Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

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.

Candidate SNP Markers of Atherogenesis Significantly Shifting the Affinity of TATA-Binding Protein for Human Gene Promoters Show Stabilizing Natural Selection as a Sum of Neutral Drift Accelerating Atherogenesis and Directional Natural Selection Slowing It

“…In addition, there is substitution A>T at position −27 of the HBB promoter (hereinafter: HBB:−27A>T) [80] (not covered by either the "1000 Genomes" project [10] or dbSNP [12]), which causes HBB overexpression that has been clinically associated with the norm of the two above-mentioned biomedical traits [80] (Table 3). Nonetheless, our keyword search via PubMed [38] led to a clinical research article [81] on the heme released by extracellular HBB (after hemolysis), as an accelerator of atherogenesis. That is why we predicted that the known SNP marker HBB:−27A>T of the norm of both malaria resistance and thalassemia [80] can also be a candidate SNP marker of accelerated atherogenesis (Table 3).…”

Role of Gender in Regulation of Redox Homeostasis in Pulmonary Arterial Hypertension

“…Another important finding was that heme could potentially cause endothelial cell dysfunction via increased iron-mediated ROS [124]. Recent research also showed that free heme in cells could trigger endoplasmic reticulum (ER) stress [125] that was also found in patients with PAH [126]. Treating rodents with the chemical chaperone phenyl-butyric acid alleviated ER stress and reduced symptoms of PAH induced by hypoxia [127], indicating that free heme could initiate PAH through ER stress.…”

Nrf2 Plays a Protective Role Against Intravascular Hemolysis-Mediated Acute Kidney Injury

“…UPR compensates stress maintaining cellular homeostasis and avoiding cell death. Heme induces ER stress by expression of activating transcription factor-4 (ATF4) as well as splicing of X-box binding protein-1 (XBP1), promoting upregulation of targets such as CHOP (C/EBP homology protein) (Gall et al, 2018). Administration of transferrin, hemopexin, and haptoglobin resulted in beneficial extravascular/intravascular hemolytic anemias (Buehler and Karnaukhova, 2018).…”