Molecular and Cellular Biochemistry

, Volume 446, Issue 1–2, pp 199–207 | Cite as

VEGF/PKD-1 signaling mediates arteriogenic gene expression and angiogenic responses in reversible human microvascular endothelial cells with extended lifespan

  • Brad Best
  • Patrick Moran
  • Bin Ren


Microvascular ECs (MVECs) are an ideal model in angiogenesis research. The aim of this study was to determine vascular endothelial growth factor (VEGF)/protein kinase D1 (PKD-1) signaling in expression of arteriogenic genes in human MVECs. To achieve this aim, we transduced specific SV40 large T antigen and telomerase into primary human dermal MVECs (HMVEC-D) to establish reversible HMVECs with extended lifespan (HMVECi-D). HMVECi-D was then exposed to VEGF/VEGF-inducer GS4012 or transduced with constitutively active protein kinase PKD-1 (PKD-CA). Quantitative RT-PCR was performed to detect arteriogenic gene expression. Furthermore, the angiogenic capacity in response to VEGF pathway was evaluated by Matrigel tube-formation and proliferation assays. We observed that VEGF/PKD-1 signaling axis significantly stimulated the expression of arteriogenic genes and promoted EC proliferation, along with downregulation of CD36 expression. Intriguingly, overexpression of PKD-CA also resulted in formation of tip cell morphology, accompanied by increased mRNA of delta-like ligand 4 (DLL4). In conclusion, we have successfully established and characterized HMVECi-D, and showed that VEGF/PKD-1 signaling axis increases angiogenic and arteriogenic gene expression. These studies suggest that the axis may regulate arteriolar differentiation through changing MVEC gene expression.


Angiogenesis Arteriolar differentiation CD36 Microvascular endothelial cells Protein kinase D VEGF 



This project is supported by the American Heart Association (13SDG14800019, B. Ren), the Ann’s Hope Foundation (FP00011709, B. Ren), an Institutional Research Grant (# 86-004-26) from American Cancer Society (B. Ren), a Career Development Award from the Central Society of Clinical and Translational Research (B. Ren), and the National Institute of Health (NHLBI R01 HL136423, B. Ren). P. Moran is supported by an Institutional Research Training Grant from NHLBI (5T35 HL072483-34).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11010_2018_3286_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 13 KB)
11010_2018_3286_MOESM2_ESM.pdf (2.2 mb)
Supplementary material 2 (PDF 2302 KB)


