Original article
Inhibition of endoplasmic reticulum stress and oxidative stress by vitamin D in endothelial cells

https://doi.org/10.1016/j.freeradbiomed.2016.07.020Get rights and content

Highlights

  • Vitamin D and its metabolites/analogs inhibited ER stress in endothelial cells.

  • Similar effects were observed with hyperglycemia-induced ER stress and SO generation.

  • Vitamin D prevented high-dextrose cell death.

  • This suggests that vitamin D has a protective effect on endothelial cells.

Abstract

Endoplasmic reticulum (ER) stress and oxidative stress promote endothelial dysfunction and atherosclerosis. Since vitamin D has been shown in several studies to lower the risk of cardiovascular disease, we examined the effects of vitamin D on ER stress and oxidative stress in endothelial cells. ER stress was measured using the placental secreted alkaline phosphatase assay and oxidative stress was measured by hydroethidine fluorescence. Expression of ER stress markers, including glucose-regulated protein 78, c-jun N-terminal kinase 1 phosphorylation, and eukaryotic initiation factor 2α phosphorylation, as well as X-box binding protein-1 splicing were measured in tunicamycin (TM)-treated human umbilical endothelial cells (HUVEC) treated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and other vitamin D analogs. When TM and 1,25-(OH)2D3 were added simultaneously, 1,25-(OH)2D3 prevented ER stress. However, the effect was much stronger when cells were pre-treated with 1,25-(OH)2D3 for 24-h. However, ER stress was not inhibited by 25-OH vitamin D3 (25-OHD3) or the vitamin D analog EB1089. Both ZK191784 and the vitamin D metabolite 24,25-dihydroxyvitamin D3 were as effective as 1,25-(OH)2D3 in preventing ER stress. Similar effects were observed dextrose-induced stress. All of the compounds tested, except for 25-OHD3, inhibited dextrose-induced (27.5 mM) oxidative stress and ER stress. Although TM with and without 1,25-(OH)2D3 had no effect on VDR expression, inhibition of VDR expression via siRNA prevented 1,25-(OH)2D3, ZK191784, EB1089, and 24,25-dihydroxyvitamin D3 from inhibiting dextrose-mediated SO generation. Furthermore, each vitamin D analog, with the exception of 25-OHD3, prevented dextrose-induced toxicity. These results suggest that vitamin D has a protective effect on vascular endothelial cells.

Introduction

Coronary artery disease is the leading cause of mortality in the United States [1]. Several risk factors predict the development of coronary artery disease, including dyslipidemia and the presence of diabetes, obesity, and the metabolic syndrome [2], [3], [4]. Recently, several studies have shown that plasma vitamin D levels are inversely correlated with atherosclerosis [5], [6], [7]. While it is not clear how vitamin D impacts the development of coronary artery disease, several studies have indicated that part of the mechanism may involve vitamin D's effects on immune cells, especially macrophages [8], [9]. In some trials however, treatment of vitamin d-deficient patients with vitamin D had little or no effect on lowering triglyceride, low-density lipoprotein (LDL) and total cholesterol levels, and raising high-density lipoprotein levels [10], [11].

Endothelial cells form an essential barrier function and are involved in regulating the passage of plasma constituents and immune cells into the extravascular space. Pro-inflammatory cytokines, free-fatty acids, and hyperglycemia have been shown to induce intracellular stress responses in endothelial cells, promoting oxidative stress and endoplasmic reticulum (ER) stress, as well as the production of chemo-attractant molecules such as monocyte chemo-attractant protein-1 (MCP-1), intercellular adhesion molecule 1 (ICAM 1), and vascular cell adhesion molecule 1 (VCAM 1) [12], [13], [14], [15]. Extravasation of monocytes and passage of oxidized LDL through a damaged endothelium allows for the formation of lipid-laden foam cells. Access to the sub-endothelial space also allows for activation of clotting and complement-based injury.

Several circulating factors have been shown to have favorable effects on endothelial barrier function. Antioxidants, estradiol, testosterone, and high-density lipoprotein (HDL) have been shown to inhibit oxidative stress, ER stress, chemo-attractant molecule expression [16], [17], [18], while promoting NO release and relaxation of vascular tone [19], [20]. Since elevated plasma vitamin D levels are inversely correlated with atherosclerosis and cardiovascular disease, we assessed the ability of vitamin D to inhibit ER stress and hyperglycemia-induced oxidative in human primary endothelial cells.

Section snippets

Materials

1,25-dihydroxy vitamin D3 (1,25-(OH)2D3), 25-hydroxyvitamin D3 (25-OHD3), and 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) were obtained from Sigma-Aldrich (St. Louis, MO.) The chemiluminescent alkaline phosphatase substrate 3-(diethylamino)-1-(2,2-dimethyl-3-H-1-benzofuran-7-yl)-propan-1-one (CSPD) was purchased from Clontech Laboratories, Inc. (Mountain View, CA). Lipofectamine and hydroethidine were obtained from Invitrogen (Carlsbad, CA.) Tunicamycin (TM) was purchased from Cayman Chemical

The effect of 1,25-(OH)2D3 on ER stress

To determine if 1,25-(OH)2D3 inhibit ER stress, HUVEC were transfected with pSEAP2.Control and treated with 0.1 μM TM and 50 nM 1,25-(OH)2D3, for 24-h (Fig. 1A). Treatment with TM decreased SEAP activity from 65,332±6183 relative light units (R.L.U.) to 6830±328 R.L.U. (p<0.0001). However, in the presence of 1,25-(OH)2D3, SEAP expression increased from 6830±328 R.L.U. to 16,581±1297 R.L.U.; p<0.0003, relative to TM-treated cells. These results suggest that 1,25-(OH)2D3 inhibits TM induced ER stress

Discussion

Several forms of cell stress have been shown to have critical roles in the initiation and progression of coronary artery disease. Oxidative stress and ER stress have both been implicated in promoting cell death, tissue damage, and organ dysfunction. Though antioxidants have been promoted and tested in clinical trials, they have not been effective at preventing the vascular dysfunction associated with coronary artery disease. This may be due to the presence of either intracellular oxidative

Acknowledgements

We would like to thank Jessica VanOmmeren, Elysia Heilig, Jessie Wang, and Sophie Wang for their assistance in the laboratory. We wish to acknowledge Schering AG (Berlin, Germany) for their generosity in providing the ZK-191784 and LEO Pharma A/S (Ballerup, Denmark) for their generosity in providing the EB1089. This work was funded by a grant from the University of Florida (Grant no. 70740) – Jacksonville.

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