File S1 - In Vivo Evidence for Platelet-Induced Physiological Angiogenesis by a COX Driven Mechanism

Table SI, Differential white cell count 24 h following platelet depletion (×103.mm−3). Table SII, Platelets VEGF content is not altered by treatment (pg VEGF.10−6 platelets; mean±SEM). Figure SI, Experimental protocol for inhibition of platelet activation with clopidogrel and/or acetylsalicylic acid (ASA; aspirin). A) Endothelial sprouting was induced by extirpation of m. tibialis anterior causing overload of m. extensor digitorum longus. 24 h after surgery oral 30 mg/kg clopidogrel/ASA as dual or single regimens was administered for 72 h. B) ASA administration was begun 24 h after initiation of prazosin treatment, lasting 72 h. Prazosin induces longitudinal splitting. In both A and B, sampling occurred on the eighth day. Figure SII, Inhibition of platelet activation with acetylsalicylic acid (ASA; aspirin) is dose-dependent. Mice were administered aspirin via drinking water for 7 days. Experimental groups were 3 control mice, 3 low dose aspirin mice (30 mg/litre) and 3 high dose aspirin mice (300 mg/litre). Following 7 days aspirin treatment, blood was collected into sodium citrate and platelet-rich plasma (PRP) was prepared by centrifugation. Platelet aggregation was monitored by light transmission aggregometry in a Born lumi-aggregometer. Following stimulation with 0.5 mM arachidonic acid, platelets from the low dose aspirin treated mice aggregated normally (A) whereas platelets from the high dose aspirin treated mice showed only a minimal shape change response (B). Platelets from all mice aggregated normally to 500 mM PAR-4 peptide (C). Figure SIII, Influence of scaling on muscle capillary supply. In response to reviewer comments we offer an explanation for the choice of capillary to fibre ratio (C∶F) as the most robust index of angiogenesis. It has been repeatedly been shown that other methods of quantifying angiogenesis (such as capillary density; Hudlická O, Brown MD, Egginton S. 1992. Angiogenesis in skeletal and cardiac muscle. Physiological Reviews 72: 369–417) have a bias due to alterations in fibre size often observed among experimental groups, that mask the changes in capillary number, whereas C∶F is much less sensitive to these scaling effects (Egginton S. 1990. Morphometric analysis of tissue capillary supply. In: Boutilier RG (ed) Vertebrate Gas Exchange from Environment to Cell. Advances in Comparative and Environmental Physiology. 6: 73–141; Hudlická O, Brown MD, Egginton S. 1998. Angiogenesis: basic concepts and methodology. Chapt. 1, pp 3–19. In: Halliday A, Hunt BJ, Poston L, Schachter M (eds) An Introduction to Vascular Biology. CUP). The current experiment is no different: (1) contralateral muscle ± platelet depletion show minor, but reciprocal differences in CD and a(f). (2) extirpation without platelet depletion has a similar CD to contralateral muscle despite muscle hypertrophy that would be expected to reduce CD if angiogenesis had not occurred (C∶F is significantly higher); in contrast extirpation with platelet depletion had both a similar CD and a(f) as contralateral muscles reflecting an absence of angiogenesis (C∶F similar). (3) in the same way, relatively small differences in fibre size explain the modest difference in CD among prazosin groups, while the C∶F is very similar (see Fig. 2 in the main text). Abbreviations: CD, capillary density; a(f), fibre cross-sectional area; C-, contralateral without platelet depletion; C+, contralateral with platelet depletion; E, extirpation; P, prazosin. (DOC)