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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2013 Jan 3;33(3):501–509. doi: 10.1161/ATVBAHA.112.300929

Pericytes from Infantile Hemangioma Display Pro-angiogenic Properties and Dysregulated Angiopoietin-1

Elisa Boscolo 1, John B Mulliken 2, Joyce Bischoff 1
PMCID: PMC3573237  NIHMSID: NIHMS435906  PMID: 23288163

Abstract

OBJECTIVE

Infantile hemangioma (IH) is a rapidly growing vascular tumor affecting newborns. It is composed of immature endothelial cells and pericytes that proliferate into a disorganized mass of blood vessels. We isolated pericytes from IH (Hem-pericytes) to test our hypothesis that Hem-pericytes are unable to stabilize blood vessels.

METHODS AND RESULTS

We injected pericytes in vivo, in combination with endothelial cells, and found that Hem-pericytes formed more microvessels compared to control retinal pericytes. We thereby analyzed pro-angiogenic properties of the Hem-pericytes. They grew fast in vitro, and were unable to stabilize endothelial cell growth and migration, and expressed high levels of VEGF-A compared to retinal pericytes. Hem-pericytes from proliferating phase IH showed lower contractility in vitro, compared to Hem-pericytes from the involuting phase and retinal pericytes. Consistent with a diminished ability to stabilize endothelium, Angiopoietin 1 (ANGPT1) was reduced in Hem-pericytes compared to retinal pericytes. Normal retinal pericytes in which ANGPT1 was silenced produced conditioned medium that stimulated endothelial cell proliferation and migration.

CONCLUSIONS

We report the first successful isolation of patient-derived pericytes from IH tissue. Hem-pericytes exhibited pro-angiogenic properties and low levels of ANGPT1, consistent with a diminished ability to stabilize blood vessels in IH.

Keywords: infantile hemangioma, pericytes, angiogenesis, VEGF-A, endothelial colony forming cells, Angiopoietin1

Blood vessels are composed of two cell types: endothelial cells that form the inner lining of the vessel wall, and perivascular cells (a term that includes pericytes and smooth muscle cells) that wrap around the vascular channel. Pericytes surround small diameter blood vessels and are embedded within the endothelial basement membrane (1). Pericytes stabilize the vessel wall by controlling endothelial cell proliferation and survival (2), lumenal size and assembly of the basement membrane (3, 4). Pericytes also regulate microvascular blood flow via contractile activity (5).

Interactions between pericytes and endothelial cells are controlled by several molecules, such as Platelet Derived Growth Factor (PDGF) -B, Vascular Endothelial Growth Factor (VEGF) -A, and Angiopoietins (6). PDGF-B is expressed by endothelial cells and promotes pericytic proliferation and migration in a paracrine manner through PDGFR-β. PDGF-B and PDGFR-β knock-out mice die at birth, and exhibit microvascular hemorrhage and edema (7, 8) as well as endothelial cell hyperplasia, enlarged blood vessels, and increased VEGF-A (9). Increased VEGF-A signaling can dismantle pericytic coverage of vascular sprouts leading to destabilization of vessels, and suppression of PDGFRβ signaling through VEGFR-2/PDGFRβ complexes (10). Pericytes express VEGF-A during angiogenic processes, such as the developing retinal vasculature where they promote endothelial cell survival (11).

Angiopoietin-1 (ANGPT1) is another molecule involved in endothelial cell survival. It is expressed by pericytes and signals by binding to the endothelial receptor tyrosine kinase TIE2 (or TEK) (12, 13). ANGPT1 stabilizes blood vessels and has been shown to counteract VEGF-induced permeability (14). ANGPT1 and TIE2 gene knockout mice show embryonic lethality with vascular defects and absence of perivascular cells, similar to what occurs in PDGF-B deficient mice (15, 16). Deletion of ANGPT1 between embryonic day 10.5 and 12.5 produces a disorganized vascular network with increased numbers of vessels with increased diameters (17, 18).

