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Article

Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice

by
Stephen G. Andrews
1,
Anthony M. Koehle
1,
Devendra Paudel
1,
Thomas Neuberger
2,3,
A. Catharine Ross
1,
Vishal Singh
1,
Teodoro Bottiglieri
4,† and
Rita Castro
1,5,*,†
1
Department of Nutritional Sciences, Penn State University, University Park, PA 16802, USA
2
Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA
3
Department of Biomedical Engineering, Penn State University, University Park, PA 16802, USA
4
Center of Metabolomics, Institute of Metabolic Disease, Baylor Scott and White Research Institute, Dallas, TX 75204, USA
5
Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Nutrients 2024, 16(3), 330; https://doi.org/10.3390/nu16030330
Submission received: 3 December 2023 / Revised: 30 December 2023 / Accepted: 9 January 2024 / Published: 23 January 2024
(This article belongs to the Special Issue Methyl Nutrients and One-Carbon Metabolism in Chronic Diseases)
Browse Figures
Versions Notes

Abstract

:
Atherosclerosis and resulting cardiovascular disease are the leading causes of death in the US. Hyperhomocysteinemia (HHcy), or the accumulation of the intermediate amino acid homocysteine, is an independent risk factor for atherosclerosis, but the intricate biological processes mediating this effect remain elusive. Several factors regulate homocysteine levels, including the activity of several enzymes and adequate levels of their coenzymes, including pyridoxal phosphate (vitamin B6), folate (vitamin B9), and methylcobalamin (vitamin B12). To better understand the biological influence of HHcy on the development and progression of atherosclerosis, apolipoprotein-E-deficient (apoE−/− mice), a model for human atherosclerosis, were fed a hyperhomocysteinemic diet (low in methyl donors and B vitamins) (HHD) or a control diet (CD). After eight weeks, the plasma, aorta, and liver were collected to quantify methylation metabolites, while plasma was also used for a broad targeted metabolomic analysis. Aortic plaque burden in the brachiocephalic artery (BCA) was quantified via 14T magnetic resonance imaging (MRI). A severe accumulation of plasma and hepatic homocysteine and an increased BCA plaque burden were observed, thus confirming the atherogenic effect of the HHD. Moreover, a decreased methylation capacity in the plasma and aorta, indirectly assessed by the ratio of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) was detected in HHD mice together with a 172-fold increase in aortic cystathionine levels, indicating increased flux through the transsulfuration pathway. Betaine and its metabolic precursor, choline, were significantly decreased in the livers of HHD mice versus CD mice. Widespread changes in the plasma metabolome of HHD mice versus CD animals were detected, including alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, triacylglycerol levels, and several indicators of dysfunctional lipid metabolism. This study confirms the relevance of severe HHcy in the progression of vascular plaque and suggests novel metabolic pathways implicated in the pathophysiology of atherosclerosis.

1. Introduction

Atherosclerosis and resulting cardiovascular disease (CVD) are the leading causes of death and morbidity in the US [1]. Hyperhomocysteinemia (HHcy), a condition defined by an accumulation of plasma homocysteine (Hcy), is an independent risk factor for atherosclerosis and CVD [2,3,4,5]. Nevertheless, the molecular basis of this association remains to be defined entirely [3]. Homocysteine is not naturally present in food; it is a by-product of methionine metabolism. This process begins with the conversion of methionine to S-adenosylmethionine (SAM), a universal methyl donor compound for various methyl transferase reactions [6,7,8] that causes the formation of the methylated substrate and of S-adenosylhomocysteine (SAH). Interestingly, excess SAH can competitively inhibit the activity of SAM-dependent methyltransferases. Consequently, the ratio of SAM to SAH (SAM:SAH) is commonly used and referred to as the methylation potential of the cell [4,6,7]. SAH undergoes further hydrolysis to form Hcy through a reversible reaction that thermodynamically favors SAH production when Hcy accumulates. Thus, HHcy causes a decrease in the SAM:SAH ratio, promoting hypomethylating stress. This imbalance may contribute to the observed vascular toxicity associated with high Hcy levels [4,6,7]. Hcy is then remethylated to methionine through a vitamin B9 (folate)-dependent pathway that requires vitamin B12 (cobalamin) as co-enzyme. In addition, and mainly in the liver, Hcy is remethylated to methionine by betaine, a choline metabolite. Moreover, Hcy is catabolized to cysteine by the vitamin-B6-dependent transsulfuration pathway. Thus, B vitamins play a significant role in regulating Hcy metabolism [9,10,11]. Accordingly, dietary manipulation of the content of methyl donors and B vitamins is an established approach to producing an accumulation of Hcy, especially in the presence of an excess of methionine [12,13,14].
The apolipoprotein-E-deficient (apoE−/−) mouse is a pre-clinical model of human atherosclerosis that mimics the human metabolic signature and accumulates aortic atherosclerotic plaque [15,16,17]. Thus, to better understand the biological processes related to the influence of HHcy in the development and progression of atherosclerosis, apoE−/− mice were fed a hyperhomocysteinemic diet (HHD) (low in methyl donors and B vitamins) or a diet with adequate levels of all micronutrients (control, CD). After confirming the presence of severe HHcy in the HHD group, the volume of the aortic atheroma was quantified using high-field magnetic resonance imaging (MRI). Lastly, the plasma, aortic, and hepatic levels of intermediates of Hcy metabolism were determined, and a targeted metabolomic analysis of the plasma was conducted.

2. Materials and Methods

2.1. Animals and Diets

Seven-week-old apoE−/− mice, purchased from Jackson Laboratory (Bar Harbor, ME, USA), were housed individually in stainless-steel wire-bottom cages in an environmentally controlled room. Only male mice were used to control for the known effect of sex hormones on atherosclerosis in this strain [18]. The animals were fed, ad libitum, water and one of the following diets that were prepared based on AIN 93G (Research Diets, New Brunswick, NJ, USA): a control diet (CD) (11 Kcal% fat, 70 Kcal% carbohydrate, 18 Kcal% protein; n = 8), or a HHcy diet (HHD) that had reduced levels of methyl donors and vitamins (folate, choline, vitamin B6, and vitamin B12) and an excess of methionine (n = 16). The composition of the CD and the altered levels of micronutrients in the HHD, shown in Table 1, were selected to match our previous studies, in which the same nutritional approach was used to induce Hcy accumulation [19,20]. Weighed portions of the diets were replaced once a week, at which time animal weights were taken. All procedures were performed in compliance with the Institutional Animal Care and Use Committee of the Pennsylvania State University, which specifically approved this study.

2.2. Biochemical Analyses: Blood Glucose and ALT Levels

Every two weeks, blood glucose was measured using a glucometer (Contour, Bayer, Tarrytown, NY, USA), following the manufacturer’s instructions.
Moreover, at the same time points, liver health was evaluated via the plasma alanine aminotransferase (ALT) levels (Randox AL146, Antrim, UK), following the manufacturer’s instructions.

2.3. Blood Collection

After 8 weeks, the mice were euthanized by carbon dioxide inhalation, and blood was collected from the portal vein into heparinized tubes and immediately placed on ice. Plasma was isolated by centrifugation at 4 °C and immediately stored at −80 °C prior to further metabolic analyses.

