The major findings of this study are as follows: 1) CSE induced cell death in VSMCs in a dose- and time-dependent manner, which was entirely abolished by ferroptosis-specific inhibitors or iron chelator, but not by inhibitors of apoptosis, pyroptosis, or necroptosis; 2) CSE-induced VSMC death was also partially abolished by the GSH precursor NAC; 3) CSE increased lipid peroxidation and Ptgs2 mRNA expression, which are key features of ferroptosis; 4) CSE markedly decreased
33 intracellular GSH levels; 5) ACR and MVK, major cytotoxic factors in CSE, induced VSMC ferroptosis; and 6) CSE induced severe damage and loss of medial VSMCs in ex vivo cultured aortas. These findings identified that ferroptosis is the mechanism responsible for CSE-induced cytotoxicity in VSMCs, and demonstrated that CSE- induced ferroptosis contributes to the development of AA and AD. Since cigarette smoke is a major risk factor for these aortic disorders, our study also suggests that ferroptosis is a novel therapeutic target for preventing cigarette smoke-related aortic disorders.
Cigarette smoking has a substantial impact on the development of AA and AD.
However, the mechanism by which smoking induces AA and AD is not yet fully understood. The loss of medial VSMCs at the arterial wall is one of the key pathophysiologic features in AA and AD, and presumably leads to arterial wall weakening and subsequent dilatation and dissection [54]. In this regard, while CSE has been shown to induce cytotoxicity in various types of cells [31], the precise mechanism of CSE-induced cytotoxicity is not yet fully understood. In the present study, we identified ferroptosis as the mechanism that is responsible for CSE-induced VSMC cytotoxicity. Previous studies suggested that apoptosis is involved in this process;
however, the presence of apoptosis in CSE-induced cell death has been questioned [35].
Indeed, we observed that CSE-induced cell death was not inhibited by inhibitors of apoptosis (Z-VAD) or other forms of cell death, such as pyroptosis (Z-YVAD) and necroptosis (Nec-1).
On the other hand, CSE-induced cell death was significantly inhibited by an iron chelator (DFO), GSH precursor (NAC), and NADPH oxidase inhibitor (DPI), which is in accordance with the notion that iron-dependent lipid peroxidation plays a critical role in the initiation of ferroptosis [37]. The TEM findings showing prominent alterations in mitochondrial morphology also support the idea that ferroptosis is involved in CSE-
34 induced cell death. Consistent with our findings, Park et al. [55] recently reported that whole cigarette smoke condensates might induce ferroptosis in bronchial epithelial cells by a KEGG pathway analysis. Yoshida et al. [42] also recently reported that CSE induces ferroptosis in lung epithelial cells, contributing to chronic obstructive pulmonary diseases' pathogenesis. Collectively, ferroptosis plays a pivotal role in cigarette smoking-related cytotoxicity in VSMCs and lung epithelial cells.
In addition to VSMC death, inflammation has been shown to play an essential role in AA's pathogenesis [56]. We determined that the expression of inflammatory cytokines is elevated in VSMCs soon after CSE treatment, suggesting that CSE stimulates chemokine release, which recruits inflammatory cells, such as monocytes/macrophages neutrophils, to the aortic wall and further enhances inflammatory responses. CSE-induced ferroptosis also triggers the extracellular release of cellular contents, such as nucleic acid and high mobility group box 1 (HMGB1), known as danger signals [57]. The released danger signals are then recognized by innate immune receptors, including Toll-like receptors on recruited macrophages, and eventually, potentiate inflammation.
Degradation of ECM by MMPs are well-established changes and contribute to the pathogenesis of AA. Histologically, elastolysis is one of the earliest observable events in aneurysmal tissue. MMP-2 and -9 are appeared to play an important role in elastic lamellae fragmentation [58]. Previous studies indicated that MMP-2 is the dominant gelatinase in small-sized aneurysms, and MMP-9 becomes important in the later stages of aneurysm formation. We had found that both MMP-2 and -9 mRNA expression levels were significantly elevated after CSE treatment in VSMCs.
Furthermore, this elevation was inhibited by Fer-1 treatment.
It is crucial to consider the activity of MMPs in relation to their inhibitor TIMPs during aneurysm progression. In previous studies, significant amounts of TIMP-1 and -
35 2 mRNA expression have been detected in human AAs [59]. In addition, increased expression levels of TIMP-1 are shown to be highly correlated to medium-large human AAs. In our experimental setting significant increase of TIMP-1 mRNA expression after CSE treatment had been observed. This results regarding the increase of MMP-2, -9, and TIMP-1 mRNA expression in VSMCs treated with CSE might indicate the importance of cigarette smoking on proteolytic state of AA.
Interestingly, CSE-induced upregulation of these cytokines and MMPs was inhibited by Fer-1. Although Fer-1 was previously thought to be a lipoxygenase (LOX) inhibitor, it is now considered to be a radical trapping agent in membrane phospholipid [60]. In this regard, lipid peroxidation has been shown to regulate redox-sensitive transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein-1 (AP-1), which play a crucial role in inflammatory cytokine expression [61]. Taken together, CSE-induced inflammation and matrix degradation contribute to the development of AA and AD.
