Chronic Cadmium Exposure Impairs Macrophage Mitochondrial Homeostasis and Promotes Macrophage Polarization Contributing to Atherosclerosis via Regulating RIPK3 Signaling

Background: Chronic cadmium (Cd) exposure can contribute to the progression of cardiovascular disease (CVD), especially atherosclerosis (AS), but the underlying mechanism is unclear. Since mitochondrial homeostasis is emerging as a core player in the development of CVD, it might serve as a potential mechanism linking Cd exposure and AS. Here, we aimed to investigate the Cd-induced AS through macrophage polarization and tried to nd out the mechanism of mitochondrial dysfunction caused by Cd exposure. Methods and results: In vitro, ow cytometry showed that Cd exposure markedly promoted M1-type polarization of macrophages, manifesting as the increasing expression of NF-kB, NLRP3 and their downstream inammatory factors, IL-1β and IL-6. Mitochondrial function test revealed that the decreasing mitochondrial membrane potential and increasing superoxide (mROS) and mitochondrial ssion were involved in Cd-induced macrophage polarization. Transmission electron microscope observation and immunouorescence both identied the decrease of mitophage after Cd exposure. And improving mitochondrial function above signicantly restored the balance of macrophage polarization. In vivo, Cd exposure was positively correlated with blood Cd concentration, and oil red O staining showed higher blood Cd signicantly increased the area of AS plaques. Besides, M1-type polarization of macrophages and mitochondrial dysfunction were observed in mouse aortic roots through immunouorescence and western blot as the dosage of Cd increasing. And the administered NAC or Mdivi-1, which decreased mROS or mitochondrial ssion, markedly attenuated AS plaques and macrophage M1-type polarization in Cd-treated group. Finally, the up-regulated expressions of RIPK3 and p-MLKL were observed both in vitro and in vivo. And knocking out RIPK3 with decreasing expression of p-MLKL followed did improve mitochondrial dysfunction caused by Cd which effectively reversed macrophage polarization. Conclusion: Cd exposure activated RIPK3 pathway and impaired mitochondrial homeostasis, resulting in macrophage polarization to a pro-inammatory phenotype and subsequent AS. These ndings suggest that improving mitochondrial homeostasis may provide a potential therapeutic target for AS induced by chronic Cd exposure.

Macrophages are the largest number of immune cells in the cardiovascular system, with functional diversity and plasticity. M1-type macrophage secretes pro-in ammatory factors, such as ROS, TNF-and IL-1β, which is well documented to damage the AS plaque stability and cause stroke [6]. On the contrary, IL-10, FGF-1, IGF-1, TGF-β and IL-12 released by M2-type macrophage contributes to the enhancement of AS plaque stability [7]. Additionally, mitochondrial membrane dynamics is tightly coupled to constant reshaping of the cellular mitochondrial network in a series of processes, involving organelle fusion and ssion (division) as well as ultrastructural remodeling of the membrane [8]. And evidences accumulated has proved that the imbalance of mitochondrial homeostasis is the initial factors of macrophage dysfunction, which emerges as the impairment of protective autophagy [9] and promotes an in ammatory pathway [10]. Given all these, it is much meaningful to investigate whether Cd exposure affects the mitochondrial homeostasis of macrophages and further promotes M1-type polarization of macrophages in AS.
In addition, as a member of the receptor-interacting protein (RIP) family of serine/threonine protein kinases, receptor interacting serine/threonine kinase 3 (RIPK3) is closely related to in ammatory activation, which contributes to the expression of NF-KB transcription factor [11]. And as we knew, the inhibition of RIPK3 suppresses RNA virus-induced activation of the NLRP3 in ammasome [12]. On the aspect of membrane integrity, p-MLKL, phosphorylated by RIPK3, transfers from cytosol to cell plasma membrane and organelles' membranes, where it directly disrupts membrane integrity [13]. Additionally, RIPK1/RIPK3 complex directly phosphorylates Drp1 at serine 616 site (p-Drp1 (Ser616) ) and triggers its translocation to mitochondria, nally increasing mitochondrial division [14]. Prior studies have suggested that RIPK3-knock-out can reduce plaque formation in the advanced stage of AS [15]. Therefore, whether RIPK3 was involved in Cd-induced macrophage mitochondrial membrane dynamic imbalance and M1type polarization in the progression of AS was a question to be studied in this research.
Thus, we hypothesized that high level blood Cd concentration could increase the expression of RIPK3-P-MLKL in macrophages and make Drp1 transfer to mitochondria, causing macrophage mitochondrial dysfunction, triggering in ammation and M1-type polarization of macrophage, nally aggravating the progression of AS.