Defective perivascular cell investment has been documented in patients with cutaneous venous malformations caused by germline (19) or somatic (20) TIE2 mutations. Diabetic retinopathy is also characterized by loss of pericytes, progressing to endothelial cell depletion and formation of acellular capillaries with increased permeability, and subsequent abnormal angiogenic rebound, leading to visual impairment (21–23). In tumor blood vessels, the close association between pericytes and endothelial cells is disrupted (24). In some tumors there is a decreased number of pericytes around feeding blood vessels, for example pericyte content in glioblastoma is about 19% the level observed in normal brain (25, 26). Excessive pericytic proliferation is a characteristic of hemangiopericytoma, a vascularized tumor in which the tumor cells are fibroblasts or pericytes (27, 28).

Infantile hemangioma (IH) is a common tumor characterized by rapid growth of disorganized blood vessels (proliferating phase) followed by a slow, spontaneous regression after 9–12 months of age (involuting phase) (29, 30). During IH growth, the densely packed proliferative blood vessels show an intense perivascular cell investment (31–34). To date, the role of pericytes in IH growth and regression has not been investigated. In previous work, we reported that stem cells (35), derived from IH patient specimens (HemSC), form IH-like lesions in mice by differentiating into both ECs and pericytes (34, 36). Inhibition of JAGGED1 signaling impaired HemSC-to-pericyte differentiation (34). Interestingly, reduced pericytic differentiation in the murine IH model is associated with decreased microvascular density. These findings suggest that pericytes in IH have a direct role in hemangiogenesis. This prompted us to study pericytes isolated directly from IH patient specimens.

Here we report for the first time the isolation of pericytes from proliferating and involuting phase IH (proliferating and involuting Hem-pericytes). Hem-pericytes cooperated with cord blood-derived endothelial colony forming cells (ECFC) to form blood vessels at high density when both cell types were implanted into immune-deficient mice. The Hem-pericytes exhibited pro-angiogenic properties in vitro:proliferated rapidly, failed to stabilize ECFC proliferation and migration, secreted high levels of the angiogenic factor VEGF-A, and expressed reduced levels of the TIE2 ligand ANGPT1. In addition, proliferating Hem-pericytes exhibited low contractile ability. Our results suggest that Hem-pericytes contribute to defective maturation of blood vessels in IH.

METHODS

Cell Isolation and Culture

Specimens of IH (n=8) were obtained under a human subject protocol approved by the Committee on Clinical Investigation, Boston Children’s Hospital. The clinical diagnosis was confirmed in the Department of Pathology, Boston Children’s Hospital. Informed consent was obtained for the specimens, according to the Declaration of Helsinki. Single cell suspensions were prepared from the proliferating and involuting phase IH specimens by digesting with collagenase (Roche, Indianapolis, IN) (37). Cells were seeded on non-coated tissue culture dishes in DMEM/10% fetal bovine serum (FBS). Cells were analyzed for pericyte/smooth muscle markers, and subsequently designated Hem-pericytes. Human umbilical cord endothelial colony forming cells (ECFC) were isolated as described (38) (39). Human retinal and placental pericytes were purchased from Cell Systems (Kirkland, WA) and PromoCell (Heidelberg, Germany) respectively, and cultured in the same conditions as Hem-pericytes.

For detailed methods, see the supplemental material, available online at http://atvb.ahajournals.org.

RESULTS

Isolation and characterization of proliferating and involuting phase IH-derived pericytes (proliferating and involuting Hem-pericytes)

Blood vessels in both proliferating and involuting phase of IH are encircled by a continuous layer of perivascular cells expressing !-smooth muscle actin (αSMA) (32–34). We previously showed that the majority of the proliferating IH perivascular cells express neural glial antigen-2 (NG2), Platelet Derived Growth Factor Receptor- (PDGFR)-β, Calponin and !SMA (34) (we provide additional images of perivascular immunostaining in Suppl. Fig. 1A). In proliferating IH tissue, some of the ECs (arrows) and αSMA+ perivascular cells (arrowheads) stained positively for Ki67 (Fig. 1A). In contrast, in the involuting phase IH tissue, Ki67+ cells were less abundant. We isolated pericytes from 4 proliferating and 4 involuting IH specimens (Table 1). The cells isolated from proliferating and involuting phase specimens showed a pericyte-like elongated morphology (Fig. 1B). The cellular phenotypes were assessed by real time PCR and immunostaining for pericyte/smooth muscle markers (Fig. 1C and 1D). Commercially available human placental and retinal pericytes served as positive controls. Cells from proliferating and involuting IH expressed PDGFRβ, NG2, Desmin, Calponin, sm22α, αSMA, smooth muscle Myosin Heavy Chain (smMHC) and CD90, but not the endothelial marker CD31. Expression levels of Calponin, αSMA and smMHC were higher in involuting phase pericytes. Blood vessels in proliferating and involuting IH tissue sections showed colocalized expression of αSMA with Calponin and of αSMA with PDGFRβ in the perivascular area (Suppl. Fig. 1B). When cultured, most of the Hem-pericytes also showed colocalization of these markers (Suppl. Fig. 1C). Based on the expression of pericytic markers, and the similarities to placental and retinal pericytes, we designated the isolated IH perivascular cells as Hem-pericytes. The Hem-pericytes used in this study are from culture passages 3 to 8. Hem-pericytes from passage 3, 6, 8 and 10 showed a consistent expression profile of pericytic markers (Suppl. Fig. 1D).

