Macrophage Ferroptosis Promotes MMP2/9 Overexpression Induced by Hemin in Hemorrhagic Plaque

Abstract

Background Intra-plaque hemorrhage (IPH) leads to rapid plaque progression and instability through upregulation of matrix metalloproteinases (MMPs) and collagen degradation. Hemoglobin-derived hemin during IPH promotes plaque instability. We investigated whether hemin affects MMP overexpression in macrophages and explored the underlying mechanisms.

Material and Methods In vivo, hemorrhagic plaque models were established in rabbits and ApoE^−/− mice. Ferrostatin-1 was used to inhibit ferroptosis. Plaque size, collagen, and MMP2/9 levels were evaluated using immunohistochemistry, H&E, Sirius Red, and Masson staining. In vitro, mouse peritoneal macrophages were extracted. Western blot and ELISA were used to measure MMP2/9 levels. Bioinformatics analysis investigated the association between MMPs and ferroptosis pathway genes. Macrophage ferroptosis was assessed by evaluating cell viability, lipid reactive oxygen species, mitochondrial ultrastructure, iron content, and COX2 levels after pretreatment with cell death inhibitors. Hemin's impact on ferroptosis and MMP expression was studied using Ferrostatin-1 and SB202190.

Results In the rabbit hemorrhagic plaques, hemin deposition and overexpression of MMP2/9 were observed, particularly in macrophage-enriched regions. In vitro, hemin induced ferroptosis and MMP2/9 expression in macrophages. Ferrostatin-1 and SB202190 inhibited hemin-induced MMP2/9 overexpression. Ferrostatin-1 inhibited p38 phosphorylation in macrophages. Ferostatin-1 inhibits macrophage ferroptosis, reduces MMP2/9 levels in plaques, and stabilizes the hemorrhagic plaques.

Conclusion Our results suggested that hemin-induced macrophage ferroptosis promotes p38 pathway activation and MMP2/9 overexpression, which may play a crucial role in increasing hemorrhagic plaque vulnerability. These findings provide insights into the pathogenesis of hemorrhagic plaques and suggest that targeting macrophage ferroptosis may be a promising strategy for stabilizing vulnerable plaque.

Keywords

intra-plaque hemorrhage - ferroptosis - macrophage - hemin - matrix metalloproteinases

Authors' Contribution

Y.T. and B.L. contributed to the conception of the study; B.L., M.L., H.W. and S.S. performed the experiment; S.G. and H.W. contributed significantly to analysis and manuscript preparation; Y.T., B.L. and J.L. performed the data analyses and wrote the manuscript.

^* These authors contributed equally to this work.