2.4. Tissue Collection

The aortas were exposed and isolated, and approximately half of the abdominal aorta was excised, immediately immersed into liquid nitrogen, and stored at −80 °C until further analysis was undertaken. The remainder of each aorta, after double perfusion (10 mL PBS followed by 10 mL neutral buffered formalin 10% (NBF, Fisher Chemical), was fixed in NBF for 24 h, and then transferred to PBS, as previously described in detail [19,20,21,22,23].
The livers were dissected, weighed, and immediately snap-frozen in liquid nitrogen or fixed in 10% NBF for 24 h, then stored in 70% ethanol until histology slides were prepared.

2.5. Liver Histologic Analysis

Paraffinized sections (5 mm thickness) from the fixed liver tissue were prepared by the Penn State Animal Diagnostic Laboratory and stained with hematoxylin and eosin (H&E). Images were generated using a Leica DMi8 via the LAS X 2021 software.

2.6. Aortic Atheroma Quantification

Magnetic resonance imaging (MRI) using an Agilent 14T microimaging system (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to quantify the volume of aortic plaque burden in the brachiocephalic artery (BCA), as previously described in detail [19,20,21,23]. Scan parameters included an echo time (TE) of 13 ms, a repetition time (TR) of 100.00 ms, eight averages, a field of view (FOV) of 12.6 × 4.2 × 4.2 mm3, and a matrix size of 630 × 210 × 210, resulting in an isotropic resolution of 20 µm. After the acquisition, MR data were reconstructed using Matlab (The MathWorks Inc., Natick, MA, USA). Zero-filling by a factor of 2 in each direction lead to a final isotropic pixel resolution of 10 µm. Data segmentation was performed using Avizo 9.5 (Thermo Fisher Scientific, Waltham, MA, USA). The lumen of the aorta, the different plaques, and the aorta wall were manually segmented to obtain the volume of the atheroma in the vessel.

2.7. Quantification of Homocysteine and Methylation Metabolites

Metabolites related to the methylation cycle, shown in Figure 1, were determined in the plasma, liver, and aorta tissue via liquid chromatography tandem mass spectrometry (LC-MS/MS). This included Hcy in the plasma and liver, as previously described [24,25], but not in the aorta due to a lack of sufficient tissue. Other metabolites, including methionine, SAM, SAH, betaine, choline, and cystathionine, were determined in all samples, as previously described [26,27]. All assays were performed with 2 levels of quality controls to monitor assay performance, which for all metabolites had an overall variance of <15%.

2.8. Targeted Metabolomic Analysis

Targeted metabolomic analysis in plasma collected at 8 weeks was performed using the commercially available MxP® Quant 500 kit (Biocrates Life Sciences, Innsbruck, Austria), following the manufacturer’s instructions provided. Added to a 96-well plate were 10 μL of plasma, calibrators (7 levels), and quality controls (3 levels). The MxP® Quant 500 kit can potentially identify 630 metabolites across 23 classes of compounds by LC-MS/MS. Peak identification was accomplished using Analyst 1.7.2 (SCIEX, Framingham, MA, USA) and multiple reaction monitoring. MS data were uploaded to MetIDQ (Biocrates, Innsbruck, Austria) for analyte quantitation. MetaboIndicatorTM (biocrates life sciences, Innsbruck, Austria) was used to calculate 232 metabolic indicators, based on the sums and/or ratios of metabolites.

2.9. Statistical Analysis and Bioinformatics

All analyses, except plasma metabolomics, were performed in GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA), with statistical significance set to p < 0.05. For the comparison of the two groups, an unpaired Student’s t-test was used.
For targeted metabolomic data, peak detection, concentration computation, and plate normalization were performed using MetIDQ Oxygen v3005 (Biocrates Life Sciences, Innsbruck, Austria). Metabolomic data were uploaded to and processed in MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) (last accessed on 8 January 2024) for statistical analysis (t-test and heatmaps). The lower limit of detection (LoDs) was calculated as 3× the median signal in the phosphate-buffered saline blanks in each plate. Analytes with more than 50% of samples below LoD in each group were filtered out, and for those with less than 50%, the <LoD values were replaced with 1/5 the minimum positive value for that variable. All data were log transformed for normalization prior to analysis.

3. Results and Discussion

3.1. General Characteristics

Sequential measurements of body weight showed that mice fed the HHD failed to gain weight compared to the CD mice (Figure 2A) despite a similar mean calorie intake in both groups. Moreover, mice in the HHD group were smaller than the CD-fed animals (Figure 2B), evidencing slowed growth and emphasizing the essential role of micronutrients for proper development [28]. Previous studies have reported similar effects on growth when animals were fed diets with suboptimal levels of B vitamins and choline [12,14,19,20].
Next, the effect of the experimental diets on liver health was investigated. The results showed that 8 weeks of HHD feeding resulted in significantly elevated ALT levels and heavier livers than CD mice (Figure 2B and Figure 2G, respectively). Accordingly, the histology analysis revealed the HHD livers’ lipid accumulation, hepatocyte ballooning, and neutrophil infiltration, features absent in the CD mice, suggesting the development of hepatic steatosis in the first group of mice (Figure 2E,F). Dietary choline deprivation has been shown to cause fatty liver due to impaired hepatic secretion of lipids into plasma lipoproteins because of the inhibition of phosphatidylcholine (PC) synthesis from choline [29]. In addition, previous studies have also indicated that acute liver toxicity in fatty liver disease is associated with atherosclerosis and CVD [30]. Moreover, a study in which mice were fed a high-methionine–low-folate diet or a control diet reported increased Hcy concentrations and hepatic steatosis in the first group of animals [31]. Lastly, in the present study, HHD-fed mice presented a persistently lower glycemia than CD mice, which reached statistical significance after 4 weeks of the dietary intervention, a phenotype that has also been reported previously [32].

3.2. Homocysteine Levels and Atherosclerotic Plaque Burden

The manipulation of the dietary content of choline and B vitamins is an established approach to promoting HHcy [12,13,14]. In the present study, a severe accumulation of plasma Hcy was achieved in the mice fed the HHD diet, with an average concentration of 247 ± 88.1 versus 4.7 ± 0.9 in the CD group (µM, mean ± SD) (Figure 3A). Interestingly, in our previous studies on apoE−/− mice, in which diets with a similar micronutrient profile but a higher-fat content were used (40–80% fat kcal versus 5% fat kcal in the present study), we observed a milder accumulation of Hcy, suggesting that a high fat content attenuates the effect of sub-optimal levels of micronutrients on the Hcy metabolism. In liver tissue, Hcy accumulation was even more pronounced than in plasma, with mean values of 543 and 31 nmol/g for HHD and CD mice, respectively, showing that the liver was the organ in which Hcy metabolism was most affected by the HDD; this was primarily due to the combined reduced activity of cobalamin/folate-dependent, choline-dependent re-methylation into methionine and the PLP-dependent transsulfuration to cysteine.
Next, the effect of the experimental diets on atherosclerosis progression was evaluated by determining the volume of BCA aortic atheroma using 14T-MR imaging. The BCA is the segment of the aorta where the first plaques develop in apoE−/− mice [23]. As shown in Figure 3B,C, HHD mice presented significantly (p < 0.05) more aortic atherosclerosis burden than the CD mice. Moreover, the increased plaque burden detected in HHD BCA was accompanied by significant plaque morphology changes, such as the presence of lipid cores, luminal wall thickening, and vessel obstruction. However, the 14T-MRI imaging approach used in the present study did not address in detail features of plaque stability and necrosis, which are significant predictors of disease outcome [23].
Many studies have used dietary approaches like ours in murine models of human atherosclerosis to explore the vascular phenotype associated with HHcy. Overall, studies in which a severe (>100 µM) accumulation of plasma Hcy was achieved reported an enhanced development of atherosclerosis, supporting the results of the present study [14]. For example, Liu et al. [33] reported that eight weeks of severe HHcy aggravated aortic plaque formation. However, the findings associated with mild HHcy are inconsistent. These include our previous studies [19,20], in which, in a twelve-week feeding trial using apoE−/− mice, we could not detect significant differences at the p < 0.05 level for BCA plaque burden between mild hyperhomocysteinemic and control mice.

