Advertisement

Biotechnology Letters

, Volume 40, Issue 8, pp 1189–1200 | Cite as

Labeling of endothelial cells with magnetic microbeads by angiophagy

  • Jessica Thomas
  • Desiree Jones
  • Leni Moldovan
  • Mirela Anghelina
  • Keith J. Gooch
  • Nicanor I. Moldovan
Original Research Paper

Abstract

Objectives

Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism ‘angiophagy’. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization.

Results

In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001).

Conclusion

We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.

Keywords

Angiophagy Electrospun scaffold Endothelial cells Magnetic microbeads Phagocytosis Poly-capro-lactone 

Notes

Acknowledgements

The authors are grateful to Ray Xu and John Lannutti from the Department of Materials Sciences and Engineering at OSU for scaffold preparation, and to Thierry Pecot for help with the software for nuclei analysis. Microscopy was performed in the Campus Microscopy and Imaging Facility of the Ohio State University. This work was supported by NIH Grant RC2 AG-036559, and by a research seed grant from OSU Center for Emergent Materials.

References

  1. Chrastina A, Massey KA, Schnitzer JE (2011) Overcoming in vivo barriers to targeted nanodelivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol 3:421–437CrossRefPubMedGoogle Scholar
  2. Chretien ML, Zhang M, Jackson MR, Kapus A, Langille BL (2010) Mechanotransduction by endothelial cells is locally generated, direction-dependent, and ligand-specific. J Cell Physiol 224:352–361CrossRefPubMedGoogle Scholar
  3. Collins C et al (2012) Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway. Curr Biol 22:2087–2094CrossRefPubMedPubMedCentralGoogle Scholar
  4. Consigny PM, Silverberg DA, Vitali NJ (1999) Use of endothelial cells containing superparamagnetic microspheres to improve endothelial cell delivery to arterial surfaces after angioplasty. J Vasc Interv Radiol 10:155–163CrossRefPubMedGoogle Scholar
  5. Dobson J (2008) Remote control of cellular behaviour with magnetic nanoparticles. Nat Nanotechnol 3:139–143CrossRefPubMedGoogle Scholar
  6. Fens MH et al (2008) Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells. Blood 111:4542–4550CrossRefPubMedGoogle Scholar
  7. Fens MH et al (2010) Erythrophagocytosis by angiogenic endothelial cells is enhanced by loss of erythrocyte deformability. Exp Hematol 38:282–291CrossRefPubMedGoogle Scholar
  8. Gao C et al (2013) Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity. Thromb Haemost 109:1079–1090CrossRefPubMedGoogle Scholar
  9. Godbey WT, Hindy SB, Sherman ME, Atala A (2004) A novel use of centrifugal force for cell seeding into porous scaffolds. Biomaterials 25:2799–2805CrossRefPubMedGoogle Scholar
  10. Grutzendler J (2013) Angiophagy: mechanism of microvascular recanalization independent of the fibrinolytic system. Stroke 44:S84-S86 doi:44/6_suppl_1/S84 [pii];10.1161/STROKEAHA.112.678730Google Scholar
  11. Grutzendler J et al (2014) Angiophagy prevents early embolus washout but recanalizes microvessels through embolus extravasation. Sci Transl Med 6:226ra231 doi:6/226/226ra31 [pii];10.1126/scitranslmed.3006585Google Scholar
  12. Hart SP, Smith JR, Dransfield I (2004) Phagocytosis of opsonized apoptotic cells: roles for ‘old-fashioned’ receptors for antibody and complement. Clin Exp Immunol 135:181–185CrossRefPubMedPubMedCentralGoogle Scholar
