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Abstract

New evidence continues to accumulate regarding a significant association between excessive inflammation and dysregulated immunity (local and systemic) and the risk of cardiovascular events in different patient cohorts. Whilst research has sought to identify novel atheroprotective therapies targeting inflammation and immunity, several marketed drugs for rheumatological conditions may serve a similar purpose. One such drug, methotrexate, has been used since 1948 for treating cancer and, more recently, for a wide range of dysimmune conditions. Over the last 30 years, epidemiological and experimental studies have shown that methotrexate is independently associated with a reduced risk of cardiovascular disease, particularly in rheumatological patients, and exerts several beneficial effects on vascular homeostasis and blood pressure control. This review article discusses the current challenges with managing cardiovascular risk and the new frontiers offered by drug discovery and drug repurposing targeting inflammation and immunity with a focus on methotrexate. Specifically, the article critically appraises the results of observational, cross-sectional and intervention studies investigating the effects of methotrexate on overall cardiovascular risk and individual risk factors. It also discusses the putative molecular mechanisms underpinning the atheroprotective effects of methotrexate and the practical advantages of using methotrexate in cardiovascular prevention, and highlights future research directions in this area.

Keywords

atherosclerosis heart disease risk factors immunity inflammation methotrexate

Introduction

Atherosclerosis is a progressive disease that is characterized by the abnormal accumulation of lipids and fibrous elements in the arterial wall, with a consequent narrowing of the arterial lumen.¹ The primary clinical manifestations of atherosclerosis, ischaemic heart disease and stroke, remain the leading cause of disability, mortality and excessive healthcare costs worldwide.² For example, in the Global Burden of Disease 2019 study, a multinational research collaboration, the global prevalence of cardiovascular disease increased from 271 million in 1990 to 523 million in 2019.³ During the same period, there was also a significant increase in cardiovascular deaths, from 12.1 to 18.6 million, and a doubling of years lived with disability, from 17.7 to 34.4 million, due to ischaemic heart disease and stroke.³ Therefore, despite the availability of medications targeting key neurohormonal pathways (e.g. the renin–angiotensin–aldosterone system), blood pressure, lipid profile and platelet aggregation, additional research is needed to investigate the significance of unconventional cellular and biochemical mechanisms underpinning atherosclerosis. There is robust evidence that such mechanisms involve or even trigger the dysregulation of the immune system and the excessive activation of specific inflammatory pathways.^4–7

A chronic local (arterial wall) and systemic pro-inflammatory and pro-oxidant state associated with a dysregulation of immune pathways plays a critical role in the pathophysiology of atherosclerosis.⁸ Such alterations also mediate the untoward effects of established cardiovascular risk factors, particularly diabetes,^9–11 arterial hypertension,^12,13 hypercholesterolaemia^14–18 and the metabolic syndrome.^19,20 Consequently, there has been an increasing focus on discovering novel immunomodulatory and anti-inflammatory agents with atheroprotective effects that could complement existing cardiovascular therapies.^21–23 An alternative approach consists of determining the atheroprotective potential of other traditional immunomodulatory and anti-inflammatory agents that are commonly prescribed in patients with autoimmune and/or inflammatory conditions.^24–26 If effective, such ‘drug repurposing’ approaches would minimize the costs and uncertainties of conventional drug discovery programmes and provide rapid public health benefits in cardiovascular prevention, defined as the combination of pharmacological and non-pharmacological strategies used to reduce the risk of cardiovascular disease in the population.^27,28

One example of such a traditional immunomodulatory and anti-inflammatory drug is methotrexate, a pteridine analogue that was initially used as an anti-cancer agent and, more recently, as a conventional synthetic disease-modifying anti-rheumatic drug (csDMARD).^29–36 Evidence generated from experimental and observational clinical studies conducted over the last 30 years suggests that treatment with methotrexate is also associated with beneficial effects on surrogate markers of atherosclerosis and cardiovascular clinical endpoints, for example, myocardial infarction and stroke.^37,38 Therefore, methotrexate could be a suitable candidate for ‘drug repurposing’ strategies aimed at enhancing the efficacy of national and international cardiovascular prevention programmes.

This review article discusses the issues that limit the efficacy of existing cardiovascular prevention strategies, particularly residual cardiovascular and inflammatory risk, and the critical pathophysiological role of dysregulated immunity and inflammation in driving the onset and the progression of atherosclerosis. Then, it critically appraises the published evidence, focusing on studies conducted over the last 5 years, regarding the potential atheroprotective effects of methotrexate in experimental and clinical studies in patients with and without autoimmune and inflammatory conditions. Finally, it discusses the potential practical advantages of methotrexate therapy over available treatments for routine cardiovascular prevention and proposes new research directions in this area, including the design of future intervention studies investigating the effects of methotrexate on cardiovascular risk.

Residual cardiovascular and inflammatory risk

A significant number of patients with previous atherosclerotic cardiovascular events, for example, acute coronary syndrome and ischaemic stroke, suffer from further events despite maximal treatment with statins, beta-blockers, antiplatelet agents, anticoagulants, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers.^39,40 This observation suggests the presence of a significant ‘residual cardiovascular risk’, that is, the component of an individual patient’s cardiovascular risk that is not influenced by existing treatments.^41–43 The increasing recognition of the critical role played by excess inflammation and dysregulated immunity in driving this residual risk has led several experts to rename this phenomenon ‘residual inflammatory risk’. The results of several observational studies support the clinical relevance of the residual inflammatory risk, potentially ascribable to the local pool of pro-inflammatory T memory cells.^44–46 For example, in a Chinese study of 5840 patients with a recent ischaemic stroke or transient ischaemic attack receiving optimal preventive treatment, the subgroup with relatively high C-reactive protein (CRP) concentrations both at baseline and at 3-month of follow-up had significantly worse outcomes at 1 year when compared to the subgroup with relatively low CRP at both timepoints (stroke recurrence: adjusted hazard ratio, aHR = 1.39, 95% CI: 1.08–1.78, p = 0.01; composite of stroke, myocardial infarction, and cardiovascular death: aHR = 1.43, 95% CI: 1.12–1.82, p = 0.004; all-cause mortality: aHR = 2.57, 95% CI: 1.50–4.41, p < 0.001; and poor functional outcome: aHR = 1.75, 95% CI: 1.34–2.28, p < 0.001).⁴⁷ In another study of 3013 patients undergoing percutaneous coronary revascularisation and receiving optimal preventive treatment, those with persistently high CRP at baseline and after 4 weeks had a significantly higher risk of major adverse cardiac and cerebrovascular events at 1 year when compared with those with low CRP at both timepoints (aHR = 2.10, 95% CI: 1.45–3.02, p < 0.001). Different risks were also observed in two other subgroups (low baseline and high CRP at follow-up: aHR = 1.91, 95% CI: 1.21–3.03, p = 0.006; high baseline and low CRP at follow-up: aHR = 1.52, 95% CI: 0.95–2.44, p = 0.08), suggesting the importance of the temporal direction in changes in inflammation in modulating residual inflammatory risk.⁴⁸ Several other recent observational trials and post-hoc analyses of intervention trials have similarly reported significant associations between residual inflammatory risk, assessed by measuring CRP or other biomarkers (e.g. the neutrophil-to-lymphocyte ratio), and adverse outcomes in patients with atherosclerosis.^49–55

