The Small GTPase Rap1A Is Central for Migration and Adhesion of Mesenchymal Stromal Cells (MSCs)

MSCs are currently intensively investigated for their tissue regenerative and immunomodulatory properties, and for many applications are applied via the intravenous route. We have previously shown that MSCs roll and adhere on endothelial cells both in vitro and in vivo , using part of the array of the adhesion receptors which are known to mediate adhesion of hematopoietic stem and progenitor cells during mobilization from bone marrow and homing after transplantation (Blood108:3938, 2006). To identify further gene products which are potentially relevant for coordinated circulation behaviour of MSCs, we kept human MSCs under shear stress by continuous stirring for 0, 3, 6 and 12 h, isolated RNA and performed cDNA microarrays. We found approx. 500 out of 21.000 sequences regulated by shear stress. Interestingly, differentiation-associated genes (osteogenic, lipogenic) were generally downregulated. Several adhesion receptors, including integrins α4 and α7 were time-dependently upregulated. Among G proteins and -associated factors, the Guanosin Nucleotide Exchange Factors (GEFs) for the small GTPase Rap1, RAP-GEF2 and PDZ-GEF1 were both strongly upregulated by shear stress, whereas Rho GTPase regulator RGS-3 was downregulated. We next isolated MSCs from murine bone marrow from Rap1A deficient or wild type mice. Wild type MSCs were found to express Rap1A RNA and protein. However, it was not possible in 5/5 cases to isolate MSCs by the culture expansion method from Rap1A−/− mouse bone marrow. Yet we were able to derive and analyze MSCs from Rap1A+/− mice, and found that their binding to immobilized HUVEC endothelial cells in laminar flow chambers was approximately threefold reduced compared to wild type controls. Similarly, shear stress resistant adhesion to both, Vascular Cell Adhesion Molecule (VCAM)-1 and Intercellular Adhesion Molecule (ICAM)-1 was about 50% decreased compared to wild type MSCs isolated from littermate mice. Moreover, SDF-1-induced adhesion strengthening on VCAM-1 and ICAM-1 was also decreased in the Rap1A+/− MSCs. Transfection of MSCs with an expression vector encoding dominant negative Rap1 (Rap1N17) decreased, whereas pretreatment of MSCs with the Rap1 specific EPAC activator, 8-Br-(4PCT)-cAMP increased adhesion of MSCs to VCAM-1. These data demonstrate an essential role of the GTPase Rap1A for integrin-mediated adhesion of MSCs. Interference with Rap1 signalling thus provides a means to modulate adhesion, and potentially, homing behaviour of MSCs.