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Design and validation of an endothelial progenitor cell capture chip and its application in patients with pulmonary arterial hypertension

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Abstract

The number of circulating endothelial progenitor cells (EPCs) inversely correlates with cardiovascular risk and clinical outcome, and thus has been proposed as a valuable biomarker for risk assessment, disease progression, and response to therapy. However, current strategies for isolation of these rare cells are limited to complex, laborious approaches. The goal of this study was the design and validation of a disposable microfluidic platform capable of selectively capturing and enumerating EPCs directly from human whole blood in healthy and diseased subjects, eliminating sample preprocessing. We then applied the “EPC capture chip” clinically and determined EPC numbers in blood from patients with pulmonary arterial hypertension (PAH). Blood was collected in tubes and injected into polymeric microfluidic chips containing microcolumns pre-coated with anti-CD34 antibody. Captured cells were immunofluorescently stained for the expression of stem and endothelial antigens, identified and counted. The EPC capture chip was validated with conventional flow cytometry counts (r = 0.83). The inter- and intra-day reliability of the microfluidic devices was confirmed at different time points in triplicates over 1–5 months. In a cohort of 43 patients with three forms of PAH (idiopathic/heritable, drug-induced, and connective tissue disease), EPC numbers are ≈50% lower in PAH subjects vs. matched controls and inversely related to two potential disease modifiers: body mass index and postmenopausal status. The EPC capture chip (5 × 30 × 0.05 mm3) requires only 200 μL of human blood and has the strong potential to serve as a rapid bedside test for the screening and monitoring of patients with PAH and other proliferative cardiovascular, pulmonary, malignant, and neurodegenerative diseases.

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References

  1. 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 275:964–966

    Article  PubMed  CAS  Google Scholar 

  2. Hristov M, Erl W, Weber PC (2003) Endothelial progenitor cells—mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol 23:1185–1189

    Article  PubMed  CAS  Google Scholar 

  3. Urbich C, Dimmeler S (2004) Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 95:343–353

    Article  PubMed  CAS  Google Scholar 

  4. 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 348:593–600

    Article  PubMed  Google Scholar 

  5. Van Craenenbroeck EM, Conraads VM, Van Bockstaele DR, Haine SE, Vermeulen K, Van Tendeloo VF, Vrints CJ, Hoymans VY (2008) Quantification of circulating endothelial progenitor cells: a methodological comparison of six flow cytometric approaches. J Immunol Methods 332:31–40

    Article  PubMed  Google Scholar 

  6. 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 353:999–1007

    Article  PubMed  CAS  Google Scholar 

  7. Plouffe BD, Kniazeva T, Mayer JE Jr, Murthy SK, Sales VL (2009) Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine. FASEB J 23:3309–3314

    Article  PubMed  CAS  Google Scholar 

  8. Gleghorn JP, Pratt ED, Denning D, Liu H, Bander NH, Tagawa ST, Nanus DM, Giannakakou PA, Kirby BJ (2010) Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody. Lab Chip 10:27–29

    Article  PubMed  CAS  Google Scholar 

  9. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A et al (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450:1235–1239

    Article  PubMed  CAS  Google Scholar 

  10. Timmermans F, Plum J, Yoder MC, Ingram DA, Vandekerckhove B, Case J (2009) Endothelial progenitor cells: identity defined? J Cell Mol Med 13:87–102

    Article  PubMed  Google Scholar 

  11. Asosingh K, Erzurum SC, Yoder MC, Tuder RM (2009) Letter by Asosingh et al. regarding article, "Circulating Endothelial Progenitor Cells in Patients with Eisenmenger Syndrome and Idiopathic Pulmonary Arterial Hypertension". Circulation 119:E230

    Article  PubMed  Google Scholar 

  12. Diller GP, Bedard E, Wort SJ, Gatzoulis MA, van Eijl S, Ali O, Wilkins MR, Wharton J, Okonko DO, Howard LS et al (2009) Response to letter regarding article, "Circulating Endothelial Progenitor Cells in Patients with Eisenmenger Syndrome and Idiopathic Pulmonary Arterial Hypertension". Circulation 119:E231–E231

    Article  Google Scholar 

  13. Fadini GP, Baesso I, Albiero M, Sartore S, Agostini C, Avogaro A (2008) Technical notes on endothelial progenitor cells: ways to escape from the knowledge plateau. Atherosclerosis 197:496–503