Figure 1. Hemangioma pericyte (Hem-pericyte) characterization.

Figure 1

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A- Proliferating and nvoluting IH tissue stained for Ki67 (green) and αSMA (red). Arrows indicate Ki67+ endothelial cells, arrowheads Ki67+ pericytes. Quantification of Ki67+ and Ki67+/αSMA+ cells in proliferating and involuting IH tissue. Data expressed as mean± SDM.

B- In vitro morphology of proliferating and involuting Hem-pericytes.

C- Real-time PCR mRNA expression levels for PDGFRβ, NG2, Desmin, Calponin, sm22α, αSMA, smMHC, CD90 and NOTCH3, in Hem-pericytes isolated from 4 different proliferating phase IH (146, 147, 154, 156) and 2 involuting phase IH (I-69, I-79). Human placental and retinal pericytes served as positive controls (n= triplicate for each cell type). Data expressed as mean± SDM.

D- Pericytes from proliferating, involuting IH, human placenta and retina, stained for PDGFRβ, NG2, Desmin, Calponin, sm22α, αSMA, smooth muscle Myosin Heavy Chain (smMHC), CD90 and endothelial marker CD31. Scale bar 100μM.

Table 1.

Clinical information for IH tumors used to isolate pericytes. Numbers indicate de-identified specimens.

Proliferating Age (months) Sex
146 3 F
147 3 M
154 4 F
156 6 F
Involuting Age (years) Sex
I-69 1 year F
I-79 1 year 7 months F
I-81 3 years F
I-82 7 years F
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Hem-pericytes form blood vessels in vivo when combined with ECFCs

We assessed whether proliferating Hem-pericytes are pro-angiogenic compared to nvoluting Hem -pericytes and retinal pericytes. Hem-pericytes were combined with cord blood ECFC in Matrigel and injected into immune-deficient nude mice (Fig. 2A). ECFC are healthy human endothelial cells with high self-renewal capacity; they were previously called endothelial progenitor cells or EPCs (38, 39). At day 7, cell/Matrigel implants were removed and vascularity was assessed qualitatively from the red appearance of the explanted Matrigel and quantitatively by counting erythrocyte-filled lumens (total microvascular density (MVD)) and human CD31+ blood vessels (human CD31-positive MVD) (Fig. 2B). Proliferating Hem-pericytes, combined with ECFC produced a significantly higher (p<0.02) number of blood vessels (Fig. 2C) and human CD31+ vessels (Fig. 2D), compared to involuting Hem-pericytes and retinal pericytes. Hem-pericytes from both proliferating and involuting IH, exhibited significantly higher (p<0.05) MVD compared to control retinal pericytes.

Figure 2. Hem-pericytes combined with ECFCs form blood vessels in Matrigel.

Figure 2

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A- Schematic of in vivo subcutaneous injection.

B- Matrigel explants at day 7: ECFC combined with proliferating (154, 146), involuting (I-79, I-69) Hem-pericytes and retinal pericytes (top row). Representative histological sections from Matrigel explants stained with hematoxylin and eosin (middle row) and anti-human specific CD31 (bottom row). Scale bar 100μM.

C- Blood vessels containing erythrocytes, indicating microvessels, were counted in hematoxylin and eosin sections. Vessels/mm2 in Hem-pericyte 154 explants set to 100%. (n=8 mice per group, 2 independent experiments, analysis carried out on 10 fields for each Matrigel explant). Data expressed as mean ± SEM. *p<0.02 compared to proliferating Hem-pericytes 154 and 146, ** p<0.05 compared to proliferating and involuting Hem-pericytes 146, 154, I-69 and I-79.

