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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

Abstract


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)


Acknowledgment

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.


Supplement

SUPPORTING INFORMATION

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.


References

[1] Acevedo A.D., Bowser S.S., Gerritsen M.E., Bizios R.. Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor. J Cell Physiol, 1993, 157: 603-614 CrossRef Google Scholar

[2] Bennett M.R., Sinha S., Owens G.K.. Vascular smooth muscle cells in atherosclerosis. Circ Res, 2016, 118: 692-702 CrossRef Google Scholar

[3] Brault S., Martinez-Bermudez A.K., Marrache A.M., Gobeil Jr F., Hou X., Beauchamp M., Quiniou C., Almazan G., Lachance C., Roberts Ii J., et al. Selective neuromicrovascular endothelial cell death by 8-iso-prostaglandin F. Stroke, 2003, 34: 776-782 CrossRef Google Scholar

[4] Capers Iv Q., Alexander R.W., Lou P., Hector De Leon P., Wilcox J.N., Ishizaka N., Howard A.B., Taylor W.R.. Monocyte chemoattractant protein-1 expression in aortic tissues of hypertensive rats. Hypertension, 1997, 30: 1397-1402 CrossRef Google Scholar

[5] Carrick D., Haig C., Maznyczka A.M., Carberry J., Mangion K., Ahmed N., Yue May V.T., McEntegart M., Petrie M.C., Eteiba H., et al. Hypertension, microvascular pathology, and prognosis after an acute myocardial infarction. Hypertension, 2018, 72: 720-730 CrossRef Google Scholar

[6] Chan C.T., Moore J.P., Budzyn K., Guida E., Diep H., Vinh A., Jones E.S., Widdop R.E., Armitage J.A., Sakkal S., et al. Reversal of vascular macrophage accumulation and hypertension by a CCR2 antagonist in deoxycorticosterone/salt-treated mice. Hypertension, 2012, 60: 1207-1212 CrossRef Google Scholar

[7] Chen C., Tso A.W.K., Cheung B.M.Y., Law L.S.C., Ong K.L., Wat N.M.S., Janus E.D., Xu A., Lam K.S.L.. Plasma concentration of pigment epithelium-derived factor is closely associated with blood pressure and predicts incident hypertension in chinese: A 10-year prospective study. Clin Endocrinol, 2012, 76: 506-513 CrossRef Google Scholar

[8] Chen, L.J., Wei, S.Y., Chiu, J.J. (2013). Mechanical regulation of epigenetics in vascular biology and pathobiology. J Cell Mol Med 17, 437–448. Google Scholar

[9] Dawson D.W., Volpert O.V., Gillis P., Crawford S.E., Xu H., Benedict W., Bouck N.P.. Pigment epithelium-derived factor: A potent inhibitor of angiogenesis. Science, 1999, 285: 245-248 CrossRef Google Scholar

[10] Dull R.O., Jo H., Sill H., Hollis T.M., Tarbell J.M.. The effect of varying albumin concentration and hydrostatic pressure on hydraulic conductivity and albumin permeability of cultured endothelial monolayers. Microvasc Res, 1991, 41: 390-407 CrossRef Google Scholar

[11] Evans P.C., Kwak B.R.. Biomechanical factors in cardiovascular disease. Cardiovasc Res, 2013, 99: 229-231 CrossRef Google Scholar

[12] Frismantiene A., Philippova M., Erne P., Resink T.J.. Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity. Cell Signal, 2018, 52: 48-64 CrossRef Google Scholar

[13] Grbovic L., Jovanovic A.. Pregnancy: Effect of the vascular endothelium on contractions induced by prostaglandin F2α in isolated pregnant guinea pig uterine artery. Hum Reprod, 1996, 11: 2041-2047 CrossRef Google Scholar

