Colony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profile

Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).

Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.

The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.

Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.

Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.

Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.

1. Cines D.B., Pollak E.S., Buck C.A., Loscalzo J., Zimmerman G.A., McEver R.P., Pober J.S., Wick T.M., Konkle B.A., Schwartz B.S., Barnathan E.S., McCrae K.R., Hug B.A., Schmidt A.M., Stern D.M. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood. 1998;91(10):3527-3561.

2. Niklason L.E., Lawson J.H. Bioengineered human blood vessels. Science. 2020;370(6513):eaaw8682. https://doi.org/10.1126/science.aaw8682

3. Ardila D.C., Liou J.J., Maestas D., Slepian M.J., Badowski M., Wagner W.R., Harris D., Vande Geest J.P. Surface Modification of Electrospun Scaffolds for Endothelialization of Tissue-Engineered Vascular Grafts Using Human Cord Blood-Derived Endothelial Cells. J. Clin. Med. 2019;8(2):185. https://doi.org/10.3390/jcm8020185

4. Yoder M.C. Human endothelial progenitor cells. Cold Spring Harb. Perspect. Med. 2012;2(7):a006692. https://doi.org/10.1101/cshperspect.a006692

5. Keighron C., Lyons C.J., Creane M., O'Brien T., Liew A. Recent Advances in Endothelial Progenitor Cells Toward Their Use in Clinical Translation. Front. Med. (Lausanne). 2018;5:354. https://doi.org/10.3389/fmed.2018.00354

6. Medina R.J., O'Neill C.L., Sweeney M., Guduric-Fuchs J., Gardiner T.A., Simpson D.A., Stitt A.W. Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities. BMC Med. Genomics. 2010;3:18. https://doi.org/10.1186/1755-8794-3-18

7. Banno K., Yoder M.C. Tissue regeneration using endothelial colonyforming cells: promising cells for vascular repair. Pediatr. Res. 2018;83(1-2):283-290. https://doi.org/10.1038/pr.2017.231

8. Tan W., Boodagh P., Selvakumar P.P., Keyser S. Strategies to counteract adverse remodeling of vascular graft: A 3D view of current graft innovations. Front. Bioeng. Biotechnol. 2023;10:1097334. https://doi.org/10.3389/fbioe.2022.1097334

9. Yoon C.H., Hur J., Park K.W., Kim J.H., Lee C.S., Oh I.Y., Kim T.Y., Cho H.J., Kang H.J., Chae I.H., Yang H.K., Oh B.H., Park Y.B., Kim H.S. Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases. Circulation. 2005;112(11):1618-1627. https://doi.org/10.1161/CIRCULATIONAHA.104.503433

10. Medina R.J., Barber C.L., Sabatier F., Dignat-George F., Melero-Martin J.M., Khosrotehrani K., Ohneda O., Randi A.M., Chan J.K.Y., Yamaguchi T., Van Hinsbergh V.W.M., Yoder M.C., Stitt A.W. Endothelial Progenitors: A Consensus Statement on Nomenclature. Stem Cells Transl. Med. 2017;6(5):1316-1320. https://doi.org/10.1002/sctm.16-0360

11. Medina R.J., O'Neill C.L., O'Doherty T.M., Knott H., Guduric-Fuchs J., Gardiner T.A., Stitt A.W. Myeloid angiogenic cells act as alternative M2 macrophages and modulate angiogenesis through interleukin-8. Mol. Med. 2011;17(9-10):1045-1055. https://doi.org/10.2119/molmed.2011.00129

12. Gifre-Renom L., Daems M., Luttun A., Jones E.A.V. Organ-Specific Endothelial Cell Differentiation and Impact of Microenvironmental Cues on Endothelial Heterogeneity. Int. J. Mol. Sci. 2022;23(3):1477. https://doi.org/10.3390/ijms23031477

13. Paik D.T., Tian L., Williams I.M., Rhee S., Zhang H., Liu C., Mishra R., Wu S.M., Red-Horse K., Wu J.C. Single-Cell RNA Sequencing Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells. Circulation. 2020;142(19):1848-1862. https://doi.org/10.1161/CIRCULATIONAHA.119.041433

