Abstract 643: CX 3 CR1 Identifies Adventitial Macrophage Progenitor Cells (AMPCS), a Local Source of Self-Renewing Macrophages in Postnatal Murine Arteries

Abstract

Background: Macrophages are integral to vascular biology and disease. Although traditional paradigms assert that vascular macrophages originate from circulating monocytes, recent data suggest that some are seeded in utero from CX 3 CR1 + progenitors, and are maintained postnatally by local self-renewal. We previously identified a novel population of locally-maintained adventitial macrophage progenitor cells (AMPCs) as a source of self-renewing macrophages in adult mouse arteries. Hypothesis: AMPCs express CX 3 CR1 and do not derive from definitive hematopoiesis. Methods: Single-cell disaggregates were prepared from postnatal murine aortas. AMPC content was assessed by macrophage colony-forming unit (CFU-M) assays. Flow cytometry together with fluorescence activated cell sorting (FACS) were used to investigate the immunophenotypic profile of AMPCs and fate-mapping to assess their origins. Results: CFU-M prevalence in C57BL/6J aortic cells was highest in neonatal mice (~100 per 10 5 cells), and diminished progressively with age (~55/10 5 at 3w, ~15/10 5 at 12w, ~5/10 5 at 52w, n>4, P<0.01). Secondary replating of single cells from aortic CFU-M revealed striking self-renewal capacity, with 1 in 10 cells forming new CFU-M (n=8). Undifferentiated CFU-M displayed >95% expression of the stem cell markers Sca-1 and c-Kit, and high levels of CX 3 CR1, but did not acquire the monocyte/macrophage markers CD11b or F4/80 until treated with macrophage-colony stimulating factor (n=6). Mice deficient in CX 3 CR1 (Cx 3 cr1 GFP/GFP ) produced fewer CFU-M than heterozygous (Cx 3 cr1 +/GFP ) littermates (8.7±0.7 vs 15.3±0.7 per 10 5 , P<0.001, n>7). FACS selection confirmed that CFU-M forming AMPCs were exclusively contained within a CX 3 CR1 + subpopulation that did not express either CD11b or F4/80 (n=3). Finally, CFU-M analysis from Flt3 Cre xRosa mT/mG mice demonstrated that AMPCs arise from a FLT3 -ve source, indicating that their origins are independent of definitive hematopoiesis (n=4). Conclusion: Clonogenic, self-renewing murine AMPCs express CX 3 CR1 but not the monocyte/macrophage markers CD11b and F4/80. The high prevalence of AMPCs in neonatal aorta is consistent with prenatal seeding from CX 3 CR1 + progenitors, independent of definitive hematopoiesis.