The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer’s tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell® tracer assays and two commercial impedance devices (xCELLigence®, ECIS®). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments.

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Objective-The cyclin-dependent kinase (CDK) inhibitor flavopiridol is currently being tested in clinical trials as anticancer drug. Beyond its cell death-inducing action, we hypothesized that flavopiridol affects inflammatory processes. Therefore, we elucidated the action of flavopiridol on leukocyte-endothelial cell interaction and endothelial activation in vivo and in vitro and studied the underlying molecular mechanisms. Methods and Results-Flavopiridol suppressed concanavalin A-induced hepatitis and neutrophil infiltration into liver tissue. Flavopiridol also inhibited tumor necrosis factor-␣-induced leukocyte-endothelial cell interaction in the mouse cremaster muscle. Endothelial cells were found to be the major target of flavopiridol, which blocked the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin), as well as NF-B-dependent transcription. Flavopiridol did not affect inhibitor of B (IB) kinase, the degradation and phosphorylation of IB␣, nuclear translocation of p65, or nuclear factor-B (NF-B) DNA-binding activity. By performing a cellular kinome array and a kinase activity panel, we found LIM domain kinase-1 (LIMK1), casein kinase 2, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), CDK4, CDK6, CDK8, and CDK9 to be influenced by flavopiridol. Using specific inhibitors, as well as RNA interference (RNAi), we revealed that only CDK9 is responsible for the action of flavopiridol. Conclusion-Our