PEDIATRICS Vol. 105 No. 1 January 2000, pp. 117-120

EXPERIENCE AND REASON:
Steroid Therapy of a Proliferating Hemangioma: Histochemical and Molecular Changes

	ABSTRACT

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Objectives.  Hemangioma is a primary tumor of the microvasculature in which angiogenesis is initially excessive, followed by regression of the newly formed vessels. Intervention is necessary in up to 20% of cases, high-dose systemic or intralesional steroids being the first-line treatment. As the mechanism of action of steroids is unknown, we undertook an investigation of the cellular and molecular effects of their action.

Study Design.  A unique opportunity to study the effect of steroid treatment was presented when biopsy material was obtained from an infant with an ulcerated proliferating hemangioma before and after intralesional triamcinolone injection, which resulted in an accelerated regression of the lesion. Histochemical quantitation of mast cells, molecular analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) for 7 growth factor transcripts and differential display RT-PCR (DD RT-PCR) were conducted.

Results.  After steroid therapy, the mast cell number increased (untreated = 2.22 ± .27 [standard error of the mean {SEM}]; treated  = 8.7 ± .71 [SEM] mast cells per field, respectively; P < .0001; n = 40 fields for each group), and the transcriptional expression of cytokines: platelet-derived growth factor-A and -B; interleukin-6; transforming growth factor-1 and -3 decreased, while that of basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor remained unaltered. Elevated urinary bFGF levels noted in cases of proliferating hemangioma, persisted even after steroid treatment. Using DD RT-PCR an amplicon that shared 100% sequence homology with the human mitochondrial cytochrome b gene was detected in the hemangioma biopsy after steroid treatment.

Conclusions.  The regression of this hemangioma subsequent to steroid therapy was accompanied by a significant increase in mast cell density, reduced transcription of several cytokines, and an enhanced expression of the mitochondrial cytochrome b gene.  Key words:  hemangioma therapy, angiogenesis, cytokines, differential display, cytochrome b.

Hemangioma, the most common tumor of infancy, affects up to 12% of whites¹ but is seen rarely in darker skin races.² The incidence increases to 23% in premature infants with a birth weight <1000 g.³ Girls are 3 times more likely to be affected.² Although a positive family history has been noted in 10% of affected individuals,⁴ based on a cohort of 118 twins, Cheung et al⁵ recently showed that hereditary factors are not crucial in causing hemangiomas.

Hemangioma is a primary tumor of the microvasculature in which angiogenesis is initially excessive but is followed by a spontaneous regression of the neovasculature.¹ The principal cellular constituents of proliferating hemangioma are endothelial cells along with accumulating mast cells, macrophages, plasma cells, and pericytes.⁶ A number of growth factors have been demonstrated as regulators of angiogenesis including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor- (TGF-), and interleukin-6 (IL-6).^7,8 Although the pathophysiology of hemangioma is poorly understood, some of these factors may be involved in both its proliferation and involution.⁶ For example, the angiogenic peptide bFGF has been shown to be elevated in the urine of infants with proliferating hemangiomas.⁹

The majority of hemangiomas require no intervention.⁴ However, treatment is necessary in 10% to 20% of cases because of their location, size, or behavior.^6,10 Various therapeutic modalities have been described including surgical excision,¹¹ laser treatment,¹² arterial embolization,¹ and cryotherapy.¹³ The mainstay treatment for hemangioma during proliferation is pharmacologic therapy with glucocorticoids or interferon-.¹⁰ High-dose systemic or intralesional steroid is the first-line treatment and a dramatic response has been observed in 30% of cases.¹⁰ The precise mechanism of action of steroids is unclear, although they are most likely to alter cellular functions by regulating cytokine expression either directly or indirectly.^14-17

Glucocorticoids are known to exert their effects by interacting directly with the glucocorticoid receptor.¹⁶ The activated steroid-receptor complex then binds to the gene it modulates at a specific sequence in its promoter known as the glucocorticoid responsive element, leading to altered gene transcription. In each cell, the number of genes directly regulated by steroids is estimated to be between 10 and 100, but many genes are indirectly regulated through an interaction with other transcription factors.¹⁸

We were presented with a unique opportunity to make clinical and experimental observations in a case of ulcerated proliferating hemangioma before and after intralesional steroid therapy. The effect of steroid therapy on the mast cell number, cytokine expression, and the identification of possible gene(s) modulated by steroids in vivo was investigated by histochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR).