3.3. Methylation Indexes and the Concentrations of Relevant Metabolites in Different Tissues

After confirming severe HHcy in the HHD group, related metabolite levels in the plasma, liver, and aortic tissue were determined (Figure 4). As anticipated, the precursor to Hcy, SAH, showed a significant elevation in all investigated tissues of HHD mice (Figure 4A). Further, the SAM:SAH ratio was significantly decreased in the plasma and aorta, indicating the presence of systemic and vascular hypomethylation in the HHD mice (Figure 4C). We have previously reported in two studies [19,20] using the same mouse model that mild HHcy was associated with systemic but not with aortic hypomethylation. This suggests that, in those studies, the levels of accumulated Hcy were insufficient to affect vascular transmethylation reactions. However, in the present study, under a severe HHcy, a significant hypomethylating environment was evident at both systemic and vascular levels. Therefore, in addition to the atherogenic effect of the HHD, we documented increased SAH and a decreased SAM:SAH ratio at the vascular level, supporting the concept that hypomethylating stress contributes to the adverse vascular consequences of HHcy [3,4,7,13,34,35,36,37,38,39]. A recent cohort study has associated elevated plasma SAH concentrations with CVD risk [40]. Plasma SAH levels were also linked to traditional CVD risk factors and subclinical atherosclerosis in individuals with low CVD risk [41]. Furthermore, plasma SAH was inversely associated with endothelial dysfunction in patients with coronary artery disease [42].
In contrast, the SAM:SAH ratio in the liver was elevated in HHD mice versus CD mice (Figure 4C). However, the excess methionine in the HHD led to a more pronounced increase in hepatic SAM than SAH (Figure 4B), compared to the plasma and aorta levels [43]. The liver is the only tissue expressing multiple isoforms of methionine-adenosyltransferase, the enzyme that converts methionine to SAM [44]. Consequently, the substantial SAM levels detected in the liver increased the SAM:SAH ratio.
The HHD notably impacted the transsulfuration pathway (Figure 4D), evident in the marked 172-fold increase in aortic cystathionine concentration, the 77-fold increase in plasma concentration, and the 31-fold increase in the liver of HHD-fed mice compared to CD mice. This substantial change across all examined tissues suggests cystathionine as a potential biomarker of hypomethylating stress. The two PLP-dependent enzymes (PLP is the active form of vitamin B6) CBS and CGL catalyze the production of cystathionine and cysteine, respectively (Figure 1). However, CGL activity is more severely affected than CBS by insufficient PLP levels [45,46]. This may be attributed to the higher affinity and increased number of PLP binding sites, with four in total, in the CBS tetramer compared to the two binding sites in the four-subunit heterodimer CGL [47]. Consequently, the HHD, which was vitamin B6 deficient, promoted a massive accumulation of cystathionine.
Finally, we determined the concentrations of betaine and its metabolic precursor, choline, in our model. The results indicated that the HHD caused a significant decrease in betaine in the liver (13 nmol/g versus 455 nmol/g in CD) (Figure 4F). Similarly, choline showed a considerable reduction (713 nmol/g in HHD versus 494 nmol/g in CD) (Figure 4G). The betaine-dependent remethylation pathway in the liver participates in homocysteine metabolism (Figure 1). As shown in Table 1, the HHD lacked choline bitartrate, which was present in the CD. Thus, the observed depletion of choline and betaine reflects its dietary origin. Additionally, the reduced betaine levels in HHD mice may have contributed to liver damage, steatosis, and the severe accumulation of Hcy, as suggested by previous studies indicating the protective role of betaine against systemic inflammation and hepatotoxicity by reducing cellular stress [48].

3.4. Targeted Metabolomic Analysis

Metabolomic profiling offers a novel approach to identifying biomarkers and studying disease mechanisms. One of the available animal models of human atherosclerosis that better mimics the human metabolite signature is the apoE−/− mice [15]. Thus, to better understand the biological processes underlying the atherosclerotic effect of severe HHcy, we assessed the metabolomic signature in plasma from the different experimental groups. The results showed significant changes in several class compounds, seen in Table 2 and Figure 5, including acylcarnitine species, amino acids, bile acids, ceramides, sphingomyelins, fatty acids, and triacylglycerols. Several amino acids were increased in the HHD group versus the CD group, suggesting protein metabolism was affected. As explained above, the severe HHcy in the HHD-fed mice was achieved by the inadequacy of the levels of micronutrients acting as co-factors in homocysteine metabolism, including vitamin B6, a micronutrient deeply involved in amino acid metabolism and degradation. PLP, the coenzyme form of vitamin B6, participates in different catalytic functions, including transaminations, α-decarboxylations, racemizations, and the transsulfuration of Hcy to cysteine [49]. Cysteine is one of the gluthathionine constituents, a major intracellular antioxidant system. PLP has recently been suggested as a scavenger of reactive oxygen species [50,51].
Additionally, the metabolomic indicators showed a various effects from HHD versus CD, as seen in Table 3 and Figure 6. These indicators are sums or sums and ratios of metabolites from the Quant 500 analysis that can reveal information on specific pathway activity and metabolic flux (Supplementary Table S1).
Acylcarnitines play an essential role in regulating lipid metabolism by permitting the transport of long-chain fatty acids into the mitochondria for β-oxidation as a source of energy [52]. A generalized depletion of plasma acylcarnitines in the HHD-fed mice was observed, suggesting a reduced β-oxidation capacity in these animals. B12 vitamin deficiency causes the accumulation of methylmalonic acid, which acts as a potent inhibitor of the rate-limiting enzyme carnitine palmitoyl transferase 1, critical for breaking long-chain fatty acids in the beta-oxidation pathway [53]. Interestingly, it has been reported that decreased plasma acylcarnitine levels in mice were associated with a defective hepatic fatty acid β-oxidation and hepatic fat deposition, thus suggesting that this may have contributed to the liver toxicity observed in the HHD group [54].
Additionally, hydrophobic bile acids, including cholic acid (CA), deoxycholic acid (DCA), and taurocholic acid (TCA), were significantly elevated in HHD mice (FDR < 0.01). Bile acids are biomarkers of lipid and carbohydrate metabolism in the liver of both mice and humans, as they are direct end products of cholesterol catabolism [46]. Alterations in bile acid metabolism are associated with vascular inflammation and atherosclerosis [55]. Moreover, bile acids, especially DCA and TCA, have been associated with liver damage [56,57,58]. Thus, the observed liver injury after 8 weeks of HHD in the present study could have contributed to the accumulation of DCA and TCA.
Moreover, in the plasma of HHD mice, the levels of 19 unsaturated ceramides and hexosyl-ceramides were significantly elevated. In contrast, four saturated ceramides decreased significantly. Ceramides are precursors to sphingolipids, and both compounds are implicated in vascular dysfunction in rodents and humans [59]. Unsaturated ceramides regulate LDL cholesterol infiltration in blood vessels, generating plaque in rodent’s vascular walls [60]. In previous studies on mice, the inhibition of ceramide synthesis limited the progression of atherosclerosis [59]. Unsaturated ceramides are known to play a substantial role in the development of cardiovascular disease. Our findings reveal a link between severe HHcy and ceramide metabolism that can influence plaque deposition, which demands further investigation.
In this study, four sphingomyelin level metabolic indicators were also significantly elevated. These include the total sum of sphingomyelins, the ratio of sphingomyelins to phosphatidylcholines, the sum of OH-deficient sphingomyelins, and the sum of long-chain fatty acid sphingomyelins. These data suggest mitochondrial dysfunction as indicated by the increase in light-chain fatty acid sphingomyelins, which have been reported to increase cardiovascular disease in humans [61]. Previous studies have suggested, however, that apoE−/− mice have altered plasma sphingomyelin processing [62]. Other studies have reported that the pharmacologic inhibition of sphingomyelin synthesis attenuated atherosclerosis progression in apoE−/− mice [63].
In our metabolomic analysis, many triacylglycerol species were significantly decreased in the plasma of HHD mice compared to CD. The reason needs to be clarified, and, to our knowledge, the effect of HHcy on triacylglycerol metabolism has not been previously reported. Triacylglycerols play an essential role in energy metabolism, as stores can be mobilized to produce free fatty acids converted to acetyl-CoA via β-oxidation. Acetyl-CoA then enters the citric acid cycle to generate ATP. The levels of free fatty acids (FA 18:1; 20:1 and 20:2) were also decreased in the HHD group compared to the CD one, consistent with the effect on triacylglycerols. It has been shown that tissue-specific gene hypomethylation correlates with lower plasma levels of triacylglycerols, suggesting that HHD-induced hypomethylating stress contributed to the reduction demonstrated in [64].
Lastly, the present metabolomic data suggest that phospholipase-A2 activity was increased under severe HHcy. Phospholipase-A2 converts phosphatidylcholines to lysophosphatidylcholines, which release fatty acids and eicosanoids [65]. Eicosanoids can contribute to tissue inflammation through downstream prostaglandin generation [66]. Accordingly, previous studies have shown that elevated PL-A2 is a marker of vascular inflammation [67]. As supporting evidence, the pharmaceutical inhibition of PL-A2 in apoE−/− mice decreased vascular inflammation. In humans, increased serum levels of PL-A2 are associated with increased severity of CVD [68]. The present findings warrant further investigation into PL-A2 as a potential bridge between vascular distress and the metabolic dysfunction associated with severe HHcy [69,70].