  13. Ishii M et al (2011) Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method. Arterioscler Thromb Vasc Biol 31:2210–2215CrossRefPubMedGoogle Scholar
  14. Jackson CJ, Garbett PK, Nissen B, Schrieber L (1990) Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium. J Cell Sci 96(Pt 2):257–262PubMedGoogle Scholar
  15. Joddar B, Sarang-Sieminski AL, Hogrebe NJ, Tennant CJ, Gooch KJ (2017) Biomaterials and the Microvasculature. In: Ducheyne P, Grainger DW, Healy KE, Hutmacher DW, Kirkpatrick CJ (eds) Comprehensive biomaterials II, vol 5. Elsevier, Oxford, pp 67–87Google Scholar
  16. Jones D et al (2015) Actin grips: circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells. Biomaterials 52:395–406. doi:S0142-9612(15)00150-7 [pii];10.1016/j.biomaterials.2015.02.034Google Scholar
  17. Kishan AP, Cosgriff-Hernandez EM (2017) Recent advancements in electrospinning design for tissue engineering applications: a review. J Biomed Mater Res A 105:2892–2905. https://doi.org/10.1002/jbm.a.36124Google Scholar
  18. Lele TP et al (2007) Tools to study cell mechanics and mechanotransduction. Methods Cell Biol 83:443–472. doi:S0091-679X(07)83019-6 [pii];10.1016/S0091-679X(07)83019-6Google Scholar
  19. Mahajan KD, Nabar GM, Xue W, Anghelina M, Moldovan NI, Chalmers JJ, Winter JO (2017) Mechanotransduction effects on endothelial cell proliferation via CD31 and VEGFR2: implications for immunomagnetic. Separation Biotechnol J. https://doi.org/10.1002/biot.201600750Google Scholar
  20. Marie-Anais F, Mazzolini J, Herit F, Niedergang F (2016) Dynamin-actin cross talk contributes to phagosome formation and closure. Traffic 17:487–499.  https://doi.org/10.1111/tra.12386 CrossRefPubMedGoogle Scholar
  21. Mirensky TL, Hibino N, Sawh-Martinez RF, Yi T, Villalona G, Shinoka T, Breuer CK (2010) Tissue-engineered vascular grafts: does cell seeding matter? J Pediatr Surg 45:1299–1305CrossRefPubMedPubMedCentralGoogle Scholar
  22. Nam J, Huang Y, Agarwal S, Lannutti J (2007) Improved cellular infiltration in electrospun fiber via engineered porosity. Tissue Eng 13:2249–2257.  https://doi.org/10.1089/ten.2006.0306 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nguyen KT, Shukla KP, Moctezuma M, Braden AR, Zhou J, Hu Z, Tang L (2009) Studies of the cellular uptake of hydrogel nanospheres and microspheres by phagocytes, vascular endothelial cells, and smooth muscle cells. J Biomed Mater Res A 88:1022–1030.  https://doi.org/10.1002/jbm.a.31734 PubMedPubMedCentralGoogle Scholar
  24. Nyangoga H, Zecheru T, Filmon R, Basle MF, Cincu C, Chappard D (2009) Synthesis and use of pHEMA microbeads with human EA.hy 926 endothelial cells. J Biomed Mater Res B Appl Biomater 89:501–507.  https://doi.org/10.1002/jbm.b.31240 CrossRefPubMedGoogle Scholar
  25. Ozdogu H, Sozer O, Boga C, Kozanoglu L, Maytalman E, Guzey M (2007) Flow cytometric evaluation of circulating endothelial cells: a new protocol for identifying endothelial cells at several stages of differentiation. Am J Hematol 82:706–711CrossRefPubMedGoogle Scholar
  26. Park DY, Jones D, Moldovan NI, Machiraju R, Pecot T (2013) Robust detection and visualization of cytoskeletal structures in fibrillar scaffolds from 3-dimensional confocal image. Paper presented at the IEEE symposium on biological data visualization 2013, Atlanta, GA, Oct 2013Google Scholar
  27. Pecot T, Singh S, Caserta E, Huang K, Machiraju R, Leone G (2012) Non-parametric cell nuclei segmentation based on a tracking over depth from 3D fluorescence confocal images. Paper presented at the 9th IEEE international symposium on biomedical imaging: from nano to macro-2012, Barcelona, Spain, May 2012Google Scholar
