Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC189
Adhesion mechanisms underlying capture and immobilisation of flowing endothelial progenitor cells by platelets
P. C. Rae1, G. B. Nash1, J. C. St John2, S. Egginton1
1. Centre for Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom. 2. Centre for Reproduction & Development, Monash Institute of Medical Research, Melbourne, Victoria, Australia.
The development of new capillaries from a pre-existing blood vessel network, angiogenesis, was long believed to be the sole mechanism of adult vasculature augmentation, through mature endothelial cell (EC) remodelling (1). However, naïve circulating endothelial progenitor cells (EPCs) have been implicated in the angiogenic response to endothelial damage and maintenance of vascular integrity (2). Several mechanisms are suggested to explain their observed effects in angiogenesis, including direct incorporation at sites of capillary growth (3) and paracrine interactions by cytokine release (4). A role for platelets in EPC recruitment has also been proposed, through the formation of platelet-EPC bridges that capture circulating EPCs from flow. Adherent platelets express surface ligands for many adhesion receptors and secrete potent chemokines, all potential mediators of platelet-EPC binding (5). In cell recruitment models such as leukocyte migration, capture occurs primarily through selectin-mediated rolling adhesion, subsequently stabilised by integrin-activating chemokine signals from the endothelium. Here, we used a flow-based in vitro adhesion assay to study mechanisms underlying adhesion of flowing EPCs to immobilised murine platelets. Platelets were isolated from 5% isoflurane-anaesthetised C57BL/6 mice and immobilised on glass capillary microslides before perfusing EPCs at wall shear stresses of 0.025 to 0.1Pa. Attachment was rare at wall shear stress above 0.025Pa. At this stress cells bound efficiently, immediately became firmly adherent, and did not start rolling even if shear stress was increased. Nevertheless, initial attachment was dependent on P-selectin, indicating that an additional adhesion mechanism was supporting immobilisation. However, when platelet αIIbβ3 integrin (GPIIbIIIa) was blocked using 10µg/ml, 25µg/ml or 50µg/ml using abciximab, EPC adhesion was not significantly reduced and cells remained stationary. Incubation of platelets with 10µg/ml and 20µg/ml each of combined clopidogrel/acetylsalicylic acid to inhibit activation through endogenous ADP or thromboxane again did not significantly affect level or type of adhesion. In addition, platelet GPVI knockdown (70% reduction determined by flow cytometry) was induced in mice over 7 days by intraperitoneal administration of 2µg/g anti-GPVI antibody. GPVI knockdown had no significant effect on EPC adhesion. Our data suggests that neither αIIbβ3 integrin nor GPVI have a role in stabilising selectin-mediated recruitment of EPCs from flow. Stationary adhesion may occur through the action of other integrin molecules, such as the β1 family. However, a selectin-initiated, integrin-stabilised mechanism (lacking the rolling adhesion seen in leukocyte recruitment) remains to be fully defined.
Where applicable, experiments conform with Society ethical requirements