D- Human CD31 stained vessels in sections were counted (n=8 mice per group, 2 independent experiments, analysis carried out on 10 fields for each Matrigel explant). Data expressed as mean ± SEM. *p<0.02 compared to proliferating Hem-pericytes 154 and 146.

E- Representative images of explant sections stained for dividing cell marker Ki67 (green), αSMA (red), and DAPI (blue). Scale bar 100μM. Quantification of Ki67+ and Ki67+/αSMA+ cells in proliferating and involuting IH tissue. Data expressed as mean ± SEM. *p<0.05 compared to proliferating Hem-pericytes 154 and 146, ** p<0.05 compared to proliferating and involuting Hem-pericytes 146, 154, I-69 and I-79.

To assess cellular proliferation, we immunostained for Ki67, a marker of dividing cells (Fig. 2E). Blood vessels formed by proliferating Hem-pericyte in combination with ECFC showed a significantly (p<0.05) higher number of Ki67+ cells in the endothelial and αSMA+ pericytic compartments compared to involuting Hem-pericyte with ECFC. This is consistent with Ki67 expression in proliferating and involuting phase IH tissue sections (Fig. 1A). Injection of Hem-pericytes alone resulted in minor ingrowth of murine (human CD31-) blood vessels into the Matrigel implant (Suppl. Fig. 2).

Hem-pericytes do not stabilize endothelial cells

We analyzed proliferation of Hem-pericytes to determine if proliferative Ki-67+ phenotype was retained in vitro. Hem-pericytes showed a significantly (p<0.05) higher proliferation rate, compared to retinal pericytes, at 48, 72 and 96 hours (Fig. 3A); furthermore, 3 out of 4 proliferating Hem-pericytes grew faster than involuting Hem-pericytes, at 72 and 96 hours.

Figure 3. Proangiogenic properties of Hem-pericytes in vitro.

Figure 3

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A- Cell proliferation evaluated at 24, 48, 72 and 96 hours for proliferating (154, 146, 147, 156), involuting (I-69, I-79, I-81, I-82) Hem-pericytes and retinal pericytes. Cell count at 24 hours after seeding was set to 100% to normalize for differences in initial adherence to the well. Data expressed as mean ± SDM.

B- Endothelial Colony Forming Cells (ECFC) proliferation analyzed after 48 hours of indirect contact with pericytes or medium alone (DMEM/10%FBS=no cells): schematic of experiment (top), quantification of ECFC proliferation (bottom). Data expressed as mean ± SEM. *p<0.05 compared to retinal pericytes, **p<0.05 compared to retinal pericytes and to control, no cells.

C- ECFC migration towards pericyte-conditioned medium or medium alone (bottom chamber) was analyzed after 4 hours: schematic of experiment (top), quantification of ECFC migration (bottom). Data expressed as mean ± SEM. *p<0.05 compared to retinal pericytes.

D- Scratch migration assay with ECFC migrating across the scraped region after 6-hour exposure to pericyte-conditioned medium. Cells fixed and nuclei stained with DAPI.

E- VEGF-A protein levels in conditioned medium of Hem-pericytes and retinal pericytes analyzed by enzyme-linked immunoadsorbant assay (ELISA) (gray bars), and VEGF-A mRNA levels in cell lysates analyzed by real-Time PCR (black bars). *p<0.05 compared to retinal pericytes.

Pericytes normally stabilize blood vessels and induce endothelial quiescence, a process referred as to vascular maturation (40). We hypothesized that Hem-pericytes cannot induce vascular maturation, but instead allow EC proliferation and migration. To test this hypothesis, we cultured pericytes at the bottom and ECFC at the top of a Transwell system. After 48 hours ECFC were counted and normalized for the number of pericytes, to correct for differences in growth rates. Proliferating Hem-pericytes but not involuting Hem-pericytes exerted a proliferative effect on ECFC when compared to medium alone (no cells) (Fig. 3B and Suppl. Fig. 3A). In contrast, retinal pericytes inhibited ECFC growth (p<0.05) compared to medium alone (no cells) and to Hem-pericytes. Migration of ECFC toward pericyte-conditioned medium was assessed in a Transwell system (Fig. 3C and Suppl. Fig. 3B) and using the scratch migration assay (Fig. 3D). In the Transwell system, ECFC migration toward Hem-pericyte-conditioned medium was no different from the control medium, but retinal pericyte-conditioned medium elicited significantly (p<0.05) lower ECFC migration (Fig. 3C). In the scratch assay, ECFC were seeded in endothelial medium and pericyte-conditioned medium was added after the scratch was made. After 6 hours, results were similar to the Transwell assay: the migration rate of ECFC in response to proliferating and involuting Hem-pericytes conditioned medium was higher than for retinal pericytes (Fig. 3D).

