC (red line) and senDIC (gray line) groups. Experimental conditions: laser power of 0.95 mW (632.8 nm); 5 scans of 10 s. c, Quantitative analysis of the DOX concentration in heart tissues from the mice in each group. ***P < 0.001. d, Left: SERS spectra of serum samples from mice treated with different concentrations of DOX. Right: changes in the serum DOX I1266/I702 ratio with the corresponding concentration in mice. e, SERS spectra of serum DOX concentrations in the control (blue line), insDIC (red line) and senDIC (gray line) groups. The experimental conditions included a laser power of 0.95 mW (632.8 nm) and 5 scans of 10 s. f, Quantitative analysis of the serum DOX concentration in each group. ***P < 0.001. g, Representative TEM images of cardiac vessels from control and insDIC or senDIC mice. Scale bars, 1…

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  Fig. 3. Cardiac ECs are reprogrammed in DIC-insensitive mice. a, A t-SNE plot showing the single-cell transcriptomes analyzed in the study, which are color-coded for clusters of different types of cells. b, A heatmap showing the mRNAs that were enriched in the top 20 genes of each cell cluster. c, A t-SNE plot showing clusters of different types of cellular heterogeneity in different groups. d, The ring heatmap shows the differences in the transcriptional profiles of CMs among the three groups. The Venn diagram shows the DEGs in the CMs of the three groups of mice. The correlation ring plot shows the correlation of the transcriptomes among the three groups of mouse CMs. A GO analysis was performed according to the principles of a false discovery rate (FDR) <0.01 and |log2fold change| >1 to filter out DEGs from the transcriptional profiles of the three groups of CMs. e, The ring heatmap shows the differences in the transcriptional profiles of fibroblasts among the three groups. The Venn diagram shows the DEGs in the fibroblasts from the three groups of mice. The correlation ring plot shows the correlation of the transcriptomes among the three groups of mouse fibroblasts. A GO analysis was…

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  Fig. 4. Cardiac microvascular ECs play a key role in determining the sensitivity to DIC. a, A t-SNE plot showing the different EC subpopulations analyzed in the study, which are color-coded for clusters of different types of cells. b, A correlation heatmap showing correlations among EC subsets. c, DEGs were screened from the transcriptional profiles of EC subsets subjected to reclustering and analyzed according to the FDR <0.01 |log2fold change| >1 principle of the GO analysis (right), and the EC subsets were named according to the GO results. The results are displayed in a t-SNE diagram (left), and different named EC subsets are distinguished by color. d, A heatmap showing mRNAs enriched in the top 50 genes of each EC subset. e, t-SNE plots showing marker gene expression in each EC subset. f, A t-SNE plot showing clusters of EC subsets with heterogeneity in different groups. g, The dynamic changes in the percentage of each class of cells in the total cells from different groups of mice. h, Monocle analyses showing CTL-ECs, INS-ECs and SEN-ECs in pseudotime. Each color indicates an EC subset. i, RNA velocity analysis was used to estimate single-cell trajectory changes via an analysis of…

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  Fig. 5. The incidence of DIC is increased by blocking the IFN-γ pathway or activating the PPAR-γ pathway. a, Immunofluorescence staining was performed to detect the levels of p-Stat1 in CD31-labeled ECs. Scale bars, 5 µm. b, Immunofluorescence staining was performed to detect the expression of PPAR-γ in CD31-labeled ECs. Scale bars, 5 µm. c, The proportions of mice in the insDIC group and senDIC group in the DOX group, DOX+Flud group and DOX+Rosi group were analyzed. d, SERS spectra of the DOX drug concentration in heart tissues from mice in the senDIC (green line), DOX+Flud (yellow line) and DOX+Rosi (red line) groups. Experimental conditions: laser power of 0.95 mW (632.8 nm); 5 scans of 10 s. e, The overall morphology of the hearts of the control, senDIC, DOX+Flud and DOX+Rosi groups after Evans blue staining. n.s., not significant. Scale bars, 1 cm. n = 6. All the data are presented as the mean ± s.e.m.

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  Fig. 6. Targeting the IFN-γ pathway or modulating the PPAR-γ pathway prevents DIC in mice. a, Immunofluorescence staining was performed to detect the levels of p-Stat1 in CD31-labeled ECs. Scale bars, 5 µm. b, Immunofluorescence staining was performed to detect the expression of PPAR-γ in CD31-labeled ECs. Scale bars, 5 µm. c, The proportions of mice in the insDIC group and insDIC group in the DOX group, DOX+IFN-γ group and DOX+T007 group were analyzed. d, SERS spectra of the DOX drug concentration in heart tissues from mice in the insDIC (green line), DOX+IFN-γ (yellow line) and DOX+T007 (red line) groups. Experimental conditions: laser power of 0.95 mW (632.8 nm); 5 scans of 10 s. e, The overall morphology of the hearts of the control, insDIC, DOX+IFN-γ and DOX+T007 groups after Evans blue staining. ***P < 0.001. Scale bars, 1 cm. n = 6. All the data are presented as the mean ± s.e.m.

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  Fig. 7. P-gp plays a vital role in DIC resistance. a, Heatmap analysis of genes involved in the IFN-γ and PPAR-γ pathways that were expressed in EC subsets from the CTL-EC, INS-EC and SEN-EC groups revealed P-gp (left), and changes in expression among the three groups were analyzed (right). b, Immunofluorescence assays of CD31-labeled ECs were performed to detect the expression of P-gp in DIC mice. Scale bars, 5 µm. c, Immunofluorescence assays of CD31-labeled ECs were performed to detect the expression of P-gp in different mouse models. Scale bars, 5 µm. d, The proportions of mice in the insDIC group and senDIC group in the DOX group and DOX+Tar group were analyzed. e, SERS spectra of the DOX drug concentration in heart tissues from mice in the insDIC (red line) and DOX+Tar (blue line) groups. Experimental conditions: laser power of 0.95 mW (632.8 nm); 5 scans of 10 s. f, SERS spectra of the serum DOX concentration in the insDIC (red line) and DOX+Tar (blue line) groups. Experimental conditions: laser power 0.95 mW (632.8 nm); 5 scans of 10 s.

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  Fig. 8. IFN-γ mediates P-gp expression and DOX transport in human ECs. a, Immunofluorescence staining was performed to detect the levels of p-Stat1 in the ECs from the mice in the DOX+IFN and DOX+IFN+Rosi groups. b, Immunofluorescence assays of CD31-labeled ECs were performed to detect the expression of P-gp in ECs from mice in the DOX+IFN and DOX+IFN+Rosi groups. Scale bars, 5 µm. c, The proportions of mice in the insDIC group and senDIC group in the DOX group, DOX+IFN group, DOX+IFN+Rosi group and DOX+IFN+Tar group were analyzed. d, Schematic of DOX drug penetration barrier model. e, DOX penetration in the DOX, DOX+IFN, DOX+IFN+Tar and DOX+IFN+Rosi groups was verified in a barrier model in vitro. f, Representative images of TUNEL staining of hearts from the control, DOX, DOX+IFN, DOX+IFN+Tar and DOX+IFN+Rosi groups. ***P < 0.001. Scale bars, 10 µm. n = 6. g, The expression of DIC markers (Atp2a2 and Qki5) in the combination groups was detected via qRT‒PCR. **P < 0.01, ***P < 0.001. n = 6. All the data are presented as the mean ± s.e.m. h, Immunofluorescence staining was performed to detect the expression of P-gp in HUVECs. Scale bars, 5 µm. ***P < 0.001. All the data are presented as…