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We performed a retrospective case series of IHs with prolonged growth to further characterize these lesions and their treatment. Recognition of this subset of hemangiomas is important for clinicians, and further study of IHs may provide clues to their pathogenesis. Infantile hemangiomas are the most common tumor of infancy and have a well-described natural history of rapid growth during early infancy followed by gradual involution. Although there are other types of vascular tumors sometimes referred to under the umbrella term hemangiomas eg, lobular capillary hemangioma [pyogenic granuloma] , hereafter in this article the term hemangioma will refer to infantile hemangiomas specifically. The duration of the growth phase can be variable, but it has long been reported that most hemangiomas reach their maximal size within the first 6 to 10 months of life.

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Hemangioma is one of the most common types of infantile vascular benign tumor. The aim of the present study was to investigate the role of B-cell lymphoma 2 Bcl-2 and tumor protein p53 p53 in the proliferation and apoptosis of hemangioma cells. A total of 38 paraffin-embedded hemangioma specimens 16 males and 22 females and another 5 paraffin-embedded healthy surrounding tissue samples, collected between January and December , were obtained from the Department of Pathology at Renmin Hospital of Wuhan University Wuhan, China.

Immunohistochemistry, hematoxylin and eosin staining, and quantum dot double staining were used to detect the expression of proliferating cell nuclear antigen PCNA , Bcl-2 and p53 in hemangioma and healthy surrounding skin tissue samples. All hemangioma specimens were classified into proliferative or the involuting stage hemangioma according to Mulliken's criteria and their expression of PCNA.

The results of the quantum dot double staining were analyzed using a multi-spectral imaging system. One-way analysis of the variance and the Student-Newman-Keuls q test were performed to statistically analyze the data. There were 24 cases of proliferative stage and 14 cases of involuting stage hemangioma among the specimens. Immunohistochemical analysis results indicated a high expression of Bcl-2 and p53 in proliferative stage hemangioma tissue samples, and low expression in involuting stage hemangioma and healthy tissue samples.

No significant difference in Bcl-2 and p53 expression was identified between the involuting hemangioma and healthy surrounding tissue samples. The higher expression of Bcl-2 and p53 in proliferative hemangioma suggests that Bcl-2 may cause an imbalance between endothelial cell proliferation and apoptosis through the inhibition of endothelial cell apoptosis. Furthermore, p53 may promote the proliferation of endothelial cells in proliferative hemangioma. Cutaneous hemangioma is one of the most common types of benign tumor 1.

The majority of types of hemangioma go through three distinct stages, the proliferation stage, the involuting phase and the involuted phase. Hemangioma typically presents itself during the proliferative phase.

During the proliferative phase, excessive proliferation of endothelial cells and nuclear hypertrophy occurs, forming lumens or lumps without cavities that are similar in size to a small capillary. The proliferative phase is characterized by rapid proliferation of capillary endothelial cells and the appearance of a vascular anomaly. Subsequently, hemangiomas enter an involuting phase during which the endothelial cells undergo apoptosis, and extracellular matrix fibers and adipose tissue replace the hemangioma.

This phase can last between a number of months and several years, following which the hemangioma enters the involuted phase 2 , 3. There are two processes that enable the formation of new blood vessels, vasculogenesis and angiogenesis. Vasculogenesis refers to the formation of early blood vessels through the induction, differentiation and aggregation of endothelial progenitor cells 4.

Angiogenesis refers to the process of forming new vessels from pre-existing vessels following birth 5. The first vessels in the embryo form through vasculogenesis and following birth angiogenesis becomes the predominant pathway used to develop blood vessels 6. Abnormalities in vasculogenesis and angiogenesis can lead to vascular diseases, of which hemangioma is a common type 7 , 8. Folkman 8 reported that an imbalance in the regulation between angiogenic factors and angiogenic inhibitors that causes uncontrolled angiogenesis may result in the occurrence of hemangioma.

Therefore, the mechanism of regulation of angiogenesis serves an important role in the pathogenesis of hemangioma. A large number of factors that positively and negatively regulate angiogenesis have different levels of expression and roles in different stages of the development of hemangioma 9 — Previous studies have demonstrated that multiple angiogenic factors, such as vascular endothelial growth factor VEGF , basic fibroblast growth factor bFGF and the estrogen receptor, are highly expressed in the proliferative stage of hemangioma development 2 , The number of mast cells is increased in proliferative hemangioma tissue compared with healthy tissue, which decreases in the involuting stage and can be restored to normal levels in the involuted stage A previous study demonstrated that the proliferation and apoptosis of endothelial cells serves an important role in the pathogenesis of hemangioma; the onset and involution of hemangioma were identified to be associated with endothelial cell proliferation and apoptosis 15 , It has been suggested that the proliferation and apoptosis of vascular endothelial cells is associated with the onset and spontaneous regression of hemangioma Specifically, it is thought that when an increased level of apoptosis occurs in endothelial cells during the second year of a child's life, cellular proliferation is offset which results in the initiation of the spontaneous regression of hemangioma.

