Skip to main content

Advertisement

SpringerLink
Log in

Labeling of endothelial cells with magnetic microbeads by angiophagy

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Chrastina A, Massey KA, Schnitzer JE (2011) Overcoming in vivo barriers to targeted nanodelivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol 3:421–437

    Article  PubMed  CAS  Google Scholar 

  • 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 224:352–361

    Article  PubMed  CAS  Google Scholar 

  • Collins C et al (2012) Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway. Curr Biol 22:2087–2094

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 10:155–163

    Article  PubMed  CAS  Google Scholar 

  • Dobson J (2008) Remote control of cellular behaviour with magnetic nanoparticles. Nat Nanotechnol 3:139–143

    Article  PubMed  CAS  Google Scholar 

  • Fens MH et al (2008) Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells. Blood 111:4542–4550

    Article  PubMed  CAS  Google Scholar 

  • Fens MH et al (2010) Erythrophagocytosis by angiogenic endothelial cells is enhanced by loss of erythrocyte deformability. Exp Hematol 38:282–291

    Article  PubMed  CAS  Google Scholar 

  • Gao C et al (2013) Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity. Thromb Haemost 109:1079–1090

    Article  PubMed  CAS  Google Scholar 

  • Godbey WT, Hindy SB, Sherman ME, Atala A (2004) A novel use of centrifugal force for cell seeding into porous scaffolds. Biomaterials 25:2799–2805

    Article  PubMed  CAS  Google Scholar 

  • 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.112.678730

  • 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.3006585

  • Hart SP, Smith JR, Dransfield I (2004) Phagocytosis of opsonized apoptotic cells: roles for ‘old-fashioned’ receptors for antibody and complement. Clin Exp Immunol 135:181–185

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 31:2210–2215

    Article  PubMed  CAS  Google Scholar 

  • 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):257–262

    PubMed  Google Scholar 

  • 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 67–87

  • 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.2015.02.034

  • 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.36124

  • 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)83019-6

  • 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.201600750

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 45:1299–1305

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 82:706–711

    Article  PubMed  Google Scholar 

  • 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 2013

  • 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 2012

  • Pislaru SV et al (2006a) Magnetic forces enable rapid endothelialization of synthetic vascular grafts. Circulation 114:I314–I318

    Article  PubMed  Google Scholar 

  • 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 48:1839–1845

    Article  PubMed  CAS  Google Scholar 

  • 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/ncomms15594

  • 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-15-01084

  • 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 104:171–177

    Article  PubMed  CAS  Google Scholar 

  • 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 22:398–410

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 9:719–727

    Article  PubMed  Google Scholar 

  • 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 31:473–481

    Article  PubMed  CAS  Google Scholar 

  • Stankus JJ, Guan J, Fujimoto K, Wagner WR (2006) Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 27:735–744

    Article  PubMed  CAS  Google Scholar 

  • 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:e1000622

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Udelsman B et al (2011) Development of an operator-independent method for seeding tissue-engineered vascular grafts. Tissue Eng Part C Methods 17:731–736

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Villalona GA et al (2010) Cell-seeding techniques in vascular tissue engineering. Tissue Eng Part B Rev 16:341–350

    Article  PubMed  PubMed Central  Google Scholar 

  • Wake K, Kawai Y, Smedsrod B (2001) Re-evaluation of the reticulo-endothelial system. Ital J Anat Embryol 106:261–269

    PubMed  CAS  Google Scholar 

  • Xie R et al (2012) Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells. Blood 119:2325–2334

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, The Ohio State University, Columbus, OH, USA

    Jessica Thomas & Keith J. Gooch

  2. Department of Internal Medicine, The Ohio State University, Columbus, OH, USA

    Desiree Jones & Mirela Anghelina

  3. Department of Surgery, Indiana University-Purdue University at Indianapolis, Indianapolis, IN, USA

    Leni Moldovan

  4. Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis, Walther Hall, R3 Research Building, Room C644, 980 W Walnut St., Indianapolis, IN, 46202, USA

    Nicanor I. Moldovan

Authors
  1. Jessica Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Desiree Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leni Moldovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mirela Anghelina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Keith J. Gooch
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nicanor I. Moldovan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicanor I. Moldovan.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thomas, J., Jones, D., Moldovan, L. et al. Labeling of endothelial cells with magnetic microbeads by angiophagy. Biotechnol Lett 40, 1189–1200 (2018). https://doi.org/10.1007/s10529-018-2581-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-018-2581-9

Keywords

Search

Navigation