Extracellular vesicles derived from HuMSCs alleviate daunorubicin-induced cardiac microvascular injury via miR-186-5p/PARP9/STAT1 signal pathway

Figures

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  Fig. 1. MSC-EVs could be internalized by daunorubicin (DNR)-damaged HCMEC A. Ultrastructure of EVs observed by transmission electron microscopy B. Western blotting of the EVs markers CD9, CD81 and calnexin in lysates from MSC and MSC-EVs C. The average particle size distribution of MSC-EVs D. Immunophenotypes of HCMEC as determined by flow cytometry E. The effects of different doses of DNR for the cell viability of HCMEC, and half of IC50 was used for further experiments F. Morphological changes and SA-β-Gal positive ratio of HCMEC under DNR intervention. Data are presented as the mean ± SD of three replicates. ∗∗∗∗P＜0.0001 G. The flow chart of the experimental design. HCMEC were subjected to DNR for 24 h when confluency reached 80 %. Afterward, the culture medium was replaced with or without MSC-EVs for the corresponding time H. PKH 26-labeled MSC-EVs (red) were internalized by DNR-damaged HCMEC.

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  Fig. 2. MSC-EVs restore the cellular function and alleviate senescence of DNR-damaged HCMEC A, B. Scratch wound Assay indicates the effects of MSC-EVs on cell migration of DNR-damaged HCMEC. Scale bar represents 100 μm C, D. Edu Proliferation Assay shows that MSC-EVs improve cell proliferation of DNR-damaged HCMEC. Scale bar represents 50 μm E, F. Tube formation assay demonstrates the effects of MSC-EVs on angiogenesis of DNR-damaged HCMEC. Scale bar represents 200 μm G, H. SA-β-Gal staining suggests that MSC-EVs alleviate cellular senescence of DNR-damaged HCMEC. Scale bar represents 50 μm I, J. The mRNA level of ICAM-1 and VE-cadherin in DNR-damaged HCMEC. K, L. Chicken Chorioallantoic Membrane (CAM) assay demonstrates the effects of MSC-EVs on angiogenesis of DNR-damaged CAM. Data are presented as the mean ± SD of three replicates. ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001, ∗∗∗∗P＜0.0001.

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  Fig. 3. Transcriptome sequencing and bioinformatics tools screen out the miR-186-5p-PARP9 axis A, B. Transcriptome samples and screening rules C, D. Heat map and GO enrichment analysis bubble map of differential genes E. GSEA enrichment analysis suggests that MSC-EVs down-regulate the type 1 interferon (IFN-I) signaling pathway in DNR-damaged HCMEC F. Heatmap of the representative genes in IFN-I pathway G, H. Effects of MSC-EVs on PARP9 expression were determined by RT-PCR and WB I. Venn diagram of 9 miRNAs targeting PARP9 by prediction tools J. The GSE211008 dataset suggests that miR-186-5p has the highest expression level among 9 predicted miRNAs in MSC-EVs K. Effects of MSC-EVs on miR-186-5p expression were determined by RT-PCR. Data are presented as the mean ± SD of three replicates. ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001.

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  Fig. 4. MSC-EVs downregulated PARP9 to restore the cellular function and alleviate senescence of DNR-damaged HCMEC PARP9 silencing improved the migration (A, B), proliferation (C, D) and attenuated cellular senescence (E, F) of DNR-damaged HCMEC G-J. The relative protein level of PARP9, STAT1 and pSTAT1 in si-PARP9 DNR-damaged HCMEC with or without MSC-EVs. Data are presented as the mean ± SD of three replicates. ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001.

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  Fig. 5. MSC-EVs disrupted the PARP9-STAT1/pSTAT1 signaling pathway by delivering miR-186-5p A. The binding site of miR-186-5p with 3′ UTR region of PARP9 B. The transfection efficiency of miR-186-5p mimics examined by RT-PCR in DNR-damaged HCMEC C. The mRNA expression level of the target gene PARP9 was downregulated by miR-186-5p mimics. miR-186-5p mimics improved the migration (D, E), proliferation (F, G) and attenuated cellular senescence (H, I) of DNR-damaged HCMEC. J-M. miR-186-5p mimics downregulated the protein expression level of PARP9, STAT1 and pSTAT1 in DNR-damaged HCMEC with or without MSC-EVs. N, O. miR-186-5p mimics improved the angiogenesis of DNR-damaged CAM. Data are presented as the mean ± SD of three replicates. ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001.