HHcy Induces Pyroptosis and Atherosclerosis via the Lipid Raft-Mediated NOX-ROS-NLRP3 Inflammasome Pathway in apoE−/− Mice

Lipid rafts play important roles in signal transduction, particularly in responses to inflammatory processes. The current study aimed to identify whether lipid raft-mediated inflammation contributes to hyperhomocysteinemia (HHcy)-accelerated atherosclerosis (AS), and to investigate the underlying mechanisms. THP-1-derived macrophages were used for in vitro experiments. ApoE−/− mice were fed a high-fat diet for 12 weeks to establish an AS model, and a high-fat plus high-methionine diet was used to induce HHcy. We found that homocysteine (Hcy) increased the expression of p22phox and p67phox and promoted their recruitment into lipid rafts (indicating the assembly of the NOX complex), thereby increasing ROS generation and NOX activity, NLRP3 inflammasome activation, and pyroptosis. Mechanistically, Hcy activated the NOX-ROS-NLRP3 inflammasome pathway and induced pyroptosis by increasing the expression of acid sphingomyelinase (ASM) to promote the formation of lipid raft clustering. Importantly, lipid raft-mediated pyroptosis was confirmed in HHcy mice, and HHcy-promoted macrophage recruitment in atherosclerotic lesions and HHcy-aggravated AS were blocked by the lipid raft disruptor methyl-β-cyclodextrin. The study findings indicate that Hcy promotes lipid raft clustering via the upregulation of ASM, which mediates the assembly of the NOX complex, causing an increase in ROS generation, NLRP3 inflammasome activation, and pyroptosis, and contributes to HHcy-induced AS.


Introduction
Atherosclerosis (AS), a chronic inflammatory cardiovascular pathological phenomenon [1], is associated with high mortality worldwide. Homocysteine (Hcy) is an intermediate product in the metabolism of sulfur-containing amino acids [2]. An abnormal increase in total plasma Hcy results in a condition known as hyperhomocysteinemia (HHcy) [3], which is recognized as an independent risk factor for AS. HHcy induces the production of reactive oxygen species (ROS) and is associated with an increased vascular oxidative burden [4]. Additionally, oxidative stress has been proposed as a primary component of AS pathogenesis. ROS generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) was shown to serve as the primary source of ROS in cardiovascular systems [5]. Emerging evidence supports the facilitative effect of ROS overproduction on inflammation and AS [6]. Findings from previous studies have confirmed that HHcy induces inflammation and circulating monocyte differentiation through NOX-mediated oxidative stress [7]. Given that ROS is a fundamental component in the activation of the Nod-like receptor 3 (NLRP3) Since NOX catalyzes NADPH to NADP + , NOX activity can be determined using the NADP + /NADPH ratio [20]. Macrophages were pretreated with MβCD (10 mmol/L) for 2 h or DES (10 µmol/L) for 15 min and then treated with Hcy (100 µmol/L) for 24 h, and the cells were lysed and mixed with 200 µL of extraction buffer for NADP + /NADPH ratio detection using an assay kit (S0179, Beyotime) according to the manufacturer's instructions.

Animal Model
Apolipoprotein E knockout (apoE −/− ) mice (8-week-old male) (Vital River, Beijing, China) were housed in a controlled facility at approximately 24°C under a 12 h light/dark cycle and provided free access to food and water. Mice were randomly divided into control, high-fat (HF), HHcy, and HHcy plus MβCD groups (n = 6 for each group). The control and HF mice were fed a regular diet and high-fat diet (HF, Dyets Inc, Wuxi, China) for 12 weeks, respectively. Mice in the HHcy and HHcy+MβCD groups were fed an HF plus high-methionine diet (D210305, Dyets Inc). MβCD was subcutaneously injected in the last 4 weeks of the treatment (2 g/kg, 2 times a week). At the end of the experiment, mice were sacrificed by exsanguination after anesthesia with the intraperitoneal injection of 80 mg/kg pentobarbital. Plasma and aorta samples were collected and stored at 80 • C for further analysis. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (No. 85-23, revised 1996). The research protocol was approved by the ethic committees of Sun Yat-sen University.

