Elsevier

Life Sciences

Volume 284, 1 November 2021, 119935
Life Sciences

HMOX1 upregulation promotes ferroptosis in diabetic atherosclerosis

https://doi.org/10.1016/j.lfs.2021.119935Get rights and content

Abstract

Objective

Atherosclerotic vascular disease remains the principal cause of death and disability among patients with type 2 diabetes. Unfortunately, the problem is not adequately resolved by therapeutic strategies with currently available drugs or approaches that solely focus on optimal glycemic control. To identify the key contributors and better understand the mechanism of diabetic atherosclerotic vascular disease, we aimed to elucidate the key genetic characteristics and pathological pathways in atherosclerotic vascular disease through nonbiased bioinformatics analysis and subsequent experimental demonstration and exploration in diabetic atherosclerotic vascular disease.

Methods and results

Sixty-eight upregulated and 23 downregulated genes were identified from the analysis of gene expression profiles (GSE30169 and GSE6584). A comprehensive bioinformatic assay further identified that ferroptosis, a new type of programmed cell death and HMOX1 (a gene that encodes heme oxygenase), were vital factors in atherosclerotic vascular disease. We further demonstrated that diabetes significantly increased ferroptosis and HMOX1 levels compared to normal controls. Importantly, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated diabetic atherosclerosis, suggesting the causative role of ferroptosis in diabetic atherosclerosis development. At the cellular level, Fer-1 ameliorated high glucose high lipid-induced lipid peroxidation and downregulated ROS production. More importantly, HMOX1 knockdown attenuated Fe2+ overload, reduced iron content and ROS, and alleviated lipid peroxidation, which led to a reduction in ferroptosis in diabetic human endothelial cells.

Conclusions

We demonstrated that HMOX1 upregulation is responsible for the increased ferroptosis in diabetic atherosclerosis development, suggesting that HMOX1 may serve as a potential therapeutic or drug development target for diabetic atherosclerosis.

Introduction

Cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide [1]. Reducing atherosclerotic cardiovascular disease is a major clinical imperative. However, when the risks of atherosclerosis concordance with diabetes, the development of atherosclerosis is accelerated and more diffusely distributed than in individuals without diabetes [2], [3], [4]. Metabolic disorders are closely involved in the pathogenesis of atherosclerosis [5], especially in the arterial wall and front line endothelial cells (ECs). To explore an effective treatment strategy for diabetic atherosclerosis, it is important to dissect pathogenic mechanisms and identify meaningful targets.

Accelerated diabetic atherosclerosis occurs when oxidized low-density lipoproteins (oxLDLs) or oxidized lipids trapped in the vessel wall induce the overlying ECs to express adhesion molecules and cytokines, promoting the recruitment of monocytes and lymphocytes to the vessel wall [6], [7]. Although oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphatidylcholine (Ox-PAPC, a component in atherosclerotic lesions [8]) is a critical factor in atherosclerosis [9], it is also a key factor associated with diabetic atherosclerosis development [10], [11]. However, the precise pathological alterations in the response of ECs to diabetic risk factors and the underlying mechanism are still poorly understood. Hence, we attempted to clarify the genetic characteristics and signaling pathways to study how atherosclerosis affects front-line ECs in human samples and then determine whether the identified genes are involved in diabetes. Completing these experiments will be helpful to enhance our understanding of diabetic atherosclerosis and to identify novel interventions preventing diabetic atherosclerosis.

We built our investigation on gene microarray technology (mRNA expression profiles), which has been widely applied in recent decades [12], [13]. In recent years, numerous studies on the gene expression profile of atherosclerosis have revealed hundreds of differentially expressed genes (DEGs), which are the basis for gene regulatory network analysis [13], [14] and avoid limitations or inconsistencies due to tissue or sample heterogeneity. In this study, we conducted in-depth analysis of two microarray datasets, GSE30169 [15] and GSE6584 [16], from the Gene Expression Omnibus database (GEO) [7] in an attempt to identify the candidate genes and pathological pathways that may contribute to diabetic atherosclerotic endothelial cell injury and could be used to prevent diabetic atherosclerosis. Moreover, the causative roles of genes identified from these databases were investigated by in vitro and in vivo diabetic animal models.

Therefore, the objectives of this study were first to identify the EC injury mode in the development of diabetic atherosclerosis and second to investigate the candidate genes and the mechanisms responsible for the development of diabetic atherosclerosis.

Section snippets

Screening of DEGs and performing pathway enrichment analysis

The GSE30169 and GSE6584 gene expression profiles and their relevant platform annotation files were obtained from the GEO database. The GSE30169 dataset was submitted by Professors Romanoski and Lusis on June 23, 2011, last updated on January 17, 2017, and stockpiled on the GPL3921 platform (HT_HG-U133A) Affymetrix HT Human Genome U133A Array (Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA). [17] GSE30169 consisted of 629 samples from primary human aortic endothelial cells (HAECs)

Bioinformatic assays of human atherosclerotic vascular injury identified ferroptosis as an important form of cell death

To identify potential novel mechanisms contributing to atherosclerotic vascular injury in a nonbiased fashion, we first gathered information from a human bioinformatic database to identify the major form of cell death in the human atherosclerotic vascular injury database (gene expression profiles of GSE30169 and GSE6584 obtained from the NCBI-GEO free database). With pĀ <Ā 0.05 and [log2 FC]Ā ā‰„Ā 0.6 as cutoff criteria, we extracted 166 and 2604 differentially expressed genes (DEGs), respectively,

Discussion

In the present study, we reported that ferroptosis contributes to the progression of diabetic atherosclerosis. The increase in HMOX1 contributes to endothelial cell ferroptosis by promoting iron overload, ROS generation and lipid peroxide (Fig. 8). A diabetic increase in HMOX-1 indicates that HMOX-1 is a novel marker for diabetic endothelial dysfunction.