[14] Group, S.R., Wright, J.T., Jr., Williamson, J.D., Whelton, P.K., Snyder, J.K., Sink, K.M., Rocco, M.V., Reboussin, D.M., Rahman, M., Oparil, S., et al. (2015). A randomized trial of intensive versus standard blood-pressure control. N Engl J Med 373, 2103–2116. Google Scholar

[15] Heo K.S., Fujiwara K., Abe J.. Shear stress and atherosclerosis. Mol Cells, 2014, 37: 435-440 CrossRef Google Scholar

[16] Hughson M.D., Gobe G.C., Hoy W.E., Manning Jr R.D., Douglas-Denton R., Bertram J.F.. Associations of glomerular number and birth weight with clinicopathological features of african americans and whites. Am J Kidney Dis, 2008, 52: 18-28 CrossRef Google Scholar

[17] Ishibashi M., Hiasa K., Zhao Q., Inoue S., Ohtani K., Kitamoto S., Tsuchihashi M., Sugaya T., Charo I.F., Kura S., et al. Critical role of monocyte chemoattractant protein-1 receptor CCR2 on monocytes in hypertension-induced vascular inflammation and remodeling. Circ Res, 2004, 94: 1203-1210 CrossRef Google Scholar

[18] Islam, M.S. (2017). Hypertension: From basic research to clinical practice. Adv Exp Med Biol 956, 1–2. Google Scholar

[19] January C.T., Wann L.S., Calkins H., Chen L.Y., Cigarroa J.E., Cleveland Jr. J.C., Ellinor P.T., Ezekowitz M.D., Field M.E., Furie K.L., et al. 2019 AHA/ACC/HRS focused update of the 2014 aha/acc/hrs guideline for the management of patients with atrial fibrillation: A report of the american college of cardiology/american heart association task force on clinical practice guidelines and the heart rhythm society. J Am Coll Cardiol, 2019, 74: 104-132 CrossRef Google Scholar

[20] Kearney P.M., Whelton M., Reynolds K., Muntner P., Whelton P.K., He J.. Global burden of hypertension: Analysis of worldwide data. Lancet, 2005, 365: 217-223 CrossRef Google Scholar

[21] Konukoglu, D., Uzun, H. (2017). Endothelial dysfunction and hypertension. Adv Exp Med Biol 956, 511–540. Google Scholar

[22] Leitinger N., Huber J., Rizza C., Mechtcheriakova D., Bochkov V., Koshelnick Y., Berliner J.A., Binder B.R.. The isoprostane 8-iso-PGF stimulates endothelial cells to bind monocytes: difference to thromboxane-mediated endothelial activation. FASEB J, 2001, 15: 1254-1256 CrossRef Google Scholar

[23] Leung C.S., Yang K.Y., Li X., Chan V.W., Ku M., Waldmann H., Hori S., Tsang J.C.H., Lo Y.M.D., Lui K.O.. Single-cell transcriptomics reveal that PD-1 mediates immune tolerance by regulating proliferation of regulatory t cells. Genome Med, 2018a, 10: 71 CrossRef Google Scholar

[24] Leung O.M., Li J., Li X., Chan V.W., Yang K.Y., Ku M., Ji L., Sun H., Waldmann H., Tian X.Y., et al. Regulatory T cells promote apelin-mediated sprouting angiogenesis in type 2 diabetes. Cell Rep, 2018b, 24: 1610-1626 CrossRef Google Scholar

[25] Levy B.I., Ambrosio G., Pries A.R., Struijker-Boudier H.A.J.. Microcirculation in hypertension: A new target for treatment. Circulation, 2001, 104: 735-740 CrossRef Google Scholar

[26] Liang C., Yang K.Y., Chan V.W., Li X., Fung T.H.W., Wu Y., Tian X.Y., Huang Y., Qin L., Lau J.Y.W., et al. CD8+ T-cell plasticity regulates vascular regeneration in type-2 diabetes. Theranostics, 2020, 10: 4217-4232 CrossRef Google Scholar

