Abstract 5257: Priming with Angiopoietin-1 Augments Angiogenic Potential of Peripheral Blood Stem Cells Mobilized with Granulocyte-Colony Stimulating Factor

Abstract

Background: We introduce the concept of ‘priming’ mobilized peripheral blood stem cells( mob -PBSCs) with G-CSF using Angiopoietin-1(Ang-1). Methods and Results: Ang-1 was administered as a priming agent because it was found that mob PBSCs highly express Tie2, Ang-1 receptor [19.2±3% vs. 1.2±0.2%, p<0.01 for mob PBSCs vs PBMCs]. We investigated whether the effect of Ang-1 on mob PBSCs differentiation is mediated via the transcriptional regulation of ets-1. The gene expression of ets-1 increased in a time dependent manner after Ang-1 stimulation(400ng/ml) in mob PBSCs. Compared with vehicle, treatment with Ang-1 for 4 hours resulted in stronger expression of endothelial markers, such as, CD31 and VE-cadherin, which were significantly reduced with antisense ets-1 oligodeoxynucleotides(ODN). These results were also confirmed at the protein level by FACS analysis. We postulated that integrins that contain an ets-1 binding motif in their promoter region may be activated by Ang-1, such as α4β1 and α5β1 integrins. RT-PCR showed increased mRNA expression of α4, α5, and β1 integrins after Ang-1 stimulation, and its attenuation after transfection with antisense ets-1 ODN. These results were also confirmed at the protein level by FACS analysis. To correlate these findings with functional changes in mob PBSCs, we performed in vitro adhesion assays. Ang-1 stimulation increased mob PBSCs adhesion to HUVECs and fibronectin, while pretreatment with anti-hTie2 or anti-integrin antibody prior to Ang-1 stimulation abrogated these results. And, we checked the effect of Ang-1 in vivo model. mob PBSCs were obtained from rabbit and primed with COMP-Ang1(400ng/ml) for 4 hours. mob PBSCs were labeled with Tc-99m HMPAO and were injected into a central artery of rabbit ear. mob PBSC engraftment was quantified by the ratio of whole rabbit ear radioactivity over total infused mob PBSC radioactivity. In contrast, cells primed with COMP-Ang1 showed markedly increased engraftment by 53±33%( p <0.05). Finally, mob PBSCs primed with Ang-1 enhanced neovascularization in a nude mouse hindlimb ischemia model. Conclusion: These results suggest that mob PBSCs priming with Ang-1 may enhance the efficacy of transplanted mob PBSCs in terms of increasing neovascularization in ischemic tissue.