Histological Analysis of the Aortic Root Plaque
The aortic roots were isolated and embedded in an optimum cutting temperature compound and cut into 6 µm thick sections. Atherosclerotic lesions were evaluated by Oil Red O staining and imaged under the microscope (OLYMPUS BX51, Tokyo, Japan). The atherosclerotic lesion area was analyzed using the Image J software (NIH, Bethesda, MD, USA).

Statistical Analysis
Data are presented as mean ± SD. All of the in vitro data were from at least three independent experiments. GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA) was used to analyze these results. Statistical comparisons between two groups were conducted using an unpaired Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA followed by the Least Significance Difference (LSD) test. Differences with P < 0.05 was considered statistically significant.

MβCD Reduced the Intracellular and Mitochondrial ROS Levels in Hcy-Treated Macrophages
To determine whether lipid rafts contribute to the production of ROS, a cholesteroldepleting reagent MβCD was used to disrupt the integrity of lipid rafts. Hcy increased both the intracellular and mitochondrial ROS levels, which was reduced by the administration of MβCD ( Figure 1A-C). These results indicated that the disruption of lipid rafts suppressed Hcy-induced ROS production.

MβCD Inhibited Hcy-induced p22 phox and p67 phox Recruitment into Lipid Rafts and Decreased NOX Activity in Macrophages
NOX is one of the key sources of ROS production in cardiovascular systems. NOXrelated oxidative stress serves as a key component in AS development. We found that Hcy significantly increased the expression of the two NOX subunits, p22 phox and p67 phox , whereas MβCD did not influence the Hcy-induced upregulation of p22 phox and p67 phox ( Figure 2A). However, Hcy-induced NOX activity was reduced by MβCD ( Figure 2B). To further determine the underlying mechanism, the lipid raft and non-lipid raft fractions were extracted to assess the recruitment of p22 phox and p67 phox in lipid rafts. Hcy promoted the recruitment of p22 phox and p67 phox into the lipid raft domain, and MβCD prevented the aggregation of NOX subunits into lipid rafts ( Figure 2C,E). Moreover, immunofluorescence staining for CTxB, a marker for lipid rafts, was performed to confirm lipid raft clustering. After Hcy treatment, weak fluorescence in a diffused punctuate staining pattern transformed to intensive fluorescence in a continuous-patch staining pattern, suggesting that Hcy promoted lipid raft clustering. We also confirmed that Hcy promoted the recruitment of p22 phox and p67 phox into lipid rafts, as evidenced by the increased colocalization of CTxB with p22 phox and p67 phox , respectively ( Figure 2D,F). These findings indicated that Hcy induces the assembly of the NOX complex in lipid rafts, thereby enhancing ROS generation.

MβCD Inhibited Hcy-induced p22 phox and p67 phox Recruitment into Lipid Rafts and Decreased NOX Activity in Macrophages
NOX is one of the key sources of ROS production in cardiovascular systems. NOXrelated oxidative stress serves as a key component in AS development. We found that Hcy significantly increased the expression of the two NOX subunits, p22 phox and p67 phox , whereas MβCD did not influence the Hcy-induced upregulation of p22 phox and p67 phox ( Figure 2A). However, Hcy-induced NOX activity was reduced by MβCD ( Figure 2B). To further determine the underlying mechanism, the lipid raft and non-lipid raft fractions were extracted to assess the recruitment of p22 phox and p67 phox in lipid rafts. Hcy promoted the recruitment of p22 phox and p67 phox into the lipid raft domain, and MβCD prevented the aggregation of NOX subunits into lipid rafts ( Figure 2C,E). Moreover, immunofluorescence staining for CTxB, a marker for lipid rafts, was performed to confirm lipid raft clustering. After Hcy treatment, weak fluorescence in a diffused punctuate staining pattern transformed to intensive fluorescence in a continuous-patch staining pattern, suggesting that Hcy promoted lipid raft clustering. We also confirmed that Hcy promoted the recruitment of p22 phox and p67 phox into lipid rafts, as evidenced by the increased colocalization of CTxB with p22 phox and p67 phox , respectively ( Figure 2D,F). These findings indicated that Hcy induces the assembly of the NOX complex in lipid rafts, thereby enhancing ROS generation. Figure 2. MβCD inhibited the Hcy-induced recruitment of p22 phox and p67 phox into lipid rafts and decreased NOX activity in macrophages. (A,C,E) Representative immunoblots and the corresponding quantification of the indicated proteins and (B) NOX activity in macrophages. (D,F) Representative confocal microscopic images of p22 phox /p67 phox and CTxB. White squares: selected magnification area. *p < 0.05, **p < 0.01 and ***p < 0.001.

