Haemangioma is the most common tumour of endothelial origin, occurring in 4-10% of Caucasian infants. It is characterized by rapid growth during the first year of postnatal life, followed by spontaneous regression from 1 to 7 years of age. The cell surface adhesion molecule CD146 has been identified as an endothelial cell marker. Despite advances in understanding the functional role of CD146 in normal endothelial cells and tumour progression, its expression and a possible role in an endothelial tumour have not been studied. As part of an investigation of endothelial cell alterations in infantile haemangioma, differential expression studies were performed with several known antigens and endothelial cell markers. Using immunohistochemical and flow cytometric analyses, cultured human dermal microvascular endothelial cells isolated from newborn foreskin (HDMEC) were compared with endothelial cells derived from haemangioma tissue (HemECs). In addition, immunohistochemistry was used to compare haemangioma tissues with normal human skin. Unexpectedly, cultured HemECs showed a significantly lower level of CD146 than HDMECs by both flow cytometric analysis and immunofluorescence staining. Using immunohistochemical studies, it was further demonstrated that endothelia in all haemangioma tissues, regardless of the tumour phase, showed negative immunoreactivity for CD146. In contrast, strong positive staining for CD146 was observed in the pericyte-like cells that surround the endothelial layers. These findings are believed to be relevant to the molecular basis of haemangioma. Furthermore, it is possible that antibodies against CD146 may be useful for separating haemangioma-derived endothelial cells from normal endothelial cells and pericytes.

BACKGROUND AND AIM OF THE STUDY: Histopathological studies of rejected orthotopic heart transplants suggest that cardiac valve endothelium is spared the inflammatory cell infiltration and tissue damage that occurs in the myocardium. To test whether this apparent protection from leukocyte invasion might be an inherent feature of the valve endothelium, leukocyte adhesion molecule expression and function were analyzed in human pulmonary valve endothelial cells (HPVEC). Use of cultured HPVEC allowed delineation of the potential contribution of functional adhesion molecules from the contribution of hemodynamic forces exerted on the leaflet surface in vivo METHODS AND RESULTS: HPVEC express E-selectin, ICAM-1, and VCAM-1 in response to the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) similarly to other types of cultured human endothelial cells. In a static cell adhesion assay, E-selectin-mediated adhesion of HL-60 cells, a human promyelocytic leukemia cell line, and U937 cells, a human monocytic cell line, was determined in cells treated with TNF-alpha for 5 h. After 24 h of TNF-alpha, adhesion of U937 cells to HPVEC was mediated primarily by VCAM-1, consistent with the high expression of VCAM-1 and diminished expression of E-selectin at 24 h. CONCLUSION: These results demonstrate that HPVEC express functional leukocyte adhesion molecules in vitro and suggest that cardiac valve endothelium is competent to initiate leukocyte adhesion. Thus, other factors, such as the hemodynamic forces exerted on the valve, may contribute to the apparent protection from inflammatory cell infiltration in vivo.

Three-dimensional scaffolds made of bioabsorbable polymeric constituents are currently being tested for use in tissue engineering of various tissues. A composite scaffold of poly-glycolic acid (PGA) non-woven mesh dip-coated in a 1% solution of poly-4-hydroxybutyrate (P4HB) was shown to be suitable as a scaffold for creation of tissue-engineered trileaflet pulmonic valve replacements in sheep [Hoerstrup, S.P., et al., Circulation 102(Suppl. 3), III44, 2000]. However, little is known about how cells seeded on PGA/P4HB respond in vitro to soluble factors supplied in the culture medium. To optimize tissue development in vitro, before implantation, we set out to develop quantitative biochemical assays to measure how cells seeded on PGA/P4HB respond to growth and differentiation factors. Herein we show that ovine aortic valvular endothelial cells and circulating endothelial progenitor cells (EPCs) seeded onto PGA/P4HB proliferate in response to vascular endothelial growth factor and transdifferentiate to a mesenchymal phenotype in response to transforming growth factor beta(1). Transdifferentiation from an endothelial to mesenchymal phenotype is a critical step during embryonic development of cardiac valves. Our results demonstrate that valvular endothelial cells and EPCs isolated from peripheral blood can recapitulate critical developmental steps on PGA/P4HB. These results demonstrate that PGA/P4HB provides a conducive environment for cellular proliferation, differentiation, and tissue development.

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.

BACKGROUND: This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach. METHODS: Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing. RESULTS: Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including alpha-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets. CONCLUSIONS: Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form "tissue" that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.