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Methylation Reactions, the Redox Balance and Atherothrombosis: The Search for a Link with Hydrogen Sulfide Roberta Lupoli, MD1 Alessandro Di Minno, PharmD2 Gaia Spadarella, MD1 Massimo Franchini, MD3 Raffaella Sorrentino, PhD2 Giuseppe Cirino, PhD2 Giovanni Di Minno, MD, PharmD1

University, Naples, Italy 2 Department of Pharmacy, “Federico II” University, Naples, Italy 3 Department of Transfusion Medicine and Hematology, Carlo Poma Hospital, Mantova, Italy

Address for correspondence Giovanni Di Minno, MD, Dipartimento di Medicina Clinica e Chirurgia, Via S. Pansini 5, 80131, Napoli, Italy (e-mail: [email protected]).

Semin Thromb Hemost 2015;41:423–432.

Abstract

Keywords

► ► ► ► ►

oxidative stress epigenetics cell proliferation inflammation translational medicine

It is now clear that homocysteine (Hcy) is irreversibly degraded to hydrogen sulfide (H2S), an endogenous gasotransmitter that causes in vivo platelet activation via upregulation of phospholipase A2 and downstream boost of the arachidonate cascade. This mechanism involves a transsulfuration pathway. Based on these new data, clinical and experimental models on the relationships between Hcy and folate pathways in vascular disease and information on the Hcy controversy have been reanalyzed in the present review. Most interventional trials focused on Hcy lowering by folate administration did not exclude patients routinely taking the arachidonate inhibitor aspirin. This may have influenced the results of some of these trials. It is also clear that nutritional intake of folate affects several enzymatic reactions of the methionine–Hcy cycle and associated one-carbon metabolism and, thereby, both methylation reactions and redox balance. Hence, it is conceivable that the abnormally high Hcy levels seen in pathologic states reflect a poorly elucidated perturbation of such reactions and of such balance. While it is unknown whether there is an interplay between H2S, methylation reactions, and redox balance, measuring the sole reduction of blood Hcy that follows folate administration may well be an oversimplified approach to a complex biologic perturbation. The need to investigate this complex framework is thoroughly discussed in this article.

In 1969, Kilmer McCully first observed that an infant with severe hyperhomocysteinemia (HHcy) leading to homocystinuria as a result of a rare condition of abnormal cobalamin metabolism exhibited severe diffuse arteriosclerosis indistinguishable from the lesions seen in some cases of homocystinuria caused by cystathionine β-synthase (CBS) deficiency.1 Because severely elevated plasma homocysteine (Hcy) concentrations were the only common metabolic abnormality in patients with similar vascular lesions and different inborn errors of Hcy metabolism, he postulated that Hcy, or one of its

derivatives, was toxic for the vascular wall (the “Hcy theory”). Seven years later, Wilcken and Wilcken showed that the concentration of Hcy-cysteine–mixed disulfide after a methionine load was slightly higher in coronary heart disease (CAD) patients than in age- and sex-matched controls.2 Major advances in the understanding of disorders of Hcy metabolism followed over the next 50 years, encompassing the rare homozygous enzyme deficiencies and the more common milder abnormalities.3 In addition, accelerated atherosclerosis and endothelial dysfunction have been associated with

published online May 14, 2015

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DOI http://dx.doi.org/ 10.1055/s-0035-1549848. ISSN 0094-6176.

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1 Department of Clinical Medicine and Surgery, “Federico II”

