Ferroptosis Signaling and Regulators in Atherosclerosis

1The main lesion of AS is lipid deposition in the arterial wall, accompanied by proliferation of smooth muscle cells and fibrous matrix, which gradually forms atherosclerotic plaques. AS is the pathological basis of cardiovascular disease, while rupture of unstable atherosclerotic plaques, platelet aggregation and thrombosis can lead to narrowing or occlusion of blood vessels, resulting in acute cardiovascular disease. Inflammation plays an important role in all stages of the atherosclerotic process, especially involved in the formation of unstable plaques.

2Ferroptosis is a regulated cell death dependent on reactive oxygen species (ROS) production and iron overload. The basic mechanism of ferroptosis is the interaction of intracellular free iron with hydrogen peroxide via the Fenton reaction, leading to the depletion of plasma membrane polyunsaturated fatty acids (PUFAs). Ferroptosis is regulated by a variety of cellular metabolic pathways, including redox homeostasis, iron handling, mitochondrial activity and the metabolism of amino acids, lipids and sugars. Metabolic processes that affect cellular susceptibility to ferroptosis include the sulfhydryl-dependent redox system and the mevalonate pathway, while the Cysteine/GSH/GPX4 axis, the NAD(P)H/FSP1/CoQ10 system, and the GCH1/BH4/DHFR system inhibit ferroptosis. The transcription factors (such as p53, Nrf2, ATF3, ATF4, YAP1, HIF1a, EPAS1/HIF2A, BACH1, TFEB, Jun, HIC1 and HNF4a) play multiple roles in the regulation of ferroptosis through transcription-dependent or non-transcriptional mechanisms. In this review, we summarize the signaling pathways of ferroptosis associated with AS in order to provide new ideas for the prevention and treatment of AS.

3Lipid peroxidation, a process of oxidative degradation of lipids, plays a key role in driving ferroptosis. The phospholipid acyl chain remodeling pathway (Lands’ cycle) is critical for achieving ferroptosis. Upon entry of extracellular free diffusible AA/AdA into the cell, acyl-CoA synthetase long chain family member 4 (ACSL4) can activate AA/Ada to generate AA/AdA- CoA, which can then be esterified to AA/AdA-PE by LPCAT3 (lysophosphatidylcholine acyltransferase 3). AA/AdA-PEs are the main target molecules for lipid peroxidation and are localized to the cytoplasmic face of the membrane during ferroptosis. AA/AdA-PE undergoes iron-dependent lipid autoxidation/peroxidation via the Fenton reaction or enzymatic catalysis (e.g., Alox15) to produce phospholipid hydroperoxides (PLOOH), which ultimately leads to the formation of a large number of byproducts, including lipid peroxides (such as 4-hydroxynonenal and malondialdehyde) and breakdown products of oxidized and modified proteins, giving rise to the disruption of membrane integrity and ferroptosis. As an amino acid reverse transporter protein distributed in the phospholipid bilayer consisting of two subunits, SLC7A11 and SLC3A2, system Xc− exchanges cysteine and glutamate intracellularly and extracellularly in a 1:1 ratio. Cysteine is involved in the synthesis of GSH and the function of GPX4 (glutathione peroxidase 4) (Figure 1).

4There are currently two explains about phenotypic features of ferroptosis observed in vitro experiments. One possibility is that the accumulation of PLOOH and breakdown products in the membrane during lipid peroxidation alters the properties of the membrane, leading to the formation of hydrophilic pores and altered permeability of water and other solutes through the cell membrane. Another possibility is that the formation of lipophilic and electrophilic substances during lipid peroxidation act as specific signaling molecules that mediate membrane permeability by modifying downstream molecules (e.g., membrane proteins).

5The regulation of iron homeostasis is particularly important for maintaining overall redox balance. On the one hand, iron is an essential element for physiological processes such as oxygen transport or energy production. On the other hand, overloaded iron acts as a catalyst for redox reactions, which can lead to oxidative stress-induced cytotoxicity. The bioavailability of intracellular iron is regulated by its import, storage and export. Iron is present in two forms, Fe3+ and Fe2+. Fe2+ can generate ROS via the Fenton reaction, leading to the accumulation of lipid peroxides, which is an important factor in the onset of ferroptosis. Extracellular Fe3+ can be bound to transferrin (TF) and transported into the cell via Trans-ferrin Receptor 1 (TFR1) on the cell membrane.