Atheroprotective strategies targeting inflammation and immunity

The structural and functional integrity of the endothelium is critical to ensure the maintenance of homeostatic mechanisms protecting against atherosclerosis, mainly through the production of the endogenous messenger nitric oxide (NO) by endothelial NO synthase (eNOS). NO regulates endothelial-dependent vasodilation, peripheral vascular resistance, arterial stiffness, blood pressure and platelet activity, preventing at the same time the adhesion of leucocytes to the arterial wall and the proliferation of vascular smooth muscle cells, critical steps involved in the pathophysiology of atherosclerosis.^56–63 According to the ‘inflammatory theory of atherosclerosis’ postulated some 40 years ago, the dysregulated production of specific cytokines by subpopulations of macrophages (M1), for example, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-12, favours excess inflammation in the arterial wall. This, in turn, favours the oxidation of specific cholesterol fractions, that is, low-density lipoprotein (LDL) cholesterol, endothelial damage, the creation of foam cells and the ultimate formation of the atherosclerotic plaque.^17,21,22,64

In support of the pathophysiological role played by pro-inflammatory cytokines in atherosclerosis, several observational studies have reported significant associations between circulating cytokines and the risk of adverse cardiovascular outcomes. For example, in a study of patients with myocardial infarction, the concentrations of TNF-α measured after an average of 9 months after the event were significantly higher in those who experienced recurrent cardiovascular events during follow-up than those who did not (2.84 pg/mL versus 2.57 pg/mL, p = 0.02). Notably, the association between TNF-α concentrations and recurrent events was also independent of conventional cardiovascular risk factors.⁶⁵ Similarly, a recent two-sample Mendelian randomization study has reported significant associations between genetically predicted TNF-α concentrations and ischaemic heart disease (odds ratio, OR = 2.25, 95% CI: 1.50–3.37, p < 0.001) and stroke (OR: 2.27, 95% CI: 1.50–3.43, p < 0.001).⁶⁶ In another prospective study investigating 3269 patients with acute coronary syndrome, plasma IL-6 concentrations ⩾5 ng/L were significantly associated with 12-month mortality in patients not undergoing revascularisation (relative risk, RR = 3.47, 95% CI: 1.95–6.21, p < 0.001) but not in those undergoing revascularisation (RR = 1.43, 95% CI: 0.64–3.21, p = 0.38).⁶⁷ Taken together, these studies highlight that the negative impact of excess inflammation is not only limited to the assessment of surrogate markers in experimental studies but also translates into a tangible increase in cardiovascular risk at the population level.

This evidence has stimulated a significant body of research over the last 10–15 years that has led to the identification of promising atheroprotective treatments targeting specific immune and inflammatory mediators. Such mediators include interleukin-1β (e.g. canakinumab),⁶⁵ the interleukin-1 receptor (e.g. anakinra),⁶⁸ the NLR family pyrin domain containing three inflammasome (e.g. MCC950 and tranilast),^69,70 TNF-α (e.g. adalimumab),⁷¹ IL-6 (e.g. tocilizumab),⁷² chemokines (e.g. maraviroc and MNL1202),^73,74 interleukin-2 (e.g. aldesleukin)⁷⁵ and CD20 (e.g. rituximab).⁷⁶ However, it is essential to highlight that these and other agents under investigation are often characterized by prohibitive costs and toxicity,^77–83 which may limit their widespread use in cardiovascular prevention, a type of treatment that can last for several decades. Recent randomized-controlled studies have also demonstrated the benefits of ‘drug repurposing’ strategies for combating atherosclerosis with colchicine, a relatively old antimalarial, antimitotic and anti-inflammatory agent targeting multiple cellular pathways that is used for acute gout and pericarditis.^84,85 A similar drug repurposing approach in the quest for alternative atheroprotective treatments will be discussed for another traditional immunomodulatory and anti-inflammatory agent, methotrexate, in the following sections.

Methotrexate pharmacology and role in cardiovascular risk in clinical studies

Methotrexate, a pteridine molecule and an analogue of the B-vitamin folic acid, has been used since 1948 for the treatment of cancer and, more recently over the last 30–40 years, as a csDMARD for a wide range of autoimmune and autoinflammatory conditions.^{34,36,86–88} In this context, it is essential to emphasize that the doses of methotrexate used for the treatment of autoimmune and inflammatory conditions, between 7.5 and 30 mg weekly, are considerably lower than those used for malignancies, up to ⩾500 mg/m².^34,86–90

Pharmacology of methotrexate

In patients with autoimmune and/or inflammatory conditions, methotrexate is normally administered once weekly either orally or subcutaneously. The mean bioavailability following oral administration has been shown to be 0.64 (range 0.21–0.96) when compared to subcutaneous administration.⁹¹ Subcutaneous methotrexate is gaining increasing popularity in clinical practice because of the higher bioavailability, as previously described, a more predictable pharmacokinetic profile, and a reduced rate of gastrointestinal toxicity when compared to oral methotrexate.⁹² Circulating methotrexate is not significantly bound to plasma proteins, ~50%, can easily distribute in the synovial fluid and is primarily eliminated by the kidney through glomerular filtration and active tubular secretion.^93,94 The plasma half-life of methotrexate ranges between 4.5 and 10 h.^93,94 However, the circulating concentrations of methotrexate are not particularly significant from a clinical standpoint as the drug enters cells via the human solute carrier superfamily of transporters before accumulating as pharmacologically active polyglutamate forms by folylpolyglutamate synthetases (Figure 1).⁹⁵ A pharmacokinetic study has shown that the median time to achieve a steady state of the different forms of methotrexate polyglutamate concentrations in red blood cells after commencing oral methotrexate ranged between 6 and 149 weeks.⁹⁶ The same study reported that the median time for the polyglutamates to become undetectable following treatment cessation ranged between 4 and 10 weeks.⁹⁶ This period is considerably longer that the half-life of circulating methotrexate, which also justifies the weekly administration schedules in patients with autoimmune and inflammatory disorders.

