, Volume 19, Issue 1, pp 67–78 | Cite as

Inflammation is associated with a reduced number of pro-angiogenic Tie-2 monocytes and endothelial progenitor cells in patients with critical limb ischemia

  • Jörn F. DopheideEmail author
  • Philipp Geissler
  • Jennifer Rubrech
  • Amelie Trumpp
  • Geraldine C. Zeller
  • Karsten Bock
  • Bernhard Dorweiler
  • Friedrich Dünschede
  • Thomas Münzel
  • Markus P. Radsak
  • Christine Espinola-Klein
Original Paper



Inflammation is the driving force in atherosclerosis. One central strategy in the treatment for PAD is the promotion of angiogenesis. Here, pro-angiogenic Tie-2-expressing monocytes (TEM) and endothelial progenitor cells (EPC) play a crucial role. Critical limb ischemia (CLI) is characterized by a severe, chronic inflammatory response; thus, progression of the disease might be related to the deleterious effects of inflammation on pro-angiogenic cells.


Forty-five patients with intermittent claudication (IC) [three groups: Rutherford (R)-1, -2, or -3; each n = 15], 20 patients with CLI [n = 20; Rutherford 4 (15 %), 5 (40 %), and 6 (45 %)], and 20 healthy controls were included in the study. Analysis of TEM and EPC was performed from whole blood by flow cytometry. Treatment for IC patients was conservative, and CLI patients underwent surgical revascularization. Follow-up was performed after mean of 7.1 months.


In comparison with healthy controls, we found increased proportions of TEM and EPC in dependence of the severity of PAD, with the highest level in patients with severe claudication (R3) (p < 0.01). In contrast, for patients with CLI, we found a significantly reduced expression of both TEM and EPC in comparison with healthy controls (p < 0.05) or IC patients (R-1, R-2, and R-3) (all p < 0.001). At follow-up, TEM and EPC in CLI patients increased significantly (both p < 0.001). Serum levels of fibrinogen and CRP were significantly increased in CLI patients (all p < 0.001), but decreased at follow-up (all p < 0.05). TEM and EPC proportions correlated inversely with levels of fibrinogen [(TEM: r = −0.266; p < 0.01) (EPC: r = −0.297; p < 0.001)], CRP (TEM: r = −0.283; p < 0.01) (EPC: r = −0.260; p < 0.01).


We found a strong association of diverse inflammatory markers with a reduced proportion of pro-angiogenic TEM or EPC in patients with CLI, giving rise to the speculation that a severe chronic inflammation might lead to deleterious effects on TEM and EPC, possibly interfering with angiogenesis, thus promoting an aggravation of the disease.


EPC TEM Peripheral arterial disease Critical limb ischemia Inflammation 



This paper contains in part data reported in the medical thesis of Jennifer Rubrech, Amelie Trumpp, and Philip Geissler. The authors like to thank Ursel Petrat, Sabine Wolk, and Andrea Drescher for excellent technical support.

Compliance with ethical standards

Conflict of interest

All authors declare that there is no conflict of interest.

Supplementary material

10456_2015_9489_MOESM1_ESM.pdf (42 kb)
Supplementary material 1 (PDF 42 kb)