4. Conclusions

In conclusion, severe HHcy promoted systemic and vascular hypomethylation and aggravated plaque formation in the BCA of apoE−/− mice. In addition, livers from these mice exhibited hepatocellular steatosis and oxidative damage, which was supported by metabolomic changes. Moreover, substantial alterations were observed in several metabolites, including cystathionine, and various metabolomic indicators affecting lipidomic profiles. Our data demonstrate the importance of maintaining adequate levels of B vitamins to prevent HHcy-induced atherosclerosis and promote cardiovascular and liver health.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu16030330/s1, Supplementary Table S1: List of metabolic indicators included in the MxP Quant 500 assay. The indicators were calculated by MetIDQ Oxygen v3005 software using Metabo INDICATOR formulas. Supplementary File S1: Typical Primex composition provided by Research Diets.

Author Contributions

Conceptualization, T.N., T.B. and R.C.; Methodology, A.M.K.; Validation, T.N.; Formal analysis, S.G.A., A.M.K., D.P. and T.B.; Investigation, S.G.A., A.M.K., V.S., T.B. and R.C.; Resources, T.N., V.S., T.B. and R.C.; Writing—original draft, S.G.A.; Writing—review & editing, D.P., A.C.R., V.S., T.B. and R.C.; Supervision, T.N., A.C.R. and R.C.; Funding acquisition, T.B., A.C.R. and R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Penn State High-Field Magnetic Resonance Imaging Facility and the Huck Institutes of the Life Sciences of the Pennsylvania State University. DP was supported by NIH grant T32DK120509.

Institutional Review Board Statement

The Institutional Animal Care and Use Committee of the Pennsylvania State University approved this study (IACUC #47911) on 23 November 2020.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors wish to thank Sean Gullette (Huck Institutes of the Life Sciences, Penn State University, USA) for his MRI technical expertise, Dian Jin (Department of Nutritional Sciences, Penn State University, USA) for her valuable help with the segmentation of the aortas, and Isabel Tavares de Almeida (University of Lisbon, Lisbon, Portugal) for her valuable support.

