Common infantile hemangioma is intriguing because of its variable presentation, rapid postnatal growth and slow regression in childhood. Interest in this tumor has increased with the recognition that it can be associated with structural anomalies in the craniofacial and ventral-caudal regions. The phenotype has expanded by characterization of rare vascular tumors that arise in the fetus and manifest at birth as rapidly involuting congenital hemangioma (RICH) or non-involuting congenital hemangioma (NICH). We describe a boy born with three RICH on the abdominal wall; one extended into the base of the umbilical cord. Two weeks later a small, infantile hemangioma arose on his neck. This patient stimulated a review of what is known about placental vascular tumors and their possible relationship to fetal and infantile hemangiomas. We suggest that chorangioma and umbilical cord hemangioma are clinically and histopathologically similar to cutaneous and hepatic RICH. These placental vascular tumors can also occur in conjunction with solitary and multiple infantile hemangiomas.

Hemogenic endothelium has been identified in embryonic dorsal aorta and in tissues generated from mouse embryonic stem cells, but to date there is no evidence for such bipotential cells in postnatal tissues or blood. Here we identify a cell population from human umbilical cord blood that gives rise to both endothelial cells and hematopoietic progenitors in vitro. Cord blood CD34+/CD133+ cells plated at high density in an endothelial basal medium formed an endothelial monolayer and a nonadherent cell population after 14-21 days. AML-1, a factor required for definitive hematopoiesis, was detected at low levels in adherent cells and at high levels in nonadherent cells. Nonadherent cells coexpressed the endothelial marker vascular endothelial (VE)-cadherin and the hematopoietic marker CD45, whereas adherent cells were composed primarily of VE-cadherin+/CD45- cells and a smaller fraction of VE-cadherin+/CD45+ cells. Both nonadherent and adherent cells produced hematopoietic colonies in methylcellulose, with the adherent cells yielding more colony-forming units (CFU)-GEMM compared with the nonadherent cells. To determine whether the adherent endothelial cells were producing hematopoietic progenitors, single cells from the adherent population were expanded in 96-well dishes for 14 days. The clonal populations expressed VE-cadherin, and a subset expressed AML-1, epsilon-globin, and gamma-globin. Three of 17 clonal cell populations gave rise to early CFU-GEMM hematopoietic progenitors and burst-forming unit-erythroid progenitors. These results provide evidence for hemogenic endothelial cells in human umbilical cord blood.

Vascularization of tissues is a major challenge of tissue engineering (TE). We hypothesize that blood-derived endothelial progenitor cells (EPCs) have the required proliferative and vasculogenic activity to create vascular networks in vivo. To test this, EPCs isolated from human umbilical cord blood or from adult peripheral blood, and human saphenous vein smooth muscle cells (HSVSMCs) as a source of perivascular cells, were combined in Matrigel and implanted subcutaneously into immunodeficient mice. Evaluation of implants at one week revealed an extensive network of human-specific lumenal structures containing erythrocytes, indicating formation of functional anastomoses with the host vasculature. Quantitative analyses showed the microvessel density was significantly superior to that generated by human dermal microvascular endothelial cells (HDMECs) but similar to that generated by human umbilical vein endothelial cells (HUVECs). We also found that as EPCs were expanded in culture, their morphology, growth kinetics, and proliferative responses toward angiogenic factors progressively resembled those of HDMECs, indicating a process of in vitro maturation. This maturation correlated with a decrease in the degree of vascularization in vivo, which could be compensated for by increasing the number of EPCs seeded into the implants. Our findings strongly support the use of human EPCs to form vascular networks in engineered organs and tissues.

In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-beta2, individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF- and TGFbeta2-induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGFbeta2, a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGFbeta2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGFbeta2 but were resistant to endothelial-inducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves.

Infantile hemangiomas are composed of endothelial cells (ECs), endothelial progenitor cells (EPCs), as well as perivascular and hematopoietic cells. Our hypothesis is that hemangioma-derived EPCs (HemEPCs) differentiate into the mature ECs that comprise the major compartment of the tumor. To test this, we isolated EPCs (CD133(+)/Ulex europeus- I(+)) and mature ECs (CD133(-)/Ulex europeus-I(+)) from proliferating hemangiomas and used a previously described property of hemangioma-derived ECs (HemECs), enhanced migratory activity in response to the angiogenesis inhibitor endostatin, to determine if HemEPCs share this abnormal behavior. Umbilical cord blood-derived EPCs (cbEPCs) were analyzed in parallel as a normal control. Our results show that HemEPCs, HemECs, and cbEPCs exhibit increased adhesion, migration, and proliferation in response to endostatin. This angiogenic response to endostatin was consistently expressed by HemEPCs over several weeks in culture, whereas HemECs and cbEPCs shifted toward the mature endothelial response to endostatin. Similar mRNA-expression patterns among HemEPCs, HemECs, and cbEPCs, revealed by microarray analyses, provided further indication of an EPC phenotype. This is the first demonstration that human EPCs, isolated from blood or from a proliferating hemangioma, are stimulated by an angiogenesis inhibitor. These findings suggest that EPCs respond differently from mature ECs when exposed to angiogenic or antiangiogenic signals.