6Fe3+, TF and TFR1 are incorporated into endosomes by endocytosis, while the binding of TF and TFR1 and the binding of iron and TF are solubilized in an acidic environment. Fe3+ is reduced to Fe2+ in the endosome by STEAP3 (iron reductase), which then mediates the release of Fe2+ from the endosome into the cytoplasm via Divalent metal transporter 1 (DMT1/SLC11A2) to form LIP. Cytoplasmic Fe2+ can be reduced by ferritin and stored as Fe3+, and Fe2+ can be transported out of the cell by ferroportin (FPN1/SLC40A1). Ferritin is an important negative regulator of ferroptosis, consisting of the ferritin heavy chain (FTH1) and the ferritin light chain (FTL). FTH1 has ferrous oxidase activity and oxidizes Fe2+ to Fe3+, which is then stored in ferritin (Figure 1).

7Ferritinophagy, the autophagic degradation process of the iron storage protein ferritin, is essential for the regulation of cellular iron levels, and requires the co-regulation of NCOA4 (nuclear receptor coactivator 4) and autophagy. Knockout of NCOA4 inhibited erastin induced ferroptosis, while overexpression significantly enhanced intracellular iron levels promoting ferroptosis. NCOA4 binds to FTH1, autophagic vesicles and autolysosomes, which induce the releases of free iron. Depletion or inhibition of NCOA4 or ATG proteins (e.g., ATG3, ATG5, ATG7 and ATG13) inhibits ferritin degradation, thereby reducing free iron levels and limiting ferroptosis (Figure 1). Lipid peroxides can be produced by three pathways: iron-catalyzed lipid autoxidation, esterification and oxidation of polyunsaturated fatty acids (PUFAs), and the production of lipid ROS associated with the Fenton reaction, all of which require iron.

8AS is characteristic by disorders of lipid metabolism, smooth muscle proliferation, endothelial dysfunction, apoptosis, necrosis, inflammation and the formation of foamy cells and lipid plaques. AS-associated inflammation is mediated by pro-inflammatory cytokines, inflammatory signaling pathways, bioactive lipids and adhesion molecules. The main cells involved in AS are endothelial cells, smooth muscle cells and macrophages, etc. The damage and dysfunction of Endothelial cells is the initiating links in AS, and cholesterol-rich low-density lipoprotein (LDL)is deposited in the inner membrane of the vascular wall of endothelial cells, where it is oxidized to oxidized LDL during increased oxidative stress, which can be recognized by macrophages and other immune cells and destroy endothelial cells. The damaged endothelium is activated and expresses cytokines, chemokines and adhesion molecules that attract circulating monocytes to the atherosclerotic lesions and adhere to the vessel wal. The main immune cells in atherosclerotic lesions are macrophages, which are divided into the pro-inflammatory macrophage phenotype M1 and the anti-inflammatory macrophage phenotype M2. Scavenger receptors (SR) on the macrophage take part in the lipids dynamic balance dependent on a number of enzymes, such as cholesterol acyltransferase-1 (ACAT1) and the cholesterol efflux transporters ABCA1 and ABCG1. Reduced phagocytosis of lipids lead to the conversion of macrophages into foam cells, which induce the formation of plaques. Macrophage-derived foam cell-dominated atherosclerotic plaques are less stable and more likely to rupture.

9Activated NRF2 improves the oxidative stress state of the body and promotes cell survival. Under non-stress conditions, NRF2 is ubiquitinated by the Keap1-CUL3 ubiquitin E3 ligase complex to mark it for degradation by the proteasome. However, redox interference leads to its functional inactivation by modifies of Keap1 when exposed to electrophilic substances or reactive oxygen species. Keap1-CUL3 ubiquitin E3 ligase activity is reduced, which promote the stabilization of NRF2. Stabilized and accumulated NRF2 translocate to the nucleus, forms a heterodimer with a small muscle tendon fibrosarcoma (Smaf), then initiates transcription of genes containing antioxidant response elements (ARE) and activates a range of cytoprotective genes. NRF2 has been shown to regulate the activity of ferroptosis and lipid peroxidation related proteins by three main pathways, iron/metal metabolism, intermediate metabolism and GSH synthesis/metabolism (Figure 2).