Figure 1.

The pharmacology of methotrexate.

The polyglutamate forms mediate the inhibitory effects of methotrexate on the biosynthesis of purines and pyrimidines. These effects involve the inhibition of the enzymes thymidylate synthase, dihydrofolate reductase and aminoimidazole carboxamide ribonucleotide (AICAR) transformylase (Figure 1) (ATIC).⁹⁷ The accumulation of the ATIC substrate, AICAR, in turn, favours the accumulation of adenosine, a critical anti-inflammatory mediator, through the inhibition of catabolic pathways mediated by adenosine deaminase and adenosine monophosphate deaminase (Figures 1 and 2).⁹⁷

Figure 2.

The effects of methotrexate on adenosine, AICAR and AMPK.

Treatment with methotrexate, particularly long-term, is known to be associated with gastroenterological, haematological, renal, neurological, pulmonary and mucocutaneous toxicity of different severity.^98,99 However, with appropriate dosing and regular clinical and biochemical monitoring, the clinical manifestations of toxicity appear to be relatively infrequent and overall benign. For example, in an observational study of 673 patients with inflammatory arthritis, including rheumatoid arthritis, about three-quarters remained on methotrexate after 5 years. In the subgroup that stopped treatment, 11% reported inefficacy and opted for withdrawal, 6% liver abnormalities and a further 6% haematological abnormalities.¹⁰⁰ In a study of 379 patients with rheumatoid arthritis receiving 1-year treatment with methotrexate with other csDMARDs and/or corticosteroids, only 2% reported serious adverse events.¹⁰¹ In a randomized-controlled study investigating the effects of 1-year treatment with methotrexate versus placebo in patients with arthritis thought to progress to rheumatoid arthritis, there were non-significant between-group differences in serious adverse events (11% versus 11%). However, patients receiving methotrexate had a higher incidence of significant (>3 × upper limit of normal) elevations in liver enzymes (incidence rate per 100 persons-years: 6.1, 95% CI: 3.2–10.4 versus 0.5, 95% CI: 0.0–27.4, p = 0.0025).¹⁰² Additionally, early reports suggesting an increased risk of liver fibrosis during methotrexate treatment have not been confirmed in recent studies.^103,104 The safety profile of methotrexate was also comprehensively investigated in a secondary analysis of the Cardiovascular Inflammation Reduction Trial (CIRT). The authors reported a mildly yet significantly higher 3-year cumulative incidence of severe adverse events in patients receiving methotrexate versus placebo (0.13, 95% CI: 0.12–0.15 versus 0.10, 95% CI: 0.08–0.11).¹⁰⁵ The safety of methotrexate reported in these studies appears similar to that reported in trials of conventional atheroprotective drugs. For example, in the Systolic Blood Pressure Intervention Trial in 9069 patients, 9.8% in the intensive treatment arm and 7.2% in the standard treatment arm reported serious adverse events.¹⁰⁶ Furthermore, in the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial investigating a total of 25,620 patients, the incidence of serious adverse events in the three treatment arms ranged between 5.0% and 12.0%.¹⁰⁷

Rheumatoid arthritis and cardiovascular disease

The pathophysiological mechanisms underlying one of the most common and studied types of autoimmune disease, rheumatoid arthritis,¹⁰⁸ exhibit significant similarities with atherosclerosis. Such similarities include the presence of a vascular and systemic pro-inflammatory and pro-oxidant state,^109–115 reduced NO synthesis and endothelial dysfunction,^116–120 arterial stiffening^121–124 and an increased risk of arterial hypertension.^125,126 A critical additional element of similarity is represented by the excess production of pro-inflammatory and pro-atherogenic cytokines, for example, TNF-α, IL-1 and IL-6, that synergistically build and maintain a pro-inflammatory microenvironment in blood vessels.^127–130 Not surprisingly, the risk of atherosclerotic cardiovascular disease in this group is significantly higher than the general population, as also shown in a systematic review and meta-analysis of six studies (RR = 1.55, 95% CI: 1.18–2.02). In subgroup analysis, the RR was higher in patients aged <60 years (RR = 1.98, 95% CI: 1.41–2.79) than in those aged ⩾60 years (RR = 1.43, 95% CI: 1.16–1.75), although the risk remained significant in both groups.¹³¹ In addition to inflammation and oxidative stress, traditional risk factors, for example, diabetes, arterial hypertension and the metabolic syndrome, have also been shown to account for the increased risk of atherosclerosis and cardiovascular disease in rheumatoid arthritis.^132,133

Methotrexate and cardiovascular risk

Several observational studies have reported that treatment with methotrexate is associated with a significant reduction of cardiovascular and all-cause mortality in patients with rheumatoid arthritis. For example, in a recent systematic review and meta-analysis of 15 studies, 7 longitudinal observational cohort, 6 retrospective cohort and 2 prospective cohort studies, the use of methotrexate was associated with a significant reduction in all-cause mortality (HR = 0.59, 95% CI: 0.50–0.71, p < 0.001). In a subgroup analysis of four studies, the use of methotrexate was also associated with a significant reduction in cardiovascular mortality (HR = 0.72, 95% CI: 0.53–0.97, p = 0.031).¹³⁴ The results of another systematic review and meta-analysis also support the possible protective role of methotrexate against cardiovascular events in rheumatoid arthritis. In 10 selected studies, 3 cohort, 2 cross-sectional, 1 case–control and four nest case–control, investigating a total of 195,416 participants, there was a significant negative association between methotrexate and cardiovascular events (RR = 0.80, 95% CI: 0.73–0.88, p < 0.001). The association was similar in a subgroup of eight studies that adjusted for concomitant cardiovascular risk factors (RR = 0.78, 95% CI: 0.71–0.86, p = 0.003).¹³⁵