Publication History

Received: 01 March 2023

Accepted: 06 September 2023

Accepted Manuscript online:
11 September 2023

Article published online:
09 October 2023

• References

• 1 Guo L, Harari E, Virmani R, Finn AV. Linking hemorrhage, angiogenesis, macrophages, and iron metabolism in atherosclerotic vascular diseases. Arterioscler Thromb Vasc Biol 2017; 37 (04) e33-e39
• 2 Turc G, Oppenheim C, Naggara O. et al; HIRISC study investigators. Relationships between recent intraplaque hemorrhage and stroke risk factors in patients with carotid stenosis: the HIRISC study. Arterioscler Thromb Vasc Biol 2012; 32 (02) 492-499
• 3 Zhao G, Tang X, Tang H. et al. Recent intraplaque hemorrhage is associated with a higher risk of ipsilateral cerebral embolism during carotid artery stenting. World Neurosurg 2020; 137: e298-e307
• 4 Li B, Gong J, Sheng S. et al. Sonodynamic therapy reduces iron retention of hemorrhagic plaque. Bioeng Transl Med 2020; 6 (01) e10193
• 5 Tziakas DN, Chalikias G, Pavlaki M. et al. Lysed erythrocyte membranes promote vascular calcification. Circulation 2019; 139 (17) 2032-2048
• 6 Jinnouchi H, Guo L, Sakamoto A. et al. Diversity of macrophage phenotypes and responses in atherosclerosis. Cell Mol Life Sci 2020; 77 (10) 1919-1932
• 7 Li B, Gong J, Sheng S. et al. Increased hepcidin in hemorrhagic plaques correlates with iron-stimulated IL-6/STAT3 pathway activation in macrophages. Biochem Biophys Res Commun 2019; 515 (02) 394-400
• 8 Hu S, Hua Y, Keep RF, Feng H, Xi G. Deferoxamine therapy reduces brain hemin accumulation after intracerebral hemorrhage in piglets. Exp Neurol 2019; 318: 244-250
• 9 Chistiakov DA, Orekhov AN, Bobryshev YV. Contribution of neovascularization and intraplaque haemorrhage to atherosclerotic plaque progression and instability. Acta Physiol (Oxf) 2015; 213 (03) 539-553
• 10 Zille M, Karuppagounder SS, Chen Y. et al. Neuronal death after hemorrhagic stroke in vitro and in vivo shares features of ferroptosis and necroptosis. Stroke 2017; 48 (04) 1033-1043
• 11 Imoto S, Kono M, Suzuki T. et al. Haemin-induced cell death in human monocytic cells is consistent with ferroptosis. Transfus Apheresis Sci 2018; 57 (04) 524-531
• 12 Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 2021; 22 (04) 266-282
• 13 Wu X, Li Y, Zhang S, Zhou X. Ferroptosis as a novel therapeutic target for cardiovascular disease. Theranostics 2021; 11 (07) 3052-3059
• 14 Bai T, Li M, Liu Y, Qiao Z, Wang Z. Inhibition of ferroptosis alleviates atherosclerosis through attenuating lipid peroxidation and endothelial dysfunction in mouse aortic endothelial cell. Free Radic Biol Med 2020; 160: 92-102
• 15 Yang K, Song H, Yin D. PDSS2 inhibits the ferroptosis of vascular endothelial cells in atherosclerosis by activating Nrf2. J Cardiovasc Pharmacol 2021; 77 (06) 767-776
• 16 Johnson JL. Metalloproteinases in atherosclerosis. Eur J Pharmacol 2017; 816 (09) 93-106
• 17 Zhang J, Dong B, Hao J, Yi S, Cai W, Luo Z. LncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in intracerebral hemorrhage rats by activating the TWEAK/Fn14/STAT3 pathway. Life Sci 2019; 237: 116929
• 18 Kolodgie FD, Gold HK, Burke AP. et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med 2003; 349 (24) 2316-2325
• 19 Chen YC, Bui AV, Diesch J. et al. A novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries using gene and microRNA expression profiling. Circ Res 2013; 113 (03) 252-265
• 20 Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol 2008; Chapter 14: 14.1.1-14.1.14
• 21 Sun X, Guo S, Yao J. et al. Rapid inhibition of atherosclerotic plaque progression by sonodynamic therapy. Cardiovasc Res 2019; 115 (01) 190-203
• 22 Chen X, Zhang B, Liu T. et al. Liproxstatin-1 attenuates morphine tolerance through inhibiting spinal ferroptosis-like cell death. ACS Chem Neurosci 2019; 10 (12) 4824-4833
• 23 Wang H, Yang Y, Sun X. et al. Sonodynamic therapy-induced foam cells apoptosis activates the phagocytic PPARγ-LXRα-ABCA1/ABCG1 pathway and promotes cholesterol efflux in advanced plaque. Theranostics 2018; 8 (18) 4969-4984
• 24 Porcu M, Anzidei M, Suri JS. et al. Carotid artery imaging: the study of intra-plaque vascularization and hemorrhage in the era of the “vulnerable” plaque. J Neuroradiol 2020; 47 (06) 464-472
• 25 Brown BA, Williams H, George SJ. Evidence for the involvement of matrix-degrading metalloproteinases (MMPs) in atherosclerosis. Prog Mol Biol Transl Sci 2017; 147: 197-237
• 26 Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol 2021; 18 (05) 280-296
• 27 Li Q, Han X, Lan X. et al. Inhibition of neuronal ferroptosis protects hemorrhagic brain. JCI Insight 2017; 2 (07) e90777
• 28 Ye Q, Zeng C, Luo C, Wu Y. Ferrostatin-1 mitigates cognitive impairment of epileptic rats by inhibiting P38 MAPK activation. Epilepsy Behav 2020; 103 (Pt A): 106670
• 29 Zhang J, Ma CR, Hua YQ. et al. Contradictory regulation of macrophages on atherosclerosis based on polarization, death and autophagy. Life Sci 2021; 276: 118957
• 30 Michel JB, Virmani R, Arbustini E, Pasterkamp G. Intraplaque haemorrhages as the trigger of plaque vulnerability. Eur Heart J 2011; 32 (16) 1977-1985 , 1985a, 1985b, 1985c
• 31 Menon AV, Liu J, Tsai HP. et al. Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease. Blood 2022; 139 (06) 936-941
• 32 Ma JL, Yang PY, Rui YC, Lu L, Kang H, Zhang J. Hemin modulates cytokine expressions in macrophage-derived foam cells via heme oxygenase-1 induction. J Pharmacol Sci 2007; 103 (03) 261-266
• 33 Jeney V, Balla G, Balla J. Red blood cell, hemoglobin and heme in the progression of atherosclerosis. Front Physiol 2014; 5: 379
• 34 Gáll T, Balla G, Balla J. Heme, heme oxygenase, and endoplasmic reticulum stress-a new insight into the pathophysiology of vascular diseases. Int J Mol Sci 2019; 20 (15) 3675
• 35 Mou Y, Wang J, Wu J. et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer. J Hematol Oncol 2019; 12 (01) 34
• 36 Imoto S, Shibuya Y, Kono M. et al. After haemin treatment intracellular non-haem iron increases prior to haem oxygenase-1 induction: a study in human monocytic cell line THP-1. Transfus Apheresis Sci 2019; 58 (06) 102662
• 37 Woo JH, Choi YS, Choi JH. Iron-storage protein ferritin is upregulated in endometriosis and iron overload contributes to a migratory phenotype. Biomedicines 2020; 8 (11) 454
• 38 Reustle A, Torzewski M. Role of p38 MAPK in atherosclerosis and aortic valve sclerosis. Int J Mol Sci 2018; 19 (12) 3761
• 39 Hattori K, Ishikawa H, Sakauchi C, Takayanagi S, Naguro I, Ichijo H. Cold stress-induced ferroptosis involves the ASK1-p38 pathway. EMBO Rep 2017; 18 (11) 2067-2078