The role of monocyte subsets in myocutaneous revascularization

Background: The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging. Materials and methods: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of wild-type mice (C57BL6) and populations of mice with genetic deletion of subset-specific chemokine ligand-receptor axes important in monocyte trafficking and function (CCL2^-/- and CX₃CR1 ^-/-) (n = 36 total; 12 mice per group, nine with flap and three unoperated controls). Planimetric analysis of digital photographic images was utilized to determine flap surface viability in wild-type and knockout mice. Real-time myocutaneous flap perfusion and functional revascularization was determined by laser speckle contrast imaging. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Macrophage quantification and localization in flap tissues was determined by F4/80 gene and protein expression. Quantitative reverse transcription-polymerase chain reaction was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression. Results: Myocutaneous flaps created on wild type and CX₃CR1^-/- mice were engrafted to the recipient site, resulting in viability. In contrast, distal full thickness cutaneous necrosis and resultant flap dehiscence was evident by d 10 in CCL2^-/- mice. Over 10 d, laser speckle contrast imaging documented immediate graded flap ischemia in all three groups of mice, functional flap revascularization in wild type and CX₃CR1^-/- mice, and lack of distal flap reperfusion in CCL2^-/- mice. Immunostaining of serial histologic specimens confirmed marked increases in microvascular density and number of macrophages in wild type mice, intermediate increases in CX ₃CR1^-/- mice, and no significant change in vessel count or macrophage quantity in CCL2^-/- mice over the study interval. Finally, quantitative reverse transcriptase polymerase chain reaction demonstrated that the loss of function of chemokine ligand and receptor genes influenced the transcription of local genes involved in monocyte chemotaxis and wound angiogenesis. Conclusions: In a graded-ischemia wound healing model, monocyte recruitment was severely impaired in CCL2^-/- mice, resulting in failure of flap revascularization and concomitant cutaneous necrosis. Analysis of CX₃CR1-deficient mice revealed adequate monocyte recruitment and revascularization for flap survival; however, the myeloid cell response and magnitude of neovascularization were dampened compared with wild type mice.