Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C44
Role of platelets and platelet-collagen interactions in physiological angiogenesis
I. M. Packham1, S. P. Watson1, R. Bicknell1, S. Egginton1
1. University of Birmingham, Birmingham, United Kingdom.
Physiological angiogenesis (capillary growth from pre-existing vessels) requires longitudinal splitting and/or endothelial sprouting (classical angiogenesis), phenotypes differing in stimuli, pathways and effector molecules involved. Platelets are pro-angiogenic in tumour and inflammatory models, with data suggesting platelet activation/granule secretion and platelet binding are necessary1. We sought to identify a platelet role in physiological angiogenesis using the two phenotypes with platelet depletion/inhibition, and investigated the requirement for platelet-collagen interactions. All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. C57BL/6 mice (n=3-6/group) were initially anaesthetised with 5% isoflurane in O2 and maintained at 2% where required. Longitudinal splitting was induced by ad libitum administration of the selective postsynaptic α1 adrenoceptor antagonist prazosin, causing chronic arteriolar vasodilatation and resultant increased downstream blood flow/shear stress. Endothelial sprouting was induced by unilateral extirpation of hindlimb m. tibialis anterior resulting in hyperplasia and hypertrophy of the synergistic m. extensor digitorum longus (EDL). Platelets or GPVI protein were depleted in vivo by 2µg/g body weight rat anti-mouse GPIbα / GPVI antibody or activity inhibited by dual clopidogrel hydrogensulfate/acetylsalicylic acid, which prevent activity by inhibiting the GPIIb/IIIa pathway. Prazosin induced a significant increase in capillary:fibre (C:F) in EDL after 7 days (P<0.05; one-way ANOVA). Angiogenesis can be determined by C:F or capillary density. C:F is less sensitive to inter-animal variability in fibre size, providing a more accurate representation. Platelet depletion did not affect the increase in C:F, demonstrating platelets are not required for shear-induced angiogenesis. However, extirpation induced significant increases in C:F compared to contralateral which were entirely absent after platelet depletion (P<0.05; one-way ANOVA). In validation, inhibition of platelet activity resulted in a similar change in response to extirpation with platelet depletion, together demonstrating platelet mediation of endothelial sprouting angiogenesis. To determine the role of platelet-collagen interactions in platelet-mediated angiogenesis we used GPVI depletion following overload, since GPVI is the major direct collagen receptor2. GPVI depletion did not affect the change in C:F normally observed after overload, suggesting platelet-collagen interactions were not required. This may suggest a different mechanism of action for platelets in mediating angiogenesis than occurs during thrombus formation, when platelets bind to areas of subendothelial collagen.
Where applicable, experiments conform with Society ethical requirements