10Iron can also be released during heme degradation by HO-1 (heme oxygenase), a stress-inducible enzyme encoded by the Hmox1 gene. HO-1 metabolite performs an essential physiological function in the vascular system. As a Metabolic product, biliverdin is rapidly reduced to bilirubin by biliverdin reductase, which is expressed in two forms: BVRa (predominant in adults and encoded by BLVRA) and BVRb (encoded by BLVRb and predominant in fetal life). Knockdown of BVR induced an increase in NRF2 transcription factor activity and HO-1 levels, accompanied by decrease of cellular heme content and increase of iron content. The binding of NRF2 to the ABCB6 promoter region was first identified in airway epithelial cells of smokers with tissue specificity in the regulation of ABCB6 by NRF2. Iron is transported from the cytoplasm to the mitochondria via mitoferrin 1 (MFRN1), where iron-containing porphyrins are synthesized to decrease iron ions. Up-regulation of ABCB6 promotes iron depletion by exporting iron-containing porphyrins, thereby inhibiting ferroptosis. The lack of ABCB6 in bone marrow cells leads to increased oxidative stress and subsequent release of platelets. Subsequent thrombocytosis increases arterial deposition of the powerful myeloid cell-attracting chemokine CCL5 (RANTES), which ultimately promotes the development of atherosclerotic lesions. In addition to ABCB6, ferrous iron chelatase (FECH) is located in the mitochondria and involved in the biosynthesis of hemoglobin. FECH inserts ferrous iron into protoporphyrin IX to produce hemoglobin cofactor. NRF2 regulates heme synthesis via ABCB6, which is responsible for transporting porphyrins from the cytosol to the mitochondrial membrane gap.

11NRF2 regulates intracellular iron homeostasis by regulating the expression of Tfr1, FTH1, FTL and FPN. It was found that p62 interacting with Keap1, promotes the translocation of NRF2 into the nucleus, up-regulate the expression of FTH, FPN and HO-1, which protects cells from ferroptosis. TfR1 was found to be highly expressed in macrophage-derived foam cells and smooth muscle cells in human carotid plaque. The expression of TfR1 was positively correlated with macrophage infiltration, ectopic lysosomal histone lyase L and ferritin expression. Ox-LDL and pro-inflammatory cytokines promote macrophage iron retention and lipid accumulation, mainly due to NRF2-mediated increased expression of Hmox1 and FPN, a specific cellular phenotype that may be associated with the development of atherosclerotic lesions and plaques instability.

12NRF2 can regulate intracellular redox homeostasis through genes encoding GSH synthesis proteins, including SLC7A11, TXNRD1, GCLC, GCLM, and GSS. SLC7A11 is an essential subunit of system Xc- that regulates the reverse transport of glutamate and cystine. In contrast, NRF2 promotes the expression of SLC7A11. Overexpression of NRF2 or knockdown of Keap1 increases SLC7A11 expression, whereas inhibition of expression of NRF2 or over-expression of Keap1 over-expression decreases SLC7A11 levels. It was found that inhibition of system Xc- induced GSH depletion increased the expression of TXNRD1, but not TXNRD2, compensated for the lack of GSH. The synthesis of GSH is divided into two steps. The first step is catalyzed by GCL (glutamate-cysteine ligase), which consists of GCLC (GCL-catalytic subunit) and GCLM (GCL-modifier subunit), and cysteine combine with glutamate to produce γ-glutamylcysteine. The second step is catalyzed by GSS (Glutathione synthetase), which adds glycine to γ-glutamylcysteine to form γ-glutamylcysteine or GSH. GPX4 converts GSH to GSSG (oxidized glutathione) to reduce lipid hydrogen peroxide (Figure 2).

13Depletion of the antioxidant GSH or ox-LDL lead to the accumulation of intracellular lipid peroxide. Over-expression of GPX4 in ApoE−/− mice attenuates the upregulation of endothelial cell adhesion molecule and monocyte-endothelial cell adhesion, thereby inhibiting the development of AS. Butyric acid treatment of VSMC not only up-regulates the expression of GPX4 but also enhances the catalytic activity of GPX4, which in turn inhibits VSMC proliferation and enhances arterial protective effect. GPX4 also down-regulates lipoxygenase (LOX) and cyclooxygenase (COX) thereby reducing the level of pro-inflammatory factors. GPX4 reduces the release of pro-inflammatory mediators and inflammatory response associated with AS, which has been proved a chronic vascular inflammatory disease.

