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Hemangiomas are the most common tumors found in children. Considerable interest in hemangiomas has been driven not just by their high prevalence and their associated complications, but also because the underlying mechanisms mediating spontaneous involution are not well understood. Clinically, many hemangiomas require treatment because of their location, size, behavior and potential for significant complications.［1,2］ Various therapeutic approaches have been described. Administration of systemic or intralesional steroids is the mainstay pharmacological therapy because little or no invasion has been associated with glucocorticoid in hemangiomas after more than 30 years, but the reported effective power varies dramatically.［3,4］ Many issues related to glucocorticoid treatment such as kinds of glucocorticoid, the form of the medication, optimal dosage, route and duration of administration remain unresolved. Also, there exists no consensus regarding appropriate selection of candidates, and therapeutic efficacy of glucocorticoids therapy cannot be reliably predicted.［4-6］ This is largely because the precise mechanism of glucocorticoids action in hemangiomas remains unclear.［1］ We therefore investigated expression patterns of the glucocorticoid receptor (GR) and its α isoform (GRα) in hemangiomas and vascular malformations, and compared these with GR and GRα expression in normal skin. Although a number of studies have evaluated expression of numerous cytokines and growth factors, to our knowledge, our results represent the first report on GR and GRα expression in children with cutaneous hemangiomas and vascular malformations.
METHODS
Study population
Specimens were obtained from 72 children with cutaneous hemangioma or vascular malformation undergoing surgery at the Second Affiliated Hospital of Shantou University from February 1999 to January 2004. Their ages ranged from 2 months to 12 years (average 2.8 years), and there were 33 boys and 39 girls. The patients were classified according to the standards of Mulliken.［7］ There were 65 hemangiomas (proliferating 33, involuting 32; ages 2 months to 12 years, average 2.5 years), and 7 vascular malformations (venous malformation; ages ranged 5 months to 12 years, average 5.1 years). Another 8 normal skin specimens from patients of comparable age were used as the controls.
Methods
Tissues were removed from the patients during surgery, fixed in neutralized 10% formalin and subsequently dehydrated in graded ethanol, then embedded in paraffin and sliced into 4 μm sections. The sections were deparaffinized and stained with hematoxylin and eosin (HE) for histological analysis. Some sections were stained with standard SP immunohistochemistry using rabbit anti-human polyclonal antibody against glucocorticoid receptor ［GR(e-20), and GRα isoforms GR(p-20); Santa Cruz Biotechnology Inc., USA］. The polyclonal antibody was used at a 1∶200 dilution in blank solution. The slides were covered with antibody solutions at 4℃ overnight and then further developed using an anti-rabbit IgG streptavidin kit (Maixin-Biotechnology Inc, Fuzhou, China). Control sections used rabbit serum instead of the primary antibody. The prepared tissue slides were observed and studied under a microscope.
Image and statistical analysis
The immunostaining of GR and GRα were performed by gray scale test with a computer-assisted image analysis system (HPIAS-1000). The data and its categorical variables were analyzed and compared by multivariate ANOVA and Hotelling T2 using a statistical software package (SPSS 10.0 for Windows). Results are expressed as mean±standard deviation (SD) and P<0.01 or >0.05 was considered statistically significant.
RESULTS
Expression of GR and GRα was detected in tissues in all phases of cutaneous hemangiomas or vascular malformations as well as in normal skin. There were no statistically significant differences in mean gray scale values of GR between cutaneous hemangiomas and vascular mamformations (P>0.05), but expression of GR and GRα in both cutaneous hemangiomas and vascular malformations were both significantly different higher than in normal skin (P<0.01). There were no statistically significant differences in gray scales of GR between proliferating and involuting hemangiomas (P>0.05), but GRα expression in proliferating hemangiomas was greater than that of involuting hemangiomas (P<0.01). GRα expression was the highest in hemangiomas, and somewhat less in vascular malformations, but both were higher than expression in normal skin (P<0.01). These results are shown in Tables 1 and 2 and in Figs. 1-8.