MβCD Inhibited Hcy-Induced NLRP3 Inflammasome Activation and Pyroptosis in Macrophages
MβCD suppressed the expression of NLRP3 and the cleavage of caspase-1, IL-1β, and GSDMD in Hcy-treated macrophages ( Figure 3A), indicating that it suppressed Hcyinduced NLRP3 inflammasome activation and pyroptosis. To further confirm the results, we assessed the co-localization of NLRP3 and cleaved caspase-1 by immunofluorescence analysis. The Hcy-induced colocalization of NLRP3 and cleaved caspase-1 was inhibited by MβCD ( Figure 3B). Moreover, pyroptosis is characterized by DNA fragmentation, membrane pore formation and proinflammatory content release [21], and DNA fragmentation can be observed by TUNEL staining. Cleaved caspase 1 + /TUNEL + cells were defined as pyroptotic cells. Additionally, MβCD reduced the pyroptotic index in Hcy-treated cells ( Figure 3C,D). MβCD suppressed the expression of NLRP3 and the cleavage of caspase-1, IL-1β, and GSDMD in Hcy-treated macrophages ( Figure 3A), indicating that it suppressed Hcy-induced NLRP3 inflammasome activation and pyroptosis. To further confirm the results, we assessed the co-localization of NLRP3 and cleaved caspase-1 by immunofluorescence analysis. The Hcy-induced colocalization of NLRP3 and cleaved caspase-1 was inhibited by MβCD ( Figure 3B). Moreover, pyroptosis is characterized by DNA fragmentation, membrane pore formation and proinflammatory content release [21], and DNA fragmentation can be observed by TUNEL staining. Cleaved caspase 1 + /TUNEL + cells were defined as pyroptotic cells. Additionally, MβCD reduced the pyroptotic index in Hcy-treated cells ( Figure 3C,D).

Disruption of Lipid Raft Clustering Suppressed Hcy-Induced NLRP3 Inflammasome Activation and Pyroptosis in Macrophages
ASM hydrolyzes membrane lipid sphingomyelin to ceramide (Cer), which initiates and facilitates the clustering of lipid rafts in the cell membrane [22]. Hcy increased the expression of ASM and NLRP3 and promoted the cleavage of caspase-1, IL-1β, and GSDMD. Importantly, the ASM-specific inhibitor DES inhibited Hcy-induced NLRP3 inflammasome activation and pyroptosis as well as NOX activity ( Figure 4A-C). To further confirm the role of lipid raft clustering, the results of gene-silencing by ASM-siRNA were

Disruption of Lipid Raft Clustering Suppressed Hcy-Induced NLRP3 Inflammasome Activation and Pyroptosis in Macrophages
ASM hydrolyzes membrane lipid sphingomyelin to ceramide (Cer), which initiates and facilitates the clustering of lipid rafts in the cell membrane [22]. Hcy increased the expression of ASM and NLRP3 and promoted the cleavage of caspase-1, IL-1β, and GSDMD. Importantly, the ASM-specific inhibitor DES inhibited Hcy-induced NLRP3 inflammasome activation and pyroptosis as well as NOX activity ( Figure 4A-C). To further confirm the role of lipid raft clustering, the results of gene-silencing by ASM-siRNA were confirmed by Western blotting analysis. Compared to the results obtained using the control-siRNA, ASM silencing significantly suppressed the expression of NLRP3 and the cleavage of caspase-1 and GSDMD induced by Hcy ( Figure 4D). These data indicate that Hcy induced NLRP3 inflammasome activation and pyroptosis by enhancing the expression of ASM to promote lipid raft clustering. confirmed by Western blotting analysis. Compared to the results obtained using the control-siRNA, ASM silencing significantly suppressed the expression of NLRP3 and the cleavage of caspase-1 and GSDMD induced by Hcy ( Figure 4D). These data indicate that Hcy induced NLRP3 inflammasome activation and pyroptosis by enhancing the expression of ASM to promote lipid raft clustering.