    Article  PubMed  CAS  Google Scholar 

  14. Friedrich EB, Walenta K, Scharlau J, Nickenig G, Werner N (2006) CD34(−)/CD133(+)/VEGFR-2(+) endothelial progenitor cell subpopulation with potent vasoregenerative capacities. Circ Res 98:E20–E25

    Article  PubMed  CAS  Google Scholar 

  15. Timmermans F, Van Hauwermeiren F, De Smedt M, Raedt R, Plasschaert F, De Buyzere ML, Gillebert TC, Plum J, Vandekerckhove B (2007) Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors. Arterioscler Thromb Vasc Biol 27:1572–1579

    Article  PubMed  CAS  Google Scholar 

  16. Diller GP, van Eijl S, Okonko DO, Howard LS, Ali O, Thum T, Wort SJ, Bedard E, Gibbs JS, Bauersachs J et al (2008) Circulating endothelial progenitor cells in patients with Eisenmenger syndrome and idiopathic pulmonary arterial hypertension. Circulation 117:3020–3030

    Article  PubMed  CAS  Google Scholar 

  17. Fadini GP, Schiavon M, Rea F, Avogaro A, Agostini C (2007) Depletion of endothelial progenitor cells may link pulmonary fibrosis and pulmonary hypertension. Am J Respir Crit Care Med 176:724–725

    PubMed  Google Scholar 

  18. Zhu JH, Wang XX, Fu GS, Shang YP, Zhang FR, Chen JZ (2008) Reduced number and activity of circulating endothelial progenitor cells in patients with idiopathic pulmonary arterial hypertension. Resp Med 102:1073–1079

    Article  Google Scholar 

  19. Asosingh K, Aldred MA, Vasanji A, Drazba J, Sharp J, Farver C, Comhair SAA, Xu WL, Licina L, Huang L et al (2008) Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension. Am J Pathol 172:615–627

    Article  PubMed  CAS  Google Scholar 

  20. Toshner M, Voswinckel R, Southwood M, Al-Lamki R, Howard LSG, Marchesan D, Yang J, Suntharalingam J, Soon E, Exley A et al (2009) Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension. Am J Respir Crit Care Med 180:780–787

    Article  PubMed  Google Scholar 

  21. Smadja DM, Mauge L, Sanchez O, Silvestre JS, Guerin C, Godier A, Henno P, Gaussem P, Israel-Biet D (2010) Distinct patterns of circulating endothelial cells in pulmonary hypertension. Eur Respir J 36:1284–1293

    Article  PubMed  CAS  Google Scholar 

  22. Plouffe BD, Njoka D, Harris J, Liao J, Horick NK, Radisic M, Murthy SK (2007) Peptide-mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow. Langmuir 23:5050–5055

    Article  PubMed  CAS  Google Scholar 

  23. Vickers JA, Caulum MM, Henry CS (2006) Generation of hydrophilic poly(dimethylsiloxane) for high-performance microchip electrophoresis. Anal Chem 78:7446–7452

    Article  PubMed  CAS  Google Scholar 

  24. Sales VL, Engelmayr GC Jr, Johnson JA Jr, Gao J, Wang Y, Sacks MS, Mayer JE Jr (2007) Protein precoating of elastomeric tissue-engineering scaffolds increased cellularity, enhanced extracellular matrix protein production, and differentially regulated the phenotypes of circulating endothelial progenitor cells. Circulation 116:I-55–I-63

    Article  CAS  Google Scholar 

  25. 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 106:2781–2786

    Article  PubMed  Google Scholar 

  26. Duda DG, Cohen KS, Scadden DT, Jain RK (2007) A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Nat Protocols 2:805–810

    Article  CAS  Google Scholar 

  27. Leor J, Marber M (2006) Endothelial progenitors: a new Tower of Babel? J Am Coll Cardiol 48:1588–1590

    Article  PubMed  Google Scholar 

  28. Erzurum S, Rounds SI, Stevens T, Aldred M, Aliotta J, Archer SL, Asosingh K, Balaban R, Bauer N, Bhattacharya J et al (2010) Strategic plan for lung vascular research: an NHLBI-ORDR workshop report. Am J Respir Crit Care Med 182:1554–1562