Because of its central role in vascular development, we measured VEGF-A expressed and secreted by Hem-pericytes. Released protein and mRNA VEGF-A levels were higher in Hem-pericytes compared to the control retinal pericytes (Fig. 3E).

Proliferating Hem-pericytes exhibit reduced contractility

Pericytes are contractile in vivo and in vitro. To assess contractile activity in Hem-pericytes, we first stained cells with phalloidin to visualize the actin cytoskeleton. The F-actin fiber density was lower in proliferating Hem-pericytes compared to involuting Hem-pericytes and retinal pericytes (Fig. 4A). We further tested Hem-pericytes for contractility activity by plating cells on a deformable silicon-based substratum coated with type I collagen for 12 hours. Contractile cells pull on the substratum, causing wrinkles. The numbers of cells with wrinkles over the total number of cells attached to the silicon substrata were counted. Representative images (Fig. 4B) and quantification (Fig. 4C and Suppl. Fig. 4A) showed that about 81% of the retinal pericytes formed wrinkles, compared to 62% of involuting Hem-pericytes and 23% of the proliferating Hem-pericytes. The significantly (p<0.05) reduced contractile phenotype of proliferating compared to involuting Hem-pericytes was consistent with the reduced F-actin fibers in proliferating Hem-pericytes.

Figure 4. Hem-pericytes exhibit low contractility.

Figure 4

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A- Representative images of proliferating, involuting Hem- and retinal pericytes stained with phalloidin to evaluate F-actin filaments. Scale bare =100μM.

B- Representative images of Hem-pericytes and retinal pericytes in silicone -based contractility assay. Arrow points to an example of a cell that has contracted and produced wrinkles in silicone substratum.

C- Quantification of wrinkle-forming cells out of total (n=12 wells per each cell type, 5 fields per well). *p<0.05 compared to involuting Hem-pericytes and retinal pericytes. Data expressed as mean ± SDM.

ANGPT1 is downregulated in Hem-pericytes

To further assess the pro-angiogenic factors produced by Hem-pericytes, pericyte-conditioned medium was screened for 43 different factors using an angiogenic protein array (Suppl. Fig. 5A). Angiopoietin-1 (ANGPT1) was not detected in Hem-pericytes, whereas retinal pericytes produced a strong signal. We screened Hem-pericytes isolated from different IH tumors for ANGPT1 mRNA and protein expression (Fig. 5A and 5B, Suppl. Fig. 5B and 5C). Both analyses showed significant (p<0.05) downregulation (mRNA levels: 79% in involuting, 96% in proliferating, protein levels: 70% in involuting, 83% in proliferating) compared to retinal pericyte ANGPT1 levels. Of note, involuting Hem-pericytes showed significantly higher (p<0.05) ANGPT1 mRNA levels compared to proliferating Hem-pericytes, suggesting involuting Hem-pericytes may tend to reach a normal pericyte phenotype. To confirm the relevance of these results, we examined the phosphorylation status of ANGPT1 receptor, TIE2, in IH patient tissue sections by immunohistochemistry. PhosphoTIE2 was detected along the endothelium in involuting phase IH, and, consistent with low ANGPT1 levels, was almost undetectable on the nascent vessels in proliferating phase IH (Fig. 5C).

Figure 5. ANGPT1 expression is downregulated in Hem-pericytes.

Figure 5

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A- Real Time PCR analysis of ANGPT1 expression in proliferating and involuting Hem-pericytes and retinal pericytes.

B- ELISA performed on the conditioned medium (48 hours) from proliferating and involuting Hem-pericytes and retinal pericytes. Data expressed as mean ± SDM. **p<0.05 compared to involuting Hem-pericytes and retinal pericytes, *p<0.05 compared to retinal pericytes.