  1. 1.
    Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG, Bell K, Caporusso J, Durand-Zaleski I, Komori K, Lammer J, Liapis C, Novo S, Razavi M, Robbs J, Schaper N, Shigematsu H, Sapoval M, White C, White J, Clement D, Creager M, Jaff M, Mohler E 3rd, Rutherford RB, Sheehan P, Sillesen H, Rosenfield K (2007) Inter-society consensus for the management of peripheral arterial disease (TASC II). Eur J Vasc Endovasc Surg 33(Suppl 1):S1–S75. doi: 10.1016/j.ejvs.2006.09.024 PubMedCrossRefGoogle Scholar
  2. 2.
    Bhatt DL, Steg PG, Ohman EM, Hirsch AT, Ikeda Y, Mas JL, Goto S, Liau CS, Richard AJ, Rother J, Wilson PW (2006) International prevalence, recognition, and treatment of cardiovascular risk factors in outpatients with atherothrombosis. JAMA 295(2):180–189. doi: 10.1001/jama.295.2.180 PubMedCrossRefGoogle Scholar
  3. 3.
    Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, Hiratzka LF, Murphy WR, Olin JW, Puschett JB, Rosenfield KA, Sacks D, Stanley JC, Taylor LM Jr, White CJ, White J, White RA, Antman EM, Smith SC Jr, Adams CD, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Hunt SA, Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B (2006) ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation 113(11):e463–e654. doi: 10.1161/CIRCULATIONAHA.106.174526 PubMedCrossRefGoogle Scholar
  4. 4.
    Adam DJ, Beard JD, Cleveland T, Bell J, Bradbury AW, Forbes JF, Fowkes FG, Gillepsie I, Ruckley CV, Raab G, Storkey H, participants Bt (2005) Bypass versus angioplasty in severe ischaemia of the leg (BASIL): multicentre, randomised controlled trial. Lancet 366(9501):1925–1934. doi: 10.1016/S0140-6736(05)67704-5 PubMedCrossRefGoogle Scholar
  5. 5.
    Fratezi AC, Albers M, De Luccia ND, Pereira CA (1995) Outcome and quality of life of patients with severe chronic limb ischaemia: a cohort study on the influence of diabetes. Eur J Vasc Endovasc Surg 10(4):459–465PubMedCrossRefGoogle Scholar
  6. 6.
    Ross R (1999) Atherosclerosis–an inflammatory disease. N Engl J Med 340(2):115–126. doi: 10.1056/NEJM199901143400207 PubMedCrossRefGoogle Scholar
  7. 7.
    Fischer MA, Stedman MR, Lii J, Vogeli C, Shrank WH, Brookhart MA, Weissman JS (2010) Primary medication non-adherence: analysis of 195,930 electronic prescriptions. J Gen Intern Med 25(4):284–290. doi: 10.1007/s11606-010-1253-9 PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Haas TL, Lloyd PG, Yang HT, Terjung RL (2012) Exercise training and peripheral arterial disease. Compr Physiol 2(4):2933–3017. doi: 10.1002/cphy.c110065 PubMedPubMedCentralGoogle Scholar
  9. 9.
    Hammer A, Steiner S (2013) Gene therapy for therapeutic angiogenesis in peripheral arterial disease—a systematic review and meta-analysis of randomized, controlled trials. Vasa 42(5):331–339. doi: 10.1024/0301-1526/a000298 PubMedCrossRefGoogle Scholar
  10. 10.
    Botham CM, Bennett WL, Cooke JP (2013) Clinical trials of adult stem cell therapy for peripheral artery disease. Methodist DeBakey Cardiovasc J 9(4):201–205PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275(5302):964–967PubMedCrossRefGoogle Scholar
  12. 12.
    Urbich C, Dimmeler S (2004) Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 95(4):343–353. doi: 10.1161/01.RES.0000137877.89448.78 PubMedCrossRefGoogle Scholar
  13. 13.
    Chavakis E, Dimmeler S (2011) Homing of progenitor cells to ischemic tissues. Antioxid Redox Signal 15(4):967–980. doi: 10.1089/ars.2010.3582 PubMedCrossRefGoogle Scholar
  14. 14.
    Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S (2000) Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 95(3):952–958PubMedGoogle Scholar
  15. 15.
    Murdoch C, Tazzyman S, Webster S, Lewis CE (2007) Expression of Tie-2 by human monocytes and their responses to angiopoietin-2. J Immunol 178(11):7405–7411PubMedCrossRefGoogle Scholar
  16. 16.
    Venneri MA, De Palma M, Ponzoni M, Pucci F, Scielzo C, Zonari E, Mazzieri R, Doglioni C, Naldini L (2007) Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer. Blood 109(12):5276–5285. doi: 10.1182/blood-2006-10-053504 PubMedCrossRefGoogle Scholar
  17. 17.
    De Palma M, Venneri MA, Galli R, Sergi Sergi L, Politi LS, Sampaolesi M, Naldini L (2005) Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8(3):211–226. doi: 10.1016/j.ccr.2005.08.002 PubMedCrossRefGoogle Scholar
  18. 18.
    Andreou I, Tousoulis D, Tentolouris C, Antoniades C, Stefanadis C (2006) Potential role of endothelial progenitor cells in the pathophysiology of heart failure: clinical implications and perspectives. Atherosclerosis 189(2):247–254. doi: 10.1016/j.atherosclerosis.2006.06.021 PubMedCrossRefGoogle Scholar
