Abstract-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.

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.

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.

Increased extracellular matrix protein production leading to structural abnormalities is a characteristic feature of chronic diabetic complications. We previously showed that high glucose in endothelial cell culture leads to the upregulation of basement membrane protein fibronectin (FN) via an endothelin (ET)-dependent pathway involving activation of NF-κB and activating protein-1 (AP-1). To delineate the mechanisms of basement membrane thickening, we used an animal model of chronic diabetes and evaluated ET-dependent activation of NF-κB and AP-1 and subsequent upregulation of FN in three target organs of chronic diabetic complications. After 3 mo of diabetes, retina, renal cortex, and myocardium demonstrated increased FN mRNA and increased ET-1 mRNA expression. Increased FN expression was shown to be dependent on ET receptor-mediated signaling, as the increase was prevented by the dual ET receptor antagonist bosentan. NF-κB activation was most pronounced in the retina, followed by kidney and heart. AP-1 activation was also most pronounced in the retina but was similar in both kidney and heart. Bosentan treatment prevented NF-κB activation in the retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to transcription factor activation may vary depending on the microenvironment of the organ.