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Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.

Fenestrae are small pores in the endothelium of renal glomerular, gastrointestinal, and endocrine gland capillaries and are involved in the bidirectional exchange of molecules between blood and tissues. Although decades of studies have characterized fenestrae at the ultrastructural level, little is known on the mechanisms by which fenestrae form. We present the development of an in vitro assay in which rapid and abundant fenestra induction enables a detailed study of their biogenesis. Through the use of agents that stabilize or disassemble actin microfilaments, we show that actin microfilament remodeling is part of fenestra biogenesis in this model. Furthermore, by using a loss-of-function approach, we show that the diaphragm protein PV-1 is necessary for fenestral pore architecture and the ordered arrangement of fenestrae in sieve plates. Together, these data provide insight into the cell biology of fenestra formation and open up the future study of the fenestra to a combined morphological and biochemical analysis.actin filaments ͉ sieve plates ͉ VEGF ͉ vascular permeability

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