14The production of GSH (GSH) and thioredoxin (TRX) eliminates peroxides and thus inhibits ferroptosis, while NADPH deficiency will lead to a decrease of GSH and TRX, promoting the accumulation of lipid ROS. The mammalian TRX system is an important reductase system. AS a system that scavenges harmful lipid peroxides, FSP1 was found to be primarily localized in lipid droplets and plasma membranes.

15Further studies have shown that FSP1 reduces coenzyme Q10 via NADPH to inhibit ferroptosis. The NADPH/FSP1/CoQ10 system is therefore a negative regulator of ferroptosis. Glucose produces NADPH via the pentose phosphate pathway (PPP), which positively regulates ferroptosis via key enzyme G6PD. NRF2 can regulate the expression of G6PD (Figure 2).

16The TRX system has been shown to play an important role in the regulation of metabolic processes, insulin signaling, regulation of blood pressure and inflammation. It has been shown that Trx2 protects intravascular homeostasis by balancing mitochondrial ROS production in endothelial cells. Down-regulation of Trx1 inhibits the expression of VCAM-1 and ICAM-1 and blocks the initiation of AS. TRX also modulates macrophage inflammation and polarization. Hypercholesterolemia causes the accumulation of lipid peroxides in the aorta of ApoE−/− mice, and the antioxidant effect of coenzyme Q10 inhibits the development of AS. In addition to the pentose phosphate pathway (PPP), NADPH production also occur through NADH phosphorylation catalyzed by NAD kinase (NADK) and the NADP-dependent conversion of isocitrate to α-KG by isocitrate dehydrogenase (IDH). NRF2 increases NADPH via G6PD, and NADPH acts as an antioxidant to cells by regulating GSH, TXN and CoQ10, which inhibits the development of AS.

17p53 can inhibit the synthesis of GSH by SLC7All in a transcription-dependent manner. p53 increases the expression of GLS2 (glutaminase2) to catalyzes the hydrolysis of glutamine, which increases cellular susceptibility to ferroptosis by downregulating cellular antioxidant through massive hydrolysis of GSH and increasing ROS levels. PTGS2 is a key enzyme in the initiation step of prostaglandin synthesis and regulates the sensitivity of cells to ferroptosis by regulating the levels of key intracellular membrane phospholipids PE. When ferroptosis inducers such as GLS2 and erastin were applied to p53 wild-type cells, the cells exhibited upregulated PTGS2 gene expression and ferroptosis, but when p53-deficient cells were induced with GLS2 and erastin, PTGS2 gene expression levels were unchanged and ferroptosis did not occur. PTGS2 has been used as a marker of ferroptosis.

18Deletion of SAT1, target gene of p53, decreases cell death induced by ROS. SATl increases the expression of ALOX15 (arachidonate-15-lipoxygenase), an iron-dependent PUFA oxidase that increases lipid peroxidation and thus promotes ferroptosis. p53 also promotes cellular ferroptosis by activating ALOX12 (arachidonate-12-lipoxygenase). 12/15-LOX promotes increased trans-endothelial transport of ox-LDL and deposition of ox-LDL in the sub-endothelial space. ALOX12 exhibits higher methylation levels in atherosclerotic plaques, particularly in endothelial cells, which is essential for p53-mediated ferroptosis following stress. The p53 gene also promotes ferroptosis through histone modification with non-dependent of p53. p53 can form a nuclear p53-USP7 protein complex with de-ubiquitin specific peptidase 7 (USP7), further reduces the level of histone H2B monoubiquitylation (H2Bub1, a histone modification) mediated expression of SLC7A11, ultimately leading to ferroptosis.

