Efficient in vivo vascularization of tissue-engineering scaffolds.

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.

Expression of angiopoietin-2 in endothelial cells is controlled by positive and negative regulatory promoter elements.

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 (-109 to +476) 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 5'-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 -4 and -7 act as positive regulators, whereas Ets-binding site -3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting. CONCLUSIONS: The data determine unique positive and negative regulatory mechanisms of endothelial cell Ang-2 expression and provide further evidence for the critical role of Ang-2 as a key autocrine regulator of vascular stability and responsiveness.

The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands with opposing functions of the receptor tyrosine kinase Tie-2 regulating endothelial cell survival and vascular maturation. Ang-1 acts in a paracrine agonistic manner, whereas Ang-2 appears to act primarily as an autocrine antagonistic regulator. To shed further light on the complexity of autocrine/paracrine agonistic/antagonistic functions of the angiopoietin/Tie-2 system, we have studied Ang-2 synthesis and secretion in different populations of wild-type and retrovirally Ang-2-transduced endothelial cells. Endogenous and overexpressed endothelial cell Ang-2 is expressed in a characteristic granular pattern indicative of a cytoplasmic storage granule. Light and electron microscopic double staining revealed Ang-2 colocalization with von Willebrand factor, identifying Ang-2 as a Weibel-Palade body molecule. Costaining with P-selectin showed that storage of Ang-2 and P-selectin in Weibel-Palade bodies is mutually exclusive. Stored Ang-2 has a long half-life of more than 18 hours and can be secreted within minutes of stimulation (eg, by phorbol 12-myristate 13-acetate [PMA], thrombin, and histamine). Collectively, the identification of Ang-2 as a stored, rapidly available molecule in endothelial cells strongly suggests functions of the angiopoietin/Tie-2 system beyond the established roles during angiogenesis likely to be involved in rapid vascular homeostatic reactions such as inflammation and coagulation.