Abstract 404: The Role of 20-HETE in the Regulation of Endothelial Progenitor Cell Stemness

Abstract

Objective: Endothelial progenitor cells (EPC) are known to contribute to neovascularization by producing various angiogenic cytokines, such as VEGF, at target sites. Factors that are capable of maintaining EPC stemness can in turn promote EPC self-renewal, thereby increasing their angiogenic functions. We recently identified the CYP4A-derived 20-HETE regulates some of the EPC pro-angiogenic properties (e.g. proliferation, migration) in vitro . In the present study, we further investigated the role of 20-HETE in the regulation of EPC stemness that are associated with their angiogenic functions. Methods: Human umbilical cord blood-derived EPC were isolated by magnetic cell sorting and cultured in an EPC enrichment media. LC/MS/MS was performed in cells harvested at various times during the EPC-EC differentiation process for 20-HETE production. Next, real-time PCR was used to assess the effects of 20-HETE (1 nM) on the expression of Oct4, Sox2, and NANOG, genes commonly associated with cell stemness. Flow cytometry was performed to quantify the population of progenitor vs differentiated EPC in the presence and absence of a 20-HETE synthesis inhibitor (DDMS; 10 μM) or a 20-HETE competitive antagonist (6, 15-20-HEDGE; 10 nM) in EPC cultures. Lastly, the effects of global increase in 20-HETE induction in vivo on bone marrow (BM)-derived EPC stemness were also examined using flow cytometry. Results: We found that differentiation of EPC to EC was associated with a time-dependent decrease in 20-HETE production. Exogenous 20-HETE significantly increased the expression of Oct4, Sox2, and NANOG by 1.5-, 2-, and 8.2-fold, respectively. DDMS and 6, 15-20-HEDGE decreased the number of progenitor cell population compared to vehicle-treated controls. In contrast, addition of 20-HETE increased the number of cell population expressing progenitor cell markers. Finally, BM-derived EPC from high 20-HETE production mice CYP4a12tg contained 50% more progenitor cells than EPC derived from control animals. Conclusion: These results implicate 20-HETE as a novel regulator of EPC stemness and an important player in the regulation of angiogenic responses that require the contribution of EPC.