	METHODS

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Case Report

A female infant, born at 34 weeks gestation (birth weight 1765 g) developed a pink macule on her left axilla that enlarged rapidly and became ulcerated. The lesion continued to proliferate and the ulcers failed to heal despite treatment with antibiotics and dressings (Fig 1A). At 3.5 months, under general anesthesia, the hemangioma was treated with intralesional triamcinolone acetonide (4mg/kg), which was repeated 5 weeks later. An incisional biopsy was obtained just before each of the steroid injections, and hence samples from the same patient before and at 5 weeks after the initial steroid injection were available for analyses. Urine samples were collected concurrently for determination of bFGF levels using an enzyme-linked immunoabsorbent assay kit (R and D Systems, Minneapolis, MN). Urinary bFGF level was 6.28 pg/mL and 5.11 pg/mL before and after treatment, respectively. Both values are above the normal levels for children (median: 1.84 pg/mL; range: .67-2.40 pg/mL).⁹

View larger version (94K): [in this window] [in a new window]   Fig. 1.   A, an ulcerated proliferating hemangioma in the left axilla of a 3.5-month-old female infant. B, intralesional triamcinolone therapy resulted in accelerated regression at 5 weeks.

Tissue Samples

Fresh operative hemangioma biopsy specimens were obtained according to a protocol approved by the Wellington Ethics Committee and used for histochemical and molecular studies.

Histochemistry

The operative samples were fixed in neutralized 4% buffered formalin and subsequently embedded in paraffin. Sections (5 µm) were cut and mounted on poly L-lysine coated glass slides (Sigma Chemical, St Louis, MO). Sections were deparaffinized, stained with hematoxylin-eosin for histologic analysis, and Csaba stain was used to identify and quantitate mast cells as described previously.¹⁹ For mast cell quantitation, 20 fields per section were counted independently by 2 observers (Q.H. and S.G.P.) at 40× magnification using an Olympus microscope (Olympus Optical Co, Ltd, Japan).

RNA Isolation and RT-PCR Analysis

RNA was isolated from the hemangioma biopsies using Trizol reagent (Gibco-Life Technol, Gaithersburg, MD) and quantitated using hypoxanthine phosphoribosyl transferase (HPRT) as the control gene as described previously.²⁰ The transcript expression of the cytokines bFGF, VEGF, platelet-derived growth factor (PDGF)-A and -B, IL-6, TGF-1 and -3, was analyzed using the Perkin-Elmer rTth kit (Norwalk, CT) as described earlier.^20,21 Briefly, a 10-minute RT step was followed by 45 cycles of 3-stage PCR with a specific annealing temperature for each primer set. The PCR products were electrophoresed on 2% agarose gels in Tris-acetic acid-ethylenediamine-tetraacetic acid buffer, stained with ethidium bromide and visualized under ultraviolet light. The design of the primers, which were synthesized by Operon Technologies (Alameda, CA) and Gibco-Life Technologies, was based on the published human sequences using the Oligo program (Table 1).

mRNA Isolation and DD-PCR

Biopsy material obtained before and after steroid therapy was snap frozen in liquid nitrogen and stored at 70°C. mRNA was isolated using an mRNA direct kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DD was performed by the modified method of Jurecic et al.²² mRNA was reverse transcribed using Superscript enzyme (Gibco-Life Technologies) with anchored poly-dT primers. The resulting cDNA obtained was PCR amplified with Taq polymerase (Qiagen) using a primer mix comprising anchored oligo-dT and arbitary primers, VEGF, PDGF-A, TGF-1, bFGF, and IL-6 (Table 1). A 3-stage PCR was conducted under the following conditions: 1) denaturing at 94°C for 30 seconds; 2) low stringency annealing temperatures that were ramped up from 41°C to 49°C for 15 seconds for the first 9 cycles and then fixed at 50°C for 26 more cycles; and 3) extension that was at 72°C for 1 minute. Samples were amplified and electrophoresed in duplicate, side by side on a 10% polyacrylamide gel in TAE buffer. The gel was stained with ethidium bromide and visualized under ultraviolet light. The differentially expressed bands were excised and DNA eluted overnight at 4°C in 50 µL of ultra-pure (Millipore Corporation, Bedford, MA) water. Approximately 5 µL of this was used for reamplification using anchored oligo-dT and each of the primers mentioned above. Reamplified products were isolated from 2% agarose gel and purified using a gel purification kit (Qiagen). The DNA extracted from the gel was analyzed using automatic sequencing by Waikato University (Hamilton, New Zealand).