DISCUSSION
Hemangiomas are primary tumors of the microvasculature characterized by excessive angiogenesis, followed by regression of the newly formed vessels. The etiology and pathogenesis remains unclear.［8,9］ Intervention is necessary in up to 20% of cases, and while numerous therapeutic strategies have been utilized,［10,11］ systemic or intralesional glucocorticoid remains the first-line treatment. Especially for hemangiomas which may interfere with normal functions (e.g., breathing, vision, hearing, eating, voiding, and movement), can produce serious disfigurement that is unlikely to resolve on its own, and can also contribute to multiple other problemats (intractable bleeding, ulceration, infection, pain, coagulation defects, heart failure, etc).［1,3,4］ In addition, recent studies suggest that hemanigiomas may lead to psychological problems.［12］ However, the reported validity of glucocorticoid treatment has varied widely,［4,13,14］ which may be related to the fact that the therapeutic regimens, strategies and patients selection are not uniform. This lack of consensus is, in turn, related to a lack of understanding of the precise mechanism of glucocorticoid action in hemangiomas.［1,6,15］
Recently, it has been proposed that proliferation and involution of hemangiomas involves a disturbance of a balance between forces that promote and inhibit vascular growth. The potential role of a number of cytokines and growth factors on acceleration of hemangioma growth have been explored; these include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF2), TGFβ1, Ang and their related receptors/genes. In addition, it is thought that involution of hemangiomas may be related to apoptosis of vascular endothelial cells.［16］ Glucocorticoids can potentially affect hemangiomas in a number of ways, for example, by decreasing transcription of many cytokines, enhancing the expression of specific genes (such as the cytb gene cluster, apoj, and IL-6), increasing the number of mast cell in the hemangioma,［1,3,6,17］ and by regulating expression of cytokines such as interferon (IFN) and tumor necrosis factor (TNF) that can cause apoptosis of vascular endothelial cells. As a result, glucocorticoids may inhibit hemangioma proliferation or accelerate hemangioma regression by a number of direct or indirect mechanisms. Clinically, local injection of sclerosing agents maybe lead to ischemia, noninfective inflammation and vascular sclerosis, and embolism. Additional mechanisms involving inhibition of cellular proliferation are also generally believed to play a role in glucocorticoid-induced degeneration or disappearance of hemangioma. It is also possible that hormone receptor abnormalities might contribute to the disease and could decrease the efficiency of hormone therapy. The biologic effects of glucocorticoids are mediated by direct binding of the ligand with the glucocorticoid receptor, and thus the interaction of glucocorticoids with its receptors is crucial and believed to be the first step leading to subsequent biologic responses.［18］ To our knowledge however, there are no reports specifically directed at determining expression patterns of the glucocorticoid receptors in children with cutaneous hemangiomas.
GR is both a nuclear receptor and also a kind of transcription factor. Recently, studies have demonstrated GR in human exists in two isoforms, GRα and GRβ. The therapeutic effects of glucocorticoids mainly result from the actions of GRα, while GRβ appears to be an endogenous inhibitor of GRα.［19］ It is important to examine GR expression patterns and their relationship to the effectiveness of glucocorticoid treatment in many tumor tissues. The further study of GR's isoforms can be expected to enhance our understanding of the causes of glucocorticoid resistance or partial resistance.［20,21］
In this study, the findings implied hemangiomas may have higher sensitivity than vascular malformations （venous malformation in this study） or normal skin to glucocorticoid treatment, and further support the notion that glucocorticoid is effective in treating hemangiomas. However, our results appear somewhat inconsistent with previous studies, which found that therapeutic effects of glucocorticoid for vascular malformations were not obvious. The reasons for these apparent discrepancies are unclear, and will require further study. Our results also imply that measuring total expression of GR may be misleading; its isoforms should be considered as well, because GRβ may inhibit the action of glucocorticoid. Hence, the understanding the effectiveness and sensitivity of glucocorticiod treatment in hemangiomas will depend upon a complete understanding of the expression patterns and regulation of the receptors. We believe that if expression of GR is limited, or predominantly GRα, then, glucocorticiod therapy might best be avoided since it is ulikely to be effective. Estimation of the potential effectiveness of glucocorticoid treatment should also take into consideration the status of the receptors. Further study of glucocorticiod receptors may lead to development of a new classification model better predicts the results of steroid therapy for this complex and unpredictable tumor.［22］
However, the contribution of gluco-corticoids and GR expression and regulation in hemangiomas is complex, and numerous other factors such as cytokines and other hormones and their receptors may interact with glucocorticoids and their receptors, and glucocorticoid itself might also regulate expression and/or activity of the GR receptor. All of these potential influences are poorly understood at present.［23-25］ Our data here proved an initial step towards a more complete understanding of the role of GR/GRα in hemangiomas.
Acknowledgement: We thank Prof. CHEN Bin for his assistance in statistics.
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