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Angiogenesis (2009) 12:197207

DOI 10.1007/s10456-009-9148-2

ORIGINAL PAPER

Vasculogenesis in infantile hemangioma


Elisa Boscolo Joyce Bischoff

Received: 18 February 2009 / Accepted: 24 April 2009 / Published online: 10 May 2009
Springer Science+Business Media B.V. 2009

Abstract Infantile hemangioma is a vascular tumor that Introduction


occurs in 510% of infants of European descent. A defining
feature of infantile hemangioma is the dramatic growth and Infantile hemangioma (IH) is a benign vascular neoplasm
development into a disorganized mass of blood vessels. of infancy that appears soon after birth in 510% of chil-
Subsequently, a slow spontaneous involution begins around dren of mixed European descent [13]. It occurs more often
1 year of age and continues for 46 years. The growth and in females when compared with males and is also more
involution of infantile hemangioma is very different from prevalent in premature infants. Most IH remain relatively
other vascular tumors and vascular malformations, which small and pose no serious threat or complication to the
do not regress and can occur at any time during childhood baby. However, a small number of IH grow so large that
or adult life. Much has been learned from careful study of they lead to tissue and organ damage and in some cases
the tissue morphology and gene expression patterns during become life-threatening. Examples of two lesions, one
the life-cycle of hemangioma. Tissue explants and tumor- small and one of moderate size, in two different infants at
derived cell populations have provided further insight to 6 months of age are shown in Fig. 1.
unravel the cellular and molecular basis of infantile hem- Depending on the size and location of the hemangioma,
angioma. A multipotent progenitor cell capable of de novo many serious problems can ensue. For example, eyelid IH
blood vessel formation has been isolated from infantile can cause deprivation amblyopia, a subglottic IH can
hemangioma, which suggests that this common tumor of compromise the airway, and extremely large IH can cause
infancy, long considered to be a model for pathologic high-output congestive heart failure [4]. Some IH ulcerate
angiogenesis, may also represent pathologic vasculogene- and bleed, which then requires transfusion and/or surgery.
sis. Whether viewed as angiogenesis or vasculogenesis, Infants with large facial hemangiomas are at risk for
infantile hemangioma represents a vascular perturbation cerebral infarction [5]. Ectopic expression of type 3 iodo-
during a critical period of post-natal growth, and as such thyronine deiodinase in IH has been shown to inactivate
provides a unique opportunity to decipher mechanisms of thyroid hormone systemically and cause hypothyroidism
human vascular development. [6]. Finally, 4080% of IH leave permanent cutaneous
residua after tumor involution, which can be particularly
Keywords Angiogenesis  Endothelial cells  disfiguring in children with facial IH.
Endothelial progenitor cells  Hemangioma  Current treatments for children with endangering IH are
Vasculogenesis  VEGF-receptors limited, and include primarily corticosteroids [7], which
have many adverse effects. Furthermore, approximately
30% of hemangiomas do not respond to corticosteroids,
prompting active investigations for new treatments. Vin-
E. Boscolo  J. Bischoff (&) cristine, a chemotherapeutic agent, has emerged as a sec-
Vascular Biology Program, Department of Surgery, Childrens
ond line treatment for IH that do not respond to steroids
Hospital Boston , Harvard Medical School, 300 Longwood Ave,
Boston, MA 02115, USA [8, 9]. Interferon-a was used for severe, problematic
e-mail: joyce.bischoff@childrens.harvard.edu hemangiomas [10, 11] but irreversible neurologic toxicity

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198 Angiogenesis (2009) 12:197207