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HHcy,4–6 which, in turn, acts as a prothrombotic marker.7 Moreover, over the last few decades, retrospective and prospective studies have consistently shown that (1) mildly elevated total Hcy (tHcy) levels (by
15 µM) are independently associated with a high risk of cardiovascular disease8; (2) a positive correlation exists between Hcy plasma levels and cerebrovascular disorders9,10; and (3) in patients with CBS deficiency chronically treated with Hcy-lowering therapy, a dramatic decrease in the tendency to arterial and venous thrombosis occurs.11 Together, such evidence argues for tHcy as being an independent graded risk factor for vascular disease in both men and women. However, the relation between elevated plasma tHcy and vascular disease is stronger in retrospective than in prospective studies,12 and a prolonged correction of HHcy by folate administration little affects cardiovascular risk in large prospective trials.13 As a consequence, there has been a debate on whether tHcy elevation is truly a risk factor or an epiphenomenon of a more complex picture.14 Here, we examine some models that provide knowledge about the relationships between the Hcy and folate pathways and vascular disease, with emphasis on the oxidative stress hypothesis. A direct connection between Hcy and the susceptibility to atherothrombosis is also provided. In vivo, Hcy is irreversibly degraded to hydrogen sulfide (H2S), a gaseous transmitter that leads to in vivo platelet activation via upregulation of phospholipase A2 and downstream boost of the arachidonic acid cascade. Platelet activation is one of the major pathways underlying an abnormally high cardiovascular risk.15 Under physiological conditions, the homeostatic equilibrium between oxidative and reductive stress (as well as between hypo- and hypermethylation) modulates and is modulated by the methionine–Hcy cycle.16 Since one-carbon

Fig. 1 Homocysteine Metabolism.

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metabolism dependent on folate availability regulates both redox and methylation status, HHcy may be presently viewed as a marker of perturbation of one-carbon metabolism.17 The relevance of such novel paradigm with respect to the data from interventional studies with folate will also be discussed.

Homocysteine Metabolism: Relationships with the Redox Balance In vivo, Hcy is irreversibly degraded either via the transsulfuration pathway or through a reaction catalyzed by 5,10methylenetetrahydrofolate reductase (MTHFR) and is remethylated back to methionine (the methionine–Hcy cycle).18 Severe HHcy (tHcy >50 µM) may be due to defects in either remethylation or transsulfuration. When circulating Hcy levels exceed 80 to 100 µM, homocystinuria occurs. Abnormal remethylation is caused by MTHFR and/or methionine synthase (MTR) deficiency due to mutations in their respective genes. MTR can also be dysfunctional due to defects in cobalamin metabolism. In MTR deficiency or dysfunction, 5-methylTHF cannot cycle through MTR, leading to 5-methyl-THF accumulation (at the expense of the other folates), and, in turn, hampering the synthesis of purines and thymidine. Remethylation defects result in high Hcy and low methionine levels. As a consequence, rapidly dividing cells (e.g., bone marrow cells) will be affected, resulting in megaloblastic anemia and pancytopenia (folate deficiency). MTHFR deficiency, which occurs in 5 to 7% of individuals, is associated with mild/moderate elevations of plasma Hcy (i.e., 15–50 μM) that neither limit the availability of folates for purines and thymidine synthesis nor lead to blood cell abnormalities. The 677TT MTHFR genotype (MTHFRþþ), associated with a thermolabile variant of the enzyme with