Atherosclerosis is the pathological basis of diabetic macrovascular complications, and it can evaluate the severity of macroangiopathy in T2DM.

Conclusions

Utilizing nonbiased in silico analysis followed by in vitro molecular investigation and in vivo concept-proven demonstration, we demonstrate that HMOX1 upregulation and resultant ferroptosis are involved in diabetic atherosclerosis. These results suggest that interventions blocking ferroptosis, such as Fer-1 administration and genetic/pharmacologic HMOX1 inhibition, may be novel approaches to attenuate diabetic atherosclerosis.

The following is the supplementary data related to this article.

Declaration of competing interest

The authors have declared that no competing interest exists.

Acknowledgements

This work was supported by the Scientific Research Project of the Shanxi Provincial Department of Health (Grant No. 2018023); Natural Science Foundation of China (81700327, 81970391, 82000799); Shanxi ā€œ1331 Projectā€ Key Subjects Construction (1331KSC); Shanxi ā€œSanjin Scholarsā€ Program; Key Laboratory of Cellular Physiology (Shanxi Medical University); Applied Basic Research Program of Shanxi Province (201801D221269); Scientific and Technological Innovation Programs of Higher Education

References (69)

  • D.P. Jones

    Redox potential of GSH/GSSG couple: assay and biological significance

    Methods Enzymol.

    (2002)
  • A.M. Pisoschi et al.

    The role of antioxidants in the chemistry of oxidative stress: a review

    Eur. J. Med. Chem.

    (2015)
  • A. Konstorum et al.

    Systems biology of ferroptosis: a modeling approach

    J. Theor. Biol.

    (2020)
  • K. Sakamoto et al.

    Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis

    Biochem. Biophys. Res. Commun.

    (2017)
  • O. Sakai et al.

    Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition

    Pathophysiology

    (2017)
  • W.S. Yang et al.

    Regulation of ferroptotic cancer cell death by GPX4

    Cell

    (2014)
  • B.R. Stockwell et al.

    A regulated cell death nexus linking metabolism, redox biology, and disease

    Cell

    (2017)
  • R.P. Brandes et al.

    NADPH oxidases in cardiovascular disease

    Free Radic. Biol. Med.

    (2010)
  • S. Xu

    Iron and atherosclerosis: the link revisited

    Trends Mol. Med.

    (2019)
  • E. Gianazza et al.

    Lipoxidation in cardiovascular diseases

    Redox Biol.

    (2019)
  • F. Wunderer et al.

    The role of hepcidin and iron homeostasis in atherosclerosis

    Pharmacol. Res.

    (2020)
  • G.Z. Liu et al.

    High glucose/High lipids impair vascular adiponectin function via inhibition of caveolin-1/AdipoR1 signalsome formation

    Free Radic. Biol. Med.

    (2015)
  • Q. Yao et al.

    Curcumin protects against diabetic cardiomyopathy by promoting autophagy and alleviating apoptosis

    J. Mol. Cell. Cardiol.

    (2018)
  • P. Joseph et al.

    Reducing the global burden of cardiovascular disease, part 1: the epidemiology and risk factors

    Circ. Res.

    (2017)
  • J. Walpot et al.

    Left ventricular mass is independently related to coronary artery atherosclerotic burden: feasibility of cardiac computed tomography to detect early geometric left ventricular changes

    J. Thorac. Imaging

    (2021)
  • P.J. Talmud

    How to identify gene-environment interactions in a multifactorial disease: CHD as an example

    Proc. Nutr. Soc.

    (2004)
  • A. Poznyak et al.

    The diabetes mellitus-atherosclerosis connection: the role of lipid and glucose metabolism and chronic inflammation

    Int. J. Mol. Sci.

    (2020)
  • A.J. Lusis

    Atherosclerosis

    Nature

    (2000)
  • J.A. Berliner et al.

    A role for oxidized phospholipids in atherosclerosis

    N. Engl. J. Med.

    (2005)
  • X. Que et al.

    Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice

    Nature

    (2018)
  • Y. Guo et al.

    Large scale comparison of gene expression levels by microarrays and RNAseq using TCGA data

    PloS one

    (2013)
  • X. Zhang et al.

    Potentially critical roles of TNPO1, RAP1B, ZDHHC17, and PPM1B in the progression of coronary atherosclerosis through microarray data analysis

    J. Cell. Biochem.

    (2019)
  • X. Tan et al.

    Identification of key pathways and genes in advanced coronary atherosclerosis using bioinformatics analysis

    Biomed. Res. Int.

    (2017)
  • N.T. Hogan et al.

    Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells

    elife

    (2017)
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