We have reported accelerated wound healing induced by intracellular
ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the
wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of
cytokines,
growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for
cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four
wounds were created on the ventral side of each ear. Two
wounds on one side were treated with
ATP-vesicles (10 mM
ATP), and the other two were treated with controls (
normal saline or
Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for
cytokines and
growth factors using real-time PCR and immunohistochemical staining. The control
wounds showed an immediate increase in proinflammatory
cytokines after
wound creation but no further increase after this initial spike. The
growth factor levels in the control
wounds remained unchanged throughout the study. Conversely, the
wounds treated with
ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1β and TNF-α expression from day 1-4, and significantly higher
VEGF-A,
VEGF-D, and
VEGFR-2 expression from day 4-6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage
collagen production and neovascularization.