Fig. 1 Two examples of


infantile hemangioma. In a, one
prominent lesion and two
smaller lesions are seen.
Multiple hemangiomas, as seen
here, occur in 20% of cases. In
b, a facial hemangioma with
potential to affect the eye is
shown

has been associated with interferon-a administration to the notion of nascent endothelia in the proliferating phase
infants with IH [12, 13], and therefore it is now rarely used. [16, 2022].
A recent report has shown the b-adrenergic receptor Growth of IH slows dramatically in the involuting
antagonist propranolol to be effective against severe IH phase, which typically begins soon after the childs first
[14]. However, the mechanism of action and potential for birthday and lasts for 46 years. As involution proceeds,
adverse effects in infants is currently unknown due to the vascular channels become more prominent and are lined
serendipitous nature of this discovery. Therefore, even with with flattened endothelial cells (Fig. 2). The clusters of
the encouraging new results with propranolol and the plump immature cells are no longer evident. There are
widely accepted use of steroids, there is still a pressing fewer interstitial cells and deposition of extracellular
need for safe and fast-acting treatments for infants with matrix occurs, with multi-laminated basement membranes
endangering IH. building up around the enlarged vessels. Pericytes, shown
by expression of the marker NG2, neural/glial antigen-2
Life cycle of IH (Fig. 2h), are embedded within the basement membranes
and surround the hemangioma vessels. Hence, it appears
IH displays a unique life-cycle of proliferation followed by that the involuting phase of IH coincides with the de novo
involution. The proliferating phase spans the first several formation of mature blood vessels (Fig. 2).
months of infancy, with most of the growth occurring by The endothelium lining these vascular channels has been
5 months of age [15]. Histological analyzes of IH tissue extensively characterized by immunohistochemical stain-
sections show that the tumors are highly cellular, with ing and shown to bear similarities with placental endo-
clusters of plump cells expressing endothelial markers and thelium [23, 24], which has brought forth much discussion
small vascular channels with barely discernable vessel and debate about whether IH arises from a displacement of
lumens (Fig. 2). Among the vascular channels there are placental cells during fetal development [25, 26]. Two
proliferating interstitial cells but little connective tissue molecular genetic studies have determined that hemangi-
[16, 17]. The human stem/progenitor cell marker CD133 oma-derived cells originate from the child and not the
can be detected on sparsely scattered subsets of cells mother [27, 28], shedding some insight on the origin of the
(Fig. 2g), indicating the undifferentiated status of cells in tumor.
the proliferating phase. The expression of angiogenic fac- A hallmark of IH is that eventually, the disorganized mass
tors and receptors, including vascular endothelial growth of vessels will regress when the child reaches 810 years of
factor (VEGF)-A [18, 19], VEGF-receptors VEGFR-1 and age. This final, involuted phase is characterized by
VEGFR-2, Tie2 and angiopoietin-2, shown by immuno- sparse, thin-walled vessels, around which the characteristic
histochemisty and in situ hybridization techniques, supports multi-laminated basement membranes remain, with large

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Angiogenesis (2009) 12:197207 199

Fig. 2 Histology and cellular


phenotyping in proliferating,
involuting and involuted phases
of IH. ac hematoxilin and
eosin staining; df
immunostaining for human
CD31, an endothelial marker;
g immunostaining for CD133, a
human stem and progenitor cell
marker CD133;
h immunostaining for NG-2, a
pericyte marker;
i immunostaining for perilipin-
A, an adipocyte marker that
prominently encircles the lipid
storage droplets in adipocytes.
Scale bars are 100 lm. (g) and
(h) contributed by Arnaud
Picard, M.D

feeding and draining vessels evident as well. However, the of new drugs and strategies for the treatment of children
bulk of involuted hemangioma tissue is composed of adi- suffering from endangering or life-threatening IH.
pocytes and connective tissue (Fig. 2). For some heman-
giomas, the mass of fat that replaces the proliferating IH can
be of a similar size. This raises intriguing questions on the Cellular components of IH
cellular origin and source of the adipocytes in this final phase
of IH. The idea that IH arises from an undifferentiated stem/
progenitor was first suggested in the nineteenth century and
Angiogenesis or vasculogenesis? further developed in the mid twentieth century as IH was
described as sequestered embryonic mesoderm or activa-
IH has long been considered as an angiogenic disease tion of dormant angioblasts [3033]. In the 1990s, Smoller
because of the tangled disorganized mass of blood vessels and Apfelberg speculated that IH arises from a primitive
in the tumor [18, 29]. The detection of angiogenic factors vascular progenitor that gives rise to endothelial cells and
such as basic fibroblast growth factor (bFGF) and VEGF-A pericytes [17]. An important approach to compliment his-
within the tumor have supported this concept [18, 19]. tological studies has been to dissect the lesions into purified
However, an alternative is that IH arises by a process more cell types that can be studied, tested and compared in the
akin to vasculogenesis, i.e., the de novo formation of laboratory. Here in we will discuss several papers that have
vessels from progenitor cells. This concept is consistent made important inroads in understanding the cellular
with the long-noted presence of immature cells in IH. The complexity of hemangioma.
immature cells could arise from stem/progenitor cells that
reside within the lesion, e.g., the dormant angioblasts Immature endothelial cells
proposed by Pack and Miller [30] or from cells recruited to
the IH from a reservoir of stem/progenitor cells such as the A pioneering study by Mulliken and colleagues showed that
placenta or bone marrow. Once tumor growth has been it was possible to isolate hemangioma-derived endothelial
initiated, angiogenesis may also contribute, with in-growth cells (HemECs) and study their phenotype in vitro [34].
of vessels from the surrounding tissue. Our view is that Dosanjh and colleagues took this a step further and com-
understanding the precise cellular mechanisms of blood pared the in vitro characteristics of HemECs with fetal skin
vessel formation in IH will be a catalyst for the discovery endothelial cells (isolated from second trimester pregnancy