Conflicts of Interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

  1. Herrington, W.; Lacey, B.; Sherliker, P.; Armitage, J.; Lewington, S. Epidemiology of Atherosclerosis and the Potential to Reduce the Global Burden of Atherothrombotic Disease. Circ. Res. 2016, 118, 535–546. [Google Scholar] [CrossRef]
  2. Ganguly, P.; Alam, S.F. Role of homocysteine in the development of cardiovascular disease. Nutr. J. 2015, 14, 6. [Google Scholar] [CrossRef]
  3. Joseph, J.; Handy, D.E.; Loscalzo, J. Quo vadis: Whither homocysteine research? Cardiovasc. Toxicol. 2009, 9, 53–63. [Google Scholar] [CrossRef]
  4. Lai, W.K.; Kan, M.Y. Homocysteine-Induced Endothelial Dysfunction. Ann. Nutr. Metab. 2015, 67, 1–12. [Google Scholar] [CrossRef]
  5. Gueant, J.L.; Gueant-Rodriguez, R.M.; Oussalah, A.; Zuily, S.; Rosenberg, I. Hyperhomocysteinemia in Cardiovascular Diseases: Revisiting Observational Studies and Clinical Trials. Thromb. Haemost. 2023, 123, 270–282. [Google Scholar] [CrossRef]
  6. Jakubowski, H. Homocysteine Modification in Protein Structure/Function and Human Disease. Physiol. Rev. 2019, 99, 555–604. [Google Scholar] [CrossRef]
  7. Loscalzo, J.; Handy, D.E. Epigenetic modifications: Basic mechanisms and role in cardiovascular disease (2013 Grover Conference series). Pulm. Circ. 2014, 4, 169–174. [Google Scholar] [CrossRef]
  8. Barroso, M.; Handy, D.E.; Castro, R. The link between hyperhomocysteinemia and hypomethylation: Implications for cardiovascular disease. J. Inborn Errors Metab. Screen. 2019, 5, e160024. [Google Scholar] [CrossRef]
  9. Lyon, P.; Strippoli, V.; Fang, B.; Cimmino, L. B Vitamins and One-Carbon Metabolism: Implications in Human Health and Disease. Nutrients 2020, 12, 2867. [Google Scholar] [CrossRef]
  10. Caldeira-Araujo, H.; Ramos, R.; Florindo, C.; Rivera, I.; Castro, R.; Tavares de Almeida, I. Homocysteine Metabolism in Children and Adolescents: Influence of Age on Plasma Biomarkers and Correspondent Genotype Interactions. Nutrients 2019, 11, 646. [Google Scholar] [CrossRef] [PubMed]
  11. Castro, R.; Barroso, M.; Rocha, M.; Esse, R.; Ramos, R.; Ravasco, P.; Rivera, I.; de Almeida, I.T. The TCN2 776CNG polymorphism correlates with vitamin B(12) cellular delivery in healthy adult populations. Clin. Biochem. 2010, 43, 645–649. [Google Scholar] [CrossRef]
  12. Troen, A.M.; Lutgens, E.; Smith, D.E.; Rosenberg, I.H.; Selhub, J. The atherogenic effect of excess methionine intake. Proc. Natl. Acad. Sci. USA 2003, 100, 15089–15094. [Google Scholar] [CrossRef]
  13. Esse, R.; Florindo, C.; Imbard, A.; Rocha, M.S.; de Vriese, A.S.; Smulders, Y.M.; Teerlink, T.; Tavares de Almeida, I.; Castro, R.; Blom, H.J. Global protein and histone arginine methylation are affected in a tissue-specific manner in a rat model of diet-induced hyperhomocysteinemia. Biochim. Biophys. Acta 2013, 1832, 1708–1714. [Google Scholar] [CrossRef]
  14. Dayal, S.; Lentz, S.R. Murine models of hyperhomocysteinemia and their vascular phenotypes. Arterioscler. Thromb. Vasc. Biol. 2008, 28, 1596–1605. [Google Scholar] [CrossRef]
  15. Saulnier-Blache, J.S.; Wilson, R.; Klavins, K.; Graham, D.; Alesutan, I.; Kastenmuller, G.; Wang-Sattler, R.; Adamski, J.; Roden, M.; Rathmann, W.; et al. Ldlr(-)(/)(-) and ApoE(-)(/)(-) mice better mimic the human metabolite signature of increased carotid intima media thickness compared to other animal models of cardiovascular disease. Atherosclerosis 2018, 276, 140–147. [Google Scholar] [CrossRef]
  16. Getz, G.S.; Reardon, C.A. Do the Apoe-/- and Ldlr-/- Mice Yield the Same Insight on Atherogenesis? Arterioscler. Thromb. Vasc. Biol. 2016, 36, 1734–1741. [Google Scholar] [CrossRef]
  17. Getz, G.S.; Reardon, C.A. ApoE knockout and knockin mice: The history of their contribution to the understanding of atherogenesis. J. Lipid. Res. 2016, 57, 758–766. [Google Scholar] [CrossRef]
  18. Caligiuri, G.; Nicoletti, A.; Zhou, X.; Tornberg, I.; Hansson, G.K. Effects of sex and age on atherosclerosis and autoimmunity in apoE-deficient mice. Atherosclerosis 1999, 145, 301–308. [Google Scholar] [CrossRef] [PubMed]
  19. Castro, R.; Whalen, C.A.; Gullette, S.; Mattie, F.J.; Florindo, C.; Heil, S.G.; Huang, N.K.; Neuberger, T.; Ross, A.C. A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes. Nutrients 2021, 13, 3576. [Google Scholar] [CrossRef] [PubMed]
  20. Whalen, C.A.; Mattie, F.J.; Florindo, C.; van Zelst, B.; Huang, N.K.; Tavares de Almeida, I.; Heil, S.G.; Neuberger, T.; Ross, A.C.; Castro, R. No Effect of Diet-Induced Mild Hyperhomocysteinemia on Vascular Methylating Capacity, Atherosclerosis Progression, and Specific Histone Methylation. Nutrients 2020, 12, 2182. [Google Scholar] [CrossRef] [PubMed]
  21. da Silva, I.V.; Whalen, C.A.; Mattie, F.J.; Florindo, C.; Huang, N.K.; Heil, S.G.; Neuberger, T.; Ross, A.C.; Soveral, G.; Castro, R. An Atherogenic Diet Disturbs Aquaporin 5 Expression in Liver and Adipocyte Tissues of Apolipoprotein E-Deficient Mice: New Insights into an Old Model of Experimental Atherosclerosis. Biomedicines 2021, 9, 150. [Google Scholar] [CrossRef] [PubMed]
  22. da Silva, I.V.; Gullette, S.; Florindo, C.; Huang, N.K.; Neuberger, T.; Ross, A.C.; Soveral, G.; Castro, R. The Effect of Nutritional Ketosis on Aquaporin Expression in Apolipoprotein E-Deficient Mice: Potential Implications for Energy Homeostasis. Biomedicines 2022, 10, 1159. [Google Scholar] [CrossRef]
  23. Castro, R.; Gullette, S.; Whalen, C.; Mattie, F.J.; Ge, X.; Ross, A.C.; Neuberger, T. High-field magnetic resonance microscopy of aortic plaques in a mouse model of atherosclerosis. MAGMA 2023, 36, 887–896. [Google Scholar] [CrossRef]
  24. Ducros, V.; Belva-Besnet, H.; Casetta, B.; Favier, A. A robust liquid chromatography tandem mass spectrometry method for total plasma homocysteine determination in clinical practice. Clin. Chem. Lab. Med. 2006, 44, 987–990. [Google Scholar] [CrossRef]
  25. Lai, S.C.; Nakayama, Y.; Sequeira, J.M.; Wlodarczyk, B.J.; Cabrera, R.M.; Finnell, R.H.; Bottiglieri, T.; Quadros, E.V. The transcobalamin receptor knockout mouse: A model for vitamin B12 deficiency in the central nervous system. FASEB J. 2013, 27, 2468–2475. [Google Scholar] [CrossRef]
  26. Kalecky, K.; Ashcraft, P.; Bottiglieri, T. One-Carbon Metabolism in Alzheimer's Disease and Parkinson’s Disease Brain Tissue. Nutrients 2022, 14, 599. [Google Scholar] [CrossRef]
  27. Rooney, M.; Bottiglieri, T.; Wasek-Patterson, B.; McMahon, A.; Hughes, C.F.; McCann, A.; Horigan, G.; Strain, J.J.; McNulty, H.; Ward, M. Impact of the MTHFR C677T polymorphism on one-carbon metabolites: Evidence from a randomised trial of riboflavin supplementation. Biochimie 2020, 173, 91–99. [Google Scholar] [CrossRef]
