| Shear stresses from regions of disease are low and reversing, and they promote a pro-inflammatory or "atheroprone" endothelial cell phenotype. Hemodynamics from regions absent of disease have comparatively higher levels of unidirectional shear stress that promotes an "atheroprotective" and anti-inflammatory phenotype. To study how the hemodynamic environment differentially dictates endothelial phenotype, atheroprone and atheroprotective shear stresses from the human vasculature were recapitulated in vitro on human ECs.;First, increased endothelial expression of GRP78 and endoplasmic reticulum (ER) stress was observed in atheroprone versus atheroprotective regions of C57/BL6 mice and in vitro. Increased GRP78 correlated with ATF6 activation of the ER stress sensing element (ERSE1) promoter by atheroprone flow signifying the activation of the unfolded protein response.;Secondly, focal activation of NF-kappaB and deposition of fibronectin (FN), are found in regions of atherosclerosis where they contribute to inflammatory signaling. We sought to elucidate the mechanism by which NF-kappaB and FN are regulated by local shear stress patterns and the role these pathways play in sustaining an atheroprone phenotype. Atheroprone flow caused a steady and sustained increase in FN expression over time significantly greater than atheroprotective flow. In vivo and In vitro, loss of PECAM-1 blocked the induction of FN and the activation of NF-kappaB by atheroprone flow. Additionally, blocking NE-KB activation attenuated the flow-induced FN expression, and siRNA against FN significantly reduced NF-kappaB activity. Thus, atheroprone shear stress creates positive feedback to maintain inflammation.;Lastly, we hypothesize that unique endothelial phenotypes by atheroprone and atheroprotective hemodynamics are rooted in distinct frequency spectrums. In order to investigate the roles of frequency, harmonics 0--8 were systematically swapped between atheroprone and atheroprotective waveforms using Fourier Transforms to create new "mutated" waveforms. NF-kappaB levels were blocked in the atheroprone waveform by raising the amplitude of the 0th or 1st harmonic. First harmonic mutations also decreased pro-inflammatory genes and proteins, such as VCAM-1, FN, and E-Selectin, while activating higher levels of KLF2 transcription. Frequency-dependent activation of NF-kappaB depends on PECAM-1, where its removal resulted in reversal of activity levels. Therefore shear stress magnitude alone does not control endothelial phenotype, but the amplitude of multiple harmonics. |
