P616Improvement of collateral growth by ex vivo stimulated macrophages

Objective: Macrophages (Mφ) play an essential role during arteriogenesis, which is defined as the remodeling of pre-existing arterioles to fully functional collateral arteries after arterial occlusion. We could show that, apart from inflammatory M1-φ, also M2-φ occur. Aim of this study was the evaluation of the functional role of different Mφ sub-populations during collateral growth. Method: Mφ derived from bone marrow of GFP mice were stimulated with IFNΓ, IL4+IL13, or IL10 ex vivo in order to polarize Mφ into different subtypes (M1, M2a or M2c, respectively) and their expression profile was evaluated by quantitative RT-PCR. 24 recipient mice received either clodronate liposomes to deplete circulating Mφ or PBS liposomes as a control group. To induce collateral growth the left femoral artery was ligated (FAL) and stimulated Mφ were administered i.v.. Functional implications on arteriogenesis were evaluated by laser doppler imaging at d3, d7, and d14. In tissue sections of adductor muscles deposition of the GFP-labeled Mφ was tracked. Results: The different stimulation protocols resulted in expression of specific markers of Mφ subtypes proving the expected ex vivo polarization. FACS analysis of peripheral blood after clodronate application confirmed depletion of endogenous Mφ. Clodronate treated mice showed a decreased reperfusion or died after FAL. Application of IL10-stimulated Mφ in the PBS group improved perfusion 14d after FAL in comparison to the other treatments (IL10: 85%; IFN: 63%; IL4+IL13: 63% recovery vs. non-ligated control). GFP+ Mφ are traceable in the perivascular space of growing collaterals. Conclusion: M2c polarized Mφ have a long-term positive effect on arteriogenesis which correlates with a well-known tissue remodeling performance of this particular subtype. Recovery of GFP+ Mφ in the perivascular space argues for recruitment of Mφ from peripheral blood. Application of specifically polarized M2c-φ populations could be a therapeutic option in PAD patients.