Method
Cell culture RAW264.7 macrophages were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidi ed atmosphere with 5% CO 2 and maintained in a logarithmic growth phase for all experiments, then RAW264.7 macrophages were exposed to CdCl 2 at different concentrations on the second day. To study the effect of mitochondrial function on the macrophages, we added 5mM N-Acetyl-L-cysteine (NAC) (Beyotime S0077) or 20μM Mdivi-1 (ab144589) to the cultures for 24h to reduce redox reactions or mitochondria division. What's more, bone marrow-derived macrophages (BMDMs) were isolated from RIPK3 -/-/ApoE -/mice to research the effects of RIPK3 on Cd-exposed macrophages.

Animal models of AS
Animal experiments were carried out according to the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Animal Ethics Committee of Southern Medical University. Seven-week-old male ApoE -/mice were fed with high-fat diets or chow diets which were provided by Guangdong Medical Laboratory Animal Center for three months, exposed to Cd in different concentrations. To study the effect of Mitochondrial function on the formation of AS plaque, we administered NAC orally (20mM) or 50mg/kg i.p.Mdivi-1 to mice exposed to high-fat with Cd diet. And RIPK3-knockout mice on a C57BL/6 background were obtained from GemPharmatech, then bred with ApoE -/mice to establish RIPK3 -/-/ApoE -/mice.

Western blot
Proteins were extracted from cells, tissues or mitochondria, while the mitochondria were isolated from RAW264.7 macrophages or aortic root using the cell mitochondria isolation kit (Beyotime C3601) or tissue mitochondria isolation kit (Beyotime C3606). After the proteins were measured by BCA ( were from Wanlei Biology. After having been incubated with the corresponding secondary antibodies (Boster BA1054) for 1.5 hours at room temperature, the immunoblot bands were detected by enhanced chemiluminescence (Engreen 29100). Prestained molecular-weight marker proteins (Thermo#26616) were used to calculate the molecular weights of proteins. The membranes were exposed to autoradiography lm following incubation with an enhanced chemiluminescence imaging system (Engreen 29100). The signal intensities were checked by Gel-Pro Analyzer 4.0 software (Media Cybernetics, Silver Spring, MD).

Flow Cytometry
Cell surfaces were stained with phycoerythrin (PE)-conjugated anti-mouse F4/80 antibody (biolegend 123110), APC-conjugated anti-mouse CD206 (MMR) antibody (141708) and FITC-conjugated anti-mouse CD86 antibody (105006). The expression of F4/80 was determined by ow cytometry to identify macrophages. And the expression of CD86 was used to delineate M1 macrophages, while the cells stained by APC-conjugated anti-mouse CD206 (MMR) antibody could be identi ed as M2 macrophages. Cell suspensions were stained for 30 minutes on ice with speci c antibodies and washed twice with 3 mL of PBS buffer supplemented with 0.5% bovine serum albumin (BSA). Finally, we analyzed it using ow cytometry (CytoFlex A00-1-1102).
Then, the samples were stained with DAPI to visualize nucleus and observed by a laser confocal microscope (LEICA SP8). All the images and staining intensities were acquired and measured using analysis software (Image J).

Mitochondrial function detection
The mitochondrial superoxide of Raw264.7 macrophages was detected by MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen™ M36008), and the Mitochondrial membrane potential was determined via mitochondrial membrane potential assay kit with JC-1 (Beyotime C2006) according to the manufacture's instruction.

Transmission electron microscope (TEM) observations
Macrophages growing to the logarithmic phase were scraped and collected to be xed with glutaraldehyde for 4 hours. Then sections (60 nm) were cut and stained with lead citrate and uranyl acetate at room temperature for 4 h. Next, samples were embedded in resin at room temperature for another 2 hours. A Hitachi H-7500 Transmission Electron Microscope (Hitachi, Ltd., Tokyo Japan) was used to observe the samples.

Oil Red O Staining
After the serial 10μm sections were cut, the OCT compound-embedded tissues were stained with Oil Red O (Solarbio 08010) to evaluate the lipid content, and Re-dyed with hematoxylin (Beyotime), differentiated by hydrochloric alcohol (Beyotime), washed by double distilled water, nally observed under microscope (OLYMPUS).

