SCIENCE CHINA Life Sciences, https://doi.org/10.1007/s11427-020-1852-x

Single cell transcriptomic analysis identifies novel vascular smooth muscle subsets under high hydrostatic pressure

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  • ReceivedSep 8, 2020
  • AcceptedNov 16, 2020
  • PublishedJan 21, 2021


Funded by

the National Key Research and Development Program of China(2018YFC1312703)

CAMS Innovation Fund for Medical Sciences(CIFMS,2016-12M1–006)

the National Natural Science Foundation of China(81630014,81825002,81800367,81870318,81670379)

and Beijing Outstanding Young Scientist Program(BJJWZYJH01201910023029)


This work was supported by the National Key Research and Development Program of China (2018YFC1312703), CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-12M1–006), the National Natural Science Foundation of China (81630014, 81825002, 81800367, 81870318, 81670379), and Beijing Outstanding Young Scientist Program (BJJWZYJH01201910023029).

Interest statement

The author(s) declare that they have no conflict of interest.



The supporting information is available online at https://doi.org/10.1007/s11427-020-1852-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    High throughput single-cell RNA-sequencing of distinct cell clusters and heterogeneity of HASMCs. A, t-distributed scholastic neighbor embedding (t-SNE) plots with HASMC clusters demarcated by colors demonstrating six distinct clusters for 7,397 cells. B, Locations within thet-SNE plot of 100 and 200 mmHg. C, t-SNE plots show six clusters distributed under 100- and 200- mmHg pressure. D, Sample distribution in different clusters. E, Proportions of cell types across the different conditions (100 or 200 mmHg).

  • Figure 2

    Definition of cell populations based on enriched gene expression pattern. A, Heat map analysis of the expression pattern of top eight gene markers differentially distinguishing six clusters on scRNA-seq analysis. The identity of each cluster was assigned by matching the cluster expression profile with established cell-specific marker gene expression. B and C, t-SNE visualization (B) and violin plots (C) show differential expression of selected marker genes: cluster 1 marker genes (CXCL2, CXCL3, CCL2) and cluster 2 marker genes (AKR1C2, AKR1C3, SERPINF1). D and E, GO enrichment analysis of differentially expressed genes in inflammatory VSMCs (D) and endothelial-function inhibitory VSMCs (E). GO terms with corrected P<0.05 were considered significantly enriched for marker genes. F and G, KEGG pathway categories of differentially expressed genes in inflammatory VSMCs (F) and endothelial-function inhibitory VSMCs (G). Size of the circles is proportional to the fold enrichment. H, Pseudotime trajectory revealing the progression of different clusters.

  • Figure 3

    Detection of inflammatory VSMC enriched genes and function with high hydrostatic pressure. A, The effect of 100/200-mmHg hydrostatic pressure on CXCL2, CXCL3, and CCL2 mRNA levels. B, Immunofluorescence staining of CCL2 (green) and CXCL2 (red) in HASMCs under 100/200-mmHg hydrostatic pressure. Scale bars: 25 μm. C, Immunofluorescence staining of CXCL3. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. D, Fluorescence intensity of CCL2, CXCL2 and CXCL3 in B and C. E, Western blot analysis of CCL2, CXCL2, and CXCL3 protein expression. Representative gel images are shown in the left panel and quantification in the right panel. F, Representative flow cytometry plots of inflammatory VSMCs in all HASMCs: proportions of CCL2+ CXCL1/2/3+ cells in total HASMCs are shown in the left panel and quantification in in right panel. G, CCL2, CXCL2 and CXCL3 levels in HASMC culture medium determined by ELISA under different hydrostatic pressure. H, Transwell migration assay of THP-1 cells with conditioned medium from HASMCs cultured for 48 h under hydrostatic stress. Representative images of Diff-quick stain of trans-migrated THP-1 cells are shown in the left panel and quantification in the right panel. Scale bars: 200 μm. I, THP-1 cell counts of migration determined by MTT assays. N>5. Data are mean±SEM.

  • Figure 4

    Detection of endothelial-function inhibitory VSMCs enriched genes and function under high hydrostatic pressure. A, qRT-PCR analysis of AKR1C2, AKR1C3 and SERPINF1 mRNA levels in HASMCs subjected to hydrostatic pressure. B, Representative immunofluorescence staining of AKR1C3 (green) and SERPINF1 (red) under 100- and 200-mmHg pressure. Nuclei were stained blue with DAPI. Scale bars: 25 μm. C, Representative immunofluorescence staining of AKR1C2. Scale bars: 50 μm. D, The quantification of B and C immunofluorescent staining. E, HASMCs were cultured under hydrostatic stress for 48 h; AKR1C2, AKR1C3 and SERPINF1 protein levels were detected by Western blot assay. The quantification is in the right panel. F, Flow cytometry show the percentage of AKR1C3+SERPINF1+ cells in all HASMCs with 100/200-mmHg treatment. G, The percentage of AKR1C2+SERPINF1+ cells under hydrostatic pressure. H, The percentage of AKR1C2+AKR1C3+ cells under hydrostatic pressure. I, ELISA of SERPINF1 secretion and 11β-PGF secretion in conditioned medium. J, Fluorescent microscope images of angiogenic tube formation of HAECs. Total tube formation is quantified in the right panel. Scale bars: 10 μm. K, For inhibition of CXCL2, CXCL3 and CCL2, their monoclonal antibodies (100 ng mL−1) were mixed and added into HAECs with HASMC medium from 200-mmHg pressure treatment. Angiogenesis was assessed after 8 h incubation. Scale bars: 5 μm. Data are mean±SEM.

  • Figure 5

    Inflammatory VSMC enriched genes (CCL2/CXCL2/CXCL3) are upregulated in hypertensive patients and animal models. A, Immunofluorescence staining of CCL2, CXCL2 (the left panel) and CXCL3 (the right panel) on paraffin-embedded normotension (NTN) and hypertension (HTN) human internal mammary arteries. B–D, Representative images of aortic immunofluorescence staining of CCL2, CXCL2 (the left panel) and CXCL3 (the right panel) in three independent hypertension models: AngII-induced hypertensive mice (B), spontaneously hypertensive rats (SHRs) (C), and Dahl/SS hypertensive rats (D). E, Relative fluorescence intensity per field of CCL2, CXCL2 and CXCL3 in arterial media from hypertensive patients and animal models. Nuclei were stained blue with DAPI. Scale bars: 25 μm. *, P<0.05; **, P<0.01; ***, P<0.001 vs. related control. Data are mean±SEM.

  • Figure 6

    Endothelial-function inhibitory VSMC enriched genes (AKR1C2/AKR1C3/SERPINF1) are accumulated in hypertensive patients and animal models. A, Immunofluorescence staining of AKR1C3, SERPINF1 (the left panel) and AKR1C2 (the right panel) in NTN and HTN human internal mammary artery. Nuclei were stained blue with DAPI. B–D, Representative images of aortic immunofluorescence co-staining of AKR1C3, SERPINF1 (the left panel) and AKR1C2 (the right panel) in three independent hypertension models: AngII-induced hypertensive mice (B), SHRs (C), and Dahl/SS hypertensive rats (D). E, Quantification of AKR1C3, SERPINF1 and AKR1C2 immunofluorescent staining in hypertensive patients and animal models. Scale bars: 25 μm. *, P<0.05; **, P<0.01 vs. related control. Data are mean±SEM.