  28. Moretti, R.; Peinkhofer, C. B Vitamins and Fatty Acids: What Do They Share with Small Vessel Disease-Related Dementia? Int. J. Mol. Sci. 2019, 20, 5797. [Google Scholar] [CrossRef]
  29. Kulinski, A.; Vance, D.E.; Vance, J.E. A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion. J. Biol. Chem. 2004, 279, 23916–23924. [Google Scholar] [CrossRef] [PubMed]
  30. Gehrke, N.; Schattenberg, J.M. Metabolic Inflammation-A Role for Hepatic Inflammatory Pathways as Drivers of Comorbidities in Nonalcoholic Fatty Liver Disease? Gastroenterology 2020, 158, 1929–1947.e6. [Google Scholar] [CrossRef] [PubMed]
  31. Ji, C.; Shinohara, M.; Vance, D.; Than, T.A.; Ookhtens, M.; Chan, C.; Kaplowitz, N. Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver. Alcohol Clin. Exp. Res. 2008, 32, 1049–1058. [Google Scholar] [CrossRef] [PubMed]
  32. Law, S.H.; Chan, H.C.; Ke, G.M.; Kamatam, S.; Marathe, G.K.; Ponnusamy, V.K.; Ke, L.Y. Untargeted Lipidomic Profiling Reveals Lysophosphatidylcholine and Ceramide as Atherosclerotic Risk Factors in apolipoprotein E Knockout Mice. Int. J. Mol. Sci. 2023, 24, 6956. [Google Scholar] [CrossRef] [PubMed]
  33. Liu, C.; Wang, Q.; Guo, H.; Xia, M.; Yuan, Q.; Hu, Y.; Zhu, H.; Hou, M.; Ma, J.; Tang, Z.; et al. Plasma S-adenosylhomocysteine is a better biomarker of atherosclerosis than homocysteine in apolipoprotein E-deficient mice fed high dietary methionine. J. Nutr. 2008, 138, 311–315. [Google Scholar] [CrossRef] [PubMed]
  34. Perla-Kajan, J.; Jakubowski, H. Dysregulation of Epigenetic Mechanisms of Gene Expression in the Pathologies of Hyperhomocysteinemia. Int. J. Mol. Sci. 2019, 20, 3140. [Google Scholar] [CrossRef] [PubMed]
  35. Castro, R.; Rivera, I.; Martins, C.; Struys, E.A.; Jansen, E.E.; Clode, N.; Graca, L.M.; Blom, H.J.; Jakobs, C.; de Almeida, I.T. Intracellular S-adenosylhomocysteine increased levels are associated with DNA hypomethylation in HUVEC. J. Mol. Med. 2005, 83, 831–836. [Google Scholar] [CrossRef]
  36. Castro, R.; Rivera, I.; Struys, E.A.; Jansen, E.E.; Ravasco, P.; Camilo, M.E.; Blom, H.J.; Jakobs, C.; Tavares de Almeida, I. Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease. Clin. Chem. 2003, 49, 1292–1296. [Google Scholar] [CrossRef]
  37. Barroso, M.; Florindo, C.; Kalwa, H.; Silva, Z.; Turanov, A.A.; Carlson, B.A.; de Almeida, I.T.; Blom, H.J.; Gladyshev, V.N.; Hatfield, D.L.; et al. Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression. J. Biol. Chem. 2014, 289, 15350–15362. [Google Scholar] [CrossRef]
  38. Barroso, M.; Rocha, M.S.; Esse, R.; Goncalves, I., Jr.; Gomes, A.Q.; Teerlink, T.; Jakobs, C.; Blom, H.J.; Loscalzo, J.; Rivera, I.; et al. Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells. Amino Acids 2012, 42, 1903–1911. [Google Scholar] [CrossRef]
  39. Esse, R.; Imbard, A.; Florindo, C.; Gupta, S.; Quinlivan, E.P.; Davids, M.; Teerlink, T.; Tavares de Almeida, I.; Kruger, W.D.; Blom, H.J.; et al. Protein arginine hypomethylation in a mouse model of cystathionine beta-synthase deficiency. FASEB J. 2014, 28, 2686–2695. [Google Scholar] [CrossRef]
  40. Xiao, Y.; Zhang, Y.; Wang, M.; Li, X.; Su, D.; Qiu, J.; Li, D.; Yang, Y.; Xia, M.; Ling, W. Plasma S-adenosylhomocysteine is associated with the risk of cardiovascular events in patients undergoing coronary angiography: A cohort study. Am. J. Clin. Nutr. 2013, 98, 1162–1169. [Google Scholar] [CrossRef]
  41. Zawada, A.M.; Rogacev, K.S.; Hummel, B.; Berg, J.T.; Friedrich, A.; Roth, H.J.; Obeid, R.; Geisel, J.; Fliser, D.; Heine, G.H. S-adenosylhomocysteine is associated with subclinical atherosclerosis and renal function in a cardiovascular low-risk population. Atherosclerosis 2014, 234, 17–22. [Google Scholar] [CrossRef] [PubMed]
  42. Xiao, Y.; Xia, J.; Cheng, J.; Huang, H.; Zhou, Y.; Yang, X.; Su, X.; Ke, Y.; Ling, W. Inhibition of S-Adenosylhomocysteine Hydrolase Induces Endothelial Dysfunction via Epigenetic Regulation of p66shc-Mediated Oxidative Stress Pathway. Circulation 2019, 139, 2260–2277. [Google Scholar] [CrossRef] [PubMed]
  43. Mato, J.M.; Martinez-Chantar, M.L.; Lu, S.C. S-adenosylmethionine metabolism and liver disease. Ann. Hepatol. 2013, 12, 183–189. [Google Scholar] [CrossRef] [PubMed]
  44. Ramani, K.; Lu, S.C. Methionine adenosyltransferases in liver health and diseases. Liver Res. 2017, 1, 103–111. [Google Scholar] [CrossRef] [PubMed]
  45. Aitken, S.M.; Lodha, P.H.; Morneau, D.J. The enzymes of the transsulfuration pathways: Active-site characterizations. Biochim. Biophys. Acta 2011, 1814, 1511–1517. [Google Scholar] [CrossRef]
  46. Honda, A.; Miyazaki, T.; Iwamoto, J.; Hirayama, T.; Morishita, Y.; Monma, T.; Ueda, H.; Mizuno, S.; Sugiyama, F.; Takahashi, S.; et al. Regulation of bile acid metabolism in mouse models with hydrophobic bile acid composition. J. Lipid. Res. 2020, 61, 54–69. [Google Scholar] [CrossRef]
  47. Gregory, J.F.; DeRatt, B.N.; Rios-Avila, L.; Ralat, M.; Stacpoole, P.W. Vitamin B6 nutritional status and cellular availability of pyridoxal 5′-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions. Biochimie 2016, 126, 21–26. [Google Scholar] [CrossRef] [PubMed]
  48. Wang, Z.; Yao, T.; Pini, M.; Zhou, Z.; Fantuzzi, G.; Song, Z. Betaine improved adipose tissue function in mice fed a high-fat diet: A mechanism for hepatoprotective effect of betaine in nonalcoholic fatty liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 2010, 298, G634–G642. [Google Scholar] [CrossRef]
  49. Ueland, P.M.; Ulvik, A.; Rios-Avila, L.; Midttun, O.; Gregory, J.F. Direct and Functional Biomarkers of Vitamin B6 Status. Annu. Rev. Nutr. 2015, 35, 33–70. [Google Scholar] [CrossRef]
  50. di Salvo, M.L.; Safo, M.K.; Contestabile, R. Biomedical aspects of pyridoxal 5′-phosphate availability. Front. Biosci. 2012, 4, 897–913. [Google Scholar]
  51. Kannan, K.; Jain, S.K. Effect of vitamin B6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H2O2-treated U937 monocytes. Free Radic. Biol. Med. 2004, 36, 423–428. [Google Scholar] [CrossRef] [PubMed]
  52. Blair, H.C.; Sepulveda, J.; Papachristou, D.J. Nature and nurture in atherosclerosis: The roles of acylcarnitine and cell membrane-fatty acid intermediates. Vasc. Pharmacol. 2016, 78, 17–23. [Google Scholar] [CrossRef] [PubMed]
  53. Boachie, J.; Adaikalakoteswari, A.; Samavat, J.; Saravanan, P. Low Vitamin B12 and Lipid Metabolism: Evidence from Pre-Clinical and Clinical Studies. Nutrients 2020, 12, 1925. [Google Scholar] [CrossRef]