Activation of oncogenes and inactivation of tumor suppressor genes can cause uncontrolled cell proliferation and apoptosis, and induce tumor formation. B-cell lymphoma 2 Bcl-2 belongs to the Bcl-2 protein family and serves an important role in regulating cell survival and apoptosis 20 , Tumor protein p53 p53 is a common tumor suppressor protein that is frequently activated when the cell is damaged, and which serves a role in various signaling pathways to regulate cell survival and death.

If a mutation of the gene encoding p53 occurs, the mutated p53 protein produced cannot trigger apoptosis, resulting in tumorigenesis 26 — Although Bcl-2 and p53 have been studied in multiple tumor types, few studies investigating their expression in tumor angiogenesis, particularly in the different stages of hemangioma, have been performed.

In the present study, immunohistochemical analysis and quantum dot technology were used to measure the expression of Bcl-2 and p53 in the proliferative and involuting stages of hemangioma, and the blood vessels of healthy skin, in order to investigate the role of Bcl-2 and p53 during the proliferative and involuting stages of hemangioma.

Formalin-fixed paraffin-embedded samples of archived cutaneous hemangioma tissue samples collected between January and December were obtained from the Department of Pathology of Renmin Hospital of Wuhan University Wuhan, China. There were samples from 38 patients with hemangioma, including 16 males and 22 females, aged between 2 months and 18 years old.

There were another 5 samples of healthy skin tissue surrounding the hemangioma. The locations of the vascular tumors included the following: Scalp, eyelids, forehead, back of the ears, neck, arms, back, legs, hands and foot. The patients did not receive any adjuvant therapy prior to surgery. OriGene Technologies, Inc.

Mouse anti-human monoclonal antibody directed against Bcl-2 was purchased from Santa Cruz Biotechnology, Inc. Wuhan, China; cat no. Specimens were classified according to Mulliken's criteria 30 combined with their expression of PCNA. The staging of specimens was determined according to the characteristics of their organizational structure. The proliferative stage was defined as hemangioma without structural changes. PCNA participates in DNA synthesis, and its expression has repeatedly been confirmed to be closely related to cellular proliferation.

As a result of its involvement in cellular proliferation, PCNA can be used in the diagnosis of malignant tumors such as hemangioma. In the present study, PCNA expression was observed to be high in proliferative stage hemangioma tissue, whilst its expression was low in involuting stage hemangioma tissue. A significant difference was found in the positive reaction of PCNA expression between proliferative stage hemangioma tissue and involuting stage hemangioma tissue.

Details of the protocol followed have been reported previously Specimens were washed with distilled water and soaked in PBS for 5 min. For antigen retrieval, slices were placed in 0. Sections were then washed with cold water in order to cool samples to room temperature, and finally washed with PBS 3x for 5 min each. Following the discard of excessive serum, the following primary antibodies were added: Bcl-2 dilution, ; cat.

Sections were then washed 3 times with TBST for 5 min each time. Following microwave antigen retrieval, specimens were washed with TBS. Subsequently, tissue sections were blocked with Immunol Staining blocking buffer cat.

Specimens were blocked with Immunol Staining blocking buffer cat. Then biotin-labeled secondary antibody cat. A wavelength of nm was used to excite the QDs with ultraviolet light, and a wavelength of nm was used to excite QDs with blue light. Cells were considered PCNA- or ppositive if brown-yellow granules appeared in the nucleus.

No brown-yellow granules were identified in the cytoplasm or nucleus of the negative control group. For each section, five complete and non-overlapping high magnification x fields were randomly selected for each sample. The average optical density, area of positive staining and total area of all cells in each field was measured, and used to calculate the positive area rate.

One-way analysis of variance and the Student-Newman-Keuls q post hoc test were used to determine the significance of differences in the average optical density and positive area rate between groups. Normality test and variance homogeneity test were conducted prior to testing. Each group met the requirements of normal distribution and homogeneity of variance. All tests were performed using SPSS software version The capillaries in the healthy skin tissue samples had thin walls and were surrounded by 1—2 endothelial cells, which were primarily thin but were thicker around the nuclei Fig.

In addition, the nuclei of the endothelial cells were flat compared with other cell types Fig. A large number of active proliferative endothelial cells shaped like a cord or mass were observed to be gathered in proliferative hemangioma tissue samples Fig.

