Toward a granular molecular-anatomic map of the blood vasculature – single-cell RNA sequencing makes the leap

  • Christer Betsholtz Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; and Department of Medicine-Huddinge, Karolinska Institutet, Huddinge, Sweden
Keywords: Single-cell RNA sequencing, endothelial cells, mural cells, zonation, organotypicity, cell stress


Single-cell RNA sequencing (scRNAseq) marks the birth of a new era in physiology and medicine. Within foreseeable future, we will know exactly what genes are expressed – and at what levels – in all the different cell types and subtypes that make up our bodies. We will also learn how a particular cell state, whether it occurs during development, tissue repair, or disease, reflects precise changes in gene expression. While profoundly impacting all areas of life science, scRNAseq may lead to a particular leap in vascular biology research. Blood vessels pervade and fulfill essential functions in all organs, but the functions differ. Innumerable organ-specific vascular adaptations and specializations are required. These, in turn, are dictated by differential gene expression by the two principal cellular building blocks of blood vessels: endothelial cells and mural cells. An organotypic vasculature is essential for functions as diverse as thinking, gas exchange, urine excretion, and xenobiotic detoxification in the brain, lung, kidney, and liver, respectively. In addition to the organotypicity, vascular cells also differ along the vascular arterio-venous axis, referred to as zonation, differences that are essential for the regulation of blood pressure and flow. Moreover, gene expression-based molecular changes dictate states of cellular activity, necessary for angiogenesis, vascular permeability, and immune cell trafficking, i.e. functions necessary for development, inflammation, and repair. These different levels of cellular heterogeneity create a nearly infinite phenotypic diversity among vascular cells. In this review, I summarize and exemplify what scRNAseq has brought to the picture in just a few years and point out where it will take us.


Download data is not yet available.


1. Aird WC. Discovery of the cardiovascular system: from Galen to William Harvey. J Thromb Haemost 2011;9:118–29. doi: 10.1111/j.1538-7836.2011.04312.x

2. Wright T. William Harvey goes back to the future. Lancet 2013;381:620–1. doi: 10.1016/S0140-6736(13)60335-9

3. Maxwell DS, Pease DC. The electron microscopy of the choroid plexus. J Biophys Biochem Cytol 1956;2:467–74. doi: 10.1083/jcb.2.4.467

4. Trier JS. The fine structure of the parathyroid gland. J Biophys Biochem Cytol 1958;4:13–22. doi: 10.1083/jcb.4.1.13

5. Edwards A, Silldforff EP, Pallone TL. The renal medullary microcirculation. Front Biosci 2000;5:E36–52. doi: 10.2741/A566

6. Majno G, Palade GE. Studies on inflammation. 1. The effect of histamine and serotonin on vascular permeability: an electron microscopic study. J Biophys Biochem Cytol. 1961;11:571–605. doi: 10.1083/jcb.11.3.571

7. Majno G, Palade GE, Schoefl GI. Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree: a topographic study. J Biophys Biochem Cytol. 1961;11:607–26. doi: 10.1083/jcb.11.3.607

8. Zimmermann KW. Der feinere bau der blutcapillaren. Z Anat Entwicklungsgesch 1923;68:29–109. doi: 10.1007/BF02593544

9. Ekman N, Lymboussaki A, Vastrik I, Sarvas K, Kaipainen A, Alitalo K. Bmx tyrosine kinase is specifically expressed in the endocardium and the endothelium of large arteries. Circulation 1997;96:1729–32. doi: 10.1161/01.CIR.96.6.1729

10. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 1998;93:741–53. doi: 10.1016/S0092-8674(00)81436-1

11. You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ, Tsai SY. Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 2005;435:98–104. doi: 10.1038/nature03511

12. Editorial. Method of the year 2013. Nat Methods 2014;11:1. doi: 10.1038/nmeth.2801

13. Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La Manno G, Jureus A, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 2015;347:1138–42. doi: 10.1126/science.aaa1934

14. Tabula Muris Consortium, Overall Coordination, Logistical Coordination, Organ Collection and Processing, Library Preparation and Sequencing, Computational Data Analysis, et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 2018;562:367–72. doi: 10.1038/s41586-018-0590-4