  19. 19.
    Coffelt SB, Chen YY, Muthana M, Welford AF, Tal AO, Scholz A, Plate KH, Reiss Y, Murdoch C, De Palma M, Lewis CE (2011) Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion. J Immunol 186(7):4183–4190. doi: 10.4049/jimmunol.1002802 PubMedCrossRefGoogle Scholar
  20. 20.
    Tendera M, Aboyans V, Bartelink ML, Baumgartner I, Clement D, Collet JP, Cremonesi A, De Carlo M, Erbel R, Fowkes FG, Heras M, Kownator S, Minar E, Ostergren J, Poldermans D, Riambau V, Roffi M, Rother J, Sievert H, van Sambeek M, Zeller T (2011) ESC Guidelines on the diagnosis and treatment of peripheral artery diseases: document covering atherosclerotic disease of extracranial carotid and vertebral, mesenteric, renal, upper and lower extremity arteries: the Task Force on the Diagnosis and Treatment of Peripheral Artery Diseases of the European Society of Cardiology (ESC). Eur Heart J 32(22):2851–2906. doi: 10.1093/eurheartj/ehr211 PubMedCrossRefGoogle Scholar
  21. 21.
    Anderson JL, Halperin JL, Albert NM, Bozkurt B, Brindis RG, Curtis LH, DeMets D, Guyton RA, Hochman JS, Kovacs RJ, Ohman EM, Pressler SJ, Sellke FW, Shen WK (2013) Management of patients with peripheral artery disease (compilation of 2005 and 2011 ACCF/AHA guideline recommendations): a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 127(13):1425–1443. doi: 10.1161/CIR.0b013e31828b82aa PubMedCrossRefGoogle Scholar
  22. 22.
    Hristov M, Schmitz S, Schuhmann C, Leyendecker T, von Hundelshausen P, Krotz F, Sohn HY, Nauwelaers FA, Weber C (2009) An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood. Cytom A 75(10):848–853. doi: 10.1002/cyto.a.20772 CrossRefGoogle Scholar
  23. 23.
    Schmidt-Lucke C, Fichtlscherer S, Aicher A, Tschope C, Schultheiss HP, Zeiher AM, Dimmeler S (2010) Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol. PLoS One 5(11):e13790. doi: 10.1371/journal.pone.0013790 PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Ross R (1999) Atherosclerosis: an inflammatory disease. N Engl J Med 340:115–126PubMedCrossRefGoogle Scholar
  25. 25.
    Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia AN, Hermiller JB, Hillegass WB, Rocha-Singh K, Moon TE, Whitehouse MJ, Annex BH, Investigators T (2002) Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet 359(9323):2053–2058PubMedCrossRefGoogle Scholar
  26. 26.
    Rajagopalan S, Mohler ER 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, Blebea J, Macko J, Kessler PD, Rasmussen HS, Annex BH (2003) Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 108(16):1933–1938. doi: 10.1161/01.CIR.0000093398.16124.29 PubMedCrossRefGoogle Scholar
  27. 27.
    Belch J, Hiatt WR, Baumgartner I, Driver IV, Nikol S, Norgren L, Van Belle E, Committees T, Investigators (2011) Effect of fibroblast growth factor NV1FGF on amputation and death: a randomised placebo-controlled trial of gene therapy in critical limb ischaemia. Lancet 377(9781):1929–1937. doi: 10.1016/S0140-6736(11)60394-2 PubMedCrossRefGoogle Scholar
  28. 28.
    Bjornheden T, Levin M, Evaldsson M, Wiklund O (1999) Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol 19(4):870–876PubMedCrossRefGoogle Scholar
  29. 29.
    Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC (2002) Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation 106(22):2781–2786PubMedCrossRefGoogle Scholar
  30. 30.
    Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro A (2005) Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 45(9):1449–1457. doi: 10.1016/j.jacc.2004.11.067 PubMedCrossRefGoogle Scholar
  31. 31.
    Li H, Zhang X, Guan X, Cui X, Wang Y, Chu H, Cheng M (2012) Advanced glycation end products impair the migration, adhesion and secretion potentials of late endothelial progenitor cells. Cardiovasc Diabetol 11:46. doi: 10.1186/1475-2840-11-46 PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    George J, Goldstein E, Abashidze S, Deutsch V, Shmilovich H, Finkelstein A, Herz I, Miller H, Keren G (2004) Circulating endothelial progenitor cells in patients with unstable angina: association with systemic inflammation. Eur Heart J 25(12):1003–1008. doi: 10.1016/j.ehj.2004.03.026 PubMedCrossRefGoogle Scholar
  33. 33.
    Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S (2004) Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation 109(13):1615–1622. doi: 10.1161/01.CIR.0000124476.32871.E3 PubMedCrossRefGoogle Scholar
  34. 34.
