Infantile hemangioma is a vascular tumor that occurs in 5-10% of infants of European descent. A defining feature of infantile hemangioma is the dramatic growth and development into a disorganized mass of blood vessels. Subsequently, a slow spontaneous involution begins around 1 year of age and continues for 4-6 years. The growth and involution of infantile hemangioma is very different from other vascular tumors and vascular malformations, which do not regress and can occur at any time during childhood or adult life. Much has been learned from careful study of the tissue morphology and gene expression patterns during the life-cycle of hemangioma. Tissue explants and tumor-derived cell populations have provided further insight to unravel the cellular and molecular basis of infantile hemangioma. A multipotent progenitor cell capable of de novo blood vessel formation has been isolated from infantile hemangioma, which suggests that this common tumor of infancy, long considered to be a model for pathologic angiogenesis, may also represent pathologic vasculogenesis. Whether viewed as angiogenesis or vasculogenesis, infantile hemangioma represents a vascular perturbation during a critical period of post-natal growth, and as such provides a unique opportunity to decipher mechanisms of human vascular development.

Infantile hemangioma is a vascular tumor that occurs in 5% to 10% of infants of European descent. A hallmark of infantile hemangioma is its life cycle, which is divided into 3 stages. The proliferating phase spans in the first year of postnatal life and is characterized by cellular masses without a defined vascular architecture and nascent blood vessels with red blood cells evident within the lumenal space. The involuting phase begins at 1 year of age and continues for 3 to 5 years. Proliferation slows or stops in this phase, and histologic examination shows that the blood vessel architecture becomes more obvious and vessel size is enlarged. The involuted phase reaches 5 to 8 years of age, at which point blood vessels are replaced with a fibrofatty residuum and capillary-sized channels. The growth and involution life cycle of infantile hemangioma are very different from other vascular tumors and vascular malformations, which do not regress and can occur at any time during childhood or adult life. Many laboratories have reported on the endothelial characteristics of the cellular masses that are prominent in the proliferating phase of infantile hemangioma, as well as their immature appearance. These studies, along with isolation and characterization of hemangioma-derived cell populations with progenitor cell properties, have lead to an emerging hypothesis that hemangioma is caused by an abnormal or delayed differentiation of mesodermal progenitor cells into the disorganized mass of blood vessels. In this paper, we discuss the literature that supports this emerging hypothesis.

Infantile hemangiomas are localized and rapidly growing regions of disorganized angiogenesis. We show that expression of vascular endothelial growth factor receptor-1 (VEGFR1) in hemangioma endothelial cells (hemECs) and hemangioma tissue is markedly reduced compared to controls. Low VEGFR1 expression in hemECs results in VEGF-dependent activation of VEGFR2 and downstream signaling pathways. In hemECs, transcription of the gene encoding VEGFR1 (FLT1) is dependent on nuclear factor of activated T cells (NFAT). Low VEGFR1 expression in hemECs is caused by reduced activity of a pathway involving beta1 integrin, the integrin-like receptor tumor endothelial marker-8 (TEM8), VEGFR2 and NFAT. In a subset of individuals with hemangioma, we found missense mutations in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations result in increased interactions among VEGFR2, TEM8 and beta1 integrin proteins and in inhibition of integrin activity. Normalization of the constitutive VEGFR2 signaling in hemECs with soluble VEGFR1 or antibodies that neutralize VEGF or stimulate beta1 integrin suggests that local administration of these or similar agents may be effective in hemangioma treatment.

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-beta (TGF-beta). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-beta-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-beta. A positive role for Notch1 was revealed in three experiments: (1) TGF-beta induced Notch1 mRNA in valve ECs, (2) a gamma-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.

Solid tumors require new blood vessels for growth and metastasis, yet the biology of tumor-specific endothelial cells is poorly understood. We have isolated tumor endothelial cells from mice that spontaneously develop prostate tumors. Clonal populations of tumor endothelial cells expressed hematopoietic and mesenchymal stem cell markers and differentiated to form cartilage- and bone-like tissues. Chondrogenic differentiation was accompanied by an upregulation of cartilage-specific col2a1 and sox9, whereas osteocalcin and the metastasis marker osteopontin were upregulated during osteogenic differentiation. In human and mouse prostate tumors, ectopic vascular calcification was predominately luminal and colocalized with the endothelial marker CD31. Thus, prostate tumor endothelial cells are atypically multipotent and can undergo a mesenchymal-like transition.

The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, can be expanded safely ex vivo, and can produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by coimplantation of human endothelial and mesenchymal progenitor cells isolated from blood and bone marrow. Evaluation of implants after 1 week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted endothelial progenitor cells were restricted to the luminal aspect of the vessels; mesenchymal progenitor cells were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from noninvasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization.

BACKGROUND: Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo. METHODS: Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence. RESULTS: Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein-positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein-positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein-expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein-positive endothelial progenitor cells. CONCLUSIONS: The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.

Common infantile hemangioma appears postnatally, grows rapidly, and regresses slowly. Two types of congenital vascular tumors present fully grown at birth and behave differently from infantile hemangioma. These rare congenital tumors have been designated rapidly involuting congenital hemangioma (RICH) and noninvoluting congenital hemangioma (NICH). RICH and NICH are similar in appearance, location, and size, and have some overlapping histologic features with infantile hemangioma. At a molecular level, neither expresses glucose transporter-1, a diagnostic marker of infantile hemangioma. To gain further insight into the molecular differences and similarities between congenital and common hemangioma, we analyzed expression of insulin-like growth factor-2, known to be highly expressed in infantile hemangioma and VEGF-receptors, by quantitative real-time PCR, in three RICH and five NICH specimens. We show that insulin-like growth factor-2 mRNA was expressed in both RICH and NICH, at a level comparable with that detected in common hemangioma over 4 y of age. In contrast, mRNA levels for membrane-associated fms-like tyrosine-kinase receptor, also known as VEGF receptor-1, were uniformly increased in congenital hemangiomas compared with proliferating or involuting phase common hemangioma. These results provide the first evidence of the molecular distinctions and similarities between congenital and postnatal hemangioma.

Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma--the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.

Rapid and complete vascularization of ischemic tissues and thick engineered tissues is likely to require vasculogenesis. Therefore, the search for clinically relevant sources of vasculogenic cells and the subsequent development of experimental models of vasculogenesis is of utmost importance. Here, we describe a methodology adapted from the Matrigel plug assay to deliver human blood-derived endothelial progenitor cells (EPCs) and mature smooth muscle cells (SMCs) subcutaneously into immunodeficient mice. One week after implantation, an extensive microvascular network composed of the human EPCs and SMCs is formed within the Matrigel. The presence of human EPC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This is a very versatile assay that allows (1) dialing the final microvessel density by varying either the total number of cells in the original cell suspension or the ratio between EPCs and SMCs, (2) studying the effect of substituting another type of perivascular cell for mature SMCs or another type of endothelial cell, (3) tracking each of the implanted cell types by labeling (e.g., GFP tagging) prior to implantation, and (4) studying the effect of genetically modifying the cells prior to implantation. Additionally, this assay is relatively simple to perform and it does not require an incision or surgical procedure. This murine model of human vasculogenesis is ideally suited for studies on the cellular and molecular components of microvessel development, pathologic neovascular responses, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.