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impaired activity and reduced 5-methyltetrahydrofolate (5-MTHF) availability,19 is the most common cause of inherited mild/moderate HHcy.20,21 A significant interaction between this MTHFR variant and folate and vitamin B12 levels is known,22 as vitamin availability by dietary intake or supplementation shows more relevant effects in the phenotypic expression of tHcy levels in subjects carrying the 677TT genotype.23 An alternate pathway of remethylation, which employs betaine as the methyl donor, is catalyzed by betaine homocysteine methyl transferase (BHMT).24 In the transsulfuration pathway (►Fig. 1), Hcy is irreversibly degraded to L-cysteine (L-Cys) and H2S through multiple steps involving CBS and cystathionine γ-lyase (CTH). The process is initiated by the irreversible conversion of Hcy and serine to cystathionine by the enzyme cystathionine β synthase (CBS), which utilizes vitamin B6 as a cofactor.25 CTH then catalyzes the conversion of cystathionine to cysteine with the release of α-ketobutyrate. Thereafter, the cysteine aminotransferase (CT) and 3-mercaptopyruvate sulfur transferase (3-MPST) enzymes convert L-Cys to generate H2S.26 CBS deficiency results in accumulation of Hcy, but in contrast to remethylation defects, methionine is increased. The major in vivo redox buffers are glutathione (γ-glutamyl cysteinyl glycine), cysteine, and thioredoxin; the thiol groups in these molecules are readily oxidized and, hence, can buffer changes in redox state. L-Cys is the major extracellular redox buffer and is present more in the oxidized form, with a ratio of reduced cysteine to oxidized cysteine disulfide in plasma of approximately 1:4, with a cytosolic ratio of reduced to oxidized glutathione disulfide (GSH/GSSG) of 50:1.27 Cysteine is derived from the transsulfuration pathway, from the diet, or from protein catabolism (in the fasting state). Cysteine synthesized via transsulfuration can be delivered to other tissues as reduced cysteine or is derived from the catabolism of glutathione. On the other hand, cysteine is a substrate for the formation of γ-glutamyl cysteine (i.e., for the rate-limiting step in glutathione synthesis). Hence, the methionine–Hcy cycle is the major system that regulates the concentration of the major intra- and extracellular redox buffers and can act as a buffer to regulate redox status. In addition to HHcy, abnormalities in the methionine–Hcy cycle, whether due to genetic, nutritional, or other factors, are associated with oxidant stress-induced (cardio)vascular pathology.28 How the remethylation and transsulfuration pathways (and the alternate pathway of remethylation) are coordinately regulated in health and disease (especially, how their regulation relates with oxidative stress) needs to be thoroughly investigated. Finally, cysteine can undergo catabolic conversion to the biologically essential compounds taurine and sulfate.

Reactive Oxygen Species and In Vivo Platelet Activation in HHcy Regardless of whether the defect is in the remethylation or the transsulfuration pathway, a high risk of arterial and venous thrombosis and juvenile atherosclerosis is frequent in patients with HHcy.29,30 In MTHFRþþ carriers with previ-

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ous early-onset (at age 50, 000 patients from high-quality randomized clinical trials document that lowering Hcy levels by low and high doses of folic acid and vitamin B12 (which are involved in remethylation of Hcy to methionine by MS) and vitamin B6 (which acts as a cofactor in the transsulfuration of Hcy to cystathionine and L-Cys) little affects such risk.48 The straightforward view that by decreasing Hcy levels addition of folic acid would reduce the risk of CAD thus appeared as an oversimplified approach to a complex biologic perturbation.49 However, the evidence from Nature’s randomized trials50 (in the setting of HHcy due to CBS deficiency, high prevalence of arterial and venous Seminars in Thrombosis & Hemostasis

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occlusive disease, dramatic decrease in vascular risk following Hcy-lowering treatments) is too strong to be ignored. As the first data from interventional trials appeared, several explanations for the poor impact of folic acid supplementation have been provided. First, such trials might have been conceivably shorter in duration than appropriate for modulating an established progressive vascular disease such as atherothrombosis.51 Second, several confounders might have affected the results of the observational (oldest) studies on Hcy. Individuals with pre-existing atherosclerosis have higher Hcy levels than those without; some drugs may increase Hcy levels (►Table 1)— suggesting the potential for attenuation of their clinical benefit—and the effect of some drugs on Hcy is reversible following treatment withdrawal. Confounding factors (e.g., the simultaneous use of statins, aspirin, and other drugs) might have led to bias and inappropriate interpretation of the data from randomized interventional trials with folate as well. Most of them did not exclude patients routinely taking the arachidonate inhibitor aspirin, an approach which may have influenced the results (in vivo platelet activation by H2S derived from Hcy degradation is mediated by enhanced release of arachidonic acid and in turn enhanced biosynthesis of platelet thromboxane A2). Third, in a meta-analysis, the effect of the MTHFRþþ variant on Hcy concentration was larger in low folate settings (Asia; difference between individuals with TT vs. CC genotype, 3·12 μmol/L; 95% confidence interval [CI], 2·23–4·01) than in areas with folate fortification (America, Australia, and New Zealand, high; 0·13 μmol/L, 0·85 to 1·11).52 The odds ratio for stroke was also higher in Asia (1·68; 95% CI, 1·44–1·97) than in America, Australia, and New Zealand (1·03; 95% CI, 0·84– 1·25). Most randomized Hcy-lowering trials have been performed in populations with high folate concentrations. The relative risk (RR) of stroke in trials of Hcy-lowering interventions (0·94; 95% CI, 0·85–1·04) was similar to that predicted for the same extent of Hcy reduction in large genetic studies in populations with similar folate status (predicted RR, 1·00; 95% CI, 0·90–1·11). While folate fortification programs in North America have been circumstantially associated with a reduction in stroke rates,53 folic acid supplementation was documented to be effective in stroke prevention in populations with no or partial folic acid fortification.54,55 Thus, the folate status of the population should be taken into account when evaluating the link between tHcy and CAD. On the other hand, the controversy over Hcy and cardiovascular risk called attention to the issue that nutritional intake of folate and of the sulfur-containing amino acids methionine and cysteine affects a variety of enzymatic reactions of the methionine–Hcy cycle and associated onecarbon metabolism and, thereby, both methylation reactions and redox balance.56 Under these circumstances, an abnormal plasma Hcy level may well reveal an underlying perturbation in the regulation of the methylation and/or the redox status that needs to be further characterized.

