Abstract

Hematopoietic Progenitor Cells (HPC) can be mobilized from bone marrow into the circulation in response to a number of stimuli including G-CSF, AMD3100 (antagonist of CXCR4-DSF-1 axis) and vascular endothelial growth factor (VEGF). The main mechanism for mobilization of HPCs upon stimulation by classical “mobilizers”as G-CSF is thought to be through extracellular matrix proteolysis in the marrow. Urokinase is a serine protease present in the marrow and contributes to mobilization of stem cells upon binding to its receptor (uPAR) and activating plasminogen that leads to matrix degradation. Our previous data show that the effect of VEGF on endothelial cell migration is exerted through activation of the uPA/uPAR system and through co-internalization of β 1 integrins. Upon internalization of these receptors, cells detach from their underlying extra-cellular matrix (ECM) as well as from stromal cells. We hypothesize that the contribution of VEGF to HPC mobilization occurs through a similar mechanism. We also want to analyze the influence of uPA/uPAR deficiencies on mobilization of Gr-1+/CD-11b+ myeloid and c-kit +/Sca-1+ (SK)cells by VEGF and AMD3100 and compare it with G-CSF as a classical “mobilizer”. Wild type, uPA knockout and uPAR knockout mice in C57BL6 background were used for in vivo experiments. We collected peripheral blood before and 2 hours after i.p. injection of VEGF-E and AMD3100 and assessed the number of SK cells and myeloid cells by FACS analysis. We also administered G-CSF for 5 days and compared blood samples before and after the experiment. To evaluate the effect of VEGF on HPC integrin expression, femurs of the respective animals were incubated with VEGF in an ex vivo experimental model and β1 expression was assessed by FACS analysis. In vivo data demonstrated a significantly reduced responsiveness of uPA−/− mice to VEGF-E in the first 2 hours after the injection. This decreased responsiveness to VEGFis observed in uPAR−/− mice but to a lesser degree than in uPA−/− mice..(40 +/−16 % and21 +/− 20% respectively vs 65 +/− 24 % in wt, means and SD). Injection of urokinase together with VEGF to uPA−/− mice rescues the lack of mobilization of SK cells. Ex vivo stimulation of uPAR knockout femoral bone marrow cells with VEGF for 20 minutes provides evidence that the internalization of β1 integrins upon VEGF stimulation is uPAR dependent. VEGF can also increase in vivo the number of Gr-1+/CD-11b+ myeloid cells after 2 hours in wt mice (96 +/− 45%) but not in urokinase deficient or urokinase receptor deficient mice (7 +/− 11% and 21+/−33%, respectively). AMD3100 has a strong effect on mobilization of SK cells in wt animals within 2 hours (increase of 2.8+/−0.78 times) but cannot mobilize these cells in uPA and uPAR deficient mice to the same extend (0.8+/−0.65 times and 0.1+/−0.07 respectively). G-CSF injection for 5 days mobilizes Gr-1+/CD-11b+and SK cells in wt and knock out mice to a similar extent, indicating that the capacity to release these cells from the bone marrow is not affected by uPA or uPAR gene deficiency. Our results demonstrate a reduced mobilization of uPA−/− and uPAR−/− HPCs and myeloid cells in response to VEGF compared to wt mice. VEGF leads to internalization of the expression of β1 integrins on the surface of SK cells in wt but not in uPAR−/− mice. In addition, we could show that the uPA/uPAR system plays a role in AMD3100-dependent mobilization of these cells. These data indicate that the uPA – uPAR system plays a pivotal role in short-term but not long-term bone marrow HPC and PMN leukocyte mobilization.

Disclosures: No relevant conflicts of interest to declare.

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