Life Cycle of Hemangioma

Proliferating Phase: Endothelial Progenitor Cells

Proliferating hemangiomas are highly cellular, with little connective tissue and barely discernible vascular lumens; proliferating endothelial cells are easily detected by histological methods. Because the tumor comprises endothelium, it is not surprising that many studies have found high levels of endothelial-expressed proteins. In Figure 2, tissue sections from proliferating and involuting hemangioma sections were double-labeled, with antibodies directed against two well-known endothelial markers. Vascular endothelial growth factor-receptor-2 (VEGF-R2), also known as kinase insert domain-containing receptor (KDR), is the major mediator of the angiogenic effects of VEGF on endothelial cells. In addition to being expressed on mature endothelial cells, KDR is also expressed by endothelial progenitor cells (EPC). CD31, also known as platelet-endothelial cell adhesion molecule-1 (PECAM-1), is a cell-cell adhesion molecule expressed on virtually all continuous endothelium. As seen in Figure 2, KDR is abundantly expressed in proliferating hemangioma; the green fluorescent signal is evident on nascent vascular structures and on cells lining vascular channels. CD31 is also expressed in proliferating phase hemangioma; the red fluorescent signal strongly highlights vascular channels. When the two signals are viewed simultaneously, it is clear that KDR and CD31 are co-localized, providing strong evidence for endothelial cells within the proliferating hemangioma. The same immunostaining procedure was performed on involuting hemangioma specimens. KDR-positive/CD31 -positive endothelial cells are evident, but the cellular density is decreased and vascular channels are more prominent. This change in endothelial morphology suggests that nascent vascular structures present in the proliferating phase differentiate into more well-defined vascular channels as the involuting phase proceeds. The speculation that proliferating hemangiomas contain primitive angioblast-like cells has been put forth by many investigators. In support of this concept, we recently reported the detection of endothelial progenitor cells (EPC) from 11 out of 12 proliferating phase hemangiomas [5]. No EPC were detected in involuting hemangiomas or in venous malformation or lymphatic malformation tissue, suggesting that the presence of EPCs in hemangioma is unique and that EPC might contribute to the pathogenesis of hemangioma. To our knowledge, our study was the first to

Figure 2 Expression of KDR/VEGF-R2 and CD31 in Hemangioma. Indirect immunofluorescence detection of KDR (panels a, c, d, f) and CD31 (panels b, c, e, f) in frozen sections from proliferating phase (a-c) and involuting phase (d-f) hemangiomas. In panels a and d, the green fluorescence (FITC) corresponding to KDR is seen. In panels b and e, the red fluorescence (TexasRed) corresponding to CD31 is seen. In panels c and f, both fluorescence signals were viewed simultaneously; cells expressing both proteins are seen as yellow or yellow/orange. (Yu and Bischoff, unpublished data.) (see color insert)

Figure 2 Expression of KDR/VEGF-R2 and CD31 in Hemangioma. Indirect immunofluorescence detection of KDR (panels a, c, d, f) and CD31 (panels b, c, e, f) in frozen sections from proliferating phase (a-c) and involuting phase (d-f) hemangiomas. In panels a and d, the green fluorescence (FITC) corresponding to KDR is seen. In panels b and e, the red fluorescence (TexasRed) corresponding to CD31 is seen. In panels c and f, both fluorescence signals were viewed simultaneously; cells expressing both proteins are seen as yellow or yellow/orange. (Yu and Bischoff, unpublished data.) (see color insert)

show direct evidence of human EPC in a human vascular tumor.

Proliferating Hemangioma: Clonal Endothelial Cells

Two experimental approaches demonstrate that hemangioma is caused by an intrinsic defect in the endothelial cells. First, Boye and colleagues isolated hemangioma-derived endothelial cells from 10 different hemangioma tissue specimens and showed that the endothelial cells were clonal by examining X-chromosome inactivation patterns [6]. Nonendothelial fibroblast-like cells that were co-isolated from the tissue and separated from the endothelial cells were not clonal, demonstrating that the clonal expansion was restricted to the endothelial cells. In addition, the hemangioma-derived endothelial cells exhibited abnormal properties in in vitro assays that measure angiogenic activity. These results substantiate the hypothesis that heman-gioma is caused by a somatic mutation in a single endothelial cell (i.e., an intrinsic defect), in a gene that controls endothelial growth and differentiation. Two other studies also support this hypothesis. Evidence for loss of heterozygosity on chromosome 5q in three out of six hemangiomas suggested a somatic mutation in this region may be the cause of sporadic hemangiomas. More recently, Walter and colleagues showed that 12 of 14 informative hemangioma lesions had nonrandom X-chromosome inactivation patterns—evidence for clonal endothelial cells—and thus, a somatic mutational event [7]. Importantly, their clonality assays were performed on DNA isolated from tissue sections, which avoids changes in cell composition that might occur during cell culture. In summary, these studies provide strong support for the hypothesis that a somatic mutation in a single endothelial cell leads to clonal expansion of dysregulated endothelial cells, resulting in hemangioma. Formal proof of the hypothesis awaits identification of the somatic mutation(s) and functional demonstration that such mutation(s) cause hemangioma.

Involuting Phase: Onset of Apoptosis

The involuting phase of hemangioma coincides with a transformation to a more lobular architecture with organized and recognizable vascular channels, which can be appreciated in Figure 2. As involution proceeds, vascular channels become larger and more prominent, and few interstitial cells are evident. Increases in perivascular cells expressing a-smooth muscle actin are consistent with the appearance of mature blood vessels. Mast cells appear to be increased in the involuting phase, and changes in extracellular matrix occur. Interferon-b in the epidermis overlying involuting and involuted hemangiomas has been proposed as a mechanism for attenuating the abnormal proliferation in heman-gioma [8]. The final, involuted phase is characterized by a few remaining thin-walled vessels that resemble normal capillaries, surrounded by fibrofatty tissue.

The cellular and biochemical mechanisms that drive the involuting phase have been difficult to study because endothelial cells from involuting hemangioma do not grow in vitro [9]. Based on studies showing that increased apop-tosis can offset cellular proliferation in murine tumor models, we examined cellular proliferation and apoptosis in a series of 16 hemangioma specimens from infants to children up to nine years of age, as well as normal infant skin for comparison [10]. Consistent with previous studies, cellular proliferation was high in hemangioma specimens obtained from children up to 24 months of age. Apoptosis was increased fivefold in hemangioma specimens from children one to four years of age compared to proliferating heman-gioma specimens from children under one year of age, late-involuting/involuted hemangioma specimens from children over four years of age, and normal skin from infants (see Figure 3). Thus, the increase in apoptosis coincided precisely with the onset of the involuting phase. Double-labeling tissue sections for apoptosis and the endothelial marker von Willebrand Factor showed that 27 to 39 percent of the apoptotic cells were endothelial. In summary, this study suggests that regression of hemangioma involves increased apoptosis even as cellular proliferation continues. One might speculate that an agent that triggers apoptosis in proliferating endothelial cells would be an effective treatment for hemangioma.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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