    Suh W, Kim KL, Choi JH, Lee YS, Lee JY, Kim JM, Jang HS, Shin IS, Lee JS, Byun J, Jeon ES, Kim DK (2004) C-reactive protein impairs angiogenic functions and decreases the secretion of arteriogenic chemo-cytokines in human endothelial progenitor cells. Biochem Biophys Res Commun 321(1):65–71. doi: 10.1016/j.bbrc.2004.06.107 PubMedCrossRefGoogle Scholar
  35. 35.
    Verma S, Kuliszewski MA, Li SH, Szmitko PE, Zucco L, Wang CH, Badiwala MV, Mickle DA, Weisel RD, Fedak PW, Stewart DJ, Kutryk MJ (2004) C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 109(17):2058–2067. doi: 10.1161/01.CIR.0000127577.63323.24 PubMedCrossRefGoogle Scholar
  36. 36.
    Werner N, Nickenig G (2006) Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy? Arterioscler Thromb Vasc Biol 26(2):257–266. doi: 10.1161/01.ATV.0000198239.41189.5d PubMedCrossRefGoogle Scholar
  37. 37.
    Zawada AM, Rogacev KS, Rotter B, Winter P, Marell RR, Fliser D, Heine GH (2011) SuperSAGE evidence for CD14++CD16+ monocytes as a third monocyte subset. Blood 118(12):e50–e61. doi: 10.1182/blood-2011-01-326827 PubMedCrossRefGoogle Scholar
  38. 38.
    Heine GH, Ulrich C, Seibert E, Seiler S, Marell J, Reichart B, Krause M, Schlitt A, Kohler H, Girndt M (2008) CD14(++)CD16+ monocytes but not total monocyte numbers predict cardiovascular events in dialysis patients. Kidney Int 73(5):622–629. doi: 10.1038/ PubMedCrossRefGoogle Scholar
  39. 39.
    Rogacev KS, Seiler S, Zawada AM, Reichart B, Herath E, Roth D, Ulrich C, Fliser D, Heine GH (2011) CD14++CD16+ monocytes and cardiovascular outcome in patients with chronic kidney disease. Eur Heart J 32(1):84–92. doi: 10.1093/eurheartj/ehq371 PubMedCrossRefGoogle Scholar
  40. 40.
    Schauer D, Starlinger P, Reiter C, Jahn N, Zajc P, Buchberger E, Bachleitner-Hofmann T, Bergmann M, Stift A, Gruenberger T, Brostjan C (2012) Intermediate monocytes but not TIE2-expressing monocytes are a sensitive diagnostic indicator for colorectal cancer. PLoS One 7(9):e44450. doi: 10.1371/journal.pone.0044450 PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Patel AS, Smith A, Nucera S, Biziato D, Saha P, Attia RQ, Humphries J, Mattock K, Grover SP, Lyons OT, Guidotti LG, Siow R, Ivetic A, Egginton S, Waltham M, Naldini L, De Palma M, Modarai B (2013) TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb. EMBO Mol Med 5(6):858–869. doi: 10.1002/emmm.201302752 PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Gill KA, Brindle NP (2005) Angiopoietin-2 stimulates migration of endothelial progenitors and their interaction with endothelium. Biochem Biophys Res Commun 336(2):392–396. doi: 10.1016/j.bbrc.2005.08.097 PubMedCrossRefGoogle Scholar
  43. 43.
    Dimmeler S, Zeiher AM (2004) Vascular repair by circulating endothelial progenitor cells: the missing link in atherosclerosis? J Mol Med 82(10):671–677. doi: 10.1007/s00109-004-0580-x PubMedCrossRefGoogle Scholar
  44. 44.
    Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T (2003) Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 348(7):593–600. doi: 10.1056/NEJMoa022287 PubMedCrossRefGoogle Scholar
  45. 45.
    Schmidt-Lucke C, Rossig L, Fichtlscherer S, Vasa M, Britten M, Kamper U, Dimmeler S, Zeiher AM (2005) Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation 111(22):2981–2987. doi: 10.1161/CIRCULATIONAHA.104.504340 PubMedCrossRefGoogle Scholar
  46. 46.
    Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Bohm M, Nickenig G (2005) Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 353(10):999–1007. doi: 10.1056/NEJMoa043814 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Jörn F. Dopheide
    • 1
    Email author
  • Philipp Geissler
    • 1
  • Jennifer Rubrech
    • 1
  • Amelie Trumpp
    • 1
  • Geraldine C. Zeller
    • 2
  • Karsten Bock
    • 1
  • Bernhard Dorweiler
    • 5
  • Friedrich Dünschede
    • 5
  • Thomas Münzel
    • 1
  • Markus P. Radsak
    • 3
    • 4
  • Christine Espinola-Klein
    • 1
  1. 1.Department of Internal Medicine IIUniversity Medical Center, Johannes Gutenberg-University MainzMainzGermany
  2. 2.Department of Internal Medicine IUniversity Medical Center, Johannes Gutenberg-University MainzMainzGermany
  3. 3.Department of Internal Medicine IIIUniversity Medical Center, Johannes Gutenberg-University MainzMainzGermany
  4. 4.Institute for ImmunologyUniversity Medical Center, Johannes Gutenberg UniversityMainzGermany
  5. 5.Division of Vascular Surgery, Department of Cardiothoracic and Vascular SurgeryUniversity Medical Center, Johannes-Gutenberg UniversityMainzGermany

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