    Article  PubMed  Google Scholar 

  29. Verma S, Kuliszewski MA, Li SH, Szmitko PE, Zucco L, Wang CH, Badiwala MV, Mickle DA, Weisel RD, Fedak PW et al (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 109:2058–2067

    Article  PubMed  CAS  Google Scholar 

  30. Zamanian RT, Hansmann G, Snook S, Lilienfeld D, Rappaport KM, Reaven GM, Rabinovitch M, Doyle RL (2009) Insulin resistance in pulmonary arterial hypertension. Eur Respir J 33:318–324

    Article  PubMed  CAS  Google Scholar 

  31. Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C, Thompson R, Lee CT, Lopaschuk GD, Puttagunta L, Bonnet S et al (2007) A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11:37–51

    Article  PubMed  CAS  Google Scholar 

  32. Sutendra G, Bonnet S, Rochefort G, Haromy A, Folmes KD, Lopaschuk GD, Dyck JR, Michelakis ED (2010) Fatty acid oxidation and malonyl-CoA decarboxylase in the vascular remodeling of pulmonary hypertension. Sci Transl Med 2:44ra58

    Article  PubMed  Google Scholar 

  33. Christou HA, Khalil RA (2010) Sex hormones and vascular protection in pulmonary arterial hypertension. J Cardiovasc Pharmacol 56:471–474

    Article  PubMed  CAS  Google Scholar 

  34. Kielstein JT, Bode-Böger SM, Hesse G, Martens-Lobenhoffer J, Takacs A, Fliser D, Hoeper MM (2005) Asymmetrical dimethylarginine in idiopathic pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol 25:1414–1418

    Article  PubMed  CAS  Google Scholar 

  35. Zhu JH, Wang XX, Zhang FR, Shang YP, Tao QM, Zhu JH, Chen JZ (2008) Safety and efficacy of autologous endothelial progenitor cells transplantation in children with idiopathic pulmonary arterial hypertension: open-label pilot study. Pediatr Transplant 12:650–655

    Article  PubMed  Google Scholar 

  36. Wang XG, Zhang FR, Shang YP, Zhu JH, Xie XD, Tao QM, Zhu JH, Chen JZ (2007) Transplantation of autologous endothelial progenitor cells may be beneficial in patients with idiopathic pulmonary arterial hypertension—a pilot randomized controlled trial. J Am Coll Cardiol 49:1566–1571

    Article  PubMed  CAS  Google Scholar 

  37. Redondo S, Hristov M, Gumbel D, Tejerina T, Weber C (2007) Biphasic effect of pioglitazone on isolated human endothelial progenitor cells: involvement of peroxisome proliferator-activated receptor-gamma and transforming growth factor-beta1. Thromb Haemost 97:979–987

    PubMed  CAS  Google Scholar 

  38. Hansmann G, Wagner RA, Schellong S, Perez VA, Urashima T, Wang L, Sheikh AY, Suen RS, Stewart DJ, Rabinovitch M (2007) Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-gamma activation. Circulation 115:1275–1284

    PubMed  CAS  Google Scholar 

  39. Hansmann G, de Jesus Perez VA, Alastalo TP, Alvira CM, Guignabert C, Bekker JM, Schellong S, Urashima T, Wang L, Morrell NW et al (2008) An antiproliferative BMP-2/PPARgamma/apoE axis in human and murine SMCs and its role in pulmonary hypertension. J Clin Invest 118:1846–1857

    Article  PubMed  CAS  Google Scholar 

  40. Hansmann G, Rabinovitch M (2010) The protective role of adiponectin in pulmonary vascular disease. Am J Physiol Lung Cell Mol Physiol 298:L1–L2

    Article  PubMed  CAS  Google Scholar 

  41. Hansmann G, Zamanian RT (2009) PPARgamma activation: a potential treatment for pulmonary hypertension. Sci Transl Med 1:12ps14

    Article  PubMed  Google Scholar 

  42. Zamanian RT, Hansmann G, Lilienfeld D, Rappaport K, Rabinovitch M, Reaven G, Doyle RL (2007) Insulin resistance and pulmonary arterial hypertension. Am J Respir Crit Care Med 175:A713, abstract

    Google Scholar 

  43. Heresi GA, Aytekin M, Newman J, Didonato J, Dweik RA (2010) Plasma levels of high-density lipoprotein cholesterol and outcomes in pulmonary arterial hypertension. Am J Respir Crit Care Med 182:661–668