  54. Bjorndal, B.; Alteras, E.K.; Lindquist, C.; Svardal, A.; Skorve, J.; Berge, R.K. Associations between fatty acid oxidation, hepatic mitochondrial function, and plasma acylcarnitine levels in mice. Nutr. Metab. 2018, 15, 10. [Google Scholar] [CrossRef] [PubMed]
  55. de Aguiar Vallim, T.Q.; Tarling, E.J.; Edwards, P.A. Pleiotropic roles of bile acids in metabolism. Cell Metab. 2013, 17, 657–669. [Google Scholar] [CrossRef] [PubMed]
  56. Zhao, Q.; Liu, F.; Cheng, Y.; Xiao, X.R.; Hu, D.D.; Tang, Y.M.; Bao, W.M.; Yang, J.H.; Jiang, T.; Hu, J.P.; et al. Celastrol Protects From Cholestatic Liver Injury Through Modulation of SIRT1-FXR Signaling. Mol. Cell Proteom. 2019, 18, 520–533. [Google Scholar] [CrossRef] [PubMed]
  57. Thomas, C.; Pellicciari, R.; Pruzanski, M.; Auwerx, J.; Schoonjans, K. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 2008, 7, 678–693. [Google Scholar] [CrossRef]
  58. Wei, S.; Ma, X.; Zhao, Y. Mechanism of Hydrophobic Bile Acid-Induced Hepatocyte Injury and Drug Discovery. Front. Pharmacol. 2020, 11, 1084. [Google Scholar] [CrossRef]
  59. Choi, R.H.; Tatum, S.M.; Symons, J.D.; Summers, S.A.; Holland, W.L. Ceramides and other sphingolipids as drivers of cardiovascular disease. Nat. Rev. Cardiol. 2021, 18, 701–711. [Google Scholar] [CrossRef]
  60. Summers, S.A. Could Ceramides Become the New Cholesterol? Cell Metab. 2018, 27, 276–280. [Google Scholar] [CrossRef]
  61. Nelson, J.C.; Jiang, X.C.; Tabas, I.; Tall, A.; Shea, S. Plasma sphingomyelin and subclinical atherosclerosis: Findings from the multi-ethnic study of atherosclerosis. Am. J. Epidemiol. 2006, 163, 903–912. [Google Scholar] [CrossRef] [PubMed]
  62. Yu, H.; Zhang, W.; Yancey, P.G.; Koury, M.J.; Zhang, Y.; Fazio, S.; Linton, M.F. Macrophage apolipoprotein E reduces atherosclerosis and prevents premature death in apolipoprotein E and scavenger receptor-class BI double-knockout mice. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 150–156. [Google Scholar] [CrossRef]
  63. Park, T.S.; Panek, R.L.; Mueller, S.B.; Hanselman, J.C.; Rosebury, W.S.; Robertson, A.W.; Kindt, E.K.; Homan, R.; Karathanasis, S.K.; Rekhter, M.D. Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice. Circulation 2004, 110, 3465–3471. [Google Scholar] [CrossRef] [PubMed]
  64. Ronn, T.; Perfilyev, A.; Jonsson, J.; Eriksson, K.F.; Jorgensen, S.W.; Brons, C.; Gillberg, L.; Vaag, A.; Stener-Victorin, E.; Ling, C. Circulating triglycerides are associated with human adipose tissue DNA methylation of genes linked to metabolic disease. Hum. Mol. Genet. 2023, 32, 1875–1887. [Google Scholar] [CrossRef] [PubMed]
  65. Dennis, E.A. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 1994, 269, 13057–13060. [Google Scholar] [CrossRef] [PubMed]
  66. Yamaguchi, A.; Botta, E.; Holinstat, M. Eicosanoids in inflammation in the blood and the vessel. Front. Pharmacol. 2022, 13, 997403. [Google Scholar] [CrossRef] [PubMed]
  67. De Stefano, A.; Mannucci, L.; Tamburi, F.; Cardillo, C.; Schinzari, F.; Rovella, V.; Nistico, S.; Bennardo, L.; Di Daniele, N.; Tesauro, M. Lp-PLA(2), a new biomarker of vascular disorders in metabolic diseases. Int. J. Immunopathol. Pharmacol. 2019, 33, 2058738419827154. [Google Scholar] [CrossRef] [PubMed]
  68. Cai, A.; Li, G.; Chen, J.; Li, X.; Li, L.; Zhou, Y. Increased serum level of Lp-PLA2 is independently associated with the severity of coronary artery diseases: A cross-sectional study of Chinese population. BMC Cardiovasc. Disord. 2015, 15, 14. [Google Scholar] [CrossRef]
  69. Ma, J.; Shen, L.; Bao, L.; Yuan, H.; Wang, Y.; Liu, H.; Wang, Q. A novel prognosis prediction model, including cytotoxic T lymphocyte-associated antigen-4, ischemia-modified albumin, lipoprotein-associated phospholipase A2, glial fibrillary acidic protein, and homocysteine, for ischemic stroke in the Chinese hypertensive population. J. Clin. Lab. Anal. 2021, 35, e23756. [Google Scholar]
  70. Li, B.; Gao, G.; Zhang, W.; Li, B.; Yang, C.; Jiang, X.; Tian, Y.; Liang, H. Metabolomics analysis reveals an effect of homocysteine on arachidonic acid and linoleic acid metabolism pathway. Mol. Med. Rep. 2018, 17, 6261–6268. [Google Scholar] [CrossRef]
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Figure 1. Schematic representation of the homocysteine metabolism. THF, tetrahydrofolate (vitamin B9); 5Me-THF (vitamin B9), 5-methyl THF (vitamin B9); Me-Cbl, methylcobalamine (vitamin B12), PLP, pyridoxal 5′-phosphate (vitamin B6); MTase, cellular methyltransferase; CBS, cystathionine-β-synthase; CGL, cystathionine-γ-lyase; BHMT, betaine–homocysteine methyltransferase.
Figure 1. Schematic representation of the homocysteine metabolism. THF, tetrahydrofolate (vitamin B9); 5Me-THF (vitamin B9), 5-methyl THF (vitamin B9); Me-Cbl, methylcobalamine (vitamin B12), PLP, pyridoxal 5′-phosphate (vitamin B6); MTase, cellular methyltransferase; CBS, cystathionine-β-synthase; CGL, cystathionine-γ-lyase; BHMT, betaine–homocysteine methyltransferase.
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Figure 2. The effect of the experimental diets (HHD, hyperhomocysteinemic diet, represented in red bars; CD, control diet, represented in blue bars) on (A) body weight progression; (B) mouse length; (C) blood glucose; (D) plasma alanine transaminase, ALT; (G) liver weight; and (E,F) liver histology (hematoxylin-eosin-stained samples at 10× amplification) from CD and HHD mice. * p < 0.05; ** p < 0.01. All error bars = ± SEM.
Figure 2. The effect of the experimental diets (HHD, hyperhomocysteinemic diet, represented in red bars; CD, control diet, represented in blue bars) on (A) body weight progression; (B) mouse length; (C) blood glucose; (D) plasma alanine transaminase, ALT; (G) liver weight; and (E,F) liver histology (hematoxylin-eosin-stained samples at 10× amplification) from CD and HHD mice. * p < 0.05; ** p < 0.01. All error bars = ± SEM.
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Figure 3. Effect of experimental diets CD (control diet) and HHD (hyperhomocysteinemic diet) on (A) the concentration of homocysteine in the plasma and liver; (B) brachiocephalic artery (BCA) atherosclerotic plaque burden. (C) Representative 14T-MRI images of the BCA from CD and HHD groups. Plaque burden was determined as the ratio of plaque volume (rendered in red) to wall volume. ** p < 0.01. All error bars = ±SEM.
