Study On The Significance Of Allograft Inflammatory Factor-1 In The Progression Of Infantile Hemangiomas

Infantile hemangiomas are benign tumors that exhibit an early and rapid proliferation phase within the first year of life, followed by a slower but steady involution phase that may last up to 5 to 10 years. Recent work has demonstrated the presence of large numbers of myeloid cells in proliferating hemangiomas and suggested that these cells might be involved in promoting growth. Ritter et al. reported that endothelial cells of hemangiomas coexpress some markers typically associated with myeloid cells, including CD14, CD45, CD32, CD15 and CD83, demonstrating a close relationship between myeloid and haemangioma endothelialcells. Furthermore, investigations into possible mechanisms of hemangioma regression suggest that this process is immune mediated. Infantile hemangiomas recruit a significant number of T cells, most of which are CD8-positive cytotoxic T cells. DNA microarray analysis has identified indoleamine 2,3-dioxygenase, an enzyme involved in the degradation of tryptophan that is critical to the function of T cells, as a potential modulator of the immune response in hemangiomas. Indoleamine 2,3-dioxygenase messenger RNA is present in high levels in proliferating hemangiomas and decreases significantly during involution, thus raising the possibility that inhibition of T cells could contribute to the slow regression of hemangiomas. All these evidences suggest that immune and immune-mediated inflammatory events may contribute to the pathogenesis of hemangiomas.The allograft inflammatory factor-1 (AIF-1) is a 17-kDa interferon (IFN)-y-inducible Ca2+-binding EF-hand protein that is encoded within the HLA class III genomic region on chromosome 6p21.3, which is known for clusters of genes involved in the inflammatory response. AIF-1 was originally cloned from activated macrophages in human and rat atherosclerotic allogenic heart grafts undergoing chronic transplant rejection. And resent research found that AIF-1 was not only a controller of various host responses to inflammatory stimuli, but also a modulator of immune responses. Therefore, we proposed that AIF-1 may play potential roles in the pathogenesis of hemangiomas. In the present study, we tried to explore the expression and possible effects of AIF-1 in the pathogenesis of hemangiomas. And there are three parts in this study.Part One: Expression of allograft inflammatory factor-1 in hemangiomas:its implication in the progression of hemangiomaObjective:To investigate the expression and location of AIF-1 in infantile hemangioma and compare the expression levels of AIF-1 among the three stages of hemangiomas. Methods:In a retrospective study of 19 hemangiomas in the oral and facial regions, we assessed lesional immunoreactivities for AIF-1. Negative controls were similarly confirmed, including 24 pyogenic granulomas,26 vascular malformations,5 samples of normal skin,10 squamous cell carcinomas of the tongue and 3 placentas. And the Double immunofluorescence labeling and series section was use to investigate the location of AIF-1 in hemangiomas and the control tissue.Results:Of all samples, intense immunoreactivity for AIF-1 was detected in 17 of 19 (89.47%) hemangioma specimens, with specific locations in endothelial cells. The intensity of AIF-1 immunostaining did not show remarkable difference among proliferating, involuting and involuted hemangiomas. However, no specific immunoreaction for AIF-1 was observed in vessels of vascular malformations, normal skin, squamous cell carcinomas, pyogenic granulomas or placental tissues.Conclusions:The exclusive expression of AIF-1 on endothelial cells of hemangiomas suggests that it possibly plays a significant role in the pathophysiology and progression of hemangioma. AIF-1 can be used as an additional biomarker for infantile hemangiomas.Part Two: The potential roles of allograft inflammatory factor-1 in the pathogenesis of hemangiomas:AIF-1 promotes the proliferation, migration and angiogenesis of human endothelial cells (HUV-EC-C) probably by up-regulation of bFGFObjective:To explore the impact of AIF-1 alterations on human endothelial cells (HUV-EC-C). Methods:Stable introduction of AIF-1 to human umbilical vein endothelial cell line (HUV-EC-C) in vitro was used to evaluate the role of AIF-1 by examining the change of cell proliferation, cell cycle, cell migration, angiogenesis and the expression level of proliferative and angiogenic related factors. Cell proliferation and cycle was measured by MTT assay and Flow Cytometry, Cell migration was assessed by the wound healing assay and the endothelial cell migration assay, and the ability of angiogenesis was investigated by the tuber formation assay and chicken chorioallantoic membrane assay. Furthermore, the expression levels of G-CSF, VEGF-a, bFGF MCP-1 and TIMP-1 was assessed by the semi-quantitative RT-PCR, Real time PCR and ELISA assay. Results:AIF-1 enhanced the proliferation,migration and angiogenesis of the endothelial cells and promoted G0/G1-to-S-phase transition, accompanied by up-regulation the expression of bFGF (P<0.05), while AIF-1 could not influence the expression of G-CSF, VEGF-a, MCP-1 and TIMP-1. Conclusions:Our studies demonstrated that AIF-1 could promote the proliferation, migration and angiogenesis of endothelial cells, probably through augmenting the expression of bFGF. The impact of AIF-1 on endothelial cells would stimulate angiogenesis, and consequently influence the progression of infantile hemangiomas.Part Three: Study on the up-stream regulator of allograft inflammatory factor-1 in infantile hemangiomasObjective:To investigate the possible up-stream regulator of AIF-1 in hemangiomas.Methods:The endothelial cells (HUV-EC-Cs) were treated by VEGF, bFGF, IGF-2 or hypoxia, respectively, for 24h or 48h. And then the mRNA of AIF-1 was assessed by RT-PCR. Results:Hypoxia, VEGF, bFGF and IGF-2, independent on the concentration and the time, could not induce the expression of AIF-1 in HUV-EC-C cells. Conclusions:Though AIF-1 is a cytokine-responsive transcription, the factors which play essential roles in the progression of infantile hemangiomas could not induce the expression of AIF-1. And further investigation is needed to determine which factor induces the expression of AIF-1 in IH.