Purified Peritoneal Macrophages Do Not Promote Angiogenesis In Vivo.

A large number of patients suffer from peripheral vascular disease not amenable to surgery, thus making medical therapies that promote neovascularization, including cell-based therapies, of interest. It is known that macrophages play an important role in angiogenesis in the context of wound healing and tumors, and that bone marrow (BM)-derived and peripheral blood (PB) mononuclear cells injected in ischemic limbs of animal models can favor development of collateral vessels. In order to determine the specific contribution of monocytes/macrophages to angiogenesis in an in vivo mouse model, we studied new vessel formation in a subcutaneously implanted Matrigel matrix plug in which peritoneal macrophages were embedded. We also tested whether macrophage exposure to GM-CSF would enhance their pro-angiogenic effect. A cohort of C57Bl/6 mice was sacrificed 6 days after intraperitoneal injection of 10% thioglycollate. Stimulated peritoneal macrophages were collected and plated in RPMI 1640 supplemented with 10% FBS (R10). Adherent macrophages were trypsinized, resuspended in 500μL of Matrigel, and injected subcutaneously in C57Bl/6 mice. The 4 experimental groups consisted of: Matrigel alone (n=5), with 5.7x10⁶ macrophages (n=4), with murine GM-CSF 500u/mL (n=5), and with both 5.7x10⁶ macrophages and murine GM-CSF 500u/mL (n=6). Matrigel plugs were resected at 21 days and endothelial cells on histological sections were stained with anti-vWF antibody. For each implant, blood vessels were counted on one entire section, excluding the periphery. The mean number of blood vessels per mm² (±SEM) was: Matrigel alone: 4.0 (±1.4), Matrigel + GM-CSF: 2.6 (±1.1), Matrigel + macrophages: 5.3 (±1.2), Matrigel + GM-CSF + macrophages: 4.7 (±0.9). There was no statistically significant difference in new vessel formation among the four groups (p>0.1 by Student T test). In a separate experiment, adherent peritoneal macrophages were cultured in R10 with or without GM-CSF 500u/mL for 24 hours. The cells were then washed thoroughly and cultured in serum-free Opti media for 18 hours. A RayBiotech antibody array testing 24 pro- and anti-angiogenic cytokines was performed using concentrated conditioned media. It demonstrated that peritoneal macrophages secrete a number of pro-angiogenic/arteriogenic cytokines such as MCP-1, VEGF, bFGF and GM-CSF, as well as anti-angiogenic cytokines such as TIMP-1 and IL-12. However, the cytokine profile was not significantly altered by stimulation of cells with GM-CSF. In summary, a purified population of peritoneal macrophages failed to significantly alter host-derived angiogenesis in a Matrigel subcutaneous implant, despite the detectable presence of pro-angiogenic cytokines. This may be explained in part by the concomitant secretion of anti-angiogenic factors. Some clinical trials have successfully achieved angiogenesis in cardiovascular disease using unfractionated BM-derived or PB mononuclear cells. Considering that the proportion of monocytes in these preparations by far outnumbers that of stem and progenitor cells with hemangioblast potential, it might be worthwhile exploring whether removing monocytes from unfractionated mononuclear cell collections would promote a distinct and possibly enhanced proangiogenic effect.

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