19MDM2 and MDMX were found to be negative regulators of p53 by regulating FSP1 expression and suggests altering the lipid distribution of cells to promote the development of ferroptosis. In addition, excess iron activates ROS production and induces lipid peroxidation in macrophage-derived foam cells, leading to instability of atherosclerotic plaques. Disturbed iron metabolism in macrophages is involved in the inflammatory response that exacerbates the severity of AS, and iron overload also closely related to the induction of macrophage polarization towards the inflammatory phenotype M1 via the ROS/acetyl-p53 pathway.

20p53 can also regulate cell cycle regulation to induce ferroptosis. p21 can inhibit ferroptosis by increasing intracellular GSH leading to increased synthesis of GPX4, while p53 directly regulates the activation of p21, although the exact mechanism is unclear. Senescence and ROS production in human aortic endothelial cells are accompanied by significant increase in p53 and p21. In addition, p21 also promote VSMC senescence. p53 inhibits ferroptosis by blocking dipeptidyl peptidase 4 (DPP4), a multifunctional serine protease involved in glucose control and a regulator of ferroptosis and lipid metabolism. DPP4 binds to nicotinamide adenine dinucleotide oxidase 1 (NOXl), leading to the production of ROS in cell membranes and plasma, resulting in a large accumulation of intracellular lipid peroxides and ferroptosis.

21The Hippo pathway consists of a series of protein kinase and transcription factors that is highly conserved from lower to higher animal. When the Hippo signaling pathway is shut down, the level of YAP phosphorylation is reduced and translocated into the nucleus, where it binds to the transcription factor TEAD, triggering the expression of a range of genes associated with cell proliferation. When the Hippo signaling pathway is turned on, MST1/2 phosphorylates and increases the interaction between MOB1 and LATS1/2 with the assistance of the co-factor SAV1. Hippo pathway plays an important role in cell growth, proliferation, metabolism and immunity, which has been a hot topic of research in the cardiovascular field (Figure 3).

22The levels of AA (arachidonic acid) and AdA (adrenic acid) are closely related to the occurrence of ferroptosis. AA coenzyme A (AA-CoA) is produced by AA/AdA catalyzed by ACSL4 (acyl-coA synthetase long-chain family member 4) and eicosatetraenoic acid coenzyme A (AdA-CoA), both of which are key substrates for the onset of ferroptosis. Ferroptosis related with cell density in epithelial cells was mediated by E-calmodulin-mediated cell-cell contact, which activates Hippo signaling via the NF2 (also known as Merlin) tumor suppressor protein, thereby inhibiting the activity of the nuclear translocation and transcriptional co-regulator YAP, which can target including ACSL4 and TFR1 to promote ferroptosis.

23NADPH oxidase dysfunction plays an important role in the pathology of development of AS, including endothelial dysfunction, inflammation and vascular remodelling. NADPH oxidase including NOX1, NOX2, NOX3 and NOX4, are the main sources of ROS during atherosclerotic formation. In renal cell carcinoma, at low cell density, activated TAZ promotes the expression of epithelial membrane protein 1 (EMP1), followed by upregulation of NOX4 levels. NOX4 increases the level of intracellular lipid reactive oxygen species and induces ferroptosis. As a subtype of NADPH oxidase, NOX4 was shown to be AS-protective. Hydrogen peroxide released by NOX4 also inhibits the proliferation rate of vascular smooth muscle cells, prevents vascular remodeling and inflammation, and maintains the expression of eNOS under vascular stress.

24In studying the role of TAZ in ferroptosis in ovarian cancer, angiopoietin like 4 was found to be a direct target gene regulated by TAZ, sensitizing ferroptosis through activation of NOX2. More importantly, ANGPTL4 maintains the integrity of the vascular endothelium by regulating vascular permeability, angiogenesis, inflammatory signaling, oxidative stress and lipid metabolism. In addition, ANGPTL4 also regulates cellular energy homeostasis and reactive oxygen species and has been implicated in the pathogenesis of AS, including endothelial dysfunction, LDL oxidation and nitric oxide reduction. Hepatic ANGPTL4 deficiency significantly reduces triacylglycerol (TAG) and cholesterol levels, providing some protection against AS. When the Hippo pathway is closed, nuclear translocation of YAP induces endothelial cell proliferation and inflammation. YAP regulates the expression of ferroptosis-related target genes, including TFR1, ACSL4, EMP1 and ANGPTL4. Ferroptosis is promoted in terms of iron homeostasis, lipid metabolism and redox regulation, but whether ferroptosis is involved between Hippo and AS remains to be investigated.