There were also irregular gaps in the sinusoid capillaries between endothelial cells, the vessel lumen and large weakly stained nuclei Fig. In involuting hemangioma tissue samples, the number of endothelial cells surrounding the capillaries was decreased, vascular lumens were enlarged, and fibrosis of the blood vessels, fatty deposits, lumen occlusion and flat endothelial cell nuclei were present Fig.

Results of hematoxylin and eosin staining. A In healthy skin tissue samples, the cross-section of a capillary consisted of 1—2 endothelial cells with light cytoplasmic staining, and thicker and flat nuclei. B In proliferative hemangioma tissue, proliferating endothelial cells were identified, and their nuclei were hypertrophic and lightly stained.

C In involuting hemangioma tissue, there were few endothelial cells, the vascular lumen was enlarged and the nuclei of endothelial cells were flat. Cells were considered PCNA-positive if brown-yellow granules were distributed diffusely in the nuclei of endothelial cells.

In proliferative hemangioma tissue samples, the nuclei of endothelial cells were large and PCNA expression was high Fig. In the involuting hemangioma tissue samples, the nuclei of endothelial cells were flat, there were few nuclei and nuclei contained a small amount of brown-yellow granules, indicating that PCNA expression was low Fig.

PCNA was detected through the immunohistochemical streptavidin-peroxidase method. The nuclei were hypertrophic and contained brownish-yellow particles, demonstrating strong PCNA expression.

The nuclei were flat and brownish-yellow particles were sparsely distributed within nuclei, demonstrating weak PCNA expression. PCNA, proliferating cell nuclear antigen. Classification indicated 24 cases of proliferative hemangioma and 14 cases of involuting hemangioma.

In addition, 5 cases had healthy skin surrounding the hemangioma and were thus used as the control group. Numerous brown-yellow granules were identified in the endochylema of endothelial cells in proliferative hemangioma tissue, indicating that Bcl-2 expression was high Fig.

No brown-yellow granules were identified in the endochylema of endothelial cells in involuting hemangioma samples Fig. Immunohistochemical streptavidin-peroxidase staining for Bcl A Expression of Bcl-2 in proliferative phase hemangioma endothelial cells. Brownish-yellow particles were densely distributed in the nuclei and cytoplasm of endothelial cells, revealing strong Bcl-2 expression.

B Expression of Bcl-2 in involuting phase hemangioma endothelial cells. Brownish-yellow particles were sparsely distributed in the nuclei and cytoplasm of endothelial cells, demonstrating little or no Bcl-2 expression.

C Expression of Bcl-2 in healthy surrounding skin tissue samples. Brownish-yellow particles were not identified in the nuclei or cytoplasm of vascular endothelial cells, demonstrating little or no Bcl-2 expression.

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Analysis of Beta-Blocker Instituted for Treatment of Hemangiomas (RABBIT Study)

To evaluate the safety and efficacy of our institutional beta-blocker protocol for treatment of complicated infantile hemangiomas IH. Based on preliminary data showing hemangioma recrudescence off-treatment, we reviewed 9 additional patients with recrudescence between August and December Propranolol appears to be associated with minor, not severe symptomatic adverse events. Propranolol appears to be effective in treating complicated IH. Recrudescence can occur off-treatment, even with discontinuing propranolol as late as 15 months of age.

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Detection of p53 and Bcl-2 expression in cutaneous hemangioma through the quantum dot technique

While ulceration is a common, predominantly benign complication in infantile hemangioma, little is known about the prognosis of ulcerated CH. However, it has been observed that ulcerated CH may be complicated by life-threatening bleeding episodes. In both cases, the CHs were fed by high-flow vessels and the ensuing massive bleeding was due to superficial vessel wall erosion induced by the ulceration. Both patients were successfully treated with intravascular embolization; one patient underwent additional hemostatic surgery.

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Hemangioma is one of the most common types of infantile vascular benign tumor. The aim of the present study was to investigate the role of B-cell lymphoma 2 Bcl-2 and tumor protein p53 p53 in the proliferation and apoptosis of hemangioma cells. A total of 38 paraffin-embedded hemangioma specimens 16 males and 22 females and another 5 paraffin-embedded healthy surrounding tissue samples, collected between January and December , were obtained from the Department of Pathology at Renmin Hospital of Wuhan University Wuhan, China. Immunohistochemistry, hematoxylin and eosin staining, and quantum dot double staining were used to detect the expression of proliferating cell nuclear antigen PCNA , Bcl-2 and p53 in hemangioma and healthy surrounding skin tissue samples.

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Read more about the Hemangioma Investigator Group here. Am J Cardiol. Mol Syndromol. Propranolol treatment of infantile hemangiomas: anticipatory guidance for parents and caretakers. Pediatr Dermatol.

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