  28. Pislaru SV et al (2006a) Magnetic forces enable rapid endothelialization of synthetic vascular grafts. Circulation 114:I314–I318CrossRefPubMedGoogle Scholar
  29. Pislaru SV, Harbuzariu A, Gulati R, Witt T, Sandhu NP, Simari RD, Sandhu GS (2006b) Magnetically targeted endothelial cell localization in stented vessels. J Am Coll Cardiol 48:1839–1845CrossRefPubMedGoogle Scholar
  30. Qiu Y et al (2017) Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions. Nat Commun 8:15594. doi:ncomms15594 [pii];10.1038/ncomms15594Google Scholar
  31. Rengarajan M, Hayer A, Theriot JA (2016) Endothelial cells use a formin-dependent phagocytosis-like process to internalize the bacterium listeria monocytogenes. PLoS Pathog 12:e1005603. https://doi.org/10.1371/journal.ppat.1005603; PPATHOGENS-D-15-01084Google Scholar
  32. Shimizu K, Ito A, Honda H (2007) Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering. J Biosci Bioeng 104:171–177CrossRefPubMedGoogle Scholar
  33. Singh S, Janoos F, Pecot T, Caserta E, Leone G, Rittscher J, Machiraju R (2011) Identifying nuclear phenotypes using semi-supervised metric learning. Inf Process Med Imaging 22:398–410CrossRefPubMedPubMedCentralGoogle Scholar
  34. Smith BR et al (2007) Localization to atherosclerotic plaque and biodistribution of biochemically derivatized superparamagnetic iron oxide nanoparticles (SPIONs) contrast particles for magnetic resonance imaging (MRI). Biomed Microdevices 9:719–727CrossRefPubMedGoogle Scholar
  35. Springhorn JP, Madri JA, Squinto SP (1995) Human capillary endothelial cells from abdominal wall adipose tissue: isolation using an anti-pecam antibody. In Vitro Cell Dev Biol Anim 31:473–481CrossRefPubMedGoogle Scholar
  36. Stankus JJ, Guan J, Fujimoto K, Wagner WR (2006) Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 27:735–744CrossRefPubMedGoogle Scholar
  37. Stefanini MO, Wu FT, Mac GF, Popel AS (2009) The presence of VEGF receptors on the luminal surface of endothelial cells affects VEGF distribution and VEGF signaling. PLoS Comput Biol 5:e1000622CrossRefPubMedPubMedCentralGoogle Scholar
  38. Terrisse AD, Puech N, Allart S, Gourdy P, Xuereb JM, Payrastre B, Sie P (2010) Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow. J Thromb Haemost 8:2810–2819.  https://doi.org/10.1111/j.1538-7836.2010.04088.x CrossRefPubMedGoogle Scholar
  39. Udelsman B et al (2011) Development of an operator-independent method for seeding tissue-engineered vascular grafts. Tissue Eng Part C Methods 17:731–736CrossRefPubMedPubMedCentralGoogle Scholar
  40. Villalona GA et al (2010) Cell-seeding techniques in vascular tissue engineering. Tissue Eng Part B Rev 16:341–350CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wake K, Kawai Y, Smedsrod B (2001) Re-evaluation of the reticulo-endothelial system. Ital J Anat Embryol 106:261–269PubMedGoogle Scholar
  42. Xie R et al (2012) Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells. Blood 119:2325–2334CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringThe Ohio State UniversityColumbusUSA
  2. 2.Department of Internal MedicineThe Ohio State UniversityColumbusUSA
  3. 3.Department of SurgeryIndiana University-Purdue University at IndianapolisIndianapolisUSA
  4. 4.Department of Biomedical EngineeringIndiana University-Purdue University at IndianapolisIndianapolisUSA

Personalised recommendations