Abstract
Platelet attachment to the subcellular matrix at injured sites of the vasculature is followed by their activation and release of microparticles. Platelet-derived microparticles (PMP) have been shown to be involved in the regulation of hemostasis. However, little is known about the role of PMP in the regulation of angiogenesis and related clinical conditions. We have recently demonstrated that platelets as a cellular system induce angiogenic responses both in vitro and in vivo. In the present study, we investigated the potential role of PMP in angiogenesis. A strong dose-dependent pro-angiogenic effect of PMP in the rat aortic ring model (5.3±2.1 mm2 surface covered with sprouting vessels versus 0.24±0.2 mm2 in the control, p<0.001) was observed. This effect was reversed by selective inhibition of VEGF, bFGF and PDGF (surface covered with vessels 0.7±0.5 mm2, 1.7±1.5 mm2, and 2.4±1.2 mm2, respectively, p<0.02 versus control), but not by inhibition of heparanase (5.1±0.8 mm2, p>0.5 versus control). PMP exert their stimulatory effect via PI3-kinase, Src kinase and ERK, whereas protein kinase C seems not to be involved, as judged by the aortic ring sprouting model. Using confocal and electron microscopy, we also demonstrate that PMP bind to non-activated endothelial cells. In addition, PMP markedly increased invasion of human endothelial cells through a layer of matrigel. This effect was abolished by an inhibitor of VEGF receptor tyrosine phosphorylation or laminaran sulfate (heparanase inhibitor). It was also partially reduced by PDGF blocking mAb, whereas blocking of bFGF had no effect. Furthermore, we have demonstrated that PMP induce angiogenesis in an in vivo model, in which beads (30 μl) of 4% agarose gel containing the substances under study were transplanted subcutaneously into mice. Image analysis of the capillary area revealed the following: control beads − 0.2±0.05 mm2, VEGF + bFGF containing beads − 4.8±1.1 mm2, PMP (100 μg/ml) containing beads − 5.1±1.3 mm2, p<0.001 versus control. The latter finding was further supported by immunohistochemical staining of the skin in the vicinity of the beads for von Willebrand factor, a marker of endothelial cells (control − 4.0±3.2, VEGF+bFGF − 12±4.4, PMP − 17±6.5 capillaries per view field, p<0.05 versus control). Finally, we explored the potential effect of PMP in a rat myocardial infarction model. Ischemia was induced by LAD ligation followed by injection of either PMP or PBS into the ischemic region. Preliminary evaluation of the LAD myocardial territory in sham-operated animals revealed 157±42.0 capillaries per view field. In contrast, number of capillaries observed 3 weeks after induction of ischemia was reduced to 34±21.5. When PMP were injected into the ischemic region, there was an increase in capillary number up to 97±27.3. In conclusion, PMP induce angiogenesis in both in vitro and in vivo models. Local injection of PMP into the ischemic myocardium may improve revascularization.
Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ3