MβCD Decreased the Levels of Plasma Inflammatory Mediators and Attenuated Atherosclerosis in HHcy Mice
Our previous findings confirmed that the activation of NLRP3 inflammasomes plays an essential role in HHcy-induced AS [9]. Here, we conducted further investigations to de-termine whether lipid rafts play a role in HHcy-accelerated AS in apoE −/− mice. Compared to HF mice, HHcy-induced mice showed higher plasma levels of the proinflammatory cytokines IL-1β and IL-18 ( Figure 5A). Similarly, Oil Red O staining showed that the plaque area of the aortic root was significantly greater in HHcy mice, and treatment with MβCD significantly decreased the plasma levels of IL-1β and IL-18 and the atherosclerotic plaque area in HHcy mice ( Figure 5A,C,D). Moreover, compared to that in HF mice, the plasma Hcy levels increased considerably in HHcy mice. MβCD did not affect the level of plasma Hcy ( Figure 5B), suggesting that MβCD exerted an anti-atherosclerotic effect by inhibiting inflammation but not decreasing the plasma level of Hcy. These results demonstrated that MβCD suppresses HHcy-induced inflammation and inhibits HHcy-induced atherosclerosis in apoE −/− mice. Our previous findings confirmed that the activation of NLRP3 inflammasomes plays an essential role in HHcy-induced AS [9]. Here, we conducted further investigations to determine whether lipid rafts play a role in HHcy-accelerated AS in apoE −/− mice. Compared to HF mice, HHcy-induced mice showed higher plasma levels of the proinflammatory cytokines IL-1β and IL-18 ( Figure 5A). Similarly, Oil Red O staining showed that the plaque area of the aortic root was significantly greater in HHcy mice, and treatment with MβCD significantly decreased the plasma levels of IL-1β and IL-18 and the atherosclerotic plaque area in HHcy mice ( Figure 5A,C,D). Moreover, compared to that in HF mice, the plasma Hcy levels increased considerably in HHcy mice. MβCD did not affect the level of plasma Hcy (Figure 5B), suggesting that MβCD exerted an anti-atherosclerotic effect by inhibiting inflammation but not decreasing the plasma level of Hcy. These results demonstrated that MβCD suppresses HHcy-induced inflammation and inhibits HHcy-induced atherosclerosis in apoE −/− mice.  HHcy induced NLRP3 inflammasome activation, as confirmed by the increased expression of NLRP3, cleaved caspase-1, IL-1β, and GSDMD, as well as the colocalization of NLRP3 inflammasome components (NLRP3 and cleaved caspase-1) detected by West-ern blotting and confocal microscopy, respectively. Consistent with our in vitro results, HHcy-induced NLRP3 inflammasome activation and pyroptosis was reversed by MβCD, indicating that lipid rafts play a vital role in the progression of HHcy-induced AS (Figure 6A,B). The recruitment and death of macrophages contribute to the formation and enlargement of AS plaques [11]. Double immunofluorescent staining with anti-NLRP3 and -F4-80 (macrophage marker) antibodies was performed to explore the recruitment of macrophages. In atherosclerotic lesions, NLRP3 and F4-80 showed marginal co-localization in control mice and relatively greater co-localization in HF mice, whereas they largely showed enhancement and colocalization in HHcy mice. After MβCD intervention, the fluorescence intensity of NLRP3 and F4-80 was weakened, and less co-localization was observed, indicating that lipid rafts are involved in HHcy-induced macrophage infiltration in the atherosclerotic lesion ( Figure 6C). HHcy induced NLRP3 inflammasome activation, as confirmed by the increased expression of NLRP3, cleaved caspase-1, IL-1β, and GSDMD, as well as the colocalization of NLRP3 inflammasome components (NLRP3 and cleaved caspase-1) detected by Western blotting and confocal microscopy, respectively. Consistent with our in vitro results, HHcy-induced NLRP3 inflammasome activation and pyroptosis was reversed by MβCD, indicating that lipid rafts play a vital role in the progression of HHcy-induced AS ( Figure  6A,B). The recruitment and death of macrophages contribute to the formation and enlargement of AS plaques [11]. Double immunofluorescent staining with anti-NLRP3 and -F4-80 (macrophage marker) antibodies was performed to explore the recruitment of macrophages. In atherosclerotic lesions, NLRP3 and F4-80 showed marginal co-localization in control mice and relatively greater co-localization in HF mice, whereas they largely showed enhancement and colocalization in HHcy mice. After MβCD intervention, the fluorescence intensity of NLRP3 and F4-80 was weakened, and less co-localization was observed, indicating that lipid rafts are involved in HHcy-induced macrophage infiltration in the atherosclerotic lesion ( Figure 6C). with ASC (green) and that of F4-80 (red) with NLRP3 (green). White squares: selected magnification area. n = 6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion
The present study is the first to provide evidence that HHcy promotes AS through lipid raft-mediated pyroptosis. Mechanistically, Hcy induces p22 phox and p67 phox recruitment into lipid rafts, leading to NOX complex assembly and ROS generation, which consequently activates the NLRP3 inflammasome and induces pyroptosis. Moreover, the destruction of lipid rafts or the silencing of ASM for interference with lipid raft clustering successfully suppressed the pro-pyroptotic effect of Hcy via inhibition of the NOX-ROS-NLRP3 pathway. Our results suggest that the targeting of lipid rafts to blunt HHcy-induced NLRP3 inflammasomes and pyroptosis could exert an atheroprotective effect.
HHcy, an independent cardiovascular risk factor, is associated with vascular inflammation and AS. HHcy enhances the adhesion between monocytes and endothelial cells and promotes monocyte differentiation, macrophage maturation, and uptake of ox-LDL by macrophages. HHcy-induced oxidative stress has been confirmed to mediate endothelial dysfunction [23]. Our previous study showed that Hcy promotes ROS generation and enhances vascular inflammation, which aggravates the progression of AS [9]. However, the mechanism underlying Hcy-induced ROS generation remains unknown. NOX is considered one of the main resources for ROS production in endothelial cells. NOX is a multi-subunit complex, composed of the cytosolic components of regulatory subunits (p47phox, p67phox, p40phox, and small G-protein Rac1/2) and the membrane components of catalytic subunits (gp91phox and p22phox); the cytoplasmic subunits are translocated to the membrane and bind to the membrane subunits to assemble the active form of the NOX complex in response to stimulus [24]. Excessive assembly and activation of the NOX complex results in the induction of chronic inflammatory diseases, such as AS [18]. Reportedly, Hcy-stimulated NOX-dependent oxidative stress results in endothelial dysfunction and induces the vascular inflammatory response [25,26]. H 2 O 2 generated by NOX is required to promote mitochondrial ROS production [27].
Lipid rafts are considered an essential platform for the aggregation and assembly of NOX subunits, and thus, form a redox signaling platform to produce ROS and conduct redox signaling. Lipid rafts are small dynamic cholesterol-and sphingolipid-enriched microdomains [28]. Compared to the surrounding membrane, lipid rafts exhibit a more ordered and tightly packed structure. Lipid rafts are involved in various biological events, such as protein transport and apoptosis, because they provide a stable platform and contribute to the mediation and amplification of various cellular signals [29,30]. In the present study, MβCD was administered to disrupt intact lipid rafts. Correspondingly, we confirmed that Hcy promoted the production of ROS, which was inhibited by MβCD. Observation of the colocalization of lipid rafts and NOX subunits via confocal microscopy is a widely accepted method for the detection of NOX subunit recruitment in lipid rafts [16]. In our study, we identified that Hcy increased the expression of p22 phox and p67 phox and substantially enhanced NOX activity, which was accompanied by increased intracellular and mitochondrial ROS generation in macrophages. Furthermore, we observed that Hcy triggered p22 phox and p67 phox aggregation and translocation, respectively, into lipid raft fractions. Meanwhile, Hcy enhanced the enrichment of p22 phox and p67 phox in lipid rafts, as evidenced by the increased colocalization of CTxB and p22 phox or p67 phox . Taken together, our results demonstrated, for the first time, that Hcy induces ROS generation via the assembly of the NOX complex in lipid rafts.