C Proliferating and involuting IH tissue stained for ANGPT1 activated- phosphoTIE2 (green), αSMA (red) and DAPI (blue). Scale bar 100μM.

D- Real-time PCR mRNA expression levels for ANGPT1 in retinal pericytes, untreated (control), or treated with short interference (si)-RNA with a non targeting (si Non Target) sequence pool or ANGPT1 targeting sequence pool (si ANGPT1). Data expressed as mean ± SDM.

E- ECFC proliferation analyzed after 48 hours of indirect contact with retinal pericytes (control, si Non Target, si ANGPT1). Values were normalized for retinal pericyte counts. Data expressed as mean ± SEM. *p<0.05.

F- ECFC migration analyzed after 4 hours of contact with retinal pericyte (control, si Non Target, si ANGPT1)-conditioned medium. Values were normalized for retinal pericyte counts. Data expressed as mean ± SEM. *p<0.05.

To test the role of ANGPT1 in the pericytes, we downregulated ANGPT1 mRNA in retinal pericytes using siRNA (Fig. 5D). Proliferation and migration of ECFC, in indirect contact with retinal pericytes or with retinal pericyte-conditioned medium, respectively, were measured as described in Fig 3B. Retinal pericytes with downregulated ANGPT1 promoted a significant increase (p<0.05) in ECFC proliferation and migration (Fig. 5E and 5F). Additionally, ANGPT1 silencing in retinal pericytes did not affect VEGF-A mRNA levels (Suppl. Fig. 5D). These results suggest that the low levels of ANGPT1 expression in Hem-pericytes can contribute to their pro-angiogenic phenotype.

DISCUSSION

We report here the first isolation of pericytes from IH specimens. Hem-pericytes, combined with ECFC, formed highly vascularized lesions in immune-deficient mice. When co-cultured, Hem-pericytes failed to stabilize the proliferation and migration of ECFC, in contrast to retinal pericytes. Proliferating Hem-pericytes showed reduced contractility compared to involuting Hem-pericytes, and retinal pericytes, as well as a reduced Phalloidin-stained actin cytoskeleton. Furthermore we found low ANGPT1 expression in Hem-pericytes, an important molecule for stabilization of blood vessels. Taken together, these results indicate that Hem-pericytes have a diminished capacity to elicit endothelial quiescence and stabilize blood vessels, a key function of pericytes in physiological angiogenesis.

IH has traditionally been considered a tumor of endothelial cells, a view supported by the abnormal properties displayed by IH-derived ECs (HemECs) (41, 42). We previously identified a stem cell (HemSC) in IH patient specimens that can form hemangioma-like blood vessels and recapitulate the IH life cycle (proliferation and involution) when injected into immune-deficient mice (35). HemSCs form functional blood vessels by differentiation into both ECs and pericytes (34, 36). When HemSC-to-pericyte differentiation was prevented, vessels did not form (34). This finding suggested that pericytes have an essential role in the formation of IH-blood vessels.

Identification of pericytes in tumors and in normal development cannot rely on a single pericytic marker; instead, a combination of markers and perivascular location are needed. Commonly used markers for tumor pericytes include αSMA (43, 44), NG2 (45), PBGFRβ (44, 46), desmin (44). In our study we extended the characterization to a total of 8 pericyte/smooth muscle cell markers in order to elucidate the specific phenotype of the pericytes in IH. Notably, after isolation and expansion in vitro, proliferating and involuting Hem-pericytes continue to express the signature markers detected in intact IH patient tumor specimens.

In physiological retinal angiogenesis, ECs sprout and form lumens, followed by recruitment of pericytes to nascent vessels by an Ang1-TIE2 dependent mechanism (47, 48). Additional in vitro studies have shown that ECs generate, in collagen matrix, vascular guidance channels that act as conduits for the subsequent recruitment of pericytes (3). In contrast, pericytes have been reported to play a lead role in an ovarian model of angiogenesis (49) where they precede the vascular sprouting tips. Our previous studies on IH led us to propose that some HemSCs first differentiate into ECs through VEGF-A signaling (36, 50), whereas remaining HemSCs differentiate into pericytes through direct contact with JAGGED1 expressed on the newly formed ECs (34).

