Cholesterol Enrichment Impairs Capacitative Calcium Entry, eNOS Phosphorylation & Shear Stress-Induced NO Production

Abstract

Endothelial dysfunction, characterized by decreased production or availability of nitric oxide (NO), is widely believed to be the hallmark of early-stage atherosclerosis. In addition, hypercholesterolemia is considered a major risk factor for development of atherosclerosis and is associated with impaired flow-induced dilation. However, the mechanism by which elevated cholesterol levels leads to decreased production of NO is unclear. NO is released in response to shear stress and agonist-evoked changes in intracellular calcium. Although calcium signaling is complex, we have previously shown that NO production by endothelial nitric oxide synthase (eNOS) is preferentially activated by calcium influx via store-operated channels. We hypothesized that cholesterol enrichment altered this signaling pathway (known as capacitive calcium entry; CCE) ultimately leading to decreased NO. Our results show that cholesterol enrichment abolished ATP-induced eNOS phosphorylation and attenuated the calcium response by the preferential inhibition of CCE. Furthermore, cholesterol enrichment also inhibited shear stress-induced NO production and eNOS phosporylation, consistent with our previous results showing a significant role for ATP autocrine stimulation and subsequent activation of CCE in the endothelial flow response.

Hypercholesterolemia‐Induced Loss of Flow‐Induced Vasodilation and Lesion Formation in Apolipoprotein E–Deficient Mice Critically Depend on Inwardly Rectifying K + Channels

“…These findings provide in vivo evidence for hypercholesterolemia‐induced suppression of eNOS, mostly mediated by the suppression of Kir channel activity. This is an effect that was previously reported in cultured aortic ECs loaded with cholesterol and exposed to shear stress . In addition, a decrease in Akt phosphorylation was previously reported in various hypercholesterolemic models …”

“…This is an effect that was previously reported in cultured aortic ECs loaded with cholesterol and exposed to shear stress. 52 In addition, a decrease in Akt phosphorylation was previously reported in various hypercholesterolemic models. [53][54][55] Another possible connection between inhibition of Kir activity and a decrease in the availability of NO is eNOS uncoupling via elevated production of reactive oxygen species.…”

Biochemical‐ and Biophysical‐Induced Barriergenesis in the Blood–Brain Barrier: A Review of Barriergenic Factors for Use in In Vitro Models

“…Andrews et al reported that the initiation of mechanotransduction in adult BMECs and fetal BMECs may be prevented by transformation into an immortalized D3 cell line. [ 260 ] It would be of interest to produce a series of thorough experiments by comparing and contrasting immortalized BMEC cell lines to the iPSC‐counterparts, in particular in regard to the use of shear stress as a barriergenic agent so as to establish within the BBB community preferred cell line.…”

Molecular chaperones in the brain endothelial barrier: neurotoxicity or neuroprotection?

“…It should be noted that apoptotic bodies (;800-5000 nm diameter) that are released from apoptotic cells exhibit ectosome characteristics but are rarely described in intracellular communication because they would be rapidly destroyed by phagocytes (87). Little is known about EVs derived from BMECs (12,85,(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98). Secreted by BMECs, EVs are heterogeneous in size and include ectosomes (;150-1000 nm diameter) and exosomes (;20-150 nm diameter) (99,100).…”