Statistical Analysis

Data were evaluated statistically by the Student's t test with a P value < .05 being considered significant.

	RESULTS

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Clinical

Injection of triamcinolone acetonide (Bristol-Myers Squibb Ltd, Auckland, New Zealand) resulted in accelerated regression of the hemangioma with healing of the ulcers (Fig 1B). Further regression was observed during the 6-month follow-up subsequent to the second steroid injection.

Histochemistry

Hematoxylin-eosin staining of the untreated hemangioma specimen showed proliferating endothelial masses within the papillary and reticular dermis. In some areas the endothelial cells were organized into capillary tubes. Histologically, apart from fibrosis, few overt changes were observed after steroid treatment (data not shown). The Csaba stain showed that virtually all of the mast cells were of biogenic amine phenotype and their number increased significantly after steroid therapy from a mean of 2.22 ± .27 (standard error of the mean [SEM]) to 8.77 ± .71 (SEM; P < .0001; n = 40 fields for each group).

RT-PCR

For the cytokines PDGF-A and -B, IL-6, TGF-1 and -3 there was a reduction in mRNA after steroid treatment, while the transcripts of bFGF and VEGF remained unchanged (Fig 2).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Ethidium bromide stained agarose gel showing RT-PCR products obtained from hemangioma biopsies before (lanes 2, 4, 6, 8, 10, 12, 14, and 16) and after (lanes 3, 5, 7, 9, 11, 13, 15, and 17) steroid therapy. 123bp DNA ladder (lane 1). Equal amounts of HPRT (lanes 2 and 3), bFGF (lanes 4 and 5) and VEGF (lanes 6 and 7) transcripts are detected before and after steroid treatment. Reduced mRNA expression of PDGF-A (lanes 8 and 9), PDGF-B (lanes 10 and 11), TGF-1 (lanes 12 and 13), TGF-3 (lanes 14 and 15), and IL-6 (lanes 16 and 17) is apparent after steroid treatment.

DD RT-PCR

DD analysis revealed the presence of an ~370-base pair (bp) amplicon exclusively in the steroid treated sample (Fig 3A). Reamplification showed that this band was amplified by anchored poly-dT and bFGF primers (Fig 3B). The sequence obtained was compared with sequences in the Gene Bank database (National Institutes of Health, Bethesda, MD) using BLAST and showed 100% homology with the human mitochondrial cytochrome b (cyt b) gene. The upregulation of cyt b in the biopsy obtained after steroid therapy was further confirmed by RT-PCR using human mitochondrial cyt b specific primers.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Lane 1 is a 123-bp DNA ladder. A, 10% polyacrylamide gel showing DD-PCR products in duplicate in the hemangioma before (lanes 2 and 3) and after (lanes 4 and 5) steroid therapy. A 370-bp band is observed only in the treated sample. B, 2% agarose gel showing the PCR products obtained after reamplification with poly-T and all the 5 sense primers used for DD-PCR (lane 2), PDGF-A (lane 3), VEGF (lane 4), TGF-1 (lane 5), bFGF (lane 6), and IL-6 (lane 7). Only lanes 2 and 6 have the 370-bp band, which was extracted and sequenced.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion Conclusion References

All hemangiomas eventually involute. However, treatment is indicated during infancy in 10% to 20% of cases. Steroids are effective in treating a number of conditions including proliferating hemangiomas. Intralesional corticosteroid is regarded as the treatment of choice for localized hemangiomas.²³ The cellular and molecular biology of hemangiomas and their spontaneous or therapy-induced regression are still largely unexplored. Understanding of the molecular pathways that are active in these tumors could lead to more effective therapies because molecules that could be contributing to the proliferation or involution of these tumors could be targeted.²⁴ It is well established that cytokines play an important role in normal physiology as well as in the pathophysiology of disease.²⁵ Cytokines are putative regulators of hemangioma proliferation and involution. Alteration of the cytokine milieu or of the expression of specific genes within the lesion by steroid therapy, provides insight into the possible mechanism by which hemangiomas respond to steroids.

Hemangioma is characterized by the proliferation of capillary endothelial cells with multilamination of the basement membrane and accumulation of cellular elements, including macrophages, plasma cells, pericytes, and mast cells.^6,26 The number of mast cells increases in late proliferating and involuting phases.^1,26 It has been suggested that one of the functions of mast cells is to release factors leading to regression of the neovessels.¹⁰ The fourfold increase of mast cell number after steroid treatment observed in this study supports this hypothesis and indicates that in this clinical condition the glucocorticoids are altering the developmental time course resulting in accelerated involution.