Importantly, current evidence suggests that other immunomodulatory and anti-inflammatory agents do not share the putative protective effects of methotrexate against atherosclerosis and cardiovascular disease.^136,137 In a systematic review and meta-analysis of eight studies investigating a total of 65,736 patients with rheumatoid arthritis, methotrexate use was associated with a significant reduction in total cardiovascular events when compared to other csDMARDs (RR = 0.72, 95% CI: 0.57–0.91, p = 0.007). A substantial reduction in the specific risk of myocardial infarction was also observed in a subgroup of three studies (RR = 0.81, 95% CI: 0.68–0.96).¹³⁸ A more recent retrospective cohort study assessing Medicare claims data in the United States for the period 2006–2015 sought to investigate whether methotrexate use might exert atheroprotective effects in patients with rheumatoid arthritis who are already receiving treatment with biologic agents. In a total of 88,255 patients receiving biologics, the additional use of methotrexate was associated with a significant reduction in the risk of a composite endpoint of myocardial infarction, stroke and fatal cardiovascular disease (aHR = 0.76, 95% CI: 0.68–0.85).¹³⁹ The effect size of the cardiovascular risk reduction with methotrexate in these studies is comparable to that observed in intervention studies of established atheroprotective agents, for example, ACE inhibitors (RR = 0.80, 95% CI: 0.70–0.91),¹⁴⁰ and statins (OR = 0.69, 95% CI: 0.64–0.75).¹⁴¹

Another recent study has sought to investigate whether methotrexate may have competitive advantages in terms of cardiovascular prevention over other csDMARDs, such as hydroxychloroquine, an agent with evidence of atheroprotective effects in animal and human studies.^26,142 Using Medicare data during the period 2008–2016, the study authors propensity score-matched 54,462 patients with rheumatoid arthritis aged ⩾65 years commenced on either hydroxychloroquine or methotrexate. No significant between-group differences were observed in the primary endpoint, sudden cardiac arrest (or ventricular arrhythmia) and major adverse cardiovascular events. However, in a subgroup of patients with heart failure, hydroxychloroquine was associated with a significantly higher risk of major adverse cardiovascular events (HR = 1.30, 95% CI: 1.08–1.56), cardiovascular mortality (HR = 1.34, 95% CI: 1.06–1.70), all-cause mortality (HR = 1.22, 95% CI: 1.04–1.43), myocardial infarction (HR = 1.74, 95% CI: 1.25–2.42) and hospitalizations for heart failure (HR = 1.29, 95% CI: 1.07–1.54) when compared to methotrexate.¹⁴³

The main study assessing the effects of methotrexate on cardiovascular prevention, the CIRT, randomized 4786 patients without autoimmune conditions but with myocardial infarction or multivessel coronary disease with either type 2 diabetes or metabolic syndrome to methotrexate (target dose of 15–20 mg/week) or placebo. There were non-significant between-group differences in the primary endpoint, a composite of nonfatal myocardial infarction, nonfatal stroke, cardiovascular death and hospitalization for unstable angina requiring urgent revascularisation (HR = 0.96, 95% CI: 0.79–1.16).¹⁴⁴ Whilst the results of this study do not support the presence of significant atheroprotective effects of methotrexate in patients without autoimmune conditions, it is essential to emphasize that, by trial design, both patients in the methotrexate and the placebo arms received treatment with the B-vitamin folic acid. Folic acid is often co-administered with methotrexate by rheumatologists, given that both compounds compete for the same transporter in the intestine and cellular uptake and that folic acid supplementation has been shown to reduce the incidence of adverse effects with methotrexate.^145–147 However, at the same time, there is robust evidence from experimental and clinical studies that treatment with folic acid per se exerts significant atheroprotective effects, including the lowering of the highly reactive and pro-atherogenic amino acid homocysteine,¹⁴⁸ improved NO synthesis and endothelial function¹⁴⁹ and reduced arterial stiffness and blood pressure.^150,151 Furthermore, in a large randomized-controlled trial conducted in China in 20,702 hypertensive patients without previous myocardial infarction or stroke, a combination treatment of folic acid with the ACE inhibitor enalapril significantly reduced the risk of overall stroke (primary endpoint, HR = 0.79, 95% CI: 0.68–0.93); first ischaemic stroke (HR = 0.76, 95% CI: 0.64–0.91) and a composite endpoint of cardiovascular death, myocardial infarction and stroke (HR = 0.80, 95% CI: 0.69–0.92). By contrast, there were non-significant between-group differences in other secondary endpoints, that is, haemorrhagic stroke (HR = 0.93, 95% CI: 0.65–1.34), myocardial infarction (HR = 1.04, 95% CI: 0.60–1.82) and all-cause mortality (HR = 0.94, 95% CI: 0.81–1.10).¹⁵² Therefore, further studies investigating the effects of methotrexate on cardiovascular prevention are warranted to determine whether the use of folic acid in the comparator group might have diluted the potential atheroprotective effects of methotrexate in the CIRT study.

Effects of methotrexate on traditional cardiovascular risk factors

Several experimental and clinical studies have investigated the effects of methotrexate on conventional cardiovascular risk factors, particularly dyslipidaemia, diabetes, arterial hypertension and the metabolic syndrome (Figure 3).

Figure 3.

Effects of methotrexate on cardiovascular risk factors (orange) and putative atheroprotective mechanisms (cyan) according to experimental and clinical studies.