Small-caliber synthetic grafts used for coronary bypass artery grafting are compromised by thrombogenicity and accelerated intimal thickening, resulting in early graft occlusion. Herein we describe the fabrication and physical properties of small-caliber blood vessels using decellularized porcine aortic segments. These vessels were further coated with human saphenous vein endothelial cells (HSVECs) for future clinical applications. Chemical staining of decellularized vessels showed that they preserved their native matrix architecture, including several collagen layers in between internal and external elastin layers. The burst pressure for the decellularized vessels was higher than 1,000 mmHg. HSVECs, seeded on the luminal side, adhered to the matrix and formed a uniform monolayer. HSVEC-seeded vessels produced prostaglandin I2 and released vasoactive agents in response to the calcium ionophore A23187. These results show that engineered blood vessels coated with the host endothelial cells possess morphologic and functional characteristics of human small-caliber vessels. Tissue-engineered vessels may potentially be useful clinically as vascular grafts.

Hemangioma is a benign tumor of infancy whose hallmark is rapid growth during the first year of life followed by slow regression during early childhood. The proliferating phase is characterized by abundant immature endothelial cells, the involuting phase by prominent endothelial-lined vascular channels and endothelial apoptosis, and the involuted phase by few remaining capillary-like vessels surrounded by loose fibrofatty tissue. Nothing is known about the mechanisms that contribute to the adipogenesis during this spontaneous regression. We postulated that mesenchymal stem cells (MSCs) reside in the tumor and preferentially differentiate into adipocytes. To test this hypothesis, we isolated MSCs from 14 proliferating and five involuting hemangiomas by taking advantage of the well known selective adhesion of MSCs to bacteriologic dishes. These hemangioma-derived MSCs (Hem-MSCs) are similar to MSCs obtained from human bone marrow, expressing the cell surface markers SH2 (CD105), SH3, SH4, CD90, CD29, smooth muscle alpha-actin, and CD133 but not the hematopoietic markers CD45 and CD14 or the hematopoietic/endothelial markers CD34, CD31, and kinase insert domain receptor (KDR). Hem-MSCs exhibited multilineage differentiation with robust adipogenic potential that correlated with the proliferating phase. The numbers of adipogenic Hem-MSCs were higher in proliferating-phase than in involuting-phase tumors and higher than in normal infantile skin. Furthermore, Hem-MSCs exhibited a random pattern of X-chromosomal inactivation, indicating that these cells are not clonally derived. In summary, we have identified MSCs as a novel cellular constituent in infantile hemangioma. These MSCs may contribute to the adipogenesis during hemangioma involution.

Vascular endothelial growth factor-receptors (VEGF-Rs) are pivotal regulators of vascular development, but a specific role for these receptors in the formation of heart valves has not been identified. We took advantage of small molecule inhibitors of VEGF-R signaling and showed that blocking VEGF-R signaling with receptor selective tyrosine kinase inhibitors, PTK 787 and AAC 787, from 17-21 hr post-fertilization (hpf) in zebrafish embryos resulted in a functional and structural defect in cardiac valve development. Regurgitation of blood between the two chambers of the heart, as well as a loss of cell-restricted expression of the valve differentiation markers notch 1b and bone morphogenetic protein-4 (bmp-4), was readily apparent in treated embryos. In addition, microangiography revealed a loss of a definitive atrioventricular constriction in treated embryos. Taken together, these data demonstrate a novel function for VEGF-Rs in the endocardial endothelium of the developing cardiac valve.

Infantile hemangioma is an endothelial tumor that grows rapidly after birth but slowly regresses during early childhood. Initial proliferation of hemangioma is characterized by clonal expansion of endothelial cells (ECs) and neovascularization. Here, we demonstrated mRNA encoding CD133-2, an important marker for endothelial progenitor cells (EPCs), predominantly in proliferating but not involuting or involuted hemangioma. Progenitor cells coexpressing CD133 and CD34 were detected by flow cytometry in 11 of 12 proliferating hemangioma specimens from children 3 to 24 months of age. Furthermore, in 4 proliferating hemangiomas, we showed that 0.14% to 1.6% of CD45(-) nucleated cells were EPCs that coexpressed CD133 and the EC marker KDR. This finding is consistent with the presence of KDR(+) immature ECs in proliferating hemangioma. Our results suggest that EPCs contribute to the early growth of hemangioma. To our knowledge, this is the first study to show direct evidence of EPCs in a human vascular tumor.