High urinary bFGF levels have been noted in individuals with proliferating hemangioma.⁹ The elevated level observed in this case persisted after steroid therapy. Concurrent with this observation was the finding that bFGF transcripts in the biopsies remained unaltered after treatment. The mRNA for another proangiogenic peptide, VEGF, was also unaltered. These results suggest that the production and metabolism of both bFGF and VEGF, which are considered to be involved in the pathogenesis of hemangioma,⁶ are relatively unaffected by the administration of triamcinolone. However, the transcripts for the cytokines PDGF-A and -B, IL-6, TGF-1 and -3 were reduced by the steroid. The decrease in transcription of the PDGFs, which are considered to promote cell proliferation, indicates that steroid may cause accelerated regression of the proliferating hemangioma via inhibition of these cytokines. The decrease in mRNA of IL-6, another growth factor that plays a role in angiogenesis,⁷ was observed after therapy. This cytokine has been reported to be attenuated by glucocorticoids.¹⁴ The reduction in the transcripts of the 2 isoforms of TGF- was not unexpected as steroids have been shown to alter the expression of this growth factor.²⁵

DD RT-PCR allows identification of differentially expressed genes in various cell types and under defined physiologic conditions. The amplicon identified, isolated, and sequenced from the hemangioma sample after steroid treatment using the DD approach was that of the human mitochondrial gene, cyt b. Sekeris²⁷ proposed that the mitochondrial genome is a primary site of action of steroid hormones. Although the influence of steroids on mitochondrial transcription is well documented, the mechanism of action has only recently been elucidated.²⁸ It has been demonstrated that mitochondrial DNA contains sequences in close proximity to the cyt b gene which share similarity to the glucocorticoid responsive elements.²⁹ It is interesting to note that an elevated expression of cyt b has been reported recently in senescent human cells³⁰ and in the involuting lactating mammary gland following weaning.³¹ These studies and our findings suggest that the steroid treatment enhances cyt b expression, which may have a role in the regression of hemangioma. Elevated cellular respiration, characterized by raised levels of mitochondrial respiratory chain components including cyt b, may be responsible for inhibiting angiogenesis as increased oxygen metabolism inhibits angiogenesis.^32,33 Further study will be required to understand the functional role of cyt b in the steroid-induced regression of hemangioma.

	CONCLUSION

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In summary, we have shown that triamcinolone, which resulted in accelerated regression of a proliferating hemangioma, increased the mast cell number, reduced the transcription of the growth factors, PDGF-A and -B, IL-6, TGF-1 and -3 but did not affect bFGF and VEGF. In addition, there was upregulation of the mitochondrial cyt b gene expression. To our knowledge, this is the first report on the cellular and molecular effects of steroid therapy on hemangioma.

	ACKNOWLEDGMENTS

This work was supported by grants from the University of Otago Internal Research Fund, the Wellington Medical Research Foundation, the Reconstructive Plastic Surgery Research Foundation, the Maurice and Phyllis Paykel Trust, Lottery Health Research, and the Health Research Council of New Zealand.

We are grateful to C. Marstella for her assistance in the preparation of this manuscript.

Qurratulain Hasan, PhD^*, Swee T. Tan, MBBS, FRACS^*, , Jason Gush, BSc^§, Sue G. Peters, BSc^*, and Paul F. Davis, PhD^*
* Department of Medicine, Wellington School of Medicine
 Swee Tan Plastic Surgery Trust
^§ Malaghan Institute of Medical Research
Wellington, New Zealand

	FOOTNOTES

Received for publication Jan 19, 1999; accepted May 4, 1999.

Reprint requests to (S.T.T.) Swee Tan Plastic Surgery Trust, Bowen Hospital, Churchill Drive, Crofton Downs, Wellington, New Zealand. E-mail: sweetan{at}plastsurg.co.nz

	ABBREVIATIONS

VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TGF-, transforming growth factor-; IL, interleukin; RT-PCR, reverse transcriptase-polymerase chain reaction; DD, differential display; HPRT, hypoxanthine phosphoribosyl transferase; PDGF, platelet-derived growth factor; SEM, standard error of the mean; bp, base pair; cyt b, cytochrome b.

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