Finding the needle in the haystack- detection of circulating endothelial cells with a new EpiSpot

Revascularisation in the setting of critical limb ischema (CLI) is the treatment objective. For the evaluation of an angiogenic effect, peripheral circulating endothelial cells (CECs) and endothelial progenitor cells in the blood stream should be detected in an EpiSpot system. The development of the assay and proof of principle with human umbilical vein endothelial cells (HUVEC) are part of this work.

Therefore, vascular endothelial growth factor (VEGF), the coagulation factor VIII (FVIII), E-Selectin, von Willebrand Faktor (vWF) and Interleukin-6 (IL-6) antibody pairs were tested and concentration and incubation times were evaluated with HUVECs in combination with PBMCs. Flow-cytometric analysis was done in parallel to determine the applicability of HUVECs as a model for circulating endothelial cells. Beside this, isolated and enriched endothelial cells (ECs) from peripheral blood were characterised as CECs by using FACS.

E-Selectin has been selected to identify ECs unambiguously in the established assay system. This was proven by titration of HUVEC in PBMCs, and recovery rates of 10 HUVEC / 200.000 PBMCs were achieved.

The developed assay-system can detect low numbers of endothelial cells in general. The amount of circulating endothelial cells in healthy volunteers is relatively low, therefore additional experiments will reveal the usefulness of the assay for CLI treatments and will allow to define the sensitivity in patient samples.

 Funding EU Horizon 2020(n° 733006 PACE)