Ly6C^high/CCR6^high Monocytes Are Required for Collateral Artery Growth

Objectives: The re-establishment of an artery network constitutes a prerequisite for recovery from peripheral artery diseases. Arteriogenesis designates the growth of preexisting collateral arteries, in which their development depends on the infiltration of immune cells. The mechanisms and factors mediating the recruitment of leukocytes, however, have not been identified.

The chemokine CCL20 has been shown to promote the migration of monocytes and macrophages. Here, we studied the role of CCL20 and its corresponding receptor CCR6 during collateral artery growth.

Methods: Femoral artery ligation (FAL) was used as a model for arteriogenesis. The recovery from peripheral occlusion of wild-type (WT) and CCR6 deficient mice (Ccr6^−/−) was assessed by dynamic laser Doppler perfusion imaging. We further performed bone marrow transplantation (BMT) between WT and Ccr6^−/− irradiated mice and combined systemic delivery of CCL20 with macrophage depletion by using osmotic minipumps and clodronate containing liposomes. Blood and tissue samples were analyzed by flow cytometry, RNA sequence analysis, and proteomic approaches.

Results: Kinetic protein profiling studies of serum samples revealed an increase of CCL20 within the first 24 hours upon FAL. This rapid but transient increase of CCL20 was paralleled by a 30-fold increase of Ly6C^high/CCR6^high monocytes in blood. RNA sequence analysis of this subset of monocytes identified a distinct cluster of genes controlling monocyte adhesion and infiltration to the site of injury. In line with this, Ccr6^−/− mice featured an impaired blood reperfusion and decline of circulating Ly6C^high monocytes after FAL.

BMT approaches between WT and Ccr6^−/− irradiated mice demonstrated that blood flow restoration accompanies an increase of bone marrow derived Ly6C^high monocytes into circulation, and more importantly, depends on the activation of the CCL20/CCR6 axis. Vice versa, we found that a systemic intravenous supply of CCL20 enhances perfusion recovery in vehicle liposomes, whereas the depletion of monocytes and macrophages by clodronate containing liposomes deteriorated perfusion.

Conclusions: The systemic increase of CCL20 results in elevated levels of circulating Ly6C^high/CCR6^high monocytes, which are required for collateral artery growth. Additive supply strategies of CCL20 in human patients suffering from peripheral artery diseases, therefore, may constitute an innovative therapeutic approach to augment collateral artery growth.