The Local Injection of Peritoneal Macrophages Induces Neovascularization in Rat Ischemic Hind Limb Muscles

Abstract

Macrophages play a pivotal role in the development of newly formed vascular networks, in addition to their normal immunological functions. This research focuses on peritoneal macrophages as a novel source in cell implantation therapy for ischemic diseases. In this study, production of angiogenic growth factors by peritoneal macrophages and its in vivo effect of neovascularization were evaluated. Mononuclear cells from the peritoneal cavity (P-MNCs) enriched with macrophages were isolated and stimulated with hypoxia and interleukin-1β (IL-1β) to mimic an ischemic tissue environment in vitro. Expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) of mRNA in P-MNCs was apparently enhanced by hypoxic stimulation, and the production of VEGF protein was also augmented by hypoxia and IL-1β. A rat ischemic hind limb model was created and P-MNCs (8 × 106/limb) were injected into the ischemic muscles. The blood flow, which was assessed using the colored microsphere method, showed that the percentage blood flow was significantly increased by P-MNCs injection 4 weeks after surgery (48.3 ± 16.8% in noninjected ischemic limb vs. 84.3 ± 13.0% in the P-MNCs-injected limb). A histological analysis revealed that the number of capillaries detected by alkaline phosphatase staining was increased in the P-MNCs group 4 weeks after injection. Furthermore, the number of α-smooth muscle actin-positive vessels also showed a significant increase following P-MNC injection. The injected P-MNCs labeled with fluorescence were detected in the interstitial space of ischemic muscles, and VEGF protein expression of the implanted cells was confirmed by immunohistochemistry. These results indicate that peritoneal macrophages stimulate capillary formation and arteriogenesis in the ischemic limbs, possibly through the production of angiogenic growth factors. These findings suggest that the physiological angiogenic property of peritoneal macrophages could therefore be utilized for neovascularization in cell implantation therapy.