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terminations) and neonatal skin endothelial cells (isolated HemECs and cord blood EPCs are similar to each other in
from newborn foreskin following routine circumcision) several in vitro assays including mRNA transcriptional
[35]. The HemEC and fetal ECs exhibited a spindle-shaped profiling for expression of cell-cell and cell-matrix adhe-
morphology when grown in vitro and produced type I col- sion molecules. Consistent with the study of Dosanjh and
lagen but not type IV collagen. Conversely, neonatal ECs colleagues [35], HemECs, as well as HemEPCs and cord
formed a cobblestone-like monolayer and secreted type IV blood EPCs, could be distinguished from neonatal human
collagen but not type I collagen. Notably, the HemECs and dermal microvascular endothelial cells in our in vitro
fetal ECs exhibited a diffuse intracellular pattern of CD31, assays. Our conclusion is that HemEPCs and HemECs are
also known as platelet endothelial adhesion molecule-1 immature endothelial cells and share properties with cord
(PECAM-1), and vWF immunostaining, which suggested blood EPCs. This raises the question of whether circulating
these were not fully differentiated endothelial cells. The EPCs are recruited into proliferating IH lesions and if so,
authors concluded their study with the speculation that IH do they contribute to growth of the tumor. In support of
may represent a dysregulated endothelial differentiation and this, Kleinman and colleagues reported an increased
maturation [35], a concept that has now been extended by number of circulating CD133?/CD34? EPCs in children
many laboratories including ours [21, 36, 37]. The dysreg- with hemangioma [41]. However, the age-matched controls
ulation could cause a delay in endothelial differentiation were somewhat older (average age 38 months) than the
and result in an ill-timed and disorderly vascular prolifer- hemangioma patients (average age 25 months). Since lev-
ation, as seen in proliferating IH. els of circulating EPCs are likely to vary during infancy
We isolated HemECs from ten different proliferating IH and early childhood, it would be important to extend these
specimens, and found these cell populations displayed studies to a larger number of more closely matched con-
many features of human ECs, for example expression of trols to verify the extent to which circulating EPCs are
endothelial markers. Together with our collaborators in the elevated in IH.
Olsen laboratory, we showed the HemECs were clonal and
exhibited increased growth and migratory properties [36]. Interstitial cells
The clonality and altered properties of the HemECs
strongly suggested an intrinsic defect in these cells, perhaps Although IH is noted for its vascularity and angiogenic
caused by a somatic mutation which would lead to clonal profile [18], some investigators have focused on the cells
expansion. Clonality was also shown in cells analyzed located between the vascular channels [17]. Smoller and
directly from tissue sections of proliferating IH by Mar- Apfelberg described these cells as interstitial tumor
chuk and colleagues [38], which argued against the clo- cells, a heterogenous mix of cells with both cuboidal and
nality being due to in vitro culture favoring growth of a spindle-shaped morphology. The interstitial cells did not
small number of cells. Marchuks finding of clonality in a express von Willebrand factor (vWF), a marker of differ-
wide swath of unselected cells recovered from tissue sec- entiated endothelial cells, but instead most of them were
tions suggests that the entire tumor might be derived from a positive for factor XIIIa, a marker of dermal dendrocytes,
single progenitor cell. some were positive for CD34, a stem cell and endothelial
marker, and even fewer were positive for a-smooth muscle
Endothelial progenitor cells actin (a-SMA), a pericyte/smooth muscle cell/myofibro-
blast marker. Importantly, most of the proliferating cells in
We subsequently set out to isolate potential precursors of the IH samples were located in the interstitial, non-vascular
HemECs from proliferating IH by selecting cells that co- areas. Based on their analyzes, the authors put forth the
expressed the human stem cell marker CD133 and an concept that proliferating phase IH is composed of a
endothelial marker; we refer to these as hemangioma- primitive progenitor cell capable of differentiating into
derived endothelial progenitor cells (HemEPCs) [22, 39]. endothelial cells and pericytes [17]. This concept was
To test whether the HemEPCs give rise to HemECs, we extended in a subsequent study that showed increased
examined cellular growth, migration and adhesion of expression of the anti-apoptotic gene bcl-2 in the interstitial
HemEPCs and HemEC in parallel, with the hypothesis that cells, suggesting that these cells might have a survival
the cells would exhibit the same properties, in particular advantage [16]. Intriguingly, their quantitative analyses
the HemEPCs would show the same stimulatory response suggested that proliferation and bcl-2 expression in the
to endostatin (an angiogenesis inhibitor) [40] that we found interstitial cells was prolonged in lesions with markedly
in HemECs [36]. The HemEPCs were stimulated by more vascular channels. A more in-depth understanding of
endostatin, but unexpectedly so were normal healthy this observation might provide a means to predict which IH
cord blood-derived EPCs [39]. In summary, HemEPCs, will grow to an endangering size.