25ATF3 was found to directly inhibit the expression of SLC7A11 in erastin-induced HT1080 cells, suggesting that ATF3 is a positive regulator of ferroptosis. Endoplasmic reticulum stress promotes the upregulation of ATF3, which increases intracellular H2O2 levels through two pathways: ATF3 activates NOX4 transcription, overproduces of superoxide, inhibits SLC7A11 transcription, and deplets cysteine and GSH. H2O2 increases intracellular Fe2+ and restricts synthesis of GSH, leading to a continuous accumulation of toxic intracellular lipid peroxides.

26Unlike ATF3, ATF4 plays a dual role in ferroptosis. Deletion of ATF4 increases ferroptosis in a variety of cancer cells. Inhibition of ATF4-HSPA5-GPX4 pathway-induced ferroptosis increases the anticancer activity of gemcitabine, which is related that ATF4 induces the binding of HSPA5 to GPX4, protecting against GPX4 protein degradation and subsequent lipid peroxidation. Also, knockdown of SLC1A5 or CHAC1 or DDIT3 inhibited erastin or RSL3 induced ferroptosis. As the first cytoplasmic NADPH phosphatase, MESH1 leads to NADPH dephosphorylation, and the knockdown of MESH1 has been shown to increase NADPH and protect against ferroptosis. The knockdown of MESH1 induces different endoplasmic reticulum stress-related genes, particularly ATF3 gene. The knockdown of ATF4 sensitizes MESH1-deficient RCC4 cells to ferroptosis, suggesting a role in MESH1 knockdown-mediated protection against ferroptosis. The phenotypic similarity of stress tolerance caused by the removal of MESH1 and the accumulation of NADPH is achieved in part through the regulation of ferroptosis by ISR.

27In general, both ATF3 and ATF4 can regulate ferroptosis. ATF3 promotes ferroptosis by depleting intracellular GSH through inhibition of the system Xc-. Knockdown of ATF4 promotes ferroptosis, and protects against degradation of GPX4 through the ATF4-HSPA5-GPX4 pathway. In addition, endoplasmic reticulum stress promotes intracellular H2O2 levels by upregulating ATF3 and enhances intracellular Fe2+ by upregulating the expression of the transferritin receptor, leading to the accumulation of toxic lipid peroxides in cells. whether the high expression of ATF3 in AS is associated with ferroptosis needs to be further explored.

28Inhibition of STAT3 phosphorylation in endothelial cells down-regulates vascular endothelial growth factor (VEGF) expression, inhibits vascular endothelial cell proliferation and migration, and delays the formation of unstable plaques in AS. STAT3 induces the proliferation and migration of vascular smooth muscle cells. STAT3 is involved in regulation of monocyte-to-macrophage differentiation, and inhibition of STAT3 activity decreases inflammation and monocyte-to-macrophage differentiation.

29Protective effect of α6β4 integrin on erastin induced adherent epithelial cells and cancer cells is that α6β4-mediated activation of Src and STAT3 inhibits the expression of ACSL4 and suppresses lipid peroxidation. An earlier study showed that the onset of ferroptosis is not dependent on lysosomes, however, lysosomes play a potential role in promoting ferroptosis. It has been shown that the transcriptional activity of NRF2 and STAT3 may jointly regulate ferroptosis-related gene expression, mainly because increased NRF2 can promote STAT3 phosphorylation, which amplifies downstream signaling and regulates SLC7A11 to inhibit ferroptosis. The NF-κB/IL-6/STAT3 signaling pathway increases hepcidin expression and decreases serum iron and transferrin saturation. Iron in macrophages is regulated by the hormone hepcidin, which reduces FPN-mediated iron export when iron is in sufficient supply. The expression of Hepcidin is associated with BMP signaling and its inhibition in vivo model has protective effect on AS, probably due to the fact that iron affects macrophage polarization and lipid metabolism levels.

30Aberrant activation of STAT3 leads to endothelial cell dysfunction, macrophage polarization, inflammation and immunity, and thus may be an important regulator of the AS process. STAT3 regulates GSH synthesis by regulating SLC7A11 in ferroptosis and also inhibits ACSL4 expression, both of which can suppress the onset of ferroptosis. STAT3 also regulates intracellular iron homeostasis. How STAT3 regulates ferroptosis in AS needs to be further investigated.

