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Heme induces endoplasmic reticulum stress (Hier stress) in human aortic smooth muscle cells

Gáll, Tamás; Pethő, Dávid; Nagy, Annamária; Hendrik, Zoltán; Méhes, Gábor; Potor, László; Gram, Magnus LU ; Åkerström, Bo LU ; Smith, Ann and Nagy, Péter, et al. (2018) In Frontiers in Physiology 9(NOV).
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... (More)

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.

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keywords
Alpha-1-microglobulin, Atherosclerosis, Heme, Heme induced endoplasmic reticulum stress, Hemopexin, Vascular smooth muscle cell
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Frontiers in Physiology
volume
9
issue
NOV
publisher
Frontiers
external identifiers
  • scopus:85057036892
ISSN
1664-042X
DOI
10.3389/fphys.2018.01595
language
English
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yes
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e2a29af4-d7d5-4c0a-931d-758c85efeb9b
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2018-12-04 08:13:04
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2019-03-06 03:00:30
@article{e2a29af4-d7d5-4c0a-931d-758c85efeb9b,
  abstract     = {<p>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.</p>},
  articleno    = {1595},
  author       = {Gáll, Tamás and Pethő, Dávid and Nagy, Annamária and Hendrik, Zoltán and Méhes, Gábor and Potor, László and Gram, Magnus and Åkerström, Bo and Smith, Ann and Nagy, Péter and Balla, György and Balla, József},
  issn         = {1664-042X},
  keyword      = {Alpha-1-microglobulin,Atherosclerosis,Heme,Heme induced endoplasmic reticulum stress,Hemopexin,Vascular smooth muscle cell},
  language     = {eng},
  month        = {11},
  number       = {NOV},
  publisher    = {Frontiers},
  series       = {Frontiers in Physiology},
  title        = {Heme induces endoplasmic reticulum stress (Hier stress) in human aortic smooth muscle cells},
  url          = {http://dx.doi.org/10.3389/fphys.2018.01595},
  volume       = {9},
  year         = {2018},
}