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Angiogenesis (2009) 12:197207 201

Pericytes Multipotent stem cells

Pericytes, sometimes called mural cells, surround the We recently reported on a multipotent progenitor-like cell
newly formed vessels in IH, however, to date there are no isolated from over thirty different proliferating IH speci-
reports of isolation and/or culture of pericytes from hem- menswe refer to the cells as hemangioma-derived stem
angioma tissue. Immunostaining shows nerve/glial antigen- cells (HemSCs) [47]. The HemSCs exhibit robust prolif-
2 (NG2), a proteoglycan and marker of pericytes [42, 43], erative and clonogenic capability and can differentiate into
prominently expressed in the perivascular cells in a pro- cells of multiple lineages, thereby fulfilling two important
liferating IH tissue section (Fig. 2g). Others have shown features of stem cells. HemSCs express VEGFR-1/Flt-1,
that the perivascular cells in IH are a-SMA- and CD146- neuropilin-1 (NRP-1), but do not express CD31/PECAM-1
positive cells [44]. Pericytes in several human tissues have or VE-cadherin, two reliable endothelial markers. Fur-
been shown to be CD146?/NG2?/PDGFR-b? with thermore, the HemSCs express CD90, a mesenchymal cell
absence of endothelial, hematopoietic and myogenic cell marker, which indicates that HemSC are phenotypically
markers. Furthermore, pericytes exhibit many of the same more similar to mesenchymal cells than to endothelial
properties as mesenchymal stem cells, including osteo- cells. It is of interest to note that CD90 was the gene most
genic, chondrogenic and adipogenic differentiation poten- differentially up-regulated in proliferating compared with
tial [45]. Recently, a subset of perivascular cells aligning involuting phase IH in a micro-array analysis in IH [48].
the vasculature of white adipose tissue were shown to be Clonal HemSCs, expanded in vitro from single cells,
adipogenic progenitor cells [46]. Therefore, one might produce human GLUT-1-positive microvessels in vivo
speculate that the pericytes in IH are also capable of multi- 714 days after sub-cutaneous implantation into immuno-
lineage differentiation potential, and specifically adipo- deficient mice. GLUT-1 is an immuno-diagnostic marker
genesis, which would provide a potential source for the for IH [49], and thus its presence indicates the vessels
adipocytes that appear in the involuted phase (Fig. 3). express an important marker of the IH phenotype. HemSCs
that had differentiated into endothelium in vivo, as deter-
mined by cell surface CD31 expression, could be retrieved
and implanted into secondary recipients, where the cells
formed blood vessels once more. This vasculogenic
potential is restricted to the HemSC because HemEC,
HemEPCs, cord blood EPCs, normal human fibroblasts and
bone marrow-derived mesenchymal stem cells do not form
vessels in this in vivo model. Over time, the human blood
vessels formed from HemSCs diminished and adipocytes
became evident, reminiscent of the involuted phase of IH.
GFP-labeled HemSCs were shown to form the adipocytes,
in addition to the endothelial lining of the vessels (Fig. 3a),
providing further confirmation that the vessels and adipo-
cytes were not murine-derived. The cellular origin of the
NG2? perivascular cells in IH has not been shown but we
speculate that HemSC may have the potential to differen-
tiate into pericytes (Fig. 3a). Based on the ability of adi-
pose perivascular cells to differentiate into fat [46], we
propose an alternative pathways in which HemSC give rise
to pericytes in the proliferating and early involuting phases
of IH, which then in turn differentiate into adipocytes
(Fig. 3b). Further studies are required to elucidate the
cellular origin and fates of the pericytes in IH.
In summary, our findings provide evidence for a stem
cell origin of hemangioma, which is in contrast to the long-
Fig. 3 Multipotent hemangioma-derived stem cells (HemSC) differ- held view that hemangioma, arises from endothelial cells
entiate in vivo and in vitro into endothelium and adipocytes. The and angiogenesis [29]. To our knowledge, the HemSC is
cellular origin of pericytes in IH has not been established, as indicated
the only post-natal, non-bone marrow-derived cell that
by the dashed line in (a) and (b). In b, we speculate that the pericytes
surrounding the IH blood vessels may differentiate into adipocytes in solely, on its own, can form functional vascular networks
the involuted phase in vivo. Our work, and that of others, has shown that rapid

