Abstract
Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.
Highlights
Vascular diseases including atherosclerosis remain one of the major causes of death worldwide (Roth et al, 2017)
We demonstrated that oxidation of low density lipoprotein can be highly catalyzed by free heme facilitating modification of plasma apolipoprotein B-100 and the end products of this process lead to vascular endothelial cell injury (Balla G. et al, 1991; Miller and Shaklai, 1994; Li et al, 2006)
Several reports have demonstrated that ER stress and unfolded protein response (UPR) are involved in cardiovascular diseases and multiple risk factors of atherosclerosis such as free cholesterol, 7-ketocholesterol, phospholipolyzed LDL or oxidized LDL have been described to trigger ER stress in macrophages, vascular endothelial cells, and human aortic smooth muscle cells (HAoSMCs) (Pedruzzi et al, 2004; Kedi et al, 2009; Sanson et al, 2009; Gora et al, 2010; Yao et al, 2010; Chistiakov et al, 2014)
Summary
Vascular diseases including atherosclerosis remain one of the major causes of death worldwide (Roth et al, 2017). Atherosclerosis is associated with various pathological manifestations like ischemic heart disease, ischemic stroke, and peripheral arterial disease remaining the leading public health issue (Herrington et al, 2016). A more complete understanding of the pathophysiology would lead to new interventions that are urgently needed especially to treat the advanced, irreversible stage of atherosclerosis. The highly oxidative environment of these complicated lesions contain lipid peroxidation products such as lipid hydroperoxides, oxysterols, aldehydes and carbonyls, which are extremely toxic to cells of the vascular wall. Our group has been interested in this area, especially the role of heme in the pathogenesis of plaque progression (Balla et al, 2007)
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