Figure 3. Effect of experimental diets CD (control diet) and HHD (hyperhomocysteinemic diet) on (A) the concentration of homocysteine in the plasma and liver; (B) brachiocephalic artery (BCA) atherosclerotic plaque burden. (C) Representative 14T-MRI images of the BCA from CD and HHD groups. Plaque burden was determined as the ratio of plaque volume (rendered in red) to wall volume. ** p < 0.01. All error bars = ±SEM.
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Figure 4. Effect of the experimental diets CD (control diet) and HHD (hyperhomocysteinemic diet) on the concentration of (A) SAM (S-adenosylmethionine); (B) SAH (S-adenosylhomocysteine); (C) SAM:SAH; (D) cystathionine; (E) methionine; (F) betaine; and (G) choline in the plasma, liver, and aorta; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All error bars = ±SEM.
Figure 4. Effect of the experimental diets CD (control diet) and HHD (hyperhomocysteinemic diet) on the concentration of (A) SAM (S-adenosylmethionine); (B) SAH (S-adenosylhomocysteine); (C) SAM:SAH; (D) cystathionine; (E) methionine; (F) betaine; and (G) choline in the plasma, liver, and aorta; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All error bars = ±SEM.
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Figure 5. Heat map of the top 50 metabolites, including methylation specific species, ceramides (Cer), triglycerides (TG), phosphatidylcholines (PC), and amino acids, significantly altered by the experimental diets. The increasing intensity of the red color indicates higher concentrations of metabolites (max +2.3), and the increasing intensity of the blue color indicates decreased concentrations of metabolites (max −3.2).
Figure 5. Heat map of the top 50 metabolites, including methylation specific species, ceramides (Cer), triglycerides (TG), phosphatidylcholines (PC), and amino acids, significantly altered by the experimental diets. The increasing intensity of the red color indicates higher concentrations of metabolites (max +2.3), and the increasing intensity of the blue color indicates decreased concentrations of metabolites (max −3.2).
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Figure 6. Heat map of the top 50 metabolic indicators of oxidative stress, lipid processing, fatty acid transport, bile acid production, and amino acid metabolism, significantly altered by the experimental diets. The increasing intensity of the red color indicates higher concentrations of metabolic indicators (max +2.1), and the increasing intensity of the blue color indicates decreased concentrations of metabolic indicators (max −2.2).
Figure 6. Heat map of the top 50 metabolic indicators of oxidative stress, lipid processing, fatty acid transport, bile acid production, and amino acid metabolism, significantly altered by the experimental diets. The increasing intensity of the red color indicates higher concentrations of metabolic indicators (max +2.1), and the increasing intensity of the blue color indicates decreased concentrations of metabolic indicators (max −2.2).
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Table 1. Composition of the experimental diets (CD, control diet; HHD, hyperhomocysteinemic diet).
Table 1. Composition of the experimental diets (CD, control diet; HHD, hyperhomocysteinemic diet).
CDHHD
gmKcalgmKcal
Casein180720180720
Corn Starch43117254311725
Maltodextrin 10155620155620
Sucrose100400100400
Cellulose350350
Primex 12522525225
Corn Oil2522525225
Mineral Mix S10001350350
Vitamin Mix V10001 2104000
Vitamin Mix V14904 3001040
L-Cystine312312
L-Methionine003.112
Choline bitartrate2.5000
Pyridoxine HCl (×103)000.20
Folic acid (×103)000.10
Cyanocobalamin, 0.1% (×10−6)0020
Succinylsulfathiazole8.608.60
1 The typical Primex composition is shown in the Supplementary File S1; 2 With adequate levels of all vitamins; 3 Without vitamin B6, B9, or B12.
Table 2. Number of significantly altered plasma metabolite concentrations between CD (control diet) and HHD (hyperhomocystenemic) mice.
Table 2. Number of significantly altered plasma metabolite concentrations between CD (control diet) and HHD (hyperhomocystenemic) mice.
Metabolite Class PotentialDetectedFDR (<0.05) (CD vs. HHD)
MetabolitesMetabolitesIncreased Decreased
LC-MS/MS MetabolitesAlkaloids1000
Amine Oxides1000
Amino Acids2020134
Amino Acids Related302478
Bile Acids1412120
Biogenic Amines9700
Carboxylic Acids7400
Cresols1100
Fatty Acids12903
Hormones4110
Indoles and Derivatives4310
Nucleobases Related2000
Sugars1101
Vitamins and Cofactors1101
FIA-MS/MS metabolitesAcylcarnitines401308
Glycerophospholipids9087326
(Lysophosphatidylcholines & Phosphatidylcholines)
Sphingolipids1510100
Cholesterol Esters222104
Ceramides2828194
Dihydroceramides8400
Glyceroceramides3434170
(Mono-, Di-, and Trihexosylceramindes)
Diacylglycerols4420016
Triacylglycerols2422370214
Total63053783289
LC-MS/MS: liquid chromatography tandem mass spectrometry; FIA-MS/MS: Flow injection analysis tandem mass spectrometry.
Table 3. Number of significantly altered metabolomic indicators (indexes based on relative metabolite concentrations) between CD (control diet) and HHD (hyperhomocysteinemic diet)-fed mice.
Table 3. Number of significantly altered metabolomic indicators (indexes based on relative metabolite concentrations) between CD (control diet) and HHD (hyperhomocysteinemic diet)-fed mice.
FDR (<0.05) CD vs. HHDRelevant to Inflammation and Atherosclerosis
Metabolic Indicator ClassSignificantIncreasedDecreased
Amino Acids13941
Amino Acid Related181174
Acylcarnitines132111
Bile Acids151320
Biogenic Amines4131
Carboxylic Acids1100
Ceramides14951
Cholesteryl Esters1011
Diglycerides4141
Fatty Acids5231
Hormones1100
Indole and Derivatives2110
Lysophosphatidylcholines5140
Phosphatidylcholines5233
Sphingomyelins4400
Triacylglycerols4042
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Andrews, S.G.; Koehle, A.M.; Paudel, D.; Neuberger, T.; Ross, A.C.; Singh, V.; Bottiglieri, T.; Castro, R. Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice. Nutrients 2024, 16, 330. https://doi.org/10.3390/nu16030330

AMA Style

Andrews SG, Koehle AM, Paudel D, Neuberger T, Ross AC, Singh V, Bottiglieri T, Castro R. Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice. Nutrients. 2024; 16(3):330. https://doi.org/10.3390/nu16030330

Chicago/Turabian Style

Andrews, Stephen G., Anthony M. Koehle, Devendra Paudel, Thomas Neuberger, A. Catharine Ross, Vishal Singh, Teodoro Bottiglieri, and Rita Castro. 2024. "Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice" Nutrients 16, no. 3: 330. https://doi.org/10.3390/nu16030330

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