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and substantial vascular network formation from normal consisted of expression analyzes on surgically removed IH
human endothelial cells requires a mesenchymal support tumor specimens while in vitro studies have benefited from
cell [5052]. Thus, the HemSC is truly a unique cell in its use of explants and purified hemangioma-derived cell
ability to form vessels in vivo when implanted alone populations. VEGF-A has understandably been a major
without a mesenchymal support cell. Whether IH repre- focus because of its enormous importance in so many
sents an environmental perturbation of a normal post-natal aspects of vascular development and pathologic angio-
stem cell or the HemSCs harbor genetic alteration(s) that genesis. Early studies showed VEGF-A protein in the
contribute to vasculogenic and adipogenic activity must be vessels of proliferating IH [18, 19, 58], and more recently
determined in future studies. VEGF-A was shown to be increased HemECs from three
different IH specimens [59]. Other studies did not reveal
Other cellular components significant expression of VEGF-A transcripts in prolifer-
ating IH [48] or in cultured HemECs [21]. However, VEGF
IH contains many additional cellular components, which secretion by stromal cells isolated from IH has been
have been primarily studied by histological analyzes of reported [58]. Perhaps the VEGF-A protein detected on
tissue sections. Mast cells have been noted in all three hemangioma vessels by immunostaining was due to VEGF
phases of IH, with the highest level appearing in the early secretion from the stromal cells and localization on endo-
involuting phase [53]. It has been suggested that mast cells thelial cells via VEGF-receptors. The stromal cells isolated
signal the beginning of involution [54], however, to date by Berard and colleagues do not react with antibodies
the precise role of mast cells in IH has not been against endothelial markers such as vWF and Ulex eu-
determined. Myeloid cells have also been detected in ropeus agglutinin (UEA-1), but express a-SMA, VEGF-A
proliferating phase IH and suggested to function in a pro- and the VEGF-A receptor KDR, also known as VEGFR-2.
angiogenic manner [55]. The potential contribution of cells The authors did not speculate on the ability of the stromal
from the bone marrow was also suggested by Nguyen and cells to become the endothelial cells in the tumor, but they
colleagues [56] based on detection of cells expressing shed light on an intriguing cellular component of IH that
HLA-DR and CD68 antigen in a close proximity to vessel may be related to the HemSC reported by our laboratory.
structures in hemangioma tissue sections. The cells were The relevance of VEGF-A in the pathogenesis of IH was
shown to be distinct from pericytes or immune cells such as also demonstrated in a study in which VEGF-A levels in
T-cells, B-cells, NK cells or mast cells. Their morphology patient serum were found to be significantly higher in
and immunophenotype was suggested to be most closely proliferating when compared with that in involuting hem-
aligned with antigen presenting cells of the monocyte/ angioma. Interestingly, after steroid therapy, VEGF-A
macrophage/dendritic lineage. Based on a hypothesis that levels were reduced when compared with that in pre-
the angiogenic vessels in proliferating IH may secrete treatment (n = 6) [60], suggesting a putative mechanism of
soluble factors that induce sensory nerve fibers growth, and action of steroids in IH.
in turn that neuropeptides may stimulate IH endothelial The role of VEGF-A has been further explored by real-
growth, Jang and colleagues looked at calcitonin gene- time quantitative PCR for VEGF-receptors in the tissue
related peptide (CGRP) and protein gene product 9.5 (PGP samples of proliferating, involuting and involuted heman-
9.5) in IH [57]. They found dramatically increased num- gioma [20]. Expression levels of VEGFR-1/Flt-1, VEGFR-
bers of CGRP? nerve fibers in proliferating IH when 2/KDR, and NRP-1, and neuropilin-2 (NRP-2) were mea-
compared with involuting IH, and suggested that the sured and normalized to GAPDH and also normalized to
hemangioma vessels and nerve fibers may regulate each VEGFR-2/KDR to account for endothelial content of the
other. In summary, the inter-relationships among these IH specimen. RNA from human neonatal foreskin and
various cell types and their functions in IH must be placenta were used for comparisons. VEGFR-1/Flt-1 was
explored to fully understand the vasculogenic mechanisms found to be consistently under-expressed in seven different
that contribute to the pathogenesis of hemangioma. IH specimens when compared to that in foreskin, placenta
and also seven different congenital hemangioma specimens
[20] whereas the other VEGF-receptors were not.
Molecular pathways in IH Decreased VEGFR-1/Flt-1 expression was also reported in
a separate study [59]. During development, VEGFR-1-/-
VEGF/VEGFRs mice die at E8.5 due to disorganized blood vessels and
endothelial cell over growth [61]two features somewhat
Many investigators have obtained clues and probed path- reminiscent of IH. Therefore, it is possible that diminished
ways to expand our nascent understanding of the vascu- or defective VEGFR-1 signaling is part of the pathogenesis
logenesis and angiogenesis in IH. In vivo studies have of IH.

