Infantile hemangioma (IH), a benign vascular tumor, is the most common tumor of infancy, with an incidence of 5%-10% at the end of the first year. The tumor displays a distinctive life cycle consisting of a proliferating phase, occurring in the first months of life, followed by an involuting phase. Thus, IH represents a unique model of postnatal vasculogenesis, angiogenesis, and vessel regression. Traditionally, corticosteroids were the drug of choice when treatment of IH was indicated. In recent years, beta-blockers, most specifically propranolol, have serendipitously been shown to be an effective pharmacological treatment. This article will focus on the mechanism of action of these two drugs, the old and the new treatments, in slowing the growth and accelerating involution of IH.

The field of tissue engineering seeks to create metabolically demanding, functional tissues, which will require blood vessel networks capable of forming rapidly in a variety of extracellular matrix (ECM) environments. We tested whether human endothelial progenitor cells (EPCs) and mesenchymal progenitor cells (MPCs) could form microvascular networks in type I collagen, fibrin and an engineered peptide hydrogel, PuraMatrix, in 7 days in vivo in immune-deficient mice. These results are compared to those previously published, based on the Matrigel ECM. Perfused blood vessels formed in all three types of ECM within 7 days. Collagen at 5 and 6 mg/ml and 10 mg/ml fibrin supported vessel formation at 30-60 vessels/mm(2), and PuraMatrix enabled vessel formation to 160 vessels/mm(2), significantly greater than collagen or fibrin. Vessels were composed of EPCs with perivascular cells on their abluminal surfaces. EPCs injected alone formed a low density of blood vessels in collagen and PuraMatrix, while MPCs injected alone resulted in sparse vessel networks in all ECMs tested. A rheometer was used to determine whether the ECMs which supported vascularization had bulk physical properties similar to or distinct from Matrigel. Collagen and fibrin were the stiffest matrices to support extensive vascularization, with storage moduli in the range 385-510 Pa, while Matrigel, at 80 Pa, and PuraMatrix, at 5 Pa, were far more compliant. Thus, EPCs and MPCs were capable of vasculogenesis in environments having disparate physical properties, although vascular density was greater in more compliant ECMs. We propose that EPC/MPC-mediated vascularization is a versatile technology which may enable the development of engineered organs.

OBJECTIVE: Cardiac valvular endothelium is unique in its ability to undergo endothelial-to-mesenchymal transformation, a differentiation process that is essential for valve development and has been proposed as mechanism for replenishing the interstitial cells of mature valves. We hypothesized that the valvular endothelium contains endothelial cells that are direct precursors to osteoblastic valvular interstitial cells (VICs). METHODS AND RESULTS: Clonal cell populations from ovine mitral valve leaflets were isolated by single cell plating. Mitral valvular endothelial and mesenchymal clones were tested for osteogenic, adipogenic, and chondrogenic differentiation, determined by the expression of lineage-specific markers. Mitral valvular endothelial clones showed a propensity for osteogenic, as well as chondrogenic differentiation that was comparable to a mitral valvular VIC clone and to bone marrow-derived mesenchymal stem cells. Osteogenic differentiation was not detected in nonvalvular endothelial cells. Regions of osteocalcin expression, a marker of osteoblastic differentiation, were detected along the endothelium of mitral valves that had been subjected in vivo to mechanical stretch. CONCLUSIONS: Mitral valve leaflets contain endothelial cells with multilineage mesenchymal differentiation potential, including osteogenic differentiation. This unique feature suggests that postnatal mitral valvular endothelium harbors a reserve of progenitor cells that can contribute to osteogenic and chondrogenic VICs.

The ability to form anastomoses with the host circulation is essential for vascular networks incorporated within cell-seeded bioengineered tissues. Here, we tested whether and how rapidly human endothelial colony forming cell (ECFC)/mesenchymal progenitor cell (MPC)-derived bioengineered vessels, originally perfused in one mouse, could become reperfused in a secondary mouse. Using in vivo labeling with a systemically injected mixture of human- and murine-specific lectins, we demonstrate that ECFC/MPC blood vessels reconnect and are perfused at day 3 after transplantation. Furthermore, we quantified the longitudinal change in perfusion volume in the same implants before and after transplantation using contrast-enhanced micro-ultrasonic imaging. Perfusion was restored at day 3 after transplantation and increased with time, suggesting an important new feature of ECFC/MPC blood vessels: the bioengineered vessels can reconnect with the vasculature when transplanted to a new site. This feature extends the potential applications of this postnatal progenitor cell-based technology for transplantable large tissue-engineered constructs.