31LSH (lymphoid-specific helicase) is a DNA methylation modifier that inhibits the progression of ferroptosis by directly upregulating lipid metabolism genes, including SCD1 (searoyl CoA desaturase) and FADS2 (fatty acid desaturase 2). SCD1 was found to have a rate-limiting effect in catalyzing monounsaturated fatty acid synthesis, which promotes tumor growth, migration and resistance to ferroptosis. SCD1 converts saturated fatty acids into monounsaturated fatty acids (MUFAs) to inhibit ferroptosis. Highly active mutations in PI3K-AKT-mTOR signaling protect cancer cells from oxidative stress and ferroptosis through SREBP1/SCD1-mediated adipogenesis. Inhibition of SCD1 through activation of AMPK/SREBP1 to promote ferroptosis. Research has shown that lead decreases SCD and FADS2 expression, leads to a significant increase in MDA and ROS content in macrophages and may lead to foam cell formation and inflammation. In vitro and in vivo results show that laminar flow protects endothelial cells by increasing endothelial cell SCD1 mRNA expression through a PPARγ-specific mechanism. Up-regulation of SCD-1 leads to desaturation of saturated fatty acids, promotes esterification and storage of saturated fatty acids, which protects HAECs from lipotoxic damage. The absence of SCD1 attenuates adipocyte inflammation and its paracrine regulation in macrophages and endothelial cells. Reduced oleic acid levels are associated with inflammatory regulation of SCD1 deficiency. Hypoxia-inducible factor (HIF) plays a crucial role in regulating the hypoxic response of tumor by altering cellular energy metabolism, including altering the expression of genes related to glucose and lipid metabolism. SCD1 and HIF-2a in human renal tubular epithelial cell lines promote tumorigenesis by maintaining cell survival, triggering cell migration and enhancing proliferation of cancer cells. The over-expression of SCD1 promotes lipid droplet (LD)-lysosome fusion through activation of TFEB nuclear translocation and its activity, which promotes lysosome biosynthesis and inhibits the formation of VSMC foam cell. Regulation of SCD1/TFEB mediated lipophagy may provide new therapeutic avenues for the treatment of AS.

32LSH induces ELAVL1 (ELAV-like) through inactivation the expression of p53 RNA-binding protein. ELAVL1 enhances the expression level of LINC00336 by interacting with LINC00336 (Long noncoding RNA), increases the expression of CBS (cystathionine-β-synthase) and inhibits ferroptosis in lung cancer. DCAF8 and WDR76 were recently reported to act as substrate connectors and molecular inhibitors of the CRL4 (Cullin-4 RING ubiquitin ligase) system for the control of LSH stability. WDR76 competitively inhibits DCAF8-targeted LSH protein hydrolysis for lipid hydroperoxide-induced ferroptosis. LINC00618 decreases the expression of SLC7A11 by interacting with LSH, thereby inhibiting ferroptosis. Furthermore, over-expression of LINC00618 increases the levels of Bax and cleaved caspase-3, promoting apoptosis. Importantly, LINC00618 was found to accelerate ferroptosis via apoptosis, but the potential interaction between ferroptosis and apoptosis remains poorly understood.

33The heat shock response (HSR) is a highly conserved stress response to maintain dynamic protein homeostasis in almost all eukaryotic cells. As molecular chaperone, heat shock proteins (HSPs) are classified mainly on the basis of their molecular size into seven large families: Hsp110, Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small HSPs (approximately 15–30 kDa). HSPB1 (also known as HSP25 in mice and HSP27 in humans) is a small heat shock protein and also HSP27 is an estrogen receptor beta (ER-β)-related protein. HSPB1 reduces cellular iron uptake by down-regulating TFR1. Protein kinase C-mediated phosphorylation of HSPB1 provides protection against ferroptosis by reducing iron-mediated production of lipid reactive oxygen species. Inhibition of the HSF1-HSPB1 pathway and HSPB1 phosphorylation increases erastin induced ferroptosis. This suggests that HSF1-HSPB1 is a negative regulator of ferroptosis.