15. Ma S, Sun S, Geng L, Song M, Wang W, Ye Y, et al. Caloric restriction reprograms the single-cell transcriptional landscape of rattus norvegicus aging. Cell 2020;180:984–1001.e22. doi: 10.1016/j.cell.2020.02.008

16. Rozenblatt-Rosen O, Shin JW, Rood JE, Hupalowska A, Human Cell Atlas Standards and Technology Working Group, Regev A, et al. Building a high-quality human cell atlas. Nat Biotechnol 2021;39:149–53. doi: 10.1038/s41587-020-00812-4

17. Tambalo M, Mitter R, Wilkinson DG. A single cell transcriptome atlas of the developing zebrafish hindbrain. Development 2020;147:dev184143.doi: 10.1242/dev.184143

18. Baran-Gale J, Chandra T, Kirschner K. Experimental design for single-cell RNA sequencing. Brief Funct Genomics 2018;17:233–9. doi: 10.1093/bfgp/elx035

19. Lafzi A, Moutinho C, Picelli S, Heyn H. Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies. Nat Protoc 2018;13:2742–57. doi: 10.1038/s41596-018-0073-y

20. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 2015;161:1202–14. doi: 10.1016/j.cell.2015.05.002

21. Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 2014;9:171–81. doi: 10.1038/nprot.2014.006

22. Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F, van der Zwan J, et al. Molecular architecture of the mouse nervous system. Cell 2018;174:999–1014.e22. doi: 10.1016/j.cell.2018.06.021

23. Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995;270:467–70. doi: 10.1126/science.270.5235.467

24. Bondjers C, Kalen M, Hellstrom M, Scheidl SJ, Abramsson A, Renner O, et al. Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells. Am J Pathol 2003;162:721–9. doi: 10.1016/S0002-9440(10)63868-0

25. Bondjers C, He L, Takemoto M, Norlin J, Asker N, Hellstrom M, et al. Microarray analysis of blood microvessels from PDGF-B and PDGF-Rbeta mutant mice identifies novel markers for brain pericytes. FASEB J 2006;20:1703–5. doi: 10.1096/fj.05-4944fje

26. Takemoto M, He L, Norlin J, Patrakka J, Xiao Z, Petrova T, et al. Large-scale identification of genes implicated in kidney glomerulus development and function. EMBO J 2006;25:1160–74. doi: 10.1038/sj.emboj.7601014

27. Barry DM, McMillan EA, Kunar B, Lis R, Zhang T, Lu T, et al. Molecular determinants of nephron vascular specialization in the kidney. Nat Commun 2019;10:5705. doi: 10.1038/s41467-019-12872-5

28. Dumas SJ, Meta E, Borri M, Goveia J, Rohlenova K, Conchinha NV, et al. Single-cell RNA sequencing reveals renal endothelium heterogeneity and metabolic adaptation to water deprivation. J Am Soc Nephrol 2020;31:118–38. doi: 10.1681/ASN.2019080832

29. He B, Chen P, Zambrano S, Dabaghie D, Hu Y, Moller-Hackbarth K, et al. Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes. Nat Commun 2021;12:2141. doi: 10.1038/s41467-021-22331-9

30. Zambrano S, He L, Kano T, Sun Y, Charrin E, Lal M, et al. Molecular insights into the early stage of glomerular injury in IgA nephropathy using single-cell RNA sequencing. Kidney Int 2022;101:752–65. doi: 10.1016/j.kint.2021.12.011

31. Stahl PL, Salmen F, Vickovic S, Lundmark A, Navarro JF, Magnusson J, et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 2016;353:78–82. doi: 10.1126/science.aaf2403

32. Ortiz C, Navarro JF, Jurek A, Martin A, Lundeberg J, Meletis K. Molecular atlas of the adult mouse brain. Sci Adv 2020;6:eabb3446. doi: 10.1126/sciadv.abb3446

33. Ratz M, von Berlin L, Larsson L, Martin M, Westholm JO, La Manno G, et al. Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics. Nat Neurosci 2022;25:285–94. doi: 10.1038/s41593-022-01011-x