Lipid profile

In a study in cholesterol-fed rabbits, treatment with lipid core nanoparticles containing methotrexate, with or without paclitaxel, caused significant regression of aortic plaque (−59%) and intima areas (−57%). These effects were associated with a substantial reduction in macrophages and TNF-α gene expression.¹⁵³ In a secondary analysis of the Optimised Treatment Algorithm for Patients With Early Rheumatoid Arthritis (OPERA) trial, comparing the effects of adalimumab and methotrexate (n = 86) versus methotrexate and placebo (n = 88), there was no significant between-group difference in the changes at 1 year versus baseline in total, LDL cholesterol, high-density lipoprotein (HDL) cholesterol and triglyceride concentrations.¹⁵⁴ In an observational study of 262 patients with psoriasis, 12-week treatment with methotrexate significantly reduced total cholesterol (p < 0.05), LDL cholesterol (p < 0.05), apolipoprotein B (p < 0.001) and lipoprotein A (p < 0.001) in annexin A6, a protein that regulates cholesterol homeostasis,¹⁵⁵ TC and CC genotype carriers of rs11960458 and apolipoprotein B (p = 0.04) in TT genotype carriers. However, methotrexate also significantly reduced the concentrations of the atheroprotective subfractions, HDL cholesterol (p = 0.007) and apolipoprotein A1 (p = 0.04) in TC genotype carriers of rs11960458. Moreover, methotrexate significantly reduced triglyceride concentrations only in the CC genotype carriers (p = 0.01).¹⁵⁶ In another study of 288 patients with psoriasis, 136 with and 152 without psoriatic arthritis, 12-week treatment with methotrexate significantly lowered apolipoprotein B (p = 0.0003), total cholesterol (p = 0.0007), triglycerides (p = 0.04), HDL cholesterol (p = 0.037) and lipoprotein A (p = 0.005) in patients with arthritis, and apolipoprotein B (p < 0.0001), total cholesterol (p < 0.0001), HDL cholesterol (p = 0.011), LDL cholesterol (p = 0.0001) and lipoprotein A (p < 0.0001) in patients without arthritis.¹⁵⁷ Interestingly, in another study in 35 patients with psoriasis, 12-week treatment with methotrexate significantly reduced the concentrations of proprotein convertase subtilisin/kexin type 9,¹⁵⁸ critically involved in cholesterol homeostasis by binding to the LDL receptor in hepatocytes and an established therapeutic target for cardiovascular prevention.^159,160 Collectively, these studies have provided conflicting results on the effects of methotrexate treatment on lipid profile and, expressly, on atheroprotective versus pro-atherogenic cholesterol fractions (Figure 3).

Type 1 and type 2 diabetes

In a systematic review and meta-analysis of 16 studies investigating patients with rheumatoid arthritis, 3 with a cross-sectional design, 1 with a nested case-control design, 8 prospective cohorts and 4 retrospective cohorts, the use of methotrexate was associated with a significant reduction in the risk of type 2 diabetes (RR = 0.13, 95% CI: 0.08–0.22). Factors significantly associated with the reduced risk of diabetes included age >60 years, rheumatoid arthritis duration ⩽2 years and the measurement of disease activity.¹⁶¹ A similar negative association between methotrexate and type 1 and type 2 diabetes has been reported in another systematic review and meta-analysis of 15 studies (seven on methotrexate) investigating a total of 552,019 patients with rheumatoid arthritis (HR = 0.81, 95% CI: 0.75–0.87). The reduced risk of type 1 and type 2 diabetes with methotrexate was similar in studies assessing comparisons with non-users of methotrexate (HR = 0.77, 95% CI: 0.67–0.88) as well as users of csDMARDs not including methotrexate or hydroxychloroquine (HR = 0.85, 95% CI: 0.74–0.98).¹⁶² Furthermore, in a nationwide population study of 69,799 patients with rheumatoid arthritis but without type 1 and type 2 diabetes at baseline, the long-term use of methotrexate (>270 days/year) was associated with a significant reduction in incident type 1 and type 2 diabetes (adjusted OR = 0.84, 95% CI: 0.78–0.92).¹⁶³ Taken together, the available evidence suggests that methotrexate treatment in rheumatoid arthritis is associated with a reduced risk of type 1 and type 2 diabetes, although the mechanisms underpinning the effects on glucose metabolism require further studies (Figure 3).

Arterial hypertension

In a repeated cross-sectional study of patients with rheumatoid arthritis, the use of methotrexate was associated with a significantly lower clinical and 24-h blood pressure when compared to other csDMARDs.¹²⁰ Another study of 21,916 patients with rheumatoid arthritis with data from administrative Veterans Affairs databases in United States investigated the changes in blood pressure after commencing methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, TNF-α inhibitors or prednisone. In this study, there was a reduction in blood pressure after starting prednisone, methotrexate and hydroxychloroquine and a more modest decline with sulfasalazine and tumour necrosis factor inhibitors. Notably, in patients commencing methotrexate, a more significant proportion had an optimal blood pressure control at 6 months versus baseline (51.0% versus 46.8%, p < 0.001). Similar associations were observed for prednisone, tumour necrosis factor inhibitors and hydroxychloroquine.¹⁶⁴ Collectively, these studies suggest that methotrexate treatment can exert ameliorative effects on blood pressure control, at least in patients with rheumatoid arthritis (Figure 3).

Metabolic syndrome

The association between methotrexate and the metabolic syndrome has been investigated in cross-sectional and intervention studies. In a cross-sectional study of 400 patients with rheumatoid arthritis, the use of methotrexate, but not other csDMARDs, was significantly and negatively associated with the risk of metabolic syndrome in multivariate regression analysis (adjusted OR = 0.52, 95% CI: 0.33–0.80, p = 0.004).¹⁶⁵ The reduced prevalence of metabolic syndrome in methotrexate users versus non-users has also been reported in another cross-sectional study investigating 100 women with rheumatoid arthritis (17% versus 35%, p = 0.046).¹⁶⁶ However, a retrospective study has failed to show any significant effect of 24-month methotrexate treatment on the prevalence of metabolic syndrome and its individual components in 70 patients with psoriatic arthritis.¹⁶⁷ Therefore, there is conflicting evidence regarding the effects of methotrexate on the risk of metabolic syndrome and its individual components (Figure 3).

Methotrexate and atheroprotection: Mechanistic insights

Several mechanisms have been postulated to account for the possible atheroprotective effects of methotrexate. Such mechanisms include cytokine modulation, the accumulation of adenosine, the activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) and the regulation of the redox balance (Figure 3).

