Abstract 1473: Notch Signaling is Required for Human Endothelial Progenitor Cell Differentiation and Vascular Repair
Introduction: Bone marrow derived endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair. Notch receptor signaling regulates differentiation of primitive hemangioblasts during vascular development but its role in adult EPCs is largely unknown.
Methods and Results: Human EPCs expressed Notch1 (N1), the Notch ligand Delta-like 1 and the Notch target gene HeyL, indicative of active Notch signaling in cultured EPCs. To block Notch signaling we inhibited Notch intramembrane processing by gamma secretases, which is required for Notch activation. The gamma secretase inhibitors (GSI), DAPT, and a structurally unrelated compound, DBZ, dose-dependently reduced EPC numbers (67±6% reduction, p<0.01) and prevented N1 processing. The specificity of Notch inhibition was verified by adenoviral transduction of a dominant negative inhibitor of Notch signaling, RBPj-κ(R218H), with identical results. Furthermore, EPC colony formation, which correlates with cardiovascular risk and prognosis, was strongly reduced after Notch inhibition (control 65±15 vs. GSI 20±6, p<0.01). Migration, adherence to activated endothelial cells (ECs) and tube formation are important functional capabilities of EPCs. Notch inhibition significantly reduced EPC migration (74±9% reduction, p<0.01), completely prevented EPC adherence to ECs (>95% reduction) and inhibited tube formation (77±12% reduction, p<0.01). EPC surface marker analysis by FACS revealed a unique phenotype shift after Notch inhibition. Expression of monocytic markers (CD45 and CD14), chemokine receptors (CXCR4) and adhesion molecules (PECAM) was impaired, but expression of endothelial markers (KDR, vWF and CD105) unchanged, indicating specific modulation of EPC differentiation. The relevance for vascular repair in vivo was evaluated in the mouse carotid injury model. Injection of control EPCs enhanced reendothelialization compared to PBS, which was completely abrogated by Notch inhibition (reendothelialized area: PBS 18±9%, EPC 48±7%, EPC+GSI 17±9%). Furthermore, homing of EPC to injured carotids was severely impaired (p<0.05).
Conclusion: These results demonstrate a requirement for Notch signaling for EPC differentiation, function and vascular repair.