34. Vanlandewijck M, He L, Mae MA, Andrae J, Ando K, Del Gaudio F, et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 2018;554:475–80. doi: 10.1038/nature25739

35. He L, Vanlandewijck M, Mae MA, Andrae J, Ando K, Del Gaudio F, et al. Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types. Sci Data 2018;5:180160. doi: 10.1038/sdata.2018.160

36. Halpern KB, Shenhav R, Massalha H, Toth B, Egozi A, Massasa EE, et al. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells. Nat Biotechnol 2018;36:962–70. doi: 10.1038/nbt.4231

37. Dobie R, Wilson-Kanamori JR, Henderson BEP, Smith JR, Matchett KP, Portman JR, et al. Single-cell transcriptomics uncovers zonation of function in the mesenchyme during liver fibrosis. Cell Rep 2019;29:1832–47.e8. doi: 10.1016/j.celrep.2019.10.024

38. Rafii S, Butler JM, Ding BS. Angiocrine functions of organ-specific endothelial cells. Nature 2016;529:316–25. doi: 10.1038/nature17040

39. Augustin HG, Koh GY. Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology. Science 2017;357:eaal2379. doi: 10.1126/science.aal2379

40. Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV. Establishment and dysfunction of the blood-brain barrier. Cell 2015;163:1064–78. doi: 10.1016/j.cell.2015.10.067

41. Kalucka J, de Rooij L, Goveia J, Rohlenova K, Dumas SJ, Meta E, et al. Single-cell transcriptome atlas of murine endothelial cells. Cell 2020;180:764–79.e20. doi: 10.1016/j.cell.2020.01.015

42. Mruk DD, Cheng CY. The mammalian blood-testis barrier: its biology and regulation. Endocr Rev 2015;36:564–91. doi: 10.1210/er.2014-1101

43. Dunton AD, Gopel T, Ho DH, Burggren W. Form and function of the vertebrate and invertebrate blood-brain barriers. Int J Mol Sci 2021;22:12111. doi: 10.3390/ijms222212111

44. Travaglini KJ, Nabhan AN, Penland L, Sinha R, Gillich A, Sit RV, et al. A molecular cell atlas of the human lung from single-cell RNA sequencing. Nature 2020;587:619–25. doi: 10.1038/s41586-020-2922-4

45. Gillich A, Zhang F, Farmer CG, Travaglini KJ, Tan SY, Gu M, et al. Capillary cell-type specialization in the alveolus. Nature 2020;586:785–9. doi: 10.1038/s41586-020-2822-7

46. Gastfriend BD, Foreman KL, Katt ME, Palecek SP, Shusta EV. Integrative analysis of the human brain mural cell transcriptome. J Cereb Blood Flow Metab 2021;41:3052–68. doi: 10.1177/0271678X211013700

47. Muhl L, Mocci G, Pietilä R, Liu J, He L, Genové G, et al. A single-cell transcriptomic inventory of murine smooth mudscle cells. Dev Cell 2022. in press

48. Goveia J, Rohlenova K, Taverna F, Treps L, Conradi LC, Pircher A, et al. An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates. Cancer Cell 2020;37:21–36.e13. doi: 10.1016/j.ccell.2019.12.001

49. Rohlenova K, Goveia J, Garcia-Caballero M, Subramanian A, Kalucka J, Treps L, et al. Single-cell RNA sequencing maps endothelial metabolic plasticity in pathological angiogenesis. Cell Metab 2020;31:862–77.e14. doi: 10.1016/j.cmet.2020.03.009

50. Xie Y, He L, Lugano R, Zhang Y, Cao H, He Q, et al. Key molecular alterations in endothelial cells in human glioblastoma uncovered through single-cell RNA sequencing. JCI Insight 2021;6:e150861. doi: 10.1172/jci.insight.150861

51. Orsenigo F, Conze LL, Jauhiainen S, Corada M, Lazzaroni F, Malinverno M, et al. Mapping endothelial-cell diversity in cerebral cavernous malformations at single-cell resolution. Elife 2020;9:e61413. doi: 10.7554/eLife.61413

52. Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, et al. Pericytes regulate the blood-brain barrier. Nature 2010;468:557–61. doi: 10.1038/nature09522

53. Mae MA, He L, Nordling S, Vazquez-Liebanas E, Nahar K, Jung B, et al. Single-cell analysis of blood-brain barrier response to pericyte loss. Circ Res 2021;128:e46–62. doi: 10.1161/CIRCRESAHA.120.317473

54. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003;161:1163–77. doi: 10.1083/jcb.200302047

55. La Manno G, Soldatov R, Zeisel A, Braun E, Hochgerner H, Petukhov V, et al. RNA velocity of single cells. Nature 2018;560:494–8. doi: 10.1038/s41586-018-0414-6

56. Rasanen M, Sultan I, Paech J, Hemanthakumar KA, Yu W, He L, et al. VEGF-B promotes endocardium-derived coronary vessel development and cardiac regeneration. Circulation 2021;143:65–77. doi: 10.1161/CIRCULATIONAHA.120.050635

57. Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS One 2010;5:e13741. doi: 10.1371/journal.pone.0013741

58. Nolan DJ, Ginsberg M, Israely E, Palikuqi B, Poulos MG, James D, et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev Cell 2013;26:204–19. doi: 10.1016/j.devcel.2013.06.017

59. Rafii S, Palikuqi B. Isolation and characterization of mouse organ-specific endothelial transcriptomes. Methods Mol Biol 2018;1846:301–8. doi: 10.1007/978-1-4939-8712-2_20

60. Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci 2014;34:11929–47. doi: 10.1523/JNEUROSCI.1860-14.2014

61. Nicin L, Abplanalp WT, Mellentin H, Kattih B, Tombor L, John D, et al. Cell type-specific expression of the putative SARS-CoV-2 receptor ACE2 in human hearts. Eur Heart J 2020;41:1804–6. doi: 10.1093/eurheartj/ehaa311

62. Teuwen LA, Geldhof V, Pasut A, Carmeliet P. COVID-19: the vasculature unleashed. Nat Rev Immunol 2020;20:389–91. doi: 10.1038/s41577-020-0343-0

63. Libby P, Luscher T. COVID-19 is, in the end, an endothelial disease. Eur Heart J 2020;41:3038–44. doi: 10.1093/eurheartj/ehaa623

64. Sluimer JC, Gasc JM, Hamming I, van Goor H, Michaud A, van den Akker LH, et al. Angiotensin-converting enzyme 2 (ACE2) expression and activity in human carotid atherosclerotic lesions. J Pathol 2008;215:273–9. doi: 10.1002/path.2357

65. Ziegler CGK, Allon SJ, Nyquist SK, Mbano IM, Miao VN, Tzouanas CN, et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell 2020;181:1016–35.e19.

66. Litvinukova M, Talavera-Lopez C, Maatz H, Reichart D, Worth CL, Lindberg EL, et al. Cells of the adult human heart. Nature 2020;588:466–72. doi: 10.1038/s41586-020-2797-4

67. McCracken IR, Saginc G, He L, Huseynov A, Daniels A, Fletcher S, et al. Lack of evidence of angiotensin-converting enzyme 2 expression and replicative infection by SARS-CoV-2 in human endothelial cells. Circulation 2021;143:865–8. doi: 10.1161/CIRCULATIONAHA.120.052824

68. Muhl L, He L, Sun Y, Andaloussi Mae M, Pietila R, Liu J, et al. The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: implications for COVID-19 vascular research. Stem Cell Reports 2022;17:1089–104. doi: 10.1016/j.stemcr.2022.03.016

69. Rajewsky N, Almouzni G, Gorski SA, Aerts S, Amit I, Bertero MG, et al. LifeTime and improving European healthcare through cell-based interceptive medicine. Nature 2020;587:377–86. doi: 10.1038/s41586-020-2715-9
How to Cite
Betsholtz C. (2022). Toward a granular molecular-anatomic map of the blood vasculature – single-cell RNA sequencing makes the leap. Upsala Journal of Medical Sciences, 127(1).