[27] McMaster W.G., Kirabo A., Madhur M.S., Harrison D.G.. Inflammation, immunity, and hypertensive end-organ damage. Circ Res, 2015, 116: 1022-1033 CrossRef Google Scholar

[28] Mehaffey E., Majid D.S.A.. Tumor necrosis factor-α, kidney function, and hypertension. Am J Physiol Renal Physiol, 2017, 313: F1005-F1008 CrossRef Google Scholar

[29] Mikolajczyk T.P., Guzik T.J.. Adaptive immunity in hypertension. Curr Hypertens Rep, 2019, 21: 68 CrossRef Google Scholar

[30] Misárková E., Behuliak M., Bencze M., Zicha J.. Excitation-contraction coupling and excitation-transcription coupling in blood vessels: Their possible interactions in hypertensive vascular remodeling. Physiol Res, 2016, 65: 173-191 CrossRef Google Scholar

[31] Nakadate, H., Minamitani, H., Aomura, S. (2010). Combinations of hydrostatic pressure and shear stress influence morphology and adhesion molecules in cultured endothelial cells. Conf Proc IEEE Eng Med Biol Soc 2010, 3812–3815. Google Scholar

[32] Nguyen T., Toussaint J., Xue Y., Raval C., Cancel L., Russell S., Shou Y., Sedes O., Sun Y., Yakobov R., et al. Aquaporin-1 facilitates pressure-driven water flow across the aortic endothelium. Am J Physiol Heart Circ Physiol, 2015, 308: H1051-H1064 CrossRef Google Scholar

[33] Ohashi T., Sakamoto N., Iwao A., Sato M.. Oxygen tension modulates Ca2+ response to flow stimulus in endothelial cells exposed to hydrostatic pressure. Technol Health Care, 2003, 11: 263-274 CrossRef Google Scholar

[34] Pedrinelli R., Ballo P., Fiorentini C., Denti S., Galderisi M., Ganau A., Germanò G., Innelli P., Paini A., Perlini S., et al. Hypertension and acute myocardial infarction. J Cardiovasc Med, 2012, 13: 194-202 CrossRef Google Scholar

[35] Prystopiuk V., Fels B., Simon C.S., Liashkovich I., Pasrednik D., Kronlage C., Wedlich-Söldner R., Oberleithner H., Fels J.. A two-phase response of endothelial cells to hydrostatic pressure. J Cell Sci, 2018, 131: jcs206920 CrossRef Google Scholar

[36] Qiu J., Zheng Y., Hu J., Liao D., Gregersen H., Deng X., Fan Y., Wang G.. Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding. J R Soc Interface, 2014, 11: 20130852 CrossRef Google Scholar

[37] Rapsomaniki E., Timmis A., George J., Pujades-Rodriguez M., Shah A.D., Denaxas S., White I.R., Caulfield M.J., Deanfield J.E., Smeeth L., et al. Blood pressure and incidence of twelve cardiovascular diseases: lifetime risks, healthy life-years lost, and age-specific associations in 1.25 million people. Lancet, 2014, 383: 1899-1911 CrossRef Google Scholar

[38] Rodriguez-Iturbe B., Pons H., Johnson R.J.. Role of the immune system in hypertension. Physiol Rev, 2017, 97: 1127-1164 CrossRef Google Scholar

[39] Rychli K., Kaun C., Hohensinner P.J., Dorfner A.J., Pfaffenberger S., Niessner A., Bauer M., Dietl W., Podesser B.K., Maurer G., et al. The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts. J Cell Mol Med, 2010, 14: 198-205 CrossRef Google Scholar

[40] Sanidas E., Papadopoulos D.P., Velliou M., Tsioufis K., Mantzourani M., Iliopoulos D., Perrea D., Barbetseas J., Papademetriou V.. The role of angiogenesis inhibitors in hypertension: following “ariadne’s Thread”. Am J Hypertens, 2018, 31: 961-969 CrossRef Google Scholar

