MFG-E8 Reduces Aortic Intimal Proliferation in a Murine Model of Transplant Vasculopathy.

Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.

MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure.

Milk fat globule epidermal growth factor-8 limits tissue damage through inflammasome modulation during renal injury.

Mediators released by apoptotic renal resident cells play a crucial role in modification of the inflammatory microenvironment. We have demonstrated that milk fat globule epidermal growth factor 8 (MFG-E8) is released by apoptotic cells, which results in reduced proinflammatory cytokine production by macrophages. The present study was designed to study the role of MFG-E8 on the modulation of tissue damage and macrophage phenotype in a renal inflammatory model, unilateral ureteral obstruction (UUO). C57BL/6 WT or MFG-E8 KO mice underwent ureteral ligation for 3, 7, and 14 d to evaluate renal injury. MFG-E8 (30 µg/kg) or vehicle was also administered i.p. MFG-E8 administration reduced kidney damage and fibrosis compared with control, whereas its absence in MFG-E8 KO mice was associated with more severe disease. Moreover, MFG-E8 administration was associated with decreased inflammasome activation in the kidney. Furthermore, adoptive transfer of MFG-E8-stimulated macrophages reduced activation of inflammasome and tissue damage. In all cases, both the systemic administration of MFG-E8 and MFG-E8-treated macrophages promoted accumulation of anti-inflammatory CD206+ macrophages. We propose that the protective role of MFG-E8 is mediated through anti-inflammatory macrophage reprogramming which results in decreased inflammasome activation, preventing severe tissue damage. These data provide valuable insight for identification of MFG-E8 as a novel target in modulation of inflammatory diseases.

MFG-E8 released by apoptotic endothelial cells triggers anti-inflammatory macrophage reprogramming.

Apoptotic endothelial cells are an important component of the "response to injury" process. Several atherosclerosis risk factors such as hyperglycemia and oxidized low-density lipoproteins, and immune injuries, such as antibodies and complement, induce endothelial cell apoptosis. While endothelial cell apoptosis is known to affect neighboring vascular wall cell biology, its consequences on macrophage reprogramming are ill defined. In this study, we report that apoptosis of human and mouse endothelial cells triggers the release of milk fat globule-epidermal growth factor 8 (MFG-E8) and reprograms macrophages into an anti-inflammatory cells. We demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from serum-starved apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if MFG-E8 is absent from the media. Macrophage treatment with recombinant MFG-E8 recapitulates the effect of conditioned media. Finally, we showed that MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Taken together, our study suggests a key role of MFG-E8 release from apoptotic endothelial cells in macrophage reprogramming and demonstrates the importance of the apoptotic microenvironment in anti-inflammatory macrophage responses.