Quantification of Genes and Proteins Associated with Endothelial Cell Function After Different Exercise-Induced Shear...

Cell culture models are used in cardiovascular research to study molecular pathways associated with endothelial shear stress (ESS). However, previous studies have limited translation from in vivo ESS, especially across exercise intensities. Using the Ibidi pump system, ESS can be replicated in vitro to study exercise-induced changes in protein and gene expression. Currently, there are no standardized protocols describing how to translate exercise-induced ESS from in vivo data to in vitro models. This protocol addresses that gap by integrating human exercise data to generate physiologically relevant ESS from 18 to 60 dyn/cm², replicating rest to high-intensity exercises. We describe the use of the Ibidi pump system to expose human umbilical vein endothelial cells (HUVEC) to exercise-induced ESS from in vivo data, followed by protein (Western blot and immunocytochemistry) and gene (reverse transcription polymerase chain reaction) analysis. The steps include ESS determination at different exercise intensities, culturing HUVEC, Ibidi pump system setup, and molecular analysis. Even though the sample experiment uses a low ESS, representative of low-intensity exercise, this model can be adapted to higher exercise intensities. Potential limitations and solutions are also discussed.