[41] Santisteban M.M., Ahmari N., Carvajal J.M., Zingler M.B., Qi Y., Kim S., Joseph J., Garcia-Pereira F., Johnson R.D., Shenoy V., et al. Involvement of bone marrow cells and neuroinflammation in hypertension. Circ Res, 2015, 117: 178-191 CrossRef Google Scholar

[42] Schwartz E.A., Bizios R., Medow M.S., Gerritsen M.E.. Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation. Circ Res, 1999, 84: 315-322 CrossRef Google Scholar

[43] Simoni J., Simoni G., Griswold J.A., Moeller J.F., Tsikouris J.P., Khanna A., Roongsritong C., Wesson D.E.. Role of free hemoglobin in 8-iso prostaglandin F2-alpha synthesis in chronic renal failure and its impact on CD163-Hb scavenger receptor and on coronary artery endothelium. ASAIO J, 2006, 52: 652-661 CrossRef Google Scholar

[44] Sinreih M., Anko M., Kene N.H., Kocbek V., Rižner T.L.. Expression of AKR1B1, AKR1C3 and other genes of prostaglandin F2α biosynthesis and action in ovarian endometriosis tissue and in model cell lines. Chem Biol Interact, 2015, 234: 320-331 CrossRef Google Scholar

[45] Sommers, S.C., Relman, A.S., Smithwick, R.H. (1958). Histologic studies of kidney biopsy specimens from patients with hypertension. Am J Pathol 34: 685–715. Google Scholar

[46] Sun L., Zhao M., Liu A., Lv M., Zhang J., Li Y., Yang X., Wu Z.. Shear stress induces phenotypic modulation of vascular smooth muscle cells via AMPK/mTOR/ULK1-mediated autophagy. Cell Mol Neurobiol, 2018, 38: 541-548 CrossRef Google Scholar

[47] Thoumine O., Nerem R.M., Girard F.R.. Oscillatory shear stress and hydrostatic pressure modulate cell-matrix attachment proteins in cultured endothelial cells. In Vitro Cell Dev Biol Anim, 1995, 31: 45-54 CrossRef Google Scholar

[48] Wang L., Zhao X.C., Cui W., Ma Y.Q., Ren H.L., Zhou X., Fassett J., Yang Y.Z., Chen Y., Xia Y.L., et al. Genetic and pharmacologic inhibition of the chemokine receptor CXCR2 prevents experimental hypertension and vascular dysfunction. Circulation, 2016, 134: 1353-1368 CrossRef Google Scholar

[49] Wang L., Zhang Y.L., Lin Q.Y., Liu Y., Guan X.M., Ma X.L., Cao H.J., Liu Y., Bai J., Xia Y.L., et al. CXCL1-CXCR2 axis mediates angiotensin II-induced cardiac hypertrophy and remodelling through regulation of monocyte infiltration. Eur Heart J, 2018, 39: 1818-1831 CrossRef Google Scholar

[50] Yoda T., Kikuchi K., Miki Y., Onodera Y., Hata S., Takagi K., Nakamura Y., Hirakawa H., Ishida T., Suzuki T., et al. 11β-Prostaglandin F, a bioactive metabolite catalyzed by AKR1C3, stimulates prostaglandin F receptor and induces slug expression in breast cancer. Mol Cell Endocrinol, 2015, 413: 236-247 CrossRef Google Scholar

[51] Yu Y., Lucitt M.B., Stubbe J., Cheng Y., Friis U.G., Hansen P.B., Jensen B.L., Smyth E.M., FitzGerald G.A.. Prostaglandin F elevates blood pressure and promotes atherosclerosis. Proc Natl Acad Sci USA, 2009, 106: 7985-7990 CrossRef ADS Google Scholar

[52] Zhou J., Li Y.S., Chien S.. Shear stress-initiated signaling and its regulation of endothelial function. Arterioscler Thromb Vasc Biol, 2014, 34: 2191-2198 CrossRef Google Scholar

  • 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.