Statistical analyses
Data were generated through at least three independent experiments and were presented as the means ± SEM, then analyzed with the method of t-test between two groups or one-way ANOVA followed by a Bonferroni comparison test among three groups. Statistical analyses were carried out using Prism 8 (GraphPad). A two-tailed P value < 0.05 was considered to be statistically signi cant.

Results
Cd promoted macrophage polarization to a pro-in ammatory phenotype To evaluate the cytotoxicity caused by Cd, CCK8 measurement was performed after exposing RAW264.7 macrophages at logarithmic growth to CdCl 2 . Cell viability of RAW264.7 macrophages was more than 90% when exposed to CdCl 2 with the concentration of ≤ 10µmol/L (Fig. 1A). Subsequently, the macrophages were treated with 0.0, 1.0, 5.0, and 10.0µmol/L CdCl 2 for qRT-PCR detection of related in ammatory factors, and the results showed that the lower concentration of CdCl 2 (1µmol/L) had the most obvious pro-in ammatory effect (Fig. 1B).To further verify the effect of Cd on macrophage polarization in AS, 1µmol/L Cd exposure was used for subsequent assays, including ow cytometry, western blot (WB) and ELISA after RAW264.7 foamed with ox-LDL (50µg/ mL) for 24h in vitro. Exposed to Cd, the surface-speci c molecule of M1-type macrophages, CD86, was markedly up-regulated while the surface-speci c molecule of M2-type macrophages, CD206, was signi cantly down-regulated ( Fig. 1C and D). Then, the results of WB showed that the expressions of in ammasome NLRP3 and its downstream proteins, IL-1 and caspase1-p20, were increased, and the expressions of pNF-KB and its downstream proteins, TNF-α and IL-6, were also increased after Cd exposure ( Fig. 1E-H). The proteins mentioned above were closely connected with M1-type polarization of macrophages [16]. Finally, the in ammatory factors, IL-1β and IL-6, in supernatant of macrophages were increased after Cd exposure measured by ELISA kit (Fig. 1I and J). Taken together, Cd promoted macrophage polarization toward the pro-in ammatory phenotype.

Cd disrupted mitochondrial homeostasis and protective autophagy
To investigate the effects of Cd exposure on the mitochondrial homeostasis and function of macrophages, we performed immuno uorescence staining on macrophages with the mitochondrial membrane protein Tom20 antibody. Among the groups we detected, the fragmentation of mitochondria was markedly increased in Cd-exposed group ( Fig. 2A). Subsequently, the MitoSOX™ Red mitochondrial superoxide indicator was used to investigate that Cd exposure enhanced the oxidative stress responses in RAW264.7 ( Fig. 2B and C). Moreover, the Cd-induced mitochondrial membrane potential reduction was presented in Fig. 2D and E. In terms of molecular mechanism, WB results revealed that Cd exposure led to the increased expression of mitochondrial division protein Drp1, decreased fusion protein Opa1, and decreased protective autophagy (the ratio of LC3II/I) in macrophages (Fig. 2F-H). In order to further verify the decreased protective autophagy level induced by decreased homeostasis, we performed immuno uorescence staining with autophagy-related protein LC3II, and the results showed that protective autophagosomes in macrophages were signi cantly decreased under Cd exposure (supplementary Fig.   S1A and B). Finally, autophagosomes observed under TEM were decreased with Cd exposure (supplementary Fig. S1C). Overall, we found Cd-induced macrophage dysfunction was characterized by mitochondrial membrane dynamic imbalance.
Improving mitochondrial homeostasis inhibited macrophage polarization toward the pro-in ammatory phenotype in vitro Based on the above experimental results, we conducted repair interventions for mitochondrial damage caused by Cd exposure: the antioxidant N-acetylcysteine (NAC) was used to alleviate mitochondrial superoxide (mROS) production, and Mdivi-1, a mitochondrial ssion inhibitor, was used to inhibit the expression of Drp1 in RAW264.7. The results showed that the above interventions effectively reduced the mitochondrial fragment of Cd-exposed macrophages (Fig. 3A), decreased the production of mROS (supplementary Fig. S2A and B), and recovered the mitochondrial membrane potential (supplementary Fig. S2E and F), indicating the restoration of mitochondrial homeostasis and function. Subsequently, WB identi ed that NAC and Mdivi-1 decreased the expression of Drp1, and increased the expression of Opa1 and the ratio of LC3II/I (Fig. 3B-D). Meanwhile, increased autophagosomes were presented by immuno uorescence with LC3II antibody after treatment of NAC and Mdivi-1(supplementary Fig. S2C and D). And it was measured by TEM that the above interventions signi cantly increased the content of autophagosomes in macrophages, while the production of mitophagy was more obvious in the Mdivi-1intervened group (Fig. 3E). Finally, the decreased expression of CD86, and the increased expression of CD206 were both demonstrated by ow cytometry with the recovery of mitochondrial homeostasis and function ( Fig. 3F and G). Moreover, the expression of NLRP3, pNF-KB and their downstream proteins, IL-1β and TNF-α, decreased in Cd-exposed group treated with NAC and Mdivi-1 (supplementary Fig. S3A and B). These results indicated that improving mitochondrial homeostasis with NAC or Mdivi-1 effectively reversed Cd-induced macrophage polarization toward the pro-in ammatory phenotype.

