Figure 3.

(a) Incubation of EPCs with neutralizing IGF-1 receptor antibodies (IGF-1Rab), 0.1 μM IR PTO-antisense oligonucleotides (IR antisense), and IR blocking antibodies (IRab). The stimulatory effect of human insulin was completely blocked by coincubation with IGF-1Rab. Coincubation with IR antisense or neutralizing IR antibodies had no effect on increased EPC outgrowth. Incubation with IGF-1Rab alone led to a decrease of EPC outgrowth, whereas incubation with IR antisense or IRab did not change EPC outgrowth. Data are given as relative number of EPC colonies of three volunteers in triplicate per culture well compared with untreated cells of each volunteer. P values as calculated by ANOVA. (b) Efficiency of IR silencing was shown by RT-PCR. EPCs treated with IR antisense (AS) had markedly suppressed IR mRNA, whereas cells treated with scrambled (Scra) oligonucleotides did not downregulate receptor mRNA. Picture shows representative data of EPCs treated with 30 nM human insulin on day 6 in culture. (c) To show the stimulatory effect of IGF-1 receptor signaling, mononuclear cells of two volunteers were treated with increasing doses of recombinant IGF-1 in the colony-forming assay. Six wells were counted for each volunteer, and data are given as relative EPC outgrowth compared with mean of untreated cells. P values as given by t test compared with controls. (d) Human insulin (30 nM) significantly increased the outgrowth of EPCs from PBMCs of 10 type 2 diabetes patients by ~208%. Addition of neutralizing IGF-1 receptor antibodies blocked the increased formation of EPC colonies, whereas addition of neutralizing IR antibodies had no effect. Data are given as absolute numbers of EPC colonies/well. (e) Identification of kinases involved in the outgrowth of EPCs from PBMCs of two healthy volunteers in triplicate. Data are shown for kinase inhibitors significantly decreasing EPC outgrowth (P < 0.05 by ANOVA) under stimulation with 30 nM insulin.