CCR2 and CCR5 Co-Expressing CD11b⁺ Monocytes Affect Neovascularization in An Ischemic Hind Limb Mouse Model,

Abstract 3270

Monocyte/myeloid -lineage cells are known to be involved in neovascularization. However, the specific monocyte /myeloid population that participates in neovascularization has not been clearly identified. Several subsets of monocyte/myeloid cells have been proposed to be important for this process. For example, CD11b⁺CXCR1^hiCCR5⁺ resident monocytes regulate tissue healing by increasing angiogenesis, and CD11b⁺CXCR1^loCCR2⁺ inflammatory monocytes are mobilized to the inflammation site. Interestingly, we found that CD11b⁺ myeloid cells isolated from ischemic muscle of post-femoral artery dissected C57BL/6 mice are positive for both CCR2 and CCR5. To study the relationship between CD11b⁺CCR2⁺CCR5⁺ monocytes and neovascularization in an ischemic hind-limb model of C57BL/6 mice, we addressed the following questions:(1) Are the number of tissue-resident CD11b⁺CCR2⁺CCR5⁺ monocytes increased in ischemic muscle, post-ischemic injury?; (2) Are CD11b⁺CCR2⁺CCR5⁺ monocytes recruited to the ischemic muscle and do they incorporate into the blood vessels in the ischemic site?; (3) Do CD11b⁺CCR2⁺CCR5⁺ monocytes modulate neovascularization in ischemic hind-limbs of C57BL/6 mice upon direct injection of these cells into ischemic muscle. Results demonstrate that numbers of CD11b⁺CCR2⁺CCR5⁺ monocytes markedly increase in ischemic muscle by day 4 after the surgery (n=5 each; 0.8±0.4×10⁵ cells/g tissue in non-surgically treated mice vs 9.5±1.7×10⁵ cells/g tissue in the ischemic muscle, p<0.05) then return to basal levels at day 21. Following immunostaining of ischemic muscles, CD11b⁺CCR2⁺CCR5⁺ monocytes localize in perivascular areas of non-surgically treated C57BL/6 mice. At day 4 after the surgery, these cells infiltrate into ischemic muscles. Infiltrated CD11b⁺CCR2⁺CCR5⁺ cells make numerous vessel-like structures 21 days after the surgery. Following direct injection of purified CD11b⁺CCR2⁺CCR5⁺ monocytes from ischemic muscle at day 4, an increase in blood flow was manifest by 28 days in the CD11b⁺CCR2⁺CCR5⁺ monocyte-injected group (n=7 each, 67.6±5.7% in the CD11b⁺CCR2⁺CCR5⁺ monocyte-injected group vs 39.9±6.7% in the control group, p<0.05). Thus, we found that CD11b⁺CCR2⁺CCR5⁺ monocytes infiltrated and formed numerous vessel-like structures in the ischemic muscle, and additional injection of CD11b⁺CCR2⁺CCR5⁺ monocytes increased the blood flow. These findings strongly suggest that CD11b⁺CCR2⁺CCR5⁺ monocytes play an important role in neovascularization.