Improving mitochondrial homeostasis counteracted atherosclerosis in vivo
In order to demonstrate the role of mitochondrial dysfunction and polarization of macrophages in the progression of Cd-induced AS, ApoE −/− mice exposed to Cd were treated with NAC or Mdivi-1 in vivo. Results showed that the blood Cd concentration was proportional to Cd exposure level (supplementary Fig. S4A). Oil red O staining of the aortic root revealed that the plaque area of the high-fat feeding group with 200mg/L Cd exposure was signi cantly increased compared with those without Cd exposure. No plaque formation was observed in the chow-diet with 200mg/L Cd exposure group. Meanwhile, NAC and Mdivi-1 effectively reduced the plaque area of the high-fat group with Cd exposure (Fig. 4A and B). Additionally, we performed immuno uorescence staining on the aortic root with the CD86 and CD206 antibodies, nding that high-fat feeding groups with elevated blood Cd level had more obvious M1-type polarization than those without Cd exposure. And the ratio of CD86 + / F4/80 + was further increased as the concentration of Cd exposure increased from 100mg/L to 200mg/L. In contrast, treatment with NAC or Mdivi-1 signi cantly decreased M1-type polarization (the ratio of CD86 + / F4/80 + ) ( Fig. 4C and D), while increasing M2-type polarization (supplementary Fig. S4B and C). Additionally, mice treated with NAC or Mdivi-1 showed a signi cant increase in the expression of Opa1 and the ratio of LC3II/I but a remarked decrease in the expression of Drp1, NLRP3 and pNF-KB (Fig. 4E-G; supplementary Fig. S5A-G). Finally, plasma ELISA results showed the decreased level of M1-type in ammatory markers, il-1 and il-6, after NAC or Mdivi-1 treatment (supplementary Fig. S5H-I). Taken together, improving mitochondrial homeostasis inhibited Cd-induced macrophage polarization to the pro-in ammatory phenotype and thus counteracted AS in vivo.
The level of RIPK3 was increased in atherosclerosis while RIPK3 knockout enhanced mitochondrial homeostasis Next, we wanted to further explore the deep mechanism of Cd-induced mitochondrial homeostasis injury. Firstly, we found that the expression of RIPK3 and its downstream protein, p-MLKL, were signi cantly increased under Cd exposure both in vivo and in vitro ( Fig. 5A-C). Meanwhile, the expression of RIPK3 in mitochondria was decreased after the mitochondrial dysfunction was restored by NAC or Mdivi-1 (supplementary Fig. S6A-F), which could be explained that the translocation of necrosomes (RIPK1-RIPK3-MLKL) from the cytosol to intracellular membranes was inhibited [17]. showed that the number of autophagosomes was indeed increased in RIPK3 −/− /ApoE −/− mice (supplementary Fig. S6H and I). These results indicated that RIPK3-p-MLKL pathway was activated under Cd exposure, and RIPK3 knockout signi cantly enhanced mitochondrial homeostasis.

