Abstract 045: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Endothelial Cells And Lead To Decreased Calcium Levels In The Endoplasmic Reticulum.

Soluble protein oligomers (SPOs) have powerful vascular effects suggesting a link between Alzheimer’s disease (AD) and cardiovascular diseases (CVD). SPOs accumulation induces endoplasmic reticulum (ER) stress hence cell death in several CVDs. Our previous data showed that in mesenteric resistance arteries (MRA), SPOs induce ER stress leading to pathologically exacerbated endothelium-dependent vasodilation. We therefore hypothesized that the SPOs will cause ER calcium dysregulation in endothelial cells isolated from MRA. To confirm the presence of ER stress and SPO subcellular localization, endothelial cells were treated with SPOs (0.1 υM) in the presence and absence of the ER stress inhibitor 4-PBA (2 mM) and vehicle (F-12 media). Immunostaining with KDEL antibody specific for ER resident chaperones Grp78/94 was performed for 19 hr prior to visualization. Endothelial cells treated with SPOs showed increased anti-KDEL fluorescence (AU: Vehicle: 4.74 ± 0.3 vs. SPO: 15.74 ± 1.7; n= 3; p= 0.018, one-way ANOVA). This is consistent with the increased Grp78/94 protein concentration during ER stress. Interestingly, 4-PBA treated cells reduced anti-KDEL fluorescence to the same level as the control (AU: Vehicle: 4.74 ± 0.3 vs. 4-PBA: 8.74 ± 2.9; n= 3; p= 0.39; one-way ANOVA) suggesting that SPOs bind to the ER-anchored proteins during ER stress. Additionally, changes in Ca 2+ concentrations have been implicated as a mediator between ER stress and SPOs. Calcium levels in endothelial cells was investigated by transfecting a plasmid expressing a Ca 2+ indicator pCMV G-CEPIA1 er for 72 hr . Cells were treated with vehicle or SPOs in the presence or absence of 4-PBA (45 min) prior to fluorescence imaging. Cells treated with SPOs+ACh had a decreased fluorescence in the ER from the basal Ca 2+ levels (%Δ Ff-F0/F0; SPO: 53% reduction vs. vehicle), but treatment with 4-PBA+ACh did not improve this response (%Δ F f -F 0 /F 0 ; SPO+4-PBA: 48.18% reduction vs. vehicle n=2). This reveals that SPOs induce ER stress and depletion of ER calcium. However, ER stress inhibitor did not improve Ca 2+ levels. These data suggest that ER stress, is the mechanism by which SPO’s exacerbate vasodilation in the mesenteric resistance artery endothelial cells regardless of changes in Ca 2+ levels.