  1. 1.
    Aird WC (2006) Mechanisms of endothelial cell heterogeneity in health and disease. Circ Res 98:159–162. CrossRefPubMedGoogle Scholar
  2. 2.
    Ren B (2015) Endothelial cells: a key player in angiogenesis and lymphangiogenesis. MOJ Cell Sci Rep. CrossRefGoogle Scholar
  3. 3.
    Nolan DJ, Ginsberg M, Israely E, Palikuqi B, Poulos MG, James D, Ding BS, Schachterle W, Liu Y, Rosenwaks Z, Butler JM, Xiang J, Rafii A, Shido K, Rabbany SY, Elemento O, Rafii S (2013) Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev Cell 26:204–219. CrossRefPubMedGoogle Scholar
  4. 4.
    Ren B, Ramanchandran R, Silverstein R (2017) Protein kinase D1-CD36 signaling axis promotes arteriolar differentiation. J Investig Med 65:807–879. CrossRefGoogle Scholar
  5. 5.
    Marcelo KL, Goldie LC, Hirschi KK (2013) Regulation of endothelial cell differentiation and specification. Circ Res 112:1272–1287. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Carmeliet P (2000) Mechanisms of angiogenesis and arteriogenesis. Nat Med 6:389–395. CrossRefPubMedGoogle Scholar
  7. 7.
    Ren B, Deng Y, Mukhopadhyay A, Lanahan AA, Zhuang ZW, Moodie KL, Mulligan-Kehoe MJ, Byzova TV, Peterson RT, Simons M (2010) ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish. J Clin Investig 120:1217–1228. CrossRefPubMedGoogle Scholar
  8. 8.
    Ren B, Hale J, Srikanthan S, Silverstein RL (2011) Lysophosphatidic acid suppresses endothelial cell CD36 expression and promotes angiogenesis via a PKD-1-dependent signaling pathway. Blood 117:6036–6045. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ren B, Best B, Ramakrishnan DP, Walcott BP, Storz P, Silverstein RL (2016) LPA/PKD-1-FoxO1 signaling axis mediates endothelial cell CD36 transcriptional repression and proangiogenic and proarteriogenic reprogramming. Arterioscler Thromb Vasc Biol 36:1197–1208. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dong L, Yuan Y, Opansky C, Chen Y, Aguilera-Barrantes I, Wu S, Yuan R, Cao Q, Cheng YC, Sahoo D, Silverstein RL, Ren B (2017) Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling. Oncotarget 8:22550–22562. PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Scholz D, Ziegelhoeffer T, Helisch A, Wagner S, Friedrich C, Podzuweit T, Schaper W (2002) Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice. J Mol Cell Cardiol 34:775–787CrossRefPubMedGoogle Scholar
  12. 12.
    Hong CC, Kume T, Peterson RT (2008) Role of crosstalk between phosphatidylinositol 3-kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways in artery-vein specification. Circ Res 103:573–579. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Moran P, Opansky C, Weihrauch D, Yuan R, Jones DW, Ramchandran R, Ren B (2017) Abstract 14944: transcriptional reprogramming of endothelial cells for arteriolar differentiation by small chemical molecule via protein kinase D1 signaling pathway. Circulation 136:A14944-A14944Google Scholar
  14. 14.
    Candal FJ, Rafii S, Parker JT, Ades EW, Ferris B, Nachman RL, Kellar KL (1996) BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics. Microvasc Res 52:221–234. CrossRefPubMedGoogle Scholar
  15. 15.
    Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352CrossRefPubMedGoogle Scholar
  16. 16.
    Salmon P, Oberholzer J, Occhiodoro T, Morel P, Lou J, Trono D (2000) Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol Ther 2:404–414. CrossRefPubMedGoogle Scholar
  17. 17.
    Papapetrou EP, Lee G, Malani N, Setty M, Riviere I, Tirunagari LM, Kadota K, Roth SL, Giardina P, Viale A, Leslie C, Bushman FD, Studer L, Sadelain M (2011) Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol 29:73–78. CrossRefPubMedGoogle Scholar
  18. 18.
    Burghoff S, Gong X, Viethen C, Jacoby C, Flogel U, Bongardt S, Schorr A, Hippe A, Homey B, Schrader J (2014) Growth and metastasis of B16-F10 melanoma cells is not critically dependent on host CD73 expression in mice. BMC Cancer 14:898. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lee GY, Kenny PA, Lee EH, Bissell MJ (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4:359–365. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hermans K, Claes F, Vandevelde W, Zheng W, Geudens I, Orsenigo F, De Smet F, Gjini E, Anthonis K, Ren B, Kerjaschki D, Autiero M, Ny A, Simons M, Dewerchin M, Schulte-Merker S, Dejana E, Alitalo K, Carmeliet P (2010) Role of synectin in lymphatic development in zebrafish and frogs. Blood 116:3356–3366. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ren B, Song K, Parangi S, Jin T, Ye M, Humphreys R, Duquette M, Zhang X, Benhaga N, Lawler J, Khosravi-Far R (2009) A double hit to kill tumor and endothelial cells by TRAIL and antiangiogenic 3TSR. Cancer Res 69:3856–3865. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Overwijk WW, Restifo NP (2001) B16 as a mouse model for human melanoma. Curr Protoc Immunol. PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Bouis D, Hospers GA, Meijer C, Molema G, Mulder NH (2001) Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis 4:91–102CrossRefPubMedGoogle Scholar
  24. 24.
    Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Lindblom P, Alva J, Nilsson AK, Karlsson L, Gaiano N, Yoon K, Rossant J, Iruela-Arispe ML, Kalen M, Gerhardt H, Betsholtz C (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445:776–780. CrossRefPubMedGoogle Scholar
  25. 25.
    Suchting S, Freitas C, le Noble F, Benedito R, Breant C, Duarte A, Eichmann A (2007) The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci USA 104:3225–3230. CrossRefPubMedGoogle Scholar
  26. 26.
    Pitulescu ME, Schmidt I, Giaimo BD, Antoine T, Berkenfeld F, Ferrante F, Park H, Ehling M, Biljes D, Rocha SF, Langen UH, Stehling M, Nagasawa T, Ferrara N, Borggrefe T, Adams RH (2017) Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nat Cell Biol. PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, Bekman E, Costa L, Henrique D, Rossant J (2004) Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev 18:2474–2478. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lobov IB, Renard RA, Papadopoulos N, Gale NW, Thurston G, Yancopoulos GD, Wiegand SJ (2007) Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc Natl Acad Sci USA 104:3219–3224. CrossRefPubMedGoogle Scholar
  29. 29.
    Ren B (2016) Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis. Front Cell Dev Biol 4:37. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Swerlick RA, Lee KH, Wick TM, Lawley TJ (1992) Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J Immunol 148:78–83PubMedGoogle Scholar
  31. 31.
    Ren B, Yee KO, Lawler J, Khosravi-Far R (2006) Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 1765:178–188. PubMedCrossRefGoogle Scholar
  32. 32.
    Febbraio M, Hajjar DP, Silverstein RL (2001) CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Investig 108:785–791. CrossRefPubMedGoogle Scholar
  33. 33.
    Voyta JC, Via DP, Butterfield CE, Zetter BR (1984) Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034–2040CrossRefPubMedGoogle Scholar
  34. 34.
    Dvorak HF, Brown LF, Detmar M, Dvorak AM (1995) Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 146:1029–1039PubMedPubMedCentralGoogle Scholar
  35. 35.
    Hong CC, Peterson QP, Hong JY, Peterson RT (2006) Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling. Curr Biol 16:1366–1372. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Peterson RT, Shaw SY, Peterson TA, Milan DJ, Zhong TP, Schreiber SL, MacRae CA, Fishman MC (2004) Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nat Biotechnol 22:595–599. CrossRefPubMedGoogle Scholar
  37. 37.
    Wong C, Jin ZG (2005) Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor. J Biol Chem 280:33262–33269. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wood BM, Bossuyt J (2017) Emergency spatiotemporal shift: the response of protein kinase D to stress signals in the cardiovascular system. Front Pharmacol 8:9. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Qin L, Zeng H, Zhao D (2006) Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cgamma phosphorylation. J Biol Chem 281:32550–32558. CrossRefPubMedGoogle Scholar
  40. 40.
    Ren B, Best B, Weihrauch D, Jones DW, Dong L, Opansky C, Yuan R, Pritchard KA, Silverstein R (2016) LPA/PKD-1-FoxO1-CD36 signaling axis regulates capillary arterialization in ischemic conditions. Circulation 134:A15673–A15673Google Scholar
  41. 41.
    Opansky C, Best B, Yuan R, Cao Q, Ren B (2016) Abstract 14437: protein kinase D1 signaling is the key to arterial differentiation of vascular endothelial cells. Circulation 134:A14437–A14437Google Scholar
  42. 42.
    Wythe JD, Dang LT, Devine WP, Boudreau E, Artap ST, He D, Schachterle W, Stainier DY, Oettgen P, Black BL, Bruneau BG, Fish JE (2013) ETS factors regulate Vegf-dependent arterial specification. Dev Cell 26:45–58. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Wong BW, Marsch E, Treps L, Baes M, Carmeliet P (2017) Endothelial cell metabolism in health and disease: impact of hypoxia. EMBO J. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Xie Y, Fan Y, Xu Q (2016) Vascular regeneration by stem/progenitor cells. Arterioscler Thromb Vasc Biol 36:e33-40. CrossRefPubMedGoogle Scholar
  45. 45.
    Annex BH (2013) Therapeutic angiogenesis for critical limb ischaemia. Nat Rev Cardiol 10:387–396. CrossRefPubMedGoogle Scholar
  46. 46.
    Simons M, Gordon E, Claesson-Welsh L (2016) Mechanisms and regulation of endothelial VEGF receptor signalling. Nat Rev Mol Cell Biol 17:611–625. CrossRefPubMedGoogle Scholar
  47. 47.
    Lipton BH, Bensch KG, Karasek MA (1991) Microvessel endothelial cell transdifferentiation: phenotypic characterization. Differentiation 46:117–133CrossRefPubMedGoogle Scholar
  48. 48.
    Mao AS, Mooney DJ (2015) Regenerative medicine: current therapies and future directions. Proc Natl Acad Sci USA 112:14452–14459. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Blood Research InstituteBloodCenter of WisconsinMilwaukeeUSA
  2. 2.Cancer CenterMedical College of WisconsinMilwaukeeUSA
  3. 3.Cardiovascular CenterMedical College of WisconsinMilwaukeeUSA
  4. 4.Department of MedicineMedical College of WisconsinMilwaukeeUSA

Personalised recommendations