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The specific IH-derived cell type in which VEGFR-1 is


down-regulated may be the HemEC. Decreased VEGFR-1
protein and mRNA, measured by ELISA and real-time
quantitative PCR, respectively, was shown in several he-
mECs compared to different types of cultured human
endothelial cells, e.g., human umbilical vein endothelial
cells (HUVECs) and human dermal microvascular endo-
thelial cells (HDMECs), cultured under the same condi-
tions [59]. The reduced expression of VEGFR-1, which
acts as a decoy receptor in some settings, could result in
increased VEGF-A binding to VEGFR-2 (Fig. 4). Evi-
dence to support this was shown by increased, constitutive
levels of phosphorylated VEGFR-2 and its target ERK in
HemECs and increased proliferation of HemECs [59]. The
presence of a potential binding site for transcription factor
NFAT (nuclear factor in activated T cells) in the promoter
region of flt-1, and the fact that NFATc1 overexpression
stimulated VEGFR-1 expression suggests that attenuated
NFAT activity is responsible for low VEGFR-1 expression
in HemECs [59]. Surprisingly NFAT transcript levels in
hemECs were comparable with that in HDMECs, but
NFAT-regulated genes as Down syndrome critical region
protein-1 (also known as Regulator of Calcineurin-1,
RCAN1) and cyclooxygenase-2 (PTGS2) were downregu-
lated in hemECs indicating that the defect involves NFAT
activity and not expression. Jinnin and colleagues went on
to show that NFAT activity was disrupted in HemECs due
to mutations in VEGFR-2 and TEM8 that affect complex
formation with b1-integrin (Fig. 4). The mutations will be
discussed in depth below.

