Shear stress-induced nitric oxide (NO) production

Abstract

Hypercholesterolemia is considered a major risk factor for the development of atherosclerosis. Early stage atherosclerosis is characterized by endothelial dysfunction, which is described by impaired or reduced availability of nitric oxide (NO). However, the mechanism by which cholesterol leads to decreased NO production remains unclear. NO is produced by the endothelium in response to agonist stimulation and shear stress, although the mechanisms of NO production have yet to be fully determined due to experimental difficulties in measuring NO, especially under flow conditions. In order to understand the effect of cholesterol enrichment on shear stress-induced NO production, we first designed a device to measure shear stress-induced NO directly and in real-time from endothelial cells in vitro. With this device we were able to establish a mechanism for shear stress-induced NO production through ATP autocrine signaling and activation of store operated calcium channels (SOCs) which are responsible for the influx of extracellular calcium following store depletion termed capacitative calcium entry (CCE). To further understand the mechanism of ATP stimulation and subsequent activation of CCE in NO production, we investigated ATP-induced eNOS phosphorylation in cells under static conditions. We found that the mechanism of ATP-induced eNOS phosphorylation is through a complex interplay between CCE and protein kinase C (PKC). In addition, our results suggest an important co-localization effect of SOCs and eNOS that is responsible for maximal activation of eNOS.We next investigated the effect of cholesterol enrichment on the shear stress-induced NO response and found that shear stress-stimulated increases in NO and eNOS phosphorylation were impaired. In addition, the reduction in the response was similar to our studies where we inhibited the ATP-CCE pathway. To further investigate the effect of cholesterol on the ATP-CCE signaling pathway, we studied ATP-induced eNOS phosphorylation and calcium signaling in cells under static conditions. Our results show that cholesterol enrichment attenuates CCE thus leads to decreased eNOS phosphorylation. Aside from impaired CCE, cholesterol reduces calcium and affects other calcium dependent signaling molecules such as PKC. Our results indicate that cholesterol enrichment leads to impairment of shear stress-induced NO production through inhibition of SOCs and CCE. These findings have important implications for the role of cholesterol in endothelial dysfunction and in the development of atherosclerosis.%%%%Ph.D., Biomedical Engineering – Drexel University, 2012