Design and validation of an endothelial progenitor cell capture chip and its application in patients with pulmonary arterial hypertension
- 376 Downloads
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.
KeywordsBioengineering Vascular disease Pulmonary Endothelium Bone marrow Progenitor cells Microfluidic device Pulmonary hypertension Biomarker Bedside test Diagnostics Biomedical engineering
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.
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
- 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 332:31–40PubMedCrossRefGoogle 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 10:27–29PubMedCrossRefGoogle 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–E231CrossRefGoogle 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-63CrossRefGoogle 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 109:2058–2067PubMedCrossRefGoogle 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 11:37–51PubMedCrossRefGoogle 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 49:1566–1571PubMedCrossRefGoogle 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 115:1275–1284PubMedGoogle 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 118:1846–1857PubMedCrossRefGoogle 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, abstractGoogle 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 119:374–381PubMedCrossRefGoogle 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 69:108–124PubMedCrossRefGoogle Scholar
- 52.Yen A (1989) Flow cytometry: advanced research and clinical applications. CRC Press, Boca RatonGoogle Scholar