Microarray analysis of human umbilical vein endothelial cells subjected to pulsatile arterial shear stress vs. steady arterial shear stress
McIntire, Larry V.
Doctor of Philosophy
Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces. I hypothesize that the temporal variations in pulsatile, arterial shear stress differentially regulate endothelial gene expression compared to steady, arterial shear stress. A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier-Stokes equation solution. Spectral analysis of the pseudo-steady state shear stress compared well with the power spectrum of published in vivo measurements of the common carotid artery flow rate. Total RNA from human umbilical vein endothelial cells (HUVEC) subjected to 24 hrs of pulsatile, arterial shear stress (average = 13 dyne/cm2, range = 7 to 25 dyne/cm 2; 1 Hz); steady, arterial shear stress (13 dyne/cm2), or static condition was collected for microarray analysis. Normalized intensities were filtered for 1.5 fold (shear vs. static) and subjected to ANOVA with Benjamini and Hochberg multiple testing correction at a false discovery rate of 5%. Compared to static condition, pulsatile arterial shear stress differentially regulated over 1,000 genes while steady arterial shear stress differentially regulated over 1,100 genes. More than 200 genes were differentially regulated by pulsatile arterial shear stress compared to steady arterial shear stress, but hierarchal cluster analysis indicates HUVEC respond similarly to both types of shear stress (Pearson correlation coefficient = 0.785). Quantitative real time polymerase chain reaction verified the trend in the gene expression results from the microarray analysis. Data mining of the differentially expressed genes with Ingenuity Pathways Analysis and with Expression Analysis Systematic Explorer identified several prominent biological themes differentially regulated by mechanical forces including glycosylphosphatidylinisotol anchor bioprocesses, nitric oxide signaling, and metal ion processes. Collectively, these results demonstrate that the common carotid flow waveform elicits subtle changes in HUVEC responses to arterial levels of shear stress. These differences reflect a shift away from quiescence and may be mediated by nitric oxide bioavailability.
Biomedical engineering; Pathology