SummaryVascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. Human genotype-phenotype studies of endothelium are limited by the unavailability of patient-specific endothelial cells. To establish a cellular platform for studying endothelial biology, we have generated vascular endothelium from human induced pluripotent stem cells (iPSCs) exhibiting the rich functional phenotypic plasticity of mature primary vascular endothelium. These endothelial cells respond to diverse proinflammatory stimuli, adopting an activated phenotype including leukocyte adhesion molecule expression, cytokine production, and support for leukocyte transmigration. They maintain dynamic barrier properties responsive to multiple vascular permeability factors. Importantly, biomechanical or pharmacological stimuli can induce pathophysiologically relevant atheroprotective or atheroprone phenotypes. Our results demonstrate that iPSC-derived endothelium possesses a repertoire of functional phenotypic plasticity and is amenable to cell-based assays probing endothelial contributions to inflammatory and cardiovascular diseases.

Rap1GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubuledependent increases in vascular permeability induced by tumor necrosis factor-␣ and transforming growth factor-␤. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endo-thelial cell borders and forms a complex with- ,- ,-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendo-thelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endo-thelial cells transduced with an adenovi-rus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leuko-cyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions. (Blood. 2008;112:2770-2779) Introduction Vascular endothelial-cadherin (VE-cad) is a transmembrane protein expressed in the vascular endothelium 1 that participates in endothelial barrier function, angiogenesis, signaling, and endothelial cell survival (reviewed in Dejana et al 2). Surface-expressed VE-cad localizes to cell-cell junctions and associates with-catenin,-catenin, plakoglobin (-catenin), and p120-catenin (p120) through its cytoplasmic tail, and with the actin cytoskeleton 3,4 in combination with vinculin and-actinin, which is thought to be critical for VE-cad adhesive interactions (reviewed in Vestweber 5). p120 is a substrate for Src family kinases and other receptor tyrosine kinases 6,7 and regulates cadherin-dependent adhesion positively and negatively, depending on the cell system under study (reviewed in Alemà and Salvatore 8). p120 associates with the juxtamembrane cytoplasmic region of VE-cad, 9 and this is crucial to maintain cadherin surface expression. 10 Overexpres-sion of VE-cad mutants that competed for p120 binding, or siRNA knockdown of p120 in endothelium, resulted in dramatically decreased surface-expressed VE-cad and concomitant increased VE-cad degradation by an endocytic pathway. In contrast, overexpression of wild-type p120 augments VE-cad surface expression and diminishes its endocytosis. 11 The precise mechanism(s) by which p120 controls the turnover and endocy-tosis of junctional VE-cad is not completely understood, 8,11 but it is conclusive that cytosolic levels of p120 regulate VE-cad surface expression in endothelial cells, and the level of E-cadherin in epithelial cells. 12 The idea that VE-ca...