Mesenchymal Stromal Cells (MSCs) Lack Adhesion to Endothelial Cells through Selectins and b2 Integrins and Interact Dominantly Using Integrin a4b1 Activated by GTPase Rap1.

Abstract 2589

Mesenchymal Stromal Cells (MSCs) are increasingly used in patients e.g. to combat Graft-versus-Host-Disease or to support hematopoietic regeneration after allogeneic Hematopoietic Stem Cell Transplantation. However, both the dynamics and the mechanisms by which intravenously transplanted MSCs (n=5 donors) interact with the vessel wall are incompletely characterized. We compared the ability of MSCs and blood leukocytes (PBMCs; n=5) to interact with known endothelial ligands using a flow chamber system and transplantation into immunodeficient mice. Although recombinant P- and E-selectin reduced the flow speed of MSCs significantly by 30±3% (means ± SD), which was reversible in the presence of function-blocking anti P- or E-selectin antibodies MSCs were induced to roll at speeds of 1–5 μm/s at much lower efficiency as PBMCs at shear stresses of 0.35 – 8 dyn/cm² (MSCs, 2±1%; PBMCs, 21±4% of interacting cells; means ± SD). In contrast, immobilized recombinant VCAM-1 Ig under these conditions dose-dependently induced interactions of MSCs with a comparable efficiency as of PBMCs, leading to stable arrest. Whereas 33 ± 8% (means ± SD) of PBMCs rolled on 20 μg/ml VCAM-1 Ig, MSCs howerver lacked rolling behaviour but always directly stopped. Analysis of the velocity vectors of individual MSCs revealed an staggering interaction of MSCs during the stopping process with prolonged distances until they reached stable arrest (58±10 vs 14±3 μm for MSCs and PBMCs, respectively (means ± SD, p=0.004). Whereas PBMCs were induced to both roll and arrest on the b2 integrin ligand ICAM-1 and on the a4b7 integrin ligand MAdCAM-1 at 0.35–2 dyn/cm², MSCs were incapable to roll or arrest under these conditions. Analysis of MSC arrest behaviour on HUVEC endothelial cells pretreated with TNF-a that expressed both VCAM-1 and ICAM-1 and coated with chemokines CXCL12 or CCL19 showed that MSCs arrested with only 14±5% of the efficiency of PBMCs at 0.35dyn/cm². Function blocking antibodies confirmed the pivotal role of a4b1/VCAM-1 dependent adhesion in stable arrest of MSCs. We found that MSCs bound to the HUVECs more rigidly than PBMCs since, when arrested cells were exposed to increased shear stress of 5 dyn/cm² only 3±4% of MSCs de-adhered, in contrast to PBMCs which 74±4% de-adhered. Chemokine-induced signals were instrumental in strengthening adhesion of MSCs since for CXCL12-induced MSCs to endothelial cells adhesion was counteracted (i) pretreatment with pertussis toxin, (ii) by application of the CXCR4 inhibitor (AMD3100) and (iii) after siRNA mediated downregulation of b arrestins 1 or 2, or the GPCR kinase (GRK)-6, which known to be involved in chemokine receptor signalling. All above treatments lead to significantly decreased numbers of MSCs that remained adherent at shear stresses between 2 and 15 dyn/cm². Since cDNA microarray analises using MSCs exposed to shear stress for 0–12h indicated, among others, upregulation of the GTPase Rap1A and its activator, PDZ-GEF1, we analyzed activation of Rap1 proteins using a pulldown assay in a CXCL12-induced manner. MSCs depleted of Rap1A and B proteins by transfection with siRNA showed a significant reduction of stably arresting cells (>10s) on HUVEC precoated with CCL19 to 66±4% of controls (p=0.04). Moreover, siRNA mediated depletion of Rap1A and B in MSCs resulted in a >60% reduction in numbers of transplanted MSCs accumulating in the lungs of NOD/SCID mice, and concomitant significant increases of numbers of MSCs detected in blood (8.9±0.4 fold; p<0.05) and other tissues such as liver (2.2±0.8 fold, p<0.05) and spleen (2.1±0.7 fold, p<0.05). Together, MSCs display major deficits in adhesion dynamics and adhesion receptor usage compared with normal leukocytes. Modulation of the GTPase Rap1 may serve as a strategy to counteract the strong activation of b1 integrins and improve the circulation behaviour of transplanted MSCs.