Substrate stiffness and frequency of cyclic stretch modulate expression of endothelial nitric oxide synthase in human endothelial cells

Objective Pulsatile flow and arterial stiffness are mediators of cardiovascular disease as they subject vascular endothelial cells to mechanical forces. These forces affect cell function through stretch-mediated ion channels and activate endothelial nitric oxide synthase (eNOS) through phosphorylation (peNOS), releasing nitric oxide (NO). This study investigated expression and activation of eNOS and NO in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs) in response to differing substrate stiffness and different cyclic strain rate. Methods Endothelial cell response was investigated with the following experiments: Experiment 1: HUVECs grown on compliant poly-di-methyl-siloxane (PDMS) substrates of constant stiffness were exposed to 5-20% magnitude cyclic stretch at 0.5 Hz or 1 Hz for 18 hrs and compared to static (no stretch) conditions. Expression of eNOS was determined using western blotting and NO release using a NO assay kit. Experiment 2: HUVECs were grown on static micropillar substrates of height 4 or 10 µM mimicking increased substrate stiffness with decreasing height. Experiment 3: HBMECs were grown on dextran hydrogels of tuneable gel stiffness with wide range of ligand density (2 mMol/L to 7mMol/L). Experiment 4: HBMECs were grown on cytosoft plates of varying elastic modulus (0.2Kpa to 64Kpa). For Experiments 2,3,4, cells were grown for 48 hrs and assessed for effect of substrate stiffness on eNOS expression. Results Experiment 1: For similar stretch magnitude, HUVECs on compliant substrates stretched at 1Hz showed increased eNOS compared to 0.5 Hz (178±32% vs 62±11%, p=0.0049); they also showed increased peNOS (188±26.31%, p=0.0029), eNOS mRNA (204±27%, p=0.0027) and NO (167±10%, p<0.0060) when all compared to the unstretched state (baseline of 100%). Experiments 2,3,4: With increasing stiffness, eNOS expression decreased in HUVECs (10 µM: 355±71%; 4 µM: 59±25%, p=0.0099) (mean±SEM% of 4 µM) and was undetectable in HBMECs on hydrogels and cytosoft plates. peNOS of HBMECs decreased with increasing hydrogel stiffness (7mMol/L: -5.72±17.55%2mMol/L, p=0.0414) (mean±SEM of % 2mMol/L) and was undetectable on cytosoft plates. Conclusion Results demonstrate that eNOS expression of human endothelial cells is modulated by substrate stiffness and pulsatile stretch.