gms | German Medical Science

Introduction: Bone marrow derived endothelial progenitor cells (EPC) are pivotal for vasculogenesis as these cells migrate to sites of ischemia or tissue damage and contribute to the new formation of vessels by self-incorporation into the endothelial layer and pro-angiogenic paracrine activity. However, mechanism of homing and migration of EPCs is a field of speculation and is controversially discussed. Hence, we investigated whether surgical wound fluid (SWF) shows homing and chemotaxis inducing properties to EPCs. Human umbilical vein endothelial cells (HUVEC) were incubated with SWF and adhesion of EPCs was determined by cytometry. Furthermore, chemotaxis inducing properties of SWF to EPCs were analysed using a modified Boyden chamber assay

Materials and methods: EPCs were isolated by positive selection with CD133 microbeads from peripheral blood mononuclear cells (PBMC) which were isolated from buffy coat by density gradient centrifugation. Enrichment of CD133+/CD34+ cells (EPCs) was controlled by cytometry (EPC population > 80 %). HUVEC were isolated from umbilical cords and passages 2-3 were used for adhesion experiments. HUVEC were incubated with SWF or serum from patients, isolated 3, 8 and 24 hours after surgical limb treatment. HUVEC activation with SWF (3h, 8h, 24h) or serum (3h, 8h, 24h) was performed for 6 hours. Thereafter EPCs were co-cultured on pre-treated HUVEC for 1 hour, non adherend EPCs were removed by washing the co-culture 3 times with PBS +/+, than all remaining cells were detached, and ratio of EPCs to HUVEC was determined after CD34/CD133 staining by flowcytometry. Chemotaxis experiments were performed using a modified Boyden Chamber of 3 μm pore size. EPCs were loaded to the upper chamber and movement of cells was compared as described in the following array: serum vs. serum, SWF vs. SWF and serum vs. SWF. Two hours after start of the experiments cells from the lower chamber were collected, stained for CD133/CD34 and total cell number was measured by flowcytometry. Results are presented as mean +/- SEM. U-Test was used for statistical evaluation, p<0.05 was considered significant.

Results: A significantly elevated EPC to HUVEC ratio could be detected after co-culture of EPCs on pre-treated HUVEC with SWF (8h) and SWF (24h) versus serum (8h) and serum (24h) (see figure 1 [Fig. 1]). Significant migration of EPCs in the Boyden chamber assay could be measured for serum/SWF (8h, 24h) vs. serum/serum (8h, 24h) and SWF/SWF (8h, 24h) (see table 1 [Tab. 1]).

Discussion: Our results show that SWF induces homing of EPCs to HUVEC and has the potency to induce chemotaxis of EPCs in a Boyden chamber assay. Further experiments with measurement of adhesion receptors on HUVEC and EPCs were performed and showed a high level of LFA-1 and VLA-4 on circulating CD133/CD34 cells as well as an up-regulation of the complement receptors ICAM and VCAM on SWF stimulated HUVEC. Blocking experiments of these receptors will show whether these receptors are involved in EPC homing to surgically caused wounds.