Deletion of RIPK3 inhibited polarity shift toward in ammatory macrophages and atherosclerosis
To understand the in vivo role of RIPK3 in the development of Cd-enhanced AS through mitochondrial homeostasis and macrophages in ammation, we employed the ApoE −/− AS mice with a deletion of RIPK3 (RIPK3 −/− /ApoE −/− ). To accelerate AS, mice were fed a high-fat diet and exposed to Cd. Oil red O staining of the aortic root showed that the area of AS plaques was signi cant decreased in RIPK3 −/ − /ApoE −/− mice compared with ApoE −/− mice ( Fig. 6A and B). Immuno uorescence staining of the aortic roots showed that the level of M1-type polarization (CD86 + /F4/80 + ) decreased while the level of M2-type polarization (CD206 + /F4/80 + ) increased in RIPK3 −/− /ApoE −/− mice compared to ApoE −/− mice (Fig. 6C-E). Additionally, deletion of RIPK3 inhibited polarity shift toward M1-type BMDMs shown by ow cytometry (Fig. 6F and G). The expressions of NLRP3 and pNF-KB were inhibited both in aortic root and in BMDMs of RIPK3 −/− /ApoE −/− mice ( Fig. 6H and I; supplementary Fig. S7A and B). Plasma ELISA was conducted to investigate that the expressions of in ammatory cytokines, IL-1β and IL-6, secreted by M1-type macrophages were decreased in RIPK3 −/− /ApoE −/− mice, too (supplementary Fig. S7C). These results indicated that the deletion of RIPK3 inhibited Cd-induced in ammatory responses and subsequent AS.