It is widely accepted that chronic progressive inflammation has deleterious and additive effects that accelerate AS and contribute to lesion rupture [31]. HHcy promotes pro-inflammatory cytokine production and lipid accumulation in macrophages and has been regarded as a major driving force in the development of atherosclerotic plaques. Pyroptosis is a form of programmed cell death, and NLRP3 inflammasome-related pyroptosis is characterized by caspase-1-dependent pore formation and pro-inflammatory factor (such as IL-1β and IL-18) release, leading to cell lysis. Previous research has shown that cholesterol crystals could trigger NLRP3 inflammasome activation, and that excessive caspase-1 expression is associated with the instability of the atherosclerotic plaque [32]. Frank et al. reported that the deficiency of caveolin-1, a lipid raft resident protein, is associated with a protective effect against AS [33]. Notably, our results showed that MβCD reduced the atherosclerotic area in HHcy mice, suggesting that lipid rafts play a vital role in HHcy-induced AS.
Lipid rafts play an essential role in many cellular function regulations, particularly in inflammation [34]. Previous studies have demonstrated that lipid rafts provide a stable platform for the assembly of the IFNR complex and are involved in IFN-stimulated inflammatory signaling [35]. Reportedly, the disruption of lipid rafts inhibits the recruitment of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) into lipid rafts, and alleviates lipopolysaccharide-mediated inflammation in macrophages [36]. Consistent with the findings from our previous study [13], the activation of the NLRP3 inflammasome could be prevented by destroying the integrity of lipid rafts. Moreover, MβCD significantly decreased the levels of pro-inflammatory factors in the peripheral blood of HHcy mice. Pyroptosis is characterized by the formation of plasma membrane pores, the release of proinflammatory cytokines, and cell lysis, and could be triggered by NLRP3 inflammasome activation [37]. Most studies have focused on the pro-apoptotic properties of lipid rafts in various diseases. A novel finding of this study is that we confirmed that lipid rafts are associated with HHcy-induced pyroptosis, and that the destruction of lipid rafts helps to efficiently attenuate pyroptosis as well as consequent atherosclerotic lesion formation in apoE −/− mice.
ASM, a major member of sphingomyelinases, can be translocated into the cell membrane to hydrolyze sphingomyelin to Cer [38,39]. Cer facilitates the combination of individual lipid rafts into larger and stable Cer-enriched membrane microdomains in response to different stimuli, further enhancing protein interactions and amplifying signal transduction [13,40]. Consistent with this, we found that Hcy increased the expression of ASM. DES, an ASM inhibitor, interrupted lipid raft clustering and inhibits the assembly of the NOX complex, as evidenced by the decreased activity of NOX. To further confirm the role of lipid raft clustering in Hcy-mediated inflammation, we induced siRNA-mediated ASM knockdown to interrupt lipid raft clustering. Our results demonstrated that, consistent with the results of treatment with DES, ASM silencing abrogated NLRP3 inflammasome activation and pyroptosis in Hcy-treated macrophages. It was reported that ASM induced by cigarette smoke markedly increases extracellular vesicle (EVs) production [41]. Recent study highlighted that monocytic-derived EVs encapsulate caspase-1 and promote macrophages apoptosis [42]. Our findings are the first to show that the inhibition of lipid raft clustering exerts a protective effect on Hcy-induced NLRP3 inflammasome activation and pyroptosis in macrophages. However, it is unclear whether EVs are involved in NLRP3 inflammasome activation and pyroptosis in HHcy model. Expanding studies are warranted to explore the effect of ASM-mediated lipid raft clustering in EVs production and dissect its role in NOX-ROS-NLRP3 activation and pyroptosis in HHcy-induced AS in vivo.

Conclusion
Taken together, this is the first study to demonstrate that Hcy promotes the assembly of the NOX complex through ASM-mediated lipid raft clustering, which increases ROS generation, NLRP3 inflammasome activation, and pyroptosis, eventually contributing to the progression of AS (Figure 7). The NOX-ROS-NLRP3 inflammasome pathway may be a potential therapeutic target against HHcy-induced pyroptosis and AS. Institutional Review Board Statement: The animal study was reviewed and approved by the Ethics Committees of Sun Yat-sen University (SYSU-IACUC-2019-000252).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available upon reasonable request from the corresponding author.

Conflicts of Interest:
The authors declare no conflicts of interest.