Folate-Dependent One-Carbon Metabolism Methyl (–CH3), methylene (–CH2–), and formyl (–CHO–) forms of folate are used in the transfer of one-carbon groups

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Table 1 Common determinants of homocysteine plasma levels Genetic Transsulfuration defects Cystathionine β-synthase defect (chromosome 21)

Homozygote: 1/340,000 born Heterozygote: 0.5% whole population Heterozygote mutation 844ins68: 10–15% whole Population in association with other risk factors

Remethylation defects 5,10-Methylenetetrahydrofolate reductase (MTHFR) defect

Homozygote: 1/3,350,000 born Heterozygote: 0.5% whole population

Thermolabile variant of MTHFR C677T (50% activity)

Homozygote: 5–20% whole population

Cobalamin/methylcobalamin conversion defect (cbl C, D, E, F, G)

a

Age/Sex

Increasing age, male sex, menopause

Nutritional

Folate þ vitamin B12 deficiency (elderly, pregnancy, malignancy) Vitamin B6 deficiency Lifestyle: excessive coffee/alcohol intake

Diseases

Bowel: malabsorption of vitamin B12 Liver failure Renal failure; renal transplantation Psoriasis: folate reduction Lymphoblastic leukemia, malignancy Hypothyroidism, diabetes mellitus, hypertension

Pharmacological

Metformin Glitazones (some) Lipid-lowering drugs (colestipol, nicotinate, fibrates) Methotrexate: 5-methyl-tetrahydrofolate reduction Interference with folate: diuretics, Anticonvulsantsa Folate antagonists Vitamin B12 antagonists (e.g., nitrate) Vitamin B6 deficit: estrogens

For example, carbamazepine, isoniazid, phenytoin.

