Labeling of endothelial cells with magnetic microbeads by angiophagy
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.
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).
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.
KeywordsAngiophagy Electrospun scaffold Endothelial cells Magnetic microbeads Phagocytosis Poly-capro-lactone
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.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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