Cytokines

Studies conducted over the last 20 years have shown the potential for methotrexate to downregulate several pro-inflammatory and pro-atherogenic cytokines, for example, TNF-α, IL-1 and IL-6,^168–175 and to upregulate anti-atherogenic cytokines, for example, IL-10,^175–181 providing a potential advantage over available therapies that target one pro-inflammatory cytokine. In more recent studies, the use of methotrexate-loaded chitosan nanoparticles has also been shown to significantly reduce the circulating concentrations of TNF-α (645 ± 37 pg/mL versus 140 ± 4 pg/mL, p < 0.001) and IL-6 (334 ± 34 pg/mL versus 62 ± 5 pg/mL, p < 0.001) in a rat model of arthritis.¹⁸² In another study, the release of another pro-inflammatory cytokine, IL-8, by monocytic MONO-MAC-6-cells activated by lysates of Fusobacterium nucleatum was significantly reduced by methotrexate, but not by other anti-inflammatory agents, that is, ibuprofen or prednisolone. However, the concentrations of IL-8 following treatment with methotrexate remained significantly higher than those measured in the absence of exposure to F. nucleatum.¹⁸³ The results of another recent study have challenged the proposition that methotrexate can upregulate anti-inflammatory cytokines. In patients with plaque psoriasis, 12-week methotrexate treatment was associated with a significant reduction of the anti-inflammatory cytokine IL-10 (p = 0.02).¹⁸⁴ Additional uncertainties regarding the clinical relevance of the modulation of cytokine pathways by methotrexate derive from a sub-analysis of the previously described CIRT study, which showed that methotrexate treatment failed to significantly reduce the concentrations of IL-1β and IL-6 during follow-up. Interestingly, in this study, methotrexate also failed to significantly reduce the concentrations of CRP. This observation might be related to the relatively low baseline concentrations of CRP (1.5 mg/L), IL-1β (1.5 pg/mL) and IL-6 (2.3 pg/mL) in CIRT participants, particularly when compared to other, successful, trials investigating treatments (canakinumab) targeting IL-1β and residual inflammatory risk (baseline CRP and IL-6 concentrations, 4.10 and 2.6 pg/mL, respectively).^144,185

Adenosine and AMPK

The methotrexate-mediated accumulation of adenosine (Figures 1 and 2) might exert significant effects on cardiovascular homeostasis, particularly vasodilatation through the inhibition of alpha-1 adrenergic vasoconstriction and the stimulation of the A_2A and A_2B receptors in the aorta,^186,187 kidney¹⁸⁸ and skeletal muscle.¹⁸⁹ The resulting increase in blood flow in the renal medulla also favours natriuresis.¹⁹⁰ The critical role of adenosine in maintaining cardiovascular homeostasis is further supported by studies reporting a significant increase in arterial stiffness and blood pressure following the pharmacological inhibition of the adenosine receptors, A₁ and A_2A.^191,192 There is also evidence that adenosine A_2B receptor activation prevents the formation of atherosclerotic lesions and reduces the plasma concentrations of cholesterol and triglycerides, possibly through the reduced activation of the transcription factor sterol regulatory element-binding protein 1 in the liver.^193,194 Additional potential beneficial effects of adenosine include promoting the differentiation of monocytes into the anti-inflammatory M2 macrophage phenotype¹⁹⁵ and upregulating cholesterol efflux transporters in macrophages. The transporters shown to be affected by adenosine include ABCA1, which effluxes cholesterol as apoA-1, a major component of HDL cholesterol, ABCG1, which effluxes cholesterol as HDL cholesterol, and sterol the cytochrome P450 enzyme 27-OH hydroxylase, which effluxes cholesterol in the form of 27-hydroxycholesterol.^196,197 These forms of cholesterol prevent lipid overload and the transformation of macrophages in foam cells, which are critically involved in the formation and progression of the atherosclerotic plaque.¹⁹⁸

AICAR per se can upregulate the AMPK,^199,200 which protects endothelial cells against oxidative stress and apoptosis and inhibits vascular smooth muscle cell proliferation.^201–203 There is also increasing evidence that AICAR and/or AMPK activation enhances vasodilation, reduces blood pressure and improves cholesterol efflux capacity.^204–211 Furthermore, AMPK exerts beneficial effects on glucose homeostasis by stimulating cellular glucose uptake and glycolysis.^212–214

Redox balance

Methotrexate has been traditionally used at high doses to induce cytotoxicity in the treatment of malignancies as well as in experimental studies investigating the effects of rescuing treatments. The cytotoxic effects of methotrexate are ascribed to the inhibition of dihydrofolate reductase, involved in the conversion of dihydrofolate into tetrahydrofolate, required for the synthesis of the nucleotides of both DNA and RNA and the de novo purine synthesis of both purine and thymidylate synthase, which further inhibits DNA synthesis (Figure 1).^32,35,215 However, at relatively high doses, methotrexate is also known to trigger a pro-oxidant state which further contributes to the structural and functional alteration of critical cell components, for example, lipids, proteins and DNA.²¹⁶

Notably, however, studies have reported that at lower doses methotrexate can exert significant anti-oxidant effects. In one study, methotrexate treatment (2 μg) in HEK293 cells was able to directly scavenge free radicals, specifically O₂^.−, consequently inhibiting the formation of malondialdehyde–acetaldehyde adducts, including proteins exerting pro-inflammatory effects that have also been detected in atherosclerotic plaques.²¹⁷ The use of a specific cell line to quantify the activation of the redox-sensitive transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), allowed to demonstrate that methotrexate also reduces the activation of the Nrf2-dependent intracellular redox signalling pathway.²¹⁸ In a more recent study in rat primary astrocytes, pre-conditioning with 10 or 20 nM methotrexate (corresponding to 5 or 10 μg of the drug) enhanced the anti-oxidant defence mechanisms in these cells, expressed as the ratio of reduced to oxidized glutathione, as well as cell viability against a higher dose of methotrexate (500 nM or 0.23 mg/L).²¹⁹ Taken together, the results of these studies suggest that, at specific doses, methotrexate can scavenge free radicals, reduce the activation of redox-sensitive intracellular signalling pathways and enhance anti-oxidant mechanisms in the context of atherosclerosis (Figure 3).

