The Rho GTPase Cdc42 Is a Key Regulator of the Genesis and Function of Bone Marrow Derived Endothelial-Like Cells

Abstract 2109

In addition to maintaining steady state hematopoiesis in the adult life, bone marrow (BM) derived cells contain endothelial progenitor cell (EPC) activity and may play a role in promoting vascular regeneration upon injury or angiogenesis during tumor progression. Whether hematopoietic stem/progenitor cells (HSPCs) in the BM can give rise to functional EPCs and if they contribute significantly to endothelial cell regeneration remain controversial questions. Cdc42 is a member of the Rho GTPase family that has been shown to play essential and unique roles in multiple lineages of blood cell regulation including HSPC proliferation and cytoskeleton organization. In addition, Cdc42 is found to regulate blood vessel permeability and endothelial lumen formation [1, 7]. By using a conditional knockout (KO) and BM transplant mouse model (i.e. Mx1-cre; Cdc42loxP/loxP mice and transplant recipients) that allows specific, inducible deletion of cdc42 gene in the bone marrow compartment and a small molecule Cdc42 activity-specific inhibitor (CASIN), we have examined the hypothesis that Cdc42 critically regulates the BM-derived EPC and/or angiogenic supporting cell production and function. First, we found that inducible Cdc42 KO in BM cells inhibited colony-forming activity of total BM cells by ~10-fold and ~40-fold in two CFU-EPC assays that have been used in the published literature - in EGM2 medium containing VEGF, FGF2, IGF1 and EGF [6] and a modified EGM2 medium containing VEGF, FGF2 and IGF1 [8], respectively. The colonies formed from Cdc42 KO BM gave a distinct small round morphology. Concomitantly, Cdc42 deletion resulted in a ~3-fold enhancement of hematopoietic colony-forming CFU-C activity of the BM cells, consistent with our previous report on hematopoietic regulation by Cdc42. Both effects on EPC and HSPC activities can be attributed to hematopoietic cell regulation by Cdc42, since similar reduction of CFU-EPC activity and increase in CFU-C activity were observed in Mx-cre; Cdc42lox/lox transplant recipient mice after polyIC induction. Second, given the controversies in the literature about cell markers of BM EPCs, FACS analysis using 5 different sets of putative EPC markers was carried out to examine the effect of Cdc42 knockout on BM EPC compartment. PolyIC induced Cdc42 deletion led to ~4-fold reduction of CD45lo/CD11b-/VE-Cad+ cells [2], ~3-fold reduction of CD45-/PDGFRa+ [5], ~3 fold reduction of CD45-/Lin-/Flk1+ [3, 4], 2-fold reduction of CD45+/Lin-/Flk1+ and 3-fold decrease of CD45-/CD31+ EC population [9]. Similar results were also obtained from the BM Cdc42 deleted transplant recipients. Third, to further distinguish EPCs of blood lineage from those of non-blood lineage in the BM cells, purified CD45+ or CD45- cells were isolated from BM cells by double FACS sorting, and their abilities to produce EC-like cells and to express endothelial markers CD31, PDGFRa and vWF in a matrigel culture, were analyzed. Cdc42 deletion led to a drastic reduction of the tube-forming and CD31+/PDGFRa+/vWF+ cells by over 100-fold, in both the CD45+ and CD45- BM cell culture. Fourth, treatment of WT BM cells with CASIN abolished colony-forming activity of BM cells in the CFU-EPC assays, mimicking that of Cdc42 knockout. Upon CASIN withdrawn, the cells could continue differentiation into adherent EC-like colonies. Finally, Cdc42 knockout resulted in a 3-fold reduction of VEGFR2 cell surface presentation, but not expression, in BM cells, suggesting Cdc42 may regulate VEGFR2 localization to impact endothelial lineage commitment. While experiments to dissect the signaling mechanisms and the requirement of Cdc42 for angiogenesis and endothelial or endothelial cell-supporting function are ongoing, these results indicate that Cdc42 is essential for BM derived EPC or EPC-like activities. Targeting Cdc42 may provide a novel strategy in modulating BM-EPC and EC regeneration.