Expression Of Angiopoietin-1, Angiopoietin-2 And Tie2 Receptor In Human Hemangioma Xenograft Animal Model On Nude Mice

PartⅠA New Human Hemangioma Xenograft Model on Nude MiceBackground and Objective: Infantile hemangioma(often abbreviated as hemangioma) is the most common benign tumor involving infants characterized by appearance during the first weeks of life, rapid growth for 6～10 months followed by spontaneous and slow regression in the subsequent 1～5 years. Though most of infantile hemangiomas can regress spontaneously, about 10％hemangiomas can bring disasters to infants because of rapid growth or special location, such as ulceration, hemorrhage, disfiguration and even life threatening. Up to date, there are all kinds of therapeutic strategies including corticosteroid, interferon-α, surgery, laser, and so on. But none of them is perfect because of limited effectiveness and side effects. The reason of clinical dilemmas lies in lack of adequate knowledge of pathogenesis of hemangioma. To investigate the underlying mechanisms of hemangioma requires a hemangioma model that can present the natural development and characteristics of human hemangioma without deviation from ethics. Unfortunately, currently available experimental models cannot meet the requirements satisfactorily and hinder researchers' footsteps toward. In this research we attempted to transplant human hemangioma into nude mice subcutaneously to establish a xenograft animal model of human hemangioma for the future investigation.Methods: An infantile hemangioma was obtained from a male baby aged 2 months according to a protocol approved by Westchina Hospital Ethics Committee. The pathological diagnosis was confirmed with Mulliken's classification.8 BALB/c nu/nu nude mice were used in this experiment. The hemangioma specimen was cut into small blocks of 5mm×4mm×3mm in size. The blocks were transplanted into 8 nude mice subcutaneously, 4 blocks each mouse. The volume of the grafted hemangiomas was measured with a vernier on day 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 90, 120 and 180 after transplantation. The growth of the grafts was observed on regular basis. 2 grafted hemangiomas were harvested on day 56, 120 and 180 after transplantation respectively. Formaldehyde-fixed and paraffin-embedded specimens were sliced into 5μm sections for HE staining and immunohistochemistry for CD31(monoclonal mouse anti-human antibody, Neomarkers, USA) and Glut1(polyclonal rabbit anti-human antibody, Neomarkers, USA).The slices were observed under Nikon E600 microscope. The pictures were taken with a SPOT Cool CCD camera and analyzed with Image pro plus 4.5 software. IOD(integrated optical density) was chosen to represent staining intensity and presented as (?)+SD. SPSS11.0 software was used to perform t text.Results: The volume changes of the grafts were not obvious in the first 3-4 weeks. Later on some of the grafts became bigger and bigger and reached the apex at the end of 2nd month after transplantation with new vessel sprouts on the periphery. After 90 days the grafts began to shrink and became pale and yellow with the texture hardening. On day 180 only a few grafts remained and fibrofatty tissue was found at dissection.HE staining showed the proliferating graft hemangiomas were crowded with a large number of plump vascular endothelial cells(VEC) with small and irregular vessel lumens. In the involuting graft hemanigoms, the VECs were sharply decreased with enlarged vessel lumens lined with flat VECs. A great deal of fibrofatty tissue was deposited in the grafts.Immunohistochemisty showed the graft hemangiomas were positive with CD31 and Glut1 staining. Both CD31 and Glut1 were located on cell membrane.Conclusion: A human hemangioma xenograft aninal model on nude mice is successfully established by transplanting human hemangioma tissue blocks into nude mice subcutaneously. And we testifies the validity of the animal model by morphologic observation, HE staining and immunohistochemistry. This model provides an excellent basis for future research. PartⅡThe Expression of Angiopoietin-1, Angiopoietin-2 and Tie2 in Human Hemangioma Xenograft Animal ModelBackground and Objective: Infantile hemangioma is characterized by abnormal vascular endothelial cell proliferation and disturbed angiogenesis. Mulliken categorized infantile hemangioma into three subgroups: proliferating, involuting and involuted hemangioma. The underlying molecular and biochemical mechanisms by which proliferating hemangiomas begin to regress spontaneously and slowly is unclear up to date. A distinctive characteristic of infantile hemangioma from other tumors is that 80％of hemangiomas can regress spontaneously which has caused a widespread dispute that is whether infantile hemangioma is a real tumor. In the past decade, many literatures have shown that the angiopoietin/Tie2 pathway plays an important role in normal and abnormal angiogenesis. In this research, we attempted to detect the dynamic expression of angiopoietin-1, angiopoietin-2 and Tie2 in different phases of hemangioma by means of the previously established animal model. Mothods: The animal models were established according to PartⅠ. Twelve specimens were harvested on day 56 and 120 after grafting respectively. 2 of 12 were fixed with 4％formaldehyde for immunohistochemistry for Ang1(goat anti-human antibody, RnD, USA), Ang2(goat anti-human antibody, RnD, USA), Tie2(goat anti-human antibody, RnD, USA) and VEGF(rabbit anti-human antibody, Boster, China). 5 of 12 were fixed with Trizol for fluorescence RT-PCR for Angl, Ang2 and Tie2. The remains were for Western blot analysis for Angl, Ang2 and Tie2. 5 normal foreskins were acquired from 5 boys(undergoing circumcision) for immunohistochemistry, RT-PCR and Western blot.Statistical analysis was performed with REST software for the RT-PCR results and SPSS11.0 software for immunohistochemistry results.Results: Fluorescence RT-PCR and Western blot showed the amount of Ang1mRNA and protein in the proliferating and involuting graft hemangiomas was much less than that of the normal foreskins. On the contrary, the amount of mRNA and protein of Ang2 and Tie2 in the proliferating and involuting graft hemangiomas was much more than that of the normal foreskins. There was no statistical difference between proliferating and involuting phases in Ang1, Ang2 and Tie2. Immunohistochemistry showed a similar result with RT-PCR and Western blot. Vasculogenesis was found in the graft hemangiomas which was a primary process of vessel formation only occurring during embryogenesis and under a pathologic condition.Conclusion: Abnormal expression of Ang1, Ang2 and Tie2 is found in infantile hemangiomas. A hypothesis is forwarded that it is possible that infantile hemangioma is not a real tumor, but rather a disorder of vasculogenesis and angiogenesis.