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Objective-Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2.Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells. Methods and Results-The human Ang-2 promoter contains a 585-bp region around the transcriptional start site (Ϫ109 to ϩ476) that is sufficient to control endothelial cell-specific and cytokine-dependent Ang-2 expression. Strong repressor elements of Ang-2-promoter activity are located in the 5Ј-region of the promoter and in the first intron. The Ets family transcription factors Ets-1 and Elf-1 act as strong enhancers of endothelial cell Ang-2-promoter activity. Ets-binding sites Ϫ4 and Ϫ7 act as positive regulators, whereas Ets-binding site Ϫ3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting. Key Words: angiogenesis Ⅲ Ang-2 Ⅲ Ets-1 Ⅲ Elf-1 Ⅲ endothelial cell A ngiogenesis, the formation of blood vessels from preexisting vessels, is controlled by a hierarchically structured signaling cascade of receptor tyrosine kinases specifically expressed in endothelial cells. 1,2 The angiopoietin-Tie ligand-receptor system plays a key critical role in the regulation of angiogenesis. The interactions of angiopoietins with the Tie-2 receptor regulate vascular maturation and vessel quiescence. 3 Angiopoietin-1 (Ang-1) consists of 4 alternatively spliced isoforms. 4 The 1.5-kb isoform codes for a multimerizing protein that acts in a paracrine manner and binds to endothelial cell-expressed Tie-2 inducing receptor phosphorylation and subsequent signal transduction. The structure of the smaller Ang-1 isoforms are consistent with dominant-negative regulatory molecules. 4 Ang-1-mediated activation of Tie-2 regulates endothelial cell survival and blood vessel maturation. Ang-1 exerts a vessel sealing effect, acts antiinflammatory, and protects against cardiac allograft arteriosclerosis. Low level constitutive Tie-2 activation may be required in the adult to maintain the mature quiescent phenotype of the resting vascular endothelium. 3 In contrast to the Tie-2 activating functions of Ang-1, Ang-2 acts primarily as functional antagonist of Ang-1/Tie-2 by binding the receptor without inducing signal transduction. 5,6 The opposing effects of Ang-1 and Ang-2 support a model of constitutive Ang-1/Tie-2 interactions controlling vascular homeostasis as default pathway 7 and Ang-2 acting as dynamically regulated antagonizing cytokine. 1,8,9 Loss of the Ang-2 gene and function is compatible with life as evidenced by the observation that Ang-2-deficient mice are born apparently normal. 6 The functionally unaffected blood vascular system of Ang-2-deficient mice has only minor abnormalities (eg, perturbed regression of hyaloid blood vessels). Yet ...

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

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