Infantile hemangioma is a benign tumor of endothelial cells (ECs) that is well known to pediatricians because of its frequency and sometimes disturbingly rapid growth. The primary defect(s) that cause hemangioma have not yet been elucidated, but recent studies have revealed that hemangiomas are composed of clonal ECs that exhibit abnormal properties in vitro. These studies support the hypothesis that hemangiomas arise when a somatic mutation occurs in a single endothelial progenitor, leading to dysregulated activity of one or more genes that control EC growth. Aberrant endothelial growth may in turn alter patterns of gene expression in nearby cells--in the overlying epidermis, for example--and account for observed extrinsic alterations that may influence hemangiogenesis. Unraveling the cellular and molecular basis of hemangioma is likely to yield critical insights into the mechanisms of EC proliferation and regression that may be applicable to other cardiovascular disorders.

Infantile hemangiomas are endothelial tumors that grow rapidly in the first year of life and regress slowly during early childhood. Although hemangiomas are well-known vascular lesions, little is known about the mechanisms that cause the excessive endothelial cell proliferation in these most common tumors of infancy. To investigate the molecular basis of hemangioma, we isolated endothelial cells from several proliferative-phase lesions and showed that these cells are clonal and exhibit abnormal properties in vitro (E. Boye, Y. Yu, G. Paranya, J. B. Mulliken, B. R. Olsen, J. Bischoff: Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest 2001, 107:745-752). Here, we analyzed mRNA expression patterns of genes required for angiogenesis, including members of the vascular endothelial growth factor (VEGF)/VEGF receptor family and the angiopoietin/Tie family, in hemangioma-derived and normal endothelial cells. KDR, Flt-1, Tie1, Tie2, and angiopoietin-2 (Ang2) were strongly expressed in cultured hemangioma-derived endothelial cells and in hemangioma tissue. In contrast, there was little expression of angiopoietin-1 (Ang1) or VEGF. We found Tie2 mRNA and protein up-regulated with a concomitant increase in cellular responsiveness to Ang1 in most hemangioma-derived endothelial cells. Ang2 mRNA was down-regulated in response to serum in hemangioma-derived endothelial cells, but not in normal endothelial cells, suggesting altered regulation. These findings implicate Tie2 and its ligands Ang1 and Ang2 in the pathogenesis of hemangioma.

Cardiac valves arise from endocardial cushions, specialized regions of the developing heart that are formed by an endothelial-to-mesenchymal cell transdifferentiation. Whether and to what extent this transdifferentiation is retained in mature heart valves is unknown. Herein we show that endothelial cells from mature valves can transdifferentiate to a mesenchymal phenotype. Using induction of alpha-smooth muscle actin (alpha-SMA), an established marker for this process, two distinct pathways of transdifferentiation were identified in clonally derived endothelial cell populations isolated from ovine aortic valve leaflets. alpha-SMA expression was induced by culturing clonal endothelial cells in medium containing either transforming growth factor-beta or low levels of serum and no basic fibroblast growth factor. Cells induced to express alpha-SMA exhibited markedly increased migration in response to platelet-derived growth factor-BB, consistent with a mesenchymal phenotype. A population of the differentiated cells co-expressed CD31, an endothelial marker, along with alpha-SMA, as seen by double-label immunofluorescence. Similarly, this co-expression of endothelial markers and alpha-SMA was detected in a subpopulation of cells in frozen sections of aortic valves, suggesting the transdifferentiation may occur in vivo. Hence, the clonal populations of valvular endothelial cells described here provide a powerful in vitro model for dissecting molecular events that regulate valvular endothelium.

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.

E-selectin is a cell adhesion molecule involved in the initial rolling and adhesion of leukocytes to the endothelium during inflammation. In addition, in vitro studies have suggested that an interaction between E-selectin and binding sites such as sialyl Lewis X-containing oligosaccharides on endothelial cells may be important for angiogenesis. In order to investigate the binding of E-selectin to endothelial cells, we developed an ELISA assay using chimeric E-selectin-Ig molecules and endothelial cells fixed on poly-L-lysine coated plates. Our results indicate that E-selectin-Ig binds to both bovine capillary endothelial cells and human dermal microvascular endothelial cells in a calcium-dependent and saturable manner. The binding is inhibited markedly by heparin and by syndecan-1 ectodomain, and moderately by chondroitin sulfate, but not by sialyl Lewis X-containing oligosaccharides. These results suggest that heparan sulfate and chondroitin sulfate proteoglycans on endothelial cells are potential ligands for E-selectin.