Genetic alterations

Most hemangiomas occur sporadically but the potential


contribution of familial and/or somatic mutations to IH has
been studied in several laboratories. Blei and colleagues
[62] postulated a familial form of IH in a study of five Fig. 4 VEGFR-1 regulation in endothelial cells. a When b1-integrin
families with multiple generations affected by IH, with in endothelial cells is activated, it promotes NFAT translocation into
evidence of an autosomal dominant inheritance. A sub- the nucleus where it binds to the VEGFR-1 promoter to stimulate
sequent linkage analysis conducted in three of these fam- VEGFR-1 transcription. b1-integrin-induced NFAT activation is
inhibited when a complex with VEGFR-2 and TEM8 is formed.
ilies established a genetic linkage with chromosome 5q Mutations in TEM8 and VEGFR-2 found in HemECs enhance affinity
[63]. The region, 5q31-33, contains three candidate genes between the complexed proteins. In b, a schematic shows HemEC
involved in blood vessel growth: FGFR4 (Fibroblast with lower VEGFR-1 levels, due to enhanced complex formation and
Growth Factor Receptor 4), PDGFR-b (Platelet Derived decreased b1-integrin activity. This leads to increased VEGF-A
availability and activation of VEGFR-2, resulting in endothelial
Growth Factor Receptor) and FLT4 (Fms-related tyrosine proliferation
kinase-4). The existence of somatic mutational events in
chromosome 5q has been explored with micro-satellite [64]. Dusp-5 was discovered as a vascular specific gene,
marker analysis. In micro-dissected tissue from non- and shown to be involved in the de-phosphorylation of
familial IH specimens, loss of heterozygosity (LOH) was MAPK [65, 66]. The reported mutation is a T ? C sub-
detected in chr5q suggesting an association with sporadic, stitution resulting in a change from serine to proline, ren-
non-familial IH [38]. A mutation was reported very dering the phosphatase unstable. The authors found the
recently in the dual specific phosphatase-5 (dusp-5), same mutation in 16/21 vascular malformations indicating
localized on chr 10q25, in one out of three IH specimens a role for Dusp-5 in the vascular development.

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204 Angiogenesis (2009) 12:197207