in several metabolic pathways.57 (1) MTHF is irreversibly converted by the Flavin Adenine dinucleotide (FAD)-dependent enzyme MTHFR to the methyl donor 5-MTHF (►Fig. 2). The latter provides the methyl group for the MS/cobalamin dependent remethylation of Hcy to methionine. The tetrahydrofolate (THF) thus generated can be converted to its immediate precursor (MTHF) by the enzyme serine hydroxyl methyl transferase with the concomitant conversion of serine to glycine. (2) MTHF donates the methyl group involved in the methylation of deoxy-uridine monophosphate to deoxythymidine monophosphate (catalyzed by thymidylate synthase). Dihydrofolate produced during thymidylate synthesis is reduced by dihydrofolate reductase to THF. (3) MTHF dehydrogenase catalyzes the conversion of MTHF to formyl THF and the transfer of the carbon atom as the formyl group during purine synthesis. Hence, via the transfer of one-carbon moieties, folate metabolism is both linked to DNA synthesis and DNA methylation. With one exception (in 102 patients with established CAD and low tHcy at baseline [2 standard deviations of controls. Significant correlations of 8-isoPGF2α with both urinary 11-dehydro-TXB2 excretion and fasting tHcy levels have been reported in that setting. The conflicting effects of H2S—both pro-thrombotic in regard to platelet activation while simultaneously protective against oxidative endothelial stress and as a vasodilator—help set the frame for evaluating whether, rather than (just) a cause, increased H2S is a marker of (and an important defense mechanism against) oxidative stress. Besides H2S, two other gasotransmitters are produced in vivo: NO and CO. CO is generated by the heme oxygenase (HO) family of enzymes (HO-1, HO-2, and HO-3).96 NO is synthesized by inducible NO synthase (iNOS), endothelial NO synthase (eNOS), and neuronal NO synthase (nNOS). The transcriptional nuclear factor kappa B (NF-κB)—a key determinant of the proliferation of vascular smooth cells whose signaling is activated by Hcy97—modulates NO production in response to Hcy.98 The activity of NO synthase is inhibited by asymmetric dimethylarginine.78 NO and H2S share many of the same regulatory roles, including vasodilation, promotion of angiogenesis, attenuation of apoptosis, and antioxidant actions. By upregulating PLA2, H2S endogenously generated within the platelets boosts the arachidonic acid cascade, the latter in turn acting as a downstream signal of the L-Cys/H2S pathway. Major effects of the L-arginine/NO pathway involve the arachidonic acid cascade as well.99 Although H2S and NO exhibit independent signaling, the cross-talk between the L-Cys/H2S and the L-arginine/NO pathways is a well-established event and is involved in multiple pathways.45,100 The extent to what such interplay is perturbed in MTHFRþþ should be clarified. Data from settings in which an oxidative stress induces depletion of B vitamins or antioxidants (e.g., prolonged states of inflammation or immune activation101 or Alzheimer disease102) support the possibility of vascular prevention by folate mechanisms independent of Hcy lowering. These involve the antioxidant potential of 5-MTHF, including the improvement of tetrahydrobiopterin (BH4) availability—a direct superoxide-scavenging capacity—and its ability to reduce superoxide generation by the “uncoupled” eNOS. By switching the enzyme to its “coupled” state, the later restores NO synthesis and NADPH oxidation in a BH4-dependent manner.103 In patients undergoing Seminars in Thrombosis & Hemostasis

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coronary bypass surgery, the intravenous infusion of 5-MTHF on saphenous veins and internal mammary arteries prevents in vivo peroxynitrite-mediated BH4 oxidation and improves eNOS coupling.104 Conversely, diminished endogenous H2S results in reduced BH4 levels, eNOS uncoupling, and profound oxidative stress.105 The plasma level of the one-carbon metabolite dimethylglycine, an independent predictor of CAD, is linked to changes in lipoprotein assembly.106 This tertiary amine is produced from betaine during the remethylation of Hcy to methionine. High plasma dimethylglycine levels may thus reflect an altered flux through BHMT, influencing liver AdoMet levels107 and thereby the availability of methyl groups for transmethylation reactions, including phosphatidylcholine synthesis, the major phospholipid in very low-density lipoprotein particles. Through its scavenging potential, H2S inhibits in vitro the atherogenic modifications of purified low-density lipoproteins.108 The so far unclear relationship between H2S, lipids, one-carbon metabolism, and CAD deserves special attention and prompts for further studies in vascular medicine. In addition to its obvious pathophysiological relevance, research targeted at estimating the role of H2S within the framework of one-carbon metabolomics is likely to gradually provide values in the form of personalized medicine. However, major achievements take often more time than anticipated by the original enthusiasm.

5 Beard RS Jr, Bearden SE. Vascular complications of cystathionine

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7 8

9

10

11

12

13

14 15 16

Conflict of interest The authors have served on advisory boards and/or have received fees as speakers at meetings unrelated to the content of the present report.

17

18

Note Roberta Lupoli and Alessandro Di Minno equally contributed to the present overview. 19

Acknowledgment Medical writing assistance was provided by Rosanna Scala.

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