Practical considerations for using methotrexate in cardiovascular prevention

One potential advantage of methotrexate, in terms of treatment adherence, is the once-weekly administration compared to the daily administration of currently available antihypertensives, antiplatelet and lipid-lowering agents. This feature is likely clinically relevant as poor treatment adherence remains a vexing issue in cardiovascular prevention. For example, a systematic review and meta-analysis of 45 prospective studies investigating a total of nearly 2 million patients reported a good adherence (defined as an intake of ⩾80%) to medications used for cardiovascular prevention in only 60% (95% CI: 52–68). In further analyses, patients with good adherence were significantly less likely to experience a cardiovascular event when compared to those with poor adherence (RR = 0.81, 95% CI: 0.76–0.86 for antihypertensive drugs).²²⁰

Future research directions

In the last 5 years, additional evidence has accumulated on the association between the use of methotrexate, surrogate markers of atherosclerosis and hard cardiovascular endpoints in experimental and clinical studies (Figure 3). However, the contrasting nature of the results observed, particularly regarding the effects on cardiovascular morbidity and mortality in observational versus intervention studies, suggests that further research is warranted to support the repurposing of methotrexate for cardiovascular prevention (Table 1). In the first instance, studies should investigate the effects of this immunomodulatory and anti-inflammatory drug on a wide range of conventional cardiovascular risk factors and determine the mediating role of pro- and anti-inflammatory cytokines, adenosine, AMPK activation and oxidative stress. In this context, the negative results of the CIRT study, whilst disappointing, have been helpful for the design of future intervention trials for at least two reasons. Firstly, the relatively low-baseline CRP concentrations in study participants and the lack of tangible effects of methotrexate on the pro-inflammatory cytokines, IL-1β and IL-6, in CIRT suggests that further studies should focus on patients with significant residual inflammatory risk, that is, higher baseline CRP, IL-1β and IL-6 concentrations. Secondly, future studies should ideally investigate the effects of methotrexate separately from those of folic acid, given the previously described effects of this B-vitamin on endothelial function, blood pressure and other markers of atherosclerosis. Such trials should ideally investigate the impact of methotrexate alone, folic acid alone and methotrexate in combination with folic acid and include a non-methotrexate/folic acid comparator arm to fully determine the atheroprotective potential of methotrexate. Finally, the peculiar pharmacology of methotrexate, which involves membrane transporters for cellular uptake, enzymes for the biotransformation into intracellular polyglutamates and other enzymes as drug targets (Figure 1), has stimulated a significant body of research to investigate whether specific genetic characteristics can influence the efficacy and safety of the drug, particularly in rheumatological disorders.^221–223 While pharmacogenetic studies investigating surrogate markers of atherosclerosis are in their infancy,²²⁴ the assessment of genetic polymorphisms might allow identifying specific subgroups that are more likely to benefit from the atheroprotective effects of methotrexate.

Table 1.

Directions for future research to investigate the role of methotrexate in cardiovascular prevention.

• Study the effects of methotrexate on surrogate markers of atherosclerosis, risk, factors and cardiovascular endpoints independently of folic acid.
• In intervention studies, include participants with high residual cardiovascular/inflammatory risk, defined using specific thresholds for CRP, IL-1 and IL-6 concentrations at baseline.
• Investigate the mediating effects of pro-inflammatory and anti-inflammatory cytokines, adenosine, AICAR and redox balance.
• Determine the potential role of genetic polymorphisms in specific transporters and enzymes in the identification of ‘high-responders’ versus ‘low-responders’ to the atheroprotective effects of methotrexate.

AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; CRP, C-reactive protein; IL, interleukin.

Conclusion

The recognition that atherosclerosis is a chronic inflammatory disease of the arterial wall, and that significant residual cardiovascular/inflammatory risk exists in many patients despite maximal treatment with atheroprotective medications justifies the search for more effective therapies that target multiple pathways, including inflammation. Recent studies have highlighted the beneficial effects of new biologics targeting specific inflammatory pathways and traditional anti-inflammatory drugs with a broader range of effects, such as colchicine and hydroxychloroquine, in reducing cardiovascular events.^26,225–227

This review has discussed the current evidence supporting the potential for methotrexate to be repurposed for the management of atherosclerotic cardiovascular disease in view of a unique combination of anti-inflammatory and, possibly, blood pressure lowering and vasculoprotective effects (Figure 3). However, one important limitation of this review is the lack of randomized-controlled studies demonstrating the efficacy of methotrexate in significantly reducing cardiovascular risk in patients with or without autoimmune and inflammatory disorders. In this context, the identification of patients that are most likely to benefit from methotrexate might require additional stratification by measuring pro-atherogenic and possibly anti-atherogenic, cytokine concentrations as well as genetic polymorphisms of relevant transporters and enzymes. Ultimately, however, only the completion of additional prospective studies that take into account the design considerations previously discussed will provide a definite answer regarding the therapeutic potential of methotrexate in cardiovascular prevention.

Footnotes

Acknowledgements

None.

Declarations

ORCID iD

Arduino A. Mangoni

References

Lusis

. Atherosclerosis. Nature 2000; 407: 233–241.

Herrington

Lacey

Sherliker

, et al. Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. Circ Res 2016; 118: 535–546.

Roth

Mensah

Johnson

. Global burden of cardiovascular diseases and risk factors, 1990. J Am Coll Cardiol 2020; 76: 2982–3021.

Weber

Habenicht

AJR

von Hundelshausen

. Novel mechanisms and therapeutic targets in atherosclerosis: inflammation and beyond. Eur Heart J 2023; 44: 2672–2681.

Libby

Buring

Badimon

, et al. Atherosclerosis. Nat Rev Dis Primers 2019; 5: 56.

Frąk

Wojtasińska

Lisińska

, et al. Pathophysiology of cardiovascular diseases: new insights into molecular mechanisms of atherosclerosis, arterial hypertension, and coronary artery disease. Biomedicines 2022; 10: 1938.

Markin

Sobenin

Grechko

, et al. Cellular mechanisms of human atherogenesis: focus on chronification of inflammation and mitochondrial mutations. Front Pharmacol 2020; 11: 642.

Wolf

Ley

. Immunity and inflammation in atherosclerosis. Circ Res 2019; 124: 315–327.

Poznyak

Grechko

Poggio

, et al. The diabetes mellitus-atherosclerosis connection: the role of lipid and glucose metabolism and chronic inflammation. Int J Mol Sci 2020; 21: 1835.

10.

Edgar

Akbar

Braithwaite

, et al. Hyperglycemia induces trained immunity in macrophages and their precursors and promotes atherosclerosis. Circulation 2021; 144: 961–982.