VEGF-A is highly expressed in HemSC (36, 50). Here we show that Hem-pericytes express increased levels of VEGF-A, compared to retinal pericytes. During retinal angiogenesis, VEGF-A is secreted from the pericytes at a basal level to contribute to EC survival. Upregulation of pericytic VEGF-A has been reported when there is contact with ECs (11). In contrast, VEGF-A has been shown to be a negative regulator of pericytic function through activation of the PDGFRβ/VEGFR-2 complex (10). Here we show that Hem-pericytes constitutively express VEGF-A, which could account for the lack of ECFC stabilization in our in vitro models of proliferation and migration.

The ANGPT/TIE2 system plays an essential role in vascular development and maintenance of normal adult vasculature (51–53). Disruption of the ANGPT/TIE2 system can lead to excessive or disrupted angiogenesis. Furthermore, a mutation that activates the TIE2 tyrosine kinase has been implicated in inherited muco-cutaneous venous malformations and sporadic venous malformations (19, 20) where enlarged blood vessels exhibit defective investment with perivascular cells. ANGPT2 expression is upregulated during tumor angiogenesis (54, 55). In contrast, ANGPT1 causes reduced tumor growth and increased pericyte coverage in the vasculature in squamous cell carcinoma and hepatic colon cancer (56, 57).

IH-derived EC (HemEC) express elevated levels of TIE2 and ANGPT2 (58, 59), which is consistent with the endothelial proliferation in IH. As shown here, Hem-pericytes are highly proliferative cells, and express reduced levels of ANGPT1. Low levels of ANGPT1 and high levels of ANGPT2 are reported as a perfect cocktail to induce uncontrolled pathological angiogenesis (60), and could explain the rapid formation of blood vessels in IH. In a recent study, deletion of ANGPT1 between embryonic day 10.5 and 12.5 caused an array of vascular defects including increased vessel number and diameter in several organs. Deletion of ANGPT1after embryonic day 13.5 resulted in viable and normal appearing mice, but in models of vascular injury, excessive angiogenesis and fibrosis (17, 18). These findings suggest that low ANGPT1 levels in IH tissue contribute to the highly angiogenic phenotype of the IH lesions.

IH lesions are characterized as fast-flow; this may be due, in part, to poor contractility of the proliferating Hem-pericytes that we report here. Consistent with this hypothesis, involuting Hem-pericytes showed improved contractile ability, comparable to retinal pericytes (Fig 4). Propranolol is a relatively new pharmacologic treatment for IH (61). Propranolol is a well-known non-selective β-blocker; however its mechanism of action in IH is still unclear. The rapid decrease in redness and size in response to propranolol has prompted the theory that propranolol induces vasoconstriction of vessels within or feeding IH (61, 62). We speculate that Hem-pericytes are an important target cell of propranolol therapy in IH.

In summary our study is the first to show that pericytes orchestrate important functions in the life cycle of IH. Hem-pericytes exhibit pro-angiogenic properties: fast proliferation, inability to stabilize ECFC proliferation and migration, and low ANGPT1 levels– properties consistent with reduced ability to affect maturation of blood vessels. We propose that normalization of ANGPT1 levels and pericytic contractility, in combination with corticosteroid- induced down regulation of VEGF-A, would promote vascular maturation and accelerate onset of the involuting phase.

Supplementary Material

Acknowledgments

This work was supported by a NIH grant HL096384-01 (J.B), the Charles Hood Foundation (E.B.). We thank Drs. Jennifer Durham and Ira Herman for their expert advice and technical assistance with the contractility assay, Harry Kozakewich, David Smadja and Lan Huang for helpful discussions, the Dana-Farber/Harvard Cancer Center (DF-HCC) for Specialized Histopathology (HSP) Core, the Cytogenetics Core of Dana Farber Harvard Cancer Center (P30 CA006516), and Kristin Johnson for the preparation of figures.

Non-standard abbreviations

IH

infantile hemangioma

Hem-pericyte

hemangioma-derived pericytes

ECFCs

cord blood-derived endothelial colony forming cells

MVD

micro vessel density

vWF

von Willebrand factor

Footnotes

AUTHOR CONTRIBUTIONS

E.B. and J.B. designed the research. E.B. performed the experiments. J.B., J.B.M. assisted with data analysis and review of the manuscript. E.B. wrote the manuscript.

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