Study On The Function Of Macrophages In The Progression Of Infantile Hemangioma

PartI. The expression and distribution of the macrophage’s biomarkers in the infantile hemangiomaObjective: It has been reported that macrophages play essential roles in the progression of caner, atherosclerosis and insulin-resistant obesity. The aim of this part was to explore the expression and distribution of the markers of macrophages, including M1and M2-polarized macrophages, and to analyze the relationship between the expression of the markers of macrophages and proliferation, adipogenic differentiation related proteins as well as signaling pathways in the infantile heamangioma.Methods:The markers of macrophages include CD68, HLA-DR and CD163, which were analyzed by IHC, and the co-expression of CD68/HLA-DR and CD68/CD163were detected by IF-P. Ki67, PPARy, phospho-Akt (Ser473) and phospho-Erkl/2(Thr202/Tyr204) were also measured using IHC. Finally, cluster analysis was performed to explore the relationship among those proteins.Results: CD68, HLA-DR and CD163could be detected in the most of cases (12/13), meanwhile the co-expression of CD68/HLA-DR for M1-polarized macrophages and CD68/CD163for M2-polarized macrophages were determined in the hemangiomas. Most of the macrophages were located in the interstitial part of the lesion. Quantitative analysis showed that M1and M2-polarized macrophages were much higher in the proliferating phase of the hemangioma. Cluster analysis showed the positive relationship between macrophage markers and VEGF, Ki67, phospho-Akt, and negative relationship with PPARy. The macrophage-related cytokines TNF-a, IL-1β, and TGF-β were found highly expressed in the proliferating phase of IHs.Conclusions:Macrophages and their subtypes were found in the infantile hemangiomas, especially in the proliferating phases, which were located in the interstitial tissue of the lesions. The marker proteins of macrophages were showed positive correlation with VEGF, Ki67and phospho-Akt, negative correlation with PPARy. All above data indicated that macrophages might participate in the progression of infantile hemangiomas. Part2. Isolation and identification of hemangiomastem cells, as well as the establishment of the hemangiomanude mice modelObjective: Recent studies suggested hemangioma stem cell (HemSC) that derived from proliferative hemangioma tissues using anti-CD133microbeads displayed abilities of robust proliferation, clone formation and pluripotency. Injecting those cells into mude mice subcutaneously could establish hemangioma nude mice model, which mimicked the human infantile hemangioma progression, becoming the powerful tool in the study of infantile hemangiomas. The aim of this part was to isolate the hemangioma stem cell using anti-CD133microbeads followed by identification, and to establish hemangioma nude mice model.Methods:Anti-CD133microbeads were used for the isolation of hemangioma stem cell in the proliferative hemangoma tissues. FACS was performed to analyze the membrane-markers of those cells. Furthermore, the proliferation assays, clone formation assays, and differentiation assays were exerted to explore the biblogical characteristics of isolated cells. The HemSCs were then injected into nude mice subcutaneously to establish hemangioma nude mice model.Results:The stem cells that isolated using anti-CD133microbeads showed short spindle and fibroblast-like morphology, as well as enhanced proliferation and clone formation properties. Stem cell transcription factors AML1and Oct-4were found positive in those cells. CD31, CD34, and CD146were found expressed weakly, and CD105and CD90were found highly expressed and STOR-1was negatively expressed in those cells using FACS analyses. Differentiation assays suggested that the cells could differentiate into osteoblast, adipocyte and endothelial cells. The hemangioma nude mice model established based on those cells displayed features such as vessels formation, adipogenesis, which was deamed to be the characteristics of infantile hemangiomas. Also the vessels that CD31identified showed to be positive of Glut-1expression, an identifiable marker of infantile hemangiomas.Conclusions: We succeeded in isolation of hemangioma stem cells, which showed enhanced proliferation, clone formation and pluripotency abilities. The isolated cells expressed the markers of mesenchymal and endothelial cells (although weakly). Finally, those cells could be used for the establishment of hemangioma nude mice model which contained Glut-1positive vessels, and adipogenic tissues after injection for1month. Part3. The effects of macrophage on the proliferation and differentiation of haemangiama stem cells as well as underlying mechanismsObjective: The aim of this part was to analyze the effects of M1and M2-polarized macrophages on hemangioma stem cells in vitro using indirect co-culture systems between HemSC and monocyte/macrophage, and to explore the mechanism that mediated these effects. Also the influence of HemSC on monocyte will be measured.Methods:The indirect co-culture system between HemSC and THP-1was establsiehd using conditioned medium (CM) as well as Transwell system. EdU incorporation and MTT assays were exerted to measure the effects of macrophage CM. on the proliferation of HemSC; Oil Red O staining was used for detection of the adipogenic differentiation ability of HemSC after macrophage CM treatment. Finally, the intracellular signaling pathways protein arrays and western blots were used to explore the underlying mechanisms.Results:CM from both Ml-and M2-polarized macrophages could promote the proliferation of HemSC and suppress its adipogenic differentiation. The results from protein array and western blots suggested that Akt and Erkl/2signaling pathways involved in the process. Suppression of Akt by LY294002prevented the enhanced proliferation of HemSC by macrophages CM, and PD98059, a specific inhibitor for Erkl/2, prevented the suppressed adipogenesis of HemSC induced by CM of macrophages. The results from Transwell system revealed that HemSC could promote the proliferation of THP-1, as well as activate the Akt signaling pathway, however, showed no effects on the differentiation of THP-1.Conclusions: Macrophages, both M1-and M2-polarized, could promote the proliferation and suppress the adipogenic differentiation of HemSC via activation of Akt and Erkl/2signaling pathways. HemSC could promote the proliferation of THP-1and activate the Akt pathway, however show no effects on the differentiation of THP-1into macropahges. Part4. The effects of macrophage in the hemangiomanude mice modelObjective: The above studies indicated that macrophage might play a very important role during the progression of infantile hemangiomas. There, the aim of this part was to establish a macrophage-involved hemangioma nude mice model, and based on which, the effects of macrophages in the transition from proliferating phase to involuting phase of IHs, as well as underlying mechanisms were explored.Methods:The macrophage-involved hemangioma nude mice model was established by injecting the mixture of HemSC and THP-1(with ratio of4:1) into nude mice subcutaneously. IHC, IF-P and real time PCR were used for detecting the expressions of macrophage markers, proliferation related proteins and adipogenic transcription factor, as well as Akt, Erkl/2signaling pathways.Results:Higher cell density and more cyclinDl positive cells were found in the models with THP-1when compared with control group (only HemSC inside). Higher microvessel density (MVD) and CD31positive vessels suggested enhanced proliferation and vessels formation in THP-1group. And the weakened lipid vacuoles formation and decreased PPARy expression showed suppressed adipogenesis of hemangioma with THP-1involved. In addition, the higher activation of Akt and Erkl/2were found in THP-1group.Conclusions: Based on the established macrophage-involved hemangioma nude mice models, we found that macrophages in the hemangioma could promote the proliferation of hemagnioma cells, enhance the vessels formation and suppress the adipogenesis of the hemangioma, as well as activate the Akt and Erkl/2signaling pathways, indicating that macrophages in the human infantile hemangiomas might promote its development in proliferating phase and suppress the involution of the lesion, which might be dependent on the activation of Akt and Erkl/2.