Hemangioma, the most common tumor of infancy, is a benign vascular neoplasm of unknown etiology. We show, for the first time to our knowledge, that endothelial cells from proliferating hemangioma are clonal, and we demonstrate that these hemangioma-derived cells differ from normal endothelial cells in their rates of proliferation and migration in vitro. Furthermore, migration of hemangioma endothelial cells is stimulated by the angiogenesis inhibitor endostatin, unlike the inhibition seen with normal endothelial cells. We conclude that hemangiomas constitute clonal expansions of endothelial cells. This is consistent with the possibility that these tumors are caused by somatic mutations in one or more genes regulating endothelial cell proliferation.

The interaction between the colon tumor cell surface and the endothelial cell layer is an important component of tumor intravasation, extravasation, and metastasis. Multiple studies suggest that tumor cells may bind to E-selectin expressed on endothelial cells during these processes. To identify possible E-selectin ligands on tumor cells that may participate in this mechanism, we used E-selectin-Ig chimera affinity chromatography to isolate glycoproteins from the human colon cancer cell line Colo-205. Binding of these cells to E-selectin was specific, required the presence of calcium, and could be blocked by antibodies against E-selectin. We identified LAMP-1 (lysosomal membrane glycoprotein-1), LAMP-2, and two high molecular weight glycoproteins (>400 kDa and 300 kDa) as the main E-selectin ligands on Colo-205 cells. Treatment of the cells with N-glycanase and O-sialoglycoprotease abolished their binding to E-selectin. The high MW glycoproteins contained sialyl Lewis X and/or sialyl Lewis A glycoconjugates, and appeared to be either alternatively spliced or alternatively glycosylated forms of MUC-1 (mucin-1).

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.

E-selectin, an endothelial-specific adhesion molecule best known for its role in leukocyte adhesion, is not detected in quiescent endothelial cells, but is induced by inflammatory stimuli. However, E-selectin is also expressed in proliferating endothelial cells under noninflammatory conditions in vivo and in vitro, suggesting that E-selectin is also regulated by growth signals. To investigate E-selectin expression in lipopolysaccharide-stimulated versus nonstimulated proliferating cells, we analyzed the distribution of E-selectin-positive human microvascular endothelial cells in G0/G1, S, and G2/M phases of the cell cycle under both conditions. Lipopolysaccharide treatment resulted in uniformly increased E-selectin expression in cells in G0/G1, S, and G2/M. In contrast, levels of E-selectin in nonstimulated proliferating cells showed a linear correlation with the percentage of cells in G2/M. E-selectin in proliferating endothelial cells was not reduced by addition of soluble tumor necrosis factor-alpha-receptor or soluble interleukin-1-receptor indicating that its expression was not due to endogenous production of either cytokine. In addition, E-selectin was increased in cells stimulated with basic fibroblast growth factor, a well-known mitogen for endothelial cells. E-selectin in proliferating endothelial cells is functional, as shown by E-selectin-dependent adhesion of the promyelocytic leukemia cell line HL-60 to subconfluent human microvascular endothelial cells. In summary, these studies indicate that E-selectin can be regulated by a non-inflammatory pathway that is related to the proliferative state of the endothelium.

OBJECTIVE: Hemangioma is an endothelial cell tumor that grows rapidly during infancy and regresses slowly during childhood. However, little is known about the natural history of this common tumor. To gain insight into the cellular mechanisms that underlie the switch from uncontrolled growth to involution of endothelium, we investigated the extent of cellular apoptosis versus proliferation in hemangioma specimens that spanned the natural life cycle of the tumor. METHODS: We analyzed apoptosis and cellular proliferation in frozen sections from 16 hemangioma specimens using the TUNEL assay to detect apoptotic cells and the Ki67 antigen to detect dividing cells. RESULTS: Apoptosis was low in proliferative phase hemangiomas but increased fivefold in involutive phase specimens obtained from children one to four years of age. Immunofluorescence double-labeling experiments showed that at least one third of the apoptotic cells were endothelial. As expected, cellular proliferation was high in specimens up to 2 years of age but decreased significantly thereafter. Apoptosis was consistently low in nine normal skin tissues (newborn to 4 years of age) obtained from discarded pathology specimens. CONCLUSIONS: These results suggest that increased apoptosis during the second year of life can offset cellular proliferation and may be involved in initiating regression of hemangioma.