Because of the accumulated evidence suggesting IH can Other pathways


be familial, in addition to the more common sporadic
forms, and in light of NFAT-VEGFR-1 dysfunctions, the Another receptor-ligand system involved in normal and
Olsen laboratory sequenced 24 genes involved in EC pro- pathogenic angiogenesis is Tie2-angiopoietins. Increased
liferation, migration and adhesion to extracellular matrix, Tie2 mRNA and protein have been shown in cultured
or in regulation of VEGF-A expression. From this effort, HemECs, with a corresponding enhanced response to an-
germline risk-factor mutations were identified in the giopoietin-1 (Ang1). Both Ang1 and angiopoietin-2 (Ang2)
integrin-like molecule TEM-8 (tumor endothelial marker- induce phosphorylation of Tie2 and are required for via-
8) and in VEGFR-2 [59]. TEM8 is selectively expressed in bility in mice [69, 70]. Both polypeptides promote angio-
tumor blood vessels; its N-terminal domain encodes an genesis in the presence of VEGF-A in vitro but in vivo they
anthrax toxin receptor (ATR) [67]. TEM8 plays a positive have opposite effects. Overexpression of Ang1 in tumor
role in angiogenesis and is over-expressed in HUVECs cells leads to increased vessel maturation and decreased
during the initiation of tube formation in vitro. The TEM8 tumor growth [71, 72]. Ang2 is antagonist of Ang1 for Tie2
mutation identified by Jinnin and colleagues is a G ? A and acts as a survival factor for ECs through PI3 K/AKT
transition that replaces alanine with threonine in the pathway. In a model of diabetic retinopathy overexpression
transmembrane domain. Introduction of this mutant TEM8 of Ang2 resulted in enhanced vascularization and impaired
into HDMECs decreased VEGFR-1 expression and pericytes recruitment [73]. As is the case for normal human
increased VEGFR-2 and ERK phosphorylation, indicating ECs, Ang2 is expressed by HemECs and appears to be
that the mutant TEM8 acts in a dominant-negative fashion. more abundantly expressed in IH tissue compared to Ang1
The VEGFR-2 mutation is a T ? C transition that replaces [21]. However, at present, the role of Tie2 and Ang2 in the
cysteine with arginine in the extracellular domain. When abnormal growth and regression of IH blood vessels
this mutant VEGFR-2 was overexpressed in HDMECs, the remains to be determined. Related studies in a murine
decrease in VEGFR-1 was limited, whereas over-expres- tumor model have shown that blocking Ang2 function with
sion of wild type VEGFR-2 in HemECs bearing the mutant soluble Tie2 receptor or down-regulating Ang2 pharma-
VEGFR-2 restored VEGFR-1 levels to normal and cologically inhibited growth of a murine endothelial tumor
decreased phosphorylated-VEGFR-2 and phosphorylated- cell line (bEnd.3 cells) [74]. The mechanism of action
ERK. These results indicate that Cys to Arg substitution in proposed involves Src-induced activation of cellular tyro-
VEGFR-2 is a loss-of-function mutation. In summary, the sine kinase signaling pathways, but there are no parallel
effects of these germline, risk-factor mutations in studies showing such a pathway is operative in IH or in
endothelial cell-based assays led to the model depicted in IH-derived cells.
Fig. 4. The mutant VEGFR-2 and TEM8 can sequester Insight into the pathogenesis of IH may also be gained
b1-integrin into a protein complex that negatively regu- from transgenic murine models. For example, sustained
lates b1-integrin activity and NFAT transcriptional AKT activation has been shown to cause vascular mal-
function, resulting in decreased expression of VEGFR-1. formations and therefore may also be involved in IH [75].
The protein complex was identified in HDMECs as well For example, sustained endothelial expression of my-
as in HemECs but it is more abundant in HemECs rAKT1 in transgenic mice results in abnormal blood ves-
because the mutant TEM8 and VEGFR-2 form the sels, with structural anomalies similar to tumor blood
complex with higher affinity resulting in NFAT activity vessels [76]. The mTOR inhibitor rapamycin inhibited the
suppression. Lower expression of the decoy VEGFR-1 in endothelial myrAKT1 and reversed the abnormal vascular
hemECs modulates the availability of VEGF-A (Fig. 4b). morphology. Since both the VEGFR-2/VEGF-A and Tie2/
VEGF-A is more available to bind to VEGFR-2 thereby Ang2 receptor/ligand systems can stimulate AKT signaling
causing a constitutive activation of VEGFR-2, elevated in endothelial cells, there may be a role for AKT in IH.
levels of phospho-ERK, and ultimately resulting in Furthermore constitutive and VEGF-A induced AKT
increased proliferation of HemEC compared to normal phosphorylation has been detected in hemECs at a higher
human endothelial cells. Interestingly, strong expression level compared with HDMECs (E. Boscolo, unpublished
of phosphorylated MAPK has been shown in benign data).
endothelial tumors such as capillary hemangioma and Hypoxia may also play a role in IH, particularly in the
pyogenic granuloma [68]. In summary, these studies early proliferating phase when only small nascent vessels
represent the most in-depth elucidation of a molecular are present within the tumor. Kleinman and colleagues
pathway in the pathogenesis of IH. This new mechanism analyzed HIF-1a expression and distribution in IH. HIF-1a
provides a strong incentive to test anti-VEGF-A agents or stabilization is increased in proliferating phase IH when
VEGFR-2 and MAP-kinase inhibitors to treat severe compared with involuting phase IH. The authors propose
cases of IH. HIF-1a induces production of VEGF-A, MMP9 and

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Angiogenesis (2009) 12:197207 205

SDF-1a, which in turn induces mobilization and homing of help with preparing the figures. Writing of this article was supported
EPC during post-natal vasculogenesis responsible for by a grant from the National Institutes of Health (P01 AR48564).
hemangioma growth [77].
Many other factors have been detected in IH and sug-
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