11.

Clement

Nanaware

Yamazaki

, et al. Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery. Immunity 2021; 54: 721–736.e10.

12.

Zhang

Zhao

Zhou

, et al. Role of inflammation, immunity, and oxidative stress in hypertension: new insights and potential therapeutic targets. Front Immunol 2023; 13: 1098725.

13.

Madhur

Elijovich

Alexander

, et al. Hypertension: do inflammation and immunity hold the key to solving this epidemic? Circ Res 2021; 128: 908–933.

14.

Galkina

Ley

. Immune and inflammatory mechanisms of atherosclerosis. Annu Rev Immunol 2009; 27: 165–197.

15.

Tall

Yvan-Charvet

. Cholesterol, inflammation and innate immunity. Nat Rev Immunol 2015; 15: 104–116.

16.

Aguilar-Ballester

Herrero-Cervera

Vinué

, et al. Impact of cholesterol metabolism in immune cell function and atherosclerosis. Nutrients 2020; 12: 20200707.

17.

Libby

Ridker

Maseri

. Inflammation and atherosclerosis. Circulation 2002; 105: 1135–1143.

18.

Gimbrone

Jr. García-Cardeña

. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 2016; 118: 620–636.

19.

Monserrat-Mesquida

Quetglas-Llabrés

Capó

, et al. Metabolic syndrome is associated with oxidative stress and proinflammatory state. Antioxidants (Basel, Switzerland) 2020; 9: 236.

20.

Manzoor

Arif

Kabir

, et al. Oxidative stress and metabolic diseases: Relevance and therapeutic strategies. Front Nutr 2022; 9: 994309.

21.

Kong

Cui

Huang

, et al. Inflammation and atherosclerosis: signaling pathways and therapeutic intervention. Signal Transduct Target Ther 2022; 7: 131.

22.

Soehnlein

Libby

. Targeting inflammation in atherosclerosis – from experimental insights to the clinic. Nat Rev Drug Discov 2021; 20: 589–610.

23.

Engelen

Robinson

AJB

Zurke

, et al. Therapeutic strategies targeting inflammation and immunity in atherosclerosis: how to proceed? Nat Rev Cardiol 2022; 19: 522–542.

24.

Chaffey

Roberti

Greaves

. Drug repurposing in cardiovascular inflammation: successes, failures, and future opportunities. Front Pharmacol 2022; 13: 1046406.

25.

Bouabdallaoui

Tardif

. Repurposing colchicine for heart disease. Annu Rev Pharmacol Toxicol 2022; 62: 121–129.

26.

Floris

Piga

Mangoni

, et al. Protective effects of hydroxychloroquine against accelerated atherosclerosis in systemic lupus erythematosus. Mediators Inflamm 2018; 2018: 3424136.

27.

Lorenzatti

Retzlaff

. Unmet needs in the management of atherosclerotic cardiovascular disease: is there a role for emerging anti-inflammatory interventions? Int J Cardiol 2016; 221: 581–586.

28.

Arnett

Blumenthal

Albert

. 2019 ACC/AHA guideline on the primary prevention of cardiovascular disease: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation 2019; 140: e596–e646.

29.

Gubner

August

Ginsberg

. Therapeutic suppression of tissue reactivity. II. Effect of aminopterin in rheumatoid arthritis and psoriasis. Am J Med Sci 1951; 221: 176–182.

30.

Black

O’Brien

Vanscott

, et al. Methotrexate therapy in psoriatic arthritis; double-blind study on 21 patients. JAMA 1964; 189: 743–747.

31.

Vanscott

Auerbach

Weinstein

. Parenteral methotrexate in psoriasis. Arch Dermatol 1964; 89: 550–556.

32.

Huennekens

. The methotrexate story: a paradigm for development of cancer chemotherapeutic agents. Adv Enzyme Regul 1994; 34: 397–419.

33.

Malaviya

. Landmark papers on the discovery of methotrexate for the treatment of rheumatoid arthritis and other systemic inflammatory rheumatic diseases: a fascinating story. Int J Rheum Dis 2016; 19: 844–851.

34.

Bedoui

Guillot

Sélambarom

, et al. Methotrexate an old drug with new tricks. Int J Mol Sci 2019; 20: 20191010.

35.

Cronstein

Aune

. Methotrexate and its mechanisms of action in inflammatory arthritis. Nat Rev Rheumatol 2020; 16: 145–154.

36.

Wilsdon

Whittle

Thynne

, et al. Methotrexate for psoriatic arthritis. Cochrane Database Syst Rev 2019; 2019: CD012722.

37.

Marks

Edwards

. Protective effect of methotrexate in patients with rheumatoid arthritis and cardiovascular comorbidity. Ther Adv Musculoskelet Dis 2012; 4: 149–157.

38.

Bălănescu

Bojincă

, et al. Cardiovascular effects of methotrexate in immune-mediated inflammatory diseases. Exp Ther Med 2019; 17: 1024–1029.

39.

van den Berg

Bhatt

Kappelle

, et al. Identification of vascular patients at very high risk for recurrent cardiovascular events: validation of the current ACC/AHA very high risk criteria. Eur Heart J 2017; 38: 3211–3218.

40.

Silverio

Cancro

Esposito

, et al. Secondary cardiovascular prevention after acute coronary syndrome: emerging risk factors and novel therapeutic targets. J Clin Med 2023; 12: 20230310.

41.

Lechner

von Schacky

McKenzie

, et al. Lifestyle factors and high-risk atherosclerosis: pathways and mechanisms beyond traditional risk factors. Eur J Prev Cardiol 2020; 27: 394–406.

42.

Dhindsa

Sandesara

Shapiro

, et al. The evolving understanding and approach to residual cardiovascular risk management. Front Cardiovasc Med 2020; 7: 88.

43.

Bubb

Nelson

Nicholls

. Targeting triglycerides to lower residual cardiovascular risk. Expert Rev Cardiovasc Ther 2022; 20: 185–191.

44.

Ridker

. How common is residual inflammatory risk? Circ Res 2017; 120: 617–619.

45.

Aday

Ridker

. Targeting residual inflammatory risk: a shifting paradigm for atherosclerotic disease. Front Cardiovasc Med 2019; 6: 16.

46.

Arnold