14. Aird W.C. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ. Res. 2007;100(2):158-173. https://doi.org/10.1161/01.RES.0000255691.76142.4a

15. Koenig A.L., Shchukina I., Amrute J., Andhey P.S., Zaitsev K., Lai L., Bajpai G., Bredemeyer A., Smith G., Jones C., Terrebonne E., Rentschler S.L., Artyomov M.N., Lavine K.J. Single-cell transcriptomics reveals celltype-specific diversification in human heart failure. Nat. Cardiovasc. Res. 2022;1(3):263-280. https://doi.org/10.1038/s44161-022-00028-6

16. Abdelgawad M.E., Desterke C., Uzan G., Naserian S. Single-cell transcriptomic profiling and characterization of endothelial progenitor cells: new approach for finding novel markers. Stem Cell Res. Ther. 2021;12(1):145. https://doi.org/10.1186/s13287-021-02185-0

17. Steinle J.J., Meininger C.J., Forough R., Wu G., Wu M.H., Granger H.J. Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway. J. Biol. Chem. 2002;277(46):43830-43835. https://doi.org/10.1074/jbc.M207221200

18. Zhang H.F., Wang Y.L., Tan Y.Z., Wang H.J., Tao P., Zhou P. Enhancement of cardiac lymphangiogenesis by transplantation of CD34+VEGFR-3+ endothelial progenitor cells and sustained release of VEGF-C. Basic Res. Cardiol. 2019;114(6):43. https://doi.org/10.1007/s00395-019-0752-z

19. Smadja D.M., Bieche I., Silvestre J.S., Germain S., Cornet A., Laurendeau I., Duong-Van-Huyen J.P., Emmerich J., Vidaud M., Aiach M., Gaussem P. Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis. Arterioscler. Thromb. Vasc. Biol. 2008;28

20. Obi S., Yamamoto K., Shimizu N., Kumagaya S., Masumura T., Sokabe T., Asahara T., Ando J. Fluid shear stress induces arterial differentiation of endothelial progenitor cells. J. Appl. Physiol. (1985). 2009;106(1):203-211. https://doi.org/10.1152/japplphysiol.00197.2008

21. Muto A., Model L., Ziegler K., Eghbalieh S.D., Dardik A. Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies. Circ. J. 2010;74(8):1501-1512. https://doi.org/10.1253/circj.cj-10-0495

22. Owens C.D., Gasper W.J., Rahman A.S., Conte M.S. Vein graft failure. J. Vasc. Surg. 2015;61(1):203-216. https://doi.org/10.1016/j.jvs.2013.08.019

23. Cai Q., Liao W., Xue F., Wang X., Zhou W., Li Y., Zeng W. Selection of different endothelialization modes and different seed cells for tissue-engineered vascular graft. Bioact. Mater. 2021;6(8):2557-2568. https://doi.org/10.1016/j.bioactmat.2020.12.021

24. Matveeva V., Khanova M., Sardin E., Antonova L., Barbarash O. Endovascular Interventions Permit Isolation of Endothelial ColonyForming Cells from Peripheral Blood. Int J Mol Sci. 2018; 19 (11): 3453. https://doi.org/10.3390/ijms19113453.

25. Velikanova E.A., Matveeva V.G., Khanova M.Yu., Antonova L.V. Effects of shear stress on the properties of colonyforming endothelial cells in comparison with coronary artery endothelial cells. Complex Issues of Cardiovascular Diseases. 2022;11(4):90-97. (In Russ.) https://doi.org/10.17802/2306-1278-2022-11-4-90-97

26. Jaffe E.A., Nachman R.L., Becker C.G., Minick C.R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 1973;52(11):2745-2756. https://doi.org/10.1172/JCI107470

27. Ucuzian A.A., Greisler H.P. In vitro models of angiogenesis. World J Surg. 2007; 31 (4): 654-63. https://doi.org/10.1007/s00268-006-0763-4.

28. Inoue M., Itoh H., Ueda M., Naruko T., Kojima A., Komatsu R., D o i K., Ogawa Y., Tamura N., Takaya K., Igaki T., Yamashita J., Chun T.H., Masatsugu K., Becker A.E., Nakao K. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation. 1998; 98 (20): 2108-16. https://doi.org/10.1161/01.cir.98.20.2108.

29. Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 1999; 56 (3): 794-814. https://doi.org/10.1046/j.1523-1755.1999.00610.x.

30. Fong G.H., Rossant J., Gertsenstein M., Breitman M.L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995; 376 (6535): 66-70. https://doi.org/10.1038/376066a0.

31. Nishi J., Minamino T., Miyauchi H., Nojima A., Tateno K., Okada S., Orimo M., Moriya J., Fong G.H., Sunagawa K., Shibuya M., Komuro I. Vascular endothelial growth factor receptor-1 regulates postnatal angiogenesis through inhibition of the excessive activation of Akt. Circ Res. 2008; 103 (3): 261-8. https://doi.org/10.1161/CIRCRESAHA.108.174128.

32. Veikkola T., Jussila L., Makinen T., Karpanen T., Jeltsch M., Petrova T.V., Kubo H., Thurston G., McDonald D.M., Achen M.G., Stacker S.A., Alitalo K. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 2001; 20 (6): 1223-1231. https://doi.org/10.1093/emboj/20.6.1223

33. Dallinga M.G., Habani Y.I., Schimmel A.W.M., Dallinga-Thie G.M., van Noorden C.J.F., Klaassen I., Schlingemann R.O. The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro. Cells. 2021;10 (4): 926. https://doi.org/10.3390/cells10040926.

34. Evans I.M., Yamaji M., Britton G., Pellet-Many C., Lockie C., Zachary I.C., Frankel P. Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells. Mol Cell Biol. 2011; 31 (6): 1174-85. https://doi.org/10.1128/MCB.00903-10.

35. Brütsch R., Liebler S.S., Wüstehube J., Bartol A., Herberich S.E., Adam M.G., Telzerow A., Augustin H.G., Fischer A. Integrin cytoplasmic domain-associated protein-1 attenuates sprouting angiogenesis. Circ Res. 2010; 107 (5): 592-601. https://doi.org/10.1161/CIRCRESA-HA.110.217257.

36. Wang Y., Yang C., Gu Q., Sims M., Gu W., Pfeffer L.M., Yue J. KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells. PLoS One. 2015; 10 (6): e0130341. https://doi.org/10.1371/journal.pone.0130341.

37. Rezzola S., Di Somma M., Corsini M., Leali D., Ravelli C., Polli V.A.B., Grillo E., Presta M., Mitola S. VEGFR2 activation mediates the pro-angiogenic activity of BMP4. Angiogenesis. 2019; 22 (4): 521-533. https://doi.org/10.1007/s10456-019-09676-y.

38. Wang H., Su Y.. Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells. Am J Physiol Cell Physiol. 2011; 300 (5): C979-88. https://doi.org/10.1152/ajpcell.00368.2010.

39. Kalucka J., de Rooij L.P.M.H., Goveia J., Rohlenova K, Dumas S.J, Meta E., Conchinha N.V., Taverna F., Teuwen L.A., Veys K., García-Caballero M., Khan S., Geldhof V., Sokol L., Chen R., Treps L., Borri M., de Zeeuw P., Dubois C., Karakach T.K., Falkenberg K.D., Parys M., Yin X., Vinckier S., Du Y., Fenton R.A., Schoonjans L., Dewerchin M., Eelen G., Thienpont B., Lin L., Bolund L., Li X., Luo Y., Carmeliet P. Single-Cell Transcriptome Atlas of Murine Endothelial Cells. Cell. 2020;180(4):764-779.e20. https://doi.org/10.1016/j.cell.2020.01.015.

40. Brulois K., Rajaraman A., Szade A., Nordling S., Bogoslowski A., Dermadi D., Rahman M., Kiefel H., O'Hara E., Koning J.J., Kawashi-ma H., Zhou B., Vestweber D., Red-Horse K., Mebius R.E., Adams R.H., Kubes P., Pan J., Butcher E.C. A molecular map of murine lymph node blood vascular endothelium at single cell resolution. Nat. Commun. 2020;11(1):3798. https://doi.org/10.1038/s41467-020-17291-5