Gene expression profile of human endothelial cells exposed to sustained fluid shear stress.

Biomechanical forces can modulate endothelial phenotype through changes in gene expression. We hypothesized that physiological laminar shear stresses (LSS) act as differentiative stimuli on endothelial cells (EC) to alter gene expression, creating an antioxidant, anti-apoptotic and anti-proliferative environment. The transcriptional profile of cultured human umbilical vein endothelial cells (HUVEC) exposed to LSS was evaluated by GeneCalling; 107 genes demonstrated at least a twofold change in expression at 24 h (LSS vs. static). These flow-responsive genes represent a limited number of functional clusters that include transcription factors, antioxidants, signaling molecules, cell cycle regulators, and genes involved in cellular differentiation. Immunohistochemistry and in situ hybridization confirmed that many of these flow-responsive genes, including the novel basic helix-loop-helix transcription factor Hath6, are expressed in EC in vivo. Thus these data identify a limited set of flow-responsive genes expressed in the endothelium that may be responsible for the establishment and maintenance of the flow-adapted endothelial phenotype in vivo.

Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents.

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.