Mechanism of Cholesterol-Induced Impairment of Store-Operated Calcium Entry in Endothelial Cells

Kelly Anne Zaccheo

doi:10.17918/00010737

Early stages of atherosclerosis are marked by endothelial dysfunction characterized by impaired NO production or bioavailability, leading to compromised endothelium-dependent vasodilation. Elevation in serum cholesterol is a high risk factor for development of atherosclerosis. Studies have shown that a rise in serum cholesterol correlates to elevated plasma membrane cholesterol levels in endothelial cells and underlying smooth muscle cells. Cholesterol is a major component of endothelial cell (EC) plasma membranes, able to pack tightly in environments with sphingolipids and longer chain or saturated phospholipids. The tight packing of these environments allow for less water penetration, making these areas more rigid than the bulk phase of the plasma membrane. Because of this, tightly packed domains are referred to as liquid ordered LO versus liquid disordered (Ld) domains which are more fluid, contain less cholesterol and more phospholipids with unsaturated tails. Ld domains allow for more water penetration. Within endothelial cells there are distinct LO regions in the plasma membrane that are high in cholesterol termed lipid rafts. While lipid rafts lay flush with the rest of the membrane, there exists a subset of lipid rafts called caveolae that are flask-like invaginations of the plasma membrane. The caveolar proteins, caveolins, serve to pinch in the membrane against a thermodynamically favorable conformation of the membrane. Both domains are concentrated with molecules vital to cell function, including G protein-coupled receptors and ion channels and serve to localize many signal processes such as endocytosis and Nitric Oxide (NO) signaling. Changes in cellular cholesterol levels can impact signaling mechanisms by disrupting the integrity of the membrane and spatial organization of signal molecules, leading to the impairment of ion channels or dynamics of membrane proteins in addition to affecting specific interactions between cholesterol and protein channels. Elevated plasma membrane cholesterol levels have been implicated as attenuating store-operated calcium entry (SOCE) upstream of NO production thought to be localized to caveolae. This study contributes to the understanding of cholesterol's role in endothelial dysfunction at the cellular level by developing a methodological approach to studying specificity of cholesterol impairment of SOCE. Changes in membrane fluidity in response to treatment with fluidizing agents and cholesterol manipulations, including a stereoisomer of cholesterol were measured using the properties of the fluorescent dye Laurdan along with novel data analysis techniques termed phasor analysis developed by the Laboratory for Fluorescence Dynamics (LFD). Verification of cholesterol manipulations were performed using liquid chromatography and mass spectroscopy. The effects of treatments on SOCE in bovine aortic endothelial cells (BAECs) were evaluated following stimulation with ATP or pharmacological treatment with thapsigargin. It was found that in BAECs, treatment with cholesterol led to increases between 2 and 3 fold of cellular cholesterol while treatment with an equal concentration of epicholesterol led to replacement of some cholesterol with epicholesterol, but did not increase total sterol concentration. Treatments with empty methyl-beta-cyclodextrin (MBCD) pulled about 20% of cholesterol out of the cells, while alcohol treatment did not significantly change cholesterol concentration. In general, treatments affected EC plasma membrane properties more than in intracellular membranes. Two different alcohols were used as two separate treatments to fluidize the membrane. Treatment of BAECs with benzyl alcohol (BA) led to a decrease in the order of BAEC membranes as did treatment with dodecanol (Dod). However, the fluidity histogram for BA was not the same shape as dodecanol, suggesting that the two alcohols do not fluidize the membrane by the same mechanism. Increasing cellular cholesterol led to an increase in order and dipolar relaxations (DR) while a slight decrease in membrane cholesterol did not significantly change fluidity or DR. Unexpectedly, substitution of some cellular cholesterol with epicholesterol lead to increases in membrane fluidity, although DR were not affected. The effects of treatments on calcium Ca²⁺ signaling were dependent on the presence of extracellular calcium. In response to ATP agonist stimulation in the presence of extracellular calcium, the most interesting findings were that BA completely inhibited the Ca²⁺ response while decreasing cholesterol with MBCD enhanced the response. When using thapsigargin to initiate calcium activated calcium release, the main difference from ATP stimulation was that epicholesterol lowered the Ca²⁺ response and cholesterol lowered it even further. When endoplasmic reticulum Ca²⁺ release was separated from SOCE both cholesterol and epicholesterol treatment attenuated both events in response to ATP and thapsigargin stimulation. Dodecanol only weakened SOCE while BA attenuated both responses, regardless of the agonist used. For BA treated cells, SOCE following ER depletion with no extracellular calcium yielded a greater Ca²⁺ response than stimulation with extracellular Ca²⁺. Based on the results, changes in fluidity alone are not enough to explain the effects of treatments on Ca²⁺ signaling. Benzyl alcohol, dodecanol and epicholesterol all increased fluidity, but yield very different Ca²⁺ signaling in response to ATP and thapsigargin. Increases in cellular cholesterol and subsequent decrease in fluidity led to attenuation of Ca²⁺ signaling except when extracellular Ca²⁺ was present. The unique results from cholesterol treated cells suggests a separate method of attenuation from other treatments. These experiments laid the foundation for further investigation into sterol specific effects on Nitric Oxide (NO) signaling in endothelial cells. Combing effects of treatments on membrane properties with their effects on sterol composition and Ca²⁺ signaling can lead to elucidation of the mechanism by which cholesterol attenuates SOCE, leading to endothelial dysfunction. Overall, insight into the development of the early stages of atherosclerosis can aid in understanding the progression of the disease and help determine possible therapeutic targets.

Mechanism of Cholesterol-Induced Impairment of Store-Operated Calcium Entry in Endothelial Cells

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Mechanism of Cholesterol-Induced Impairment of Store-Operated Calcium Entry in Endothelial Cells

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