Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer

Abstract

Purpose

To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL).

Methods and Results

A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS.

Conclusion

The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Graphical Abstract

Keywords:

• citrate coated nanoparticles
• atherosclerosis
• blood flow
• endothelial barrier
• permeability
• internalization

Abbreviations

ECs, endothelial cells; LSS, laminar shear stress; S, static; VSOPs, very small superparamagnetic iron oxide nanoparticles; qPEA, quantitative particle exclusion assay; HF, halofuginone lactate; HUVECs, human umbilical vein endothelial cells; ESL, endothelial surface layer; GCX, glycocalyx; WGA, wheat germ agglutinin.

Acknowledgments

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) SFB 1340 (project # 372486779) and SFB 958 (project Z02 to J.S.). S.G.T. received a travel grant from Deutsches Zentrum für Herz-Kreislauf-Forschung-Promotion of Young Scientists: Visiting Scientist Programme. For portions of the work performed at Northeastern University, funding was provided by the United States National Science Foundation (NSF) through the CAREER Award CMMI-1846962 (granted to E.E.E.) and the United States National Institutes of Health (NIH) through K01-HL125499 (granted to E.E.E.).

The authors would like to thank the Advanced Medical Bioimaging Core Facility (AMBIO) at the Charité - Universitätsmedizin Berlin for their support in CLSM and SDCM image acquisition and the Institute for Chemical Imaging of Living Systems Core Facility at Northeastern University for providing confocal imaging capabilities. The authors also acknowledge financial support from the Open Access Publication Fund of Charité – Universitätsmedizin Berlin.

Disclosure

The authors report no conflicts of interest in this work. A.M.W. and A.L. contributed equally to this work and are co-senior authors.