GPX4 converts lipid hydroperoxides to nontoxic lipid alcohol, which utilizes GSH as a cosubstrate. Pharmacological inhibition or genetic deletion of GPX4 promotes ferroptosis. RSL-3 is a small molecule that is a well-known ferroptosis inducer through its direct inhibitory effect on GPX4, resulting in lipid ROS accumulation. Moreover, system Xc inhibitor Erastin could induce ferroptosis through inhibiting GSH synthesis.
We did not find a change in GPX4 protein level after CSE treatments. Moreover, GPX4 overexpression in VSMCs did not rescue CSE-induced ferroptosis. Indeed, CSE treatment had exhausted the intracellular GSH level even before the cellular morphologic changes occur, suggesting that GSH depletion is the primary mechanism involved in the induction of VSMC ferroptosis. Moreover, this result is consistent with the study describing cigarette smoke-induced depletion of GSH, reducing the defenses against the oxidant-induced cellular injury, and causes an increase of cell death [62].
36 Furthermore, previous studies have suggested that ACR and MVK, primary cytotoxic compounds in CSE, can form conjugates with GSH rapidly [63-64]. In line with previous findings, we observed depletion of GSH in response to CSE exposure within one hour. Furthermore, NAC treatment rescued cells from death, further supporting the notion that GSH levels are a critical determinant for ferroptosis.
Our ex vivo experiment results showed that CSE caused medial VSMC loss, which was determined by EVG, and further confirmed by electron microscopy analysis.
TEM results revealed severe mitochondrial damage and myofibril loss in medial VSMCs of CSE-treated aortas. All of these manifestations were partially restored by Fer-1. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death and suggest that ferroptosis is a potential therapeutic target for preventing AA and AD.
The cigarettes used to generate CSE contains 17 mg tar per cigarette [31], indicating that the dosage of CSE (0.8 mg/mL) used in this study is approximately 235 times higher than that of one cigarette if the smoke was absorbed and distributed in 5000 mL blood. In general, chronic smokers are known to smoke more than 20 cigarettes per day, and thus the CSE dosage is just ten times higher than that in the blood of chronic smokers. In this regard, lipid peroxidants, a hallmark of ferroptosis, are increased in the plasma, urine, and arterial tissue of chronic smokers. Moreover, ACR and MVK have been reported to be stable and cumulative [34]. Therefore, we assume that CSE-induced ferroptosis can occur in the aortic walls of chronic smokers and contribute to AA and AD development.
We observed an increase in ferroptotic VSMCs and the corresponding decrease in medial thickness in CSE treated ex-vivo aortas in contrast to control aortas. Previous studies were demonstrated that VSMC apoptosis is the plausible mechanism of medial
37 layer degeneration [65-66]. However, in this study, we had found that VSMC ferroptosis might occur in chronic smokers' aortic walls.
Lipid peroxidation is one of the hallmark events that occur during ferroptosis. In association with this, our results also showed high levels of lipid peroxidation in both in VSMCs and in ex vivo aorta treated with CSE, which were significantly inhibited by Fer-1. According to previous studies, these results showed that lipid peroxidation occurred in greater extents in smokers than non-smokers [67-68]. This phenomenon might also further confirm ferroptosis involvement in aortic medial VSMC loss due to cigarette smoking.
Several limitations of this study should be noted. First, we clearly showed that CSE induced VSMC ferroptosis in vitro and caused medial VSMC loss in ex vivo aortas;
however, the in vivo effect of CSE on the development of aortic aneurysm remains to be examined. Although no previous report has described animal models of AA and AD induced by CSE treatment, chronic CSE treatment for 2–5 months has been shown to accelerate atherogenesis in atherosclerosis-prone mice and rabbits [69-70]. Interestingly, the atherosclerotic lesions in CSE-treated rabbits were significantly decreased by treatment with vitamin E [70], a potent inhibitor of ferroptosis [71]. Moreover, Sawada et al. [72] reported that iron is involved in abdominal AA formation's pathophysiology with oxidative stress and inflammation. Thus, it is likely that ferroptosis is involved in the development of smoking-related atherosclerosis and AA. Second, although we showed that intracellular GSH depletion after CSE exposure leads to lipid peroxidation, the subcellular locations, such as plasma membrane, mitochondria, and endoplasmic reticulum, where lipid peroxidation occurs during ferroptosis have not been identified [73]. GSH is freely distributed in the cytosol and can be compartmentalized in mitochondria and endoplasmic reticulum [74]. On the other hand, a reduced form of GSH is predominantly detected in cytoplasm and mitochondria [75], suggesting that
38 CSE-induced lipid peroxidation occurs in mitochondria and/or plasma membrane. In this regard, Gao et al. [76] reported that mitochondria play a crucial role in ferroptosis induced by cysteine deprivation, a precursor of GSH synthesis. Because we observed plasma membrane rupture shown by SYTOX staining and significant damage of mitochondria, we postulate that CSE-induced lipid peroxidation and subsequent ferroptosis occur in the mitochondria. Further investigations are necessary to elucidate the precise mechanism underlying CSE-induced ferroptosis and its role in the pathophysiology of AA and AD.