34HSP27 is a novel biomarker of AS that predicts adverse cardiovascular events. HSP27 prevents the formation of foam cell and plaques in female but not male mice. Intracellular HSPB1 stabilizes plaques by reducing vascular smooth muscle cell apoptosis. Vascular wall infiltration of LDL may reduce VSMC adhesion and migration by inducing changes in HSP27/pHSP27. ROS were found to activate p38/MAPK phosphorylation, mediating the activation of HSF1, which in turn inhibits the development of ferroptosis. Another study showed that the lipid peroxidation metabolite 4-HNE could also mediate the activation of HSF1 through p38/MAPK phosphorylation, promoting the transcription of PROM2 (prominin2) and resisting the development of ferroptosis. Studies have shown that prominin2 promotes resistance to ferroptosis in mammary epithelial cells and breast cancer cells. Mechanistically, prominin2 promotes the formation of ferritin-containing multi-vesicular vesicles (MVBs) and exosomes transports iron out of cells. Oentin-1 inhibits VSMC migration by inactivating the NOX-ROS-p38-HSP27 pathway. PA (Palmitic acid) cause lipotoxic injury in cardiomyocytes, and over-expression of HSF1 restores the disturbance of iron homeostasis by mediating the expression of iron metabolism-related proteins such as TFRC, SLC40A1 and FTH1. Thus HSF1 appears to protect cardiomyocytes from PA induced ferroptosis by regulating the expression of iron-associated proteins and GPX4.

35Heat shock protein A family (HSP70) member 5 (HSPA5, also known as BIP or GRP78) is a member of the HSP70 family and is mainly distributed in the endoplasmic reticulum. As mentioned above, ATF4 also induces the binding of HSPA5 to GPX4, thereby protecting GPX4 protein degradation and inhibiting ferroptosis. HSP70 facilitates over-expression of monocyte adhesion molecules and progression of AS. However, in parallel to the inhibition of HSP90 activity, a reduction in arterial wall inflammation and oxidative stress was also observed in association with increased HSP70 expression. HSP70 induces Treg to act as an anti-inflammatory agent. When proteins carry a specific KFERQ-like motif to be degraded by CMA, GPX4 has a potential CMA-targeting motif that is recognized by HSPA8 and then transferred from the cytoplasm to the lysosomes via LAMP2A, ultimately causing protein degradation. HSP90-mediated LAMP2A protein stability promotes erastin induced ferroptosis by forming the HSPA8-LAMP2A-GPX4 protein complex to promote CMA-mediated GPX4 degradation. LAMP2A and HSPA8-mediated CMA promote erastin induced GPX4 degradation via lysosomes, and HSP90 has been shown to increase the stability of LAMP2A protein, thereby linking autophagy and ferroptosis, known as chaperone-mediated autophagy.

36Over-expression of HSP90 is associated with the characteristics of plaque instability. Inhibition of HSP90 reduces inflammatory responses and oxidative stress. HO-1 protein transcription is also regulated by HSF1 and NRF2. And HSF1 is also involved in nuclear translocation and stabilization of p53. Over-expression of HSP27 is protective against AS and is also a negative regulator of ferroptosis. Although HSP70 can inhibit ferroptosis via the ATF4-HSPA5-GPX4 pathway, the roles of HSP70 in AS are currently controversial.

37The pathogenesis of AS is complex and involves three major cell types including endothelial cells, smooth muscle cells and macrophages. The development of ferroptosis is associated with restricted GSH synthesis, disturbances in iron homeostasis, accumulation of lipid peroxides and fatty acid synthesis, which are also closely linked to the development of AS. In this review, we describe the role of ferroptosis signalling pathways and transcription factors and other regulatory molecules in the development of ferroptosis, with a focus on the linkage of these signalling pathways and transcription factors and other regulatory factors between ferroptosis and AS. The blood vessel is not just a simple anatomical organ, but one with complex functions. AS is the basis for many cardiovascular pathologies, including myocardial infarction and cerebral infarction, and ferroptosis also plays a very important role in many systemic cardiovascular diseases. Current research into ferroptosis in AS is still at a very early stage. What role does iron death play in the pathogenesis of atherosclerosis, and in the involvement of other organ lesions based on atherosclerosis. We hope that this review will provide new insights into the relationship between AS and ferroptosis.