Discussion
In this study, we focused on the non-essential elements of the human body, Cd, which was widely existing in the nature. Our ndings showed that Cd disrupted macrophage mitochondrial homeostasis through RIPK3 signaling pathway and caused M1-type polarization of macrophage, nally aggravating the progression of AS (Fig. 7). To the best of our knowledge, we demonstrated the rst time that the mechanism of Cd-induced AS is the imbalance of macrophage mitochondrial homeostasis via RIPK3 pathway.
Previous studies on Cd-induced in ammation of macrophages reported that oral exposure to Cd (3 hours) triggered an acute in ammatory response in the intestines of mice, initiated by the over-expression of tissue macrophage in ammatory protein-2 mRNA which was produced by both intestinal epithelial cells and macrophages. But this study showed no signi cant changes of cytokines, IL-1β and TNF-α [18]. In contrast, Marina Ninkov et al studied the effects of oral Cd exposure on intestinal immunity, suggesting that Cd consumption resulted in the changes of intestinal ora as reduction of Lactobacillus strain, and intestinal in ammation as increasing pro-in ammation cytokines (TNF, IL-1β, IFN-γ, IL-17) [19]. Furthermore, Kielldahl et al found that blood Cd was associated with pro-in ammatory macrophage density in the sections of carotid plaques with most frequent rupture which was previously shown to contain most Cd [20]. The studies mentioned above indicated that Cd can induce in ammatory response in macrophages which was the same as the result of this article. However, the pro-in ammatory properties of Cd is still controversial. Till now, the only one article that mentioned the effect of Cd on macrophage polarization revealed that in chronic obstructive pulmonary disease COPD patients, Cd exposure inhibited the NF-ĸB pathway, dose-dependently inhibited LPS-induced immunoreaction by macrophages, and inhibited M1-type macrophage behavior with less effect on M2-type polarization. This article became a new evidence that Cd could lead to immune dysfunction, further increasing the susceptibility to COPD infection [21]. Furthermore, another study came up to be the rst report investigating the effects of Cd on the in ammatory responses and oxidative stress together in vitro system, and the result showed that murine macrophage exposed to Cd signi cantly decreased the in ammatory responses but increased the oxidative stress, a little different from studies explained above [22]. The difference may be due to Cd concentration and treatment time that high-level Cd exposure for long time can impair cell viability and function so that the high concentration of Cd in smokers' lungs impairs the normal pro-in ammatory function of macrophages and worsens the infection of COPD.
However, the concentration of blood Cd is not high, as a result, the function of macrophages in cardiovascular system hasn't been impaired.
As to the mechanism of Cd-induced in ammation, an existing study on Cd-induced liver injury showed that liver injury was mediated by in ammatory mediators activated by reactive oxygen species [23]. Glycine, an antioxidant, could reduce the secretion of in ammatory cytokines IL-6, TNF-α and IL-1 by macrophage U937 cells [24]. In addition, another study has shown that the enhanced REDOX reaction in peritoneal macrophages of mice exposed to chronic low levels of Cd induced lipid peroxidation, and increased the expressions of cyclooxygenase-2 and inducible nitric oxide synthase, nally causing in ammatory response [25]. Therefore, we can conclude that Cd-induced oxidative stress is related to in ammatory response, and the former is the upstream pathway of the latter which is the same as the result of our research. Furthermore, the main sources of intracellular ROS are membrane-derived and mitochondrion-derived. The former is mainly dependent on NAPDH oxidase, while the latter is associated with mitochondrial membrane potential damage. As to the mitochondrion-derived ROS, studies have shown that mitochondria in THP-1 macrophages could be targets of Cd toxicity: Cd binded to thiol proteins on the mitochondrial membrane, and then changed the mitochondrial membrane permeability to inhibit the mitochondrial respiratory chain response, or damaged the function of electron transfer chain complex III, and nally induced the production of ROS [26]. However, the mitochondrial damage and subsequent macrophage polarization involved in the process of Cd-induced AS remain unclear. In this study, we lled this gap in knowledge by reporting that chronic Cd exposure impaired macrophage mitochondrial homeostasis and promoted macrophage polarization contributing to AS through RIPK3 pathway.
The molecular mechanism of in ammatory reaction includes the discovery by Ja Shil Hyun et al in 2007 that 20-60µM concentration of CdCl 2 signi cantly induced the increase of IL-8 secretion through NF-KB activation in human intestinal epithelial cell Caco-2 [27]. Moreover, vitro studies showed that Cd activated NF-KB pathway through increasing oxidative stress level [28]. In addition, Wirth's team has successively found that the activation of heat shock response induced by Cd effectively protect the lung function of mice. Compared with HSF-/-mice, NF-KB pathway was suppressed in wild-type mice, leading to lower in ltrating macrophages and neutrophils [29,30]. These studies showed that the activation of NF-KB was one of the molecular mechanisms by which Cd promoted in ammation, the same as our results. Besides, Kunpeng Wu' s team found that the M1-type polarization of macrophages required NLRP3 in ammasome activation [16]. But no researches related to Cd-induced macrophage polarization have paid attention to NLRP3 in ammasome except this study.
In addition, as described above, RIPK3 was associated with mitochondrial function, and the expressions of NF-KB and NLRP3, but there has been no study revealing that Cd could impair macrophage mitochondrial homeostasis and promote macrophage polarization contributing to atherosclerosis via regulating RIPK3 signaling. So trying to verify the polarization effect of macrophages induced by Cd in AS, exploring the mechanism from the perspective of mitochondrial homeostasis and dysfunction, nally knocking out RIPK3 gene from the upstream of mitochondria, made this study more innovative.
In addition to AS, Cd exposure can also worsen other CVD progression. It has been reported that Cd induced hypertension by decreased endothelial nitric oxide synthase protein level [31]. Additionally, macrophages create a bene cial microenvironment for the survival of myocardial cells, which is crucial for the survival and regeneration of myocardial cells after MI [32]. And local in ammatory response and changes in immune microenvironment are considered to be one of the main reasons for the di culty in myocardial repair and regeneration after myocardial tissue injury [33]. Therefore, the in uence of Cd exposure on the immune microenvironment in the heart region may be the key interference factor in the myocardial repair after MI in Cd-exposed area. At the same time, many known studies have reported that bone and kidney were two of the important target organs of multi-organ damage caused by Cd poisoning, among which osteoporosis is the main manifestation of bone damage caused by Cd [34]. Moreover, existing studies have shown that with the growth of age, the lost human calcium from bones would deposit on the cardiovascular system and then induce vascular calci cation [35]. Although there is a strong relationship between osteoporosis and vascular calci cation, there is a lack of research on the mechanism of vascular calci cation caused by Cd exposure.
Finally, in addition to the environmental pollutant, Cd, which was the focus of this paper, other heavy metal pollution, such as lead, mercury, and arsenic, may also have an impact on the cardiovascular

Conclusion
Our ndings demonstrated that mitochondrial homeostasis played an important role in Cd-induced in ammatory response in macrophages. We further revealed that RIPK3 regulated Cd-induced mitochondrial dysfunction via Drp1 protein, leading to increasing expressions of NF-KB and NLRP3 proteins. Finally, Cd exposure induced M1-type polarization of macrophages in cardiovascular system which was conducive to AS. These results revealed a novel mechanism for Cd-induced in ammation and offered new insights into the pathophysiology of AS caused by heavy metal pollution.     . Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7
The mechanism of Cd-induced AS via RIPK3-regulated mitochondrial homeostasis