Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.
Concurrent Stimulation of Cannabinoid CB1 and Dopamine D2 Receptors Augments cAMP Accumulation in Striatal Neurons: Evidence for a GsLinkage to the CB1 Receptor