OBJECTIVE: The aim of our study is to determine the cellular and molecular origin for the pericytes in infantile hemangioma (IH) and their functional role in the formation of pathological blood vessels. METHODS AND RESULTS: Here we show that IH-derived stem cells (HemSCs) form pericyte-like cells. With in vitro studies, we demonstrate that HemSC-to-pericyte differentiation depends on direct contact with endothelial cells. JAGGED1 expressed ectopically in fibroblasts was sufficient to induce HemSCs to acquire a pericyte-like phenotype, indicating a critical role for JAGGED1. In vivo, we blocked pericyte differentiation with recombinant JAGGED1, and we observed reduced formation of blood vessels, with an evident lack of pericytes. Silencing JAGGED1 in the endothelial cells reduced blood vessel formation and resulted in a paucity of pericytes. CONCLUSIONS: Our data show that endothelial JAGGED1 controls HemSC-to-pericyte differentiation in a murine model of IH and suggests that pericytes have a fundamental role in formation of blood vessels in IH.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a member of the VEGFR family, and binds to VEGF-A, VEGF-B, and placental growth factor. VEGFR-1 contributes to tumor growth and metastasis, but its role in the pathological formation of blood vessels is still poorly understood. Herein, we used infantile hemangioma (IH), the most common tumor of infancy, as a means to study VEGFR-1 activation in pathological vasculogenesis. IH arises from stem cells (HemSCs) that can form the three most prominent cell types in the tumor: endothelial cells, pericytes, and adipocytes. HemSCs can recapitulate the IH life cycle when injected in immuncompromised mice, and are targeted by corticosteroids, the traditional treatment for IH. We report here that VEGF-A or VEGF-B induces VEGFR-1-mediated ERK1/2 phosphorylation in HemSCs and promotes differentiation of HemSCs to endothelial cells. Studies of VEGFR-2 phosphorylation status and down-regulation of neuropilin-1 in the HemSCs demonstrate that VEGFR-2 and NRP1 are not needed for VEGF-A- or VEGF-B-induced ERK1/2 activation. U0216-mediated blockade of ERK1/2 phosphorylation or shRNA-mediated suppression of VEGFR-1 prevents HemSC-to-EC differentiation. Furthermore, the down-regulation of VEGFR-1 in the HemSCs results in decreased formation of blood vessels in vivo and reduced ERK1/2 activation. Thus, our study reveals a critical role for VEGFR-1 in the HemSC-to-EC differentiation that underpins pathological vasculogenesis in IH.

UNLABELLED: ACKGROUND:: Arteriovenous malformations cause significant morbidity, primarily because they expand over time and recur after treatment. The authors hypothesized that neovascularization might contribute to arteriovenous malformation progression. METHODS: Arteriovenous malformation tissue was collected prospectively from 12 patients after resection. Schobinger stage was determined by clinical history. Neovascularization in stage II lesions (n=7) was compared with stage III arteriovenous malformations (n=5) that had progressed. Specimens were analyzed using immunohistochemistry for CD31, Ki67, and CD34/CD133. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to determine mRNA expression of factors that recruit endothelial progenitor cells: vascular endothelial growth factor (VEGF), stromal cell-derived factor-1α (SDF-1α), and hypoxia-inducible factor-1α (HIF-1α). VEGF receptors (VEGFR1, VEGFR2, neuropilin 1, and neuropilin 2) also were quantified using quantitative real-time reverse-transcriptase polymerase chain reaction. RESULTS: Stage III arteriovenous malformations showed greater microvessel density (5.8 percent) than stage II lesions (1.3 percent) (p=0.004); no difference in proliferating endothelial cells was noted (p=0.67). CD133CD34 endothelial progenitor cells were elevated in stage III (0.53 percent) compared with stage II arteriovenous malformations (0.25 percent) (p=0.03). HIF-1α and SDF-1α were increased 7.6- and 7.9-fold in stage III compared with stage II lesions (1.7-fold and 3.3-fold), respectively (p=0.02). Neuropilin 1 and neuropilin 2 expression was greater in stage III (5.8-fold and 4.6-fold) than stage II arteriovenous malformations (3.0-fold and 2.4-fold) (p=0.03). CONCLUSIONS: Higher-staged arteriovenous malformations exhibit increased expression of endothelial progenitor cells and factors that stimulate their recruitment. Neovascularization by vasculogenesis may be involved in progression of arteriovenous malformation.

Infantile hemangioma (IH) is a common childhood vascular tumor. Although benign, some hemangiomas cause deformation and destruction of features or endanger life. The current treatments, corticosteroid or propranolol, are administered for several months and can have adverse effects on the infant. We designed a high-throughput screen to identify the Food and Drug Administration-approved drugs that could be used to treat this tumor. Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, was identified, based on its ability to inhibit proliferation of a hemangioma-derived stem cell population, human vasculogenic cells, which we had previously discovered. In vitro and in vivo studies show that Rapamycin reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells in vivo. Longitudinal in vivo imaging of blood flow through vessels formed with hemangioma stem cells shows that Rapamycin also leads to regression of hemangioma blood vessels, consistent with its known anti-angiogenic activity. Finally, we demonstrate that Rapamycin-induced loss of stemness can work in concert with corticosteroid, the current standard therapy for problematic hemangioma, to block hemangioma formation in vivo. Our studies reveal that Rapamycin targets the self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells, and suggests a novel therapeutic strategy to prevent formation of this disfiguring and endangering childhood tumor.

The endothelium covering the aortic, pulmonary, mitral, and tricuspid valves looks much like the endothelium throughout the vasculature, in terms of general morphology and expression of many endothelial markers. Closer examination, however, reveals important differences and hints of a unique phenotype that reflects the valvular endothelium's embryonic history, and potentially, its ability to maintain integrity and function over a life span of dynamic mechanical stress. A well-studied property that sets the cardiac valvular endothelium apart is the ability to transition from an endothelial to a mesenchymal phenotype-an event known as epithelial to mesenchymal transition (EMT). EMT is a critical step during embryonic valvulogenesis, it can occur in post-natal valves and has recently been implicated in the adaptive response of mitral valve leaflets exposed to a controlled in vivo setting designed to mimic the leaflet tethering that occurs in ischemic mitral regurgitation. In this review, we will discuss what is known about valvular endothelial cells, with a particular focus on post-natal, adult valves. We will put forth the idea that at subset of valvular endothelial cells are progenitor cells, which may serve to replenish valvular cells during normal cellular turnover and in response to injury and disease.

BACKGROUND: Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. MATERIALS AND METHODS: HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. RESULTS: HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. CONCLUSIONS: HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.