    Article  PubMed  CAS  Google Scholar 

  44. Brock M, Trenkmann M, Gay RE, Michel BA, Gay S, Fischler M, Ulrich S, Speich R, Huber LC (2009) Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II through a novel STAT3-microRNA cluster 17/92 pathway. Circ Res 104:1184–1191

    Article  PubMed  CAS  Google Scholar 

  45. Chan SY, Zhang Y-Y, Hemann C, Mahoney CE, Zweier JL, Loscalzo J (2009) MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab 10:273–284

    Article  PubMed  CAS  Google Scholar 

  46. Tofovic SP (2010) Estrogens and development of pulmonary hypertension—interaction of estradiol metabolism and pulmonary vascular disease. J Cardiovasc Pharmacol 56:696–708

    Article  PubMed  CAS  Google Scholar 

  47. Diller GP, Thum T, Wilkins MR, Wharton J (2010) Endothelial progenitor cells in pulmonary arterial hypertension. Trends Cardiovasc Med 20:22–29

    Article  PubMed  CAS  Google Scholar 

  48. Smadja DM, Gaussem P, Mauge L, Israel-Biet D, Dignat-George F, Peyrard S, Agnoletti G, Vouhe PR, Bonnet D, Levy M (2009) Circulating endothelial cells a new candidate biomarker of irreversible pulmonary hypertension secondary to congenital heart disease. Circulation 119:374–381

    Article  PubMed  Google Scholar 

  49. Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones PL, Maitland ML, Michelakis ED, Morrell NW et al (2009) Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 54:S10–S19

    Article  PubMed  CAS  Google Scholar 

  50. Lee ST, Chu K, Jung KH, Park HK, Kim DH, Bahn JJ, Kim JH, Oh MJ, Lee SK, Kim M et al (2009) Reduced circulating angiogenic cells in Alzheimer disease. Neurology 72:1858–1863

    Article  PubMed  Google Scholar 

  51. Dome B, Timar J, Ladanyi A, Paku S, Renyi-Vamos F, Klepetko W, Lang G, Dome P, Bogos K, Tovari J (2009) Circulating endothelial cells, bone marrow-derived endothelial progenitor cells and proangiogenic hematopoietic cells in cancer: from biology to therapy. Crit Rev Oncol Hematol 69:108–124

    Article  PubMed  Google Scholar 

  52. Yen A (1989) Flow cytometry: advanced research and clinical applications. CRC Press, Boca Raton

    Google Scholar 

  53. Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, Elliott CG, Gaine SP, Gladwin MT, Jing ZC, Krowka MJ, Langleben D, Nakanishi N, Souza R (2009) Updated Clinical Classification of pulmonary hypertension. J Am Coll Cardiol 54:S43–54

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank the organizers of the Pulmonary Hypertension Association’s Research Room, Garden Grove, CA for their help with setting up the laboratory equipment and the PAH patients and volunteers for participating in the study.

Funding

This work was supported by NIH grant R01 EB009327 (S.K.M) and IGERT NSF/NCI grant NSF-DGE-0504331 (B.D.P.).

Potential conflict of interest

None

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Authors and Affiliations

  1. Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA

    Georg Hansmann & Alexander von Gise

  2. Department of Chemical Engineering, Northeastern University, 360 Huntington Ave, 342 Snell Engineering Center, Boston, MA, 02115, USA

    Brian D. Plouffe, Adam Hatch & Shashi K. Murthy

  3. Division of Newborn Medicine, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA

    Hannes Sallmon

  4. Vera Moulton Wall Center for Pulmonary Vascular Disease and Division of Pulmonary and Critical Care Medicine, Stanford University, Stanford, CA, USA

    Roham T. Zamanian

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  1. Georg Hansmann
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  2. Brian D. Plouffe
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  3. Adam Hatch
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  4. Alexander von Gise
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  5. Hannes Sallmon
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  6. Roham T. Zamanian
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  7. Shashi K. Murthy
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Corresponding authors

Correspondence to Georg Hansmann or Shashi K. Murthy.

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Georg Hansmann and Brian D. Plouffe contributed equally to this work.

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Hansmann, G., Plouffe, B.D., Hatch, A. et al. Design and validation of an endothelial progenitor cell capture chip and its application in patients with pulmonary arterial hypertension. J Mol Med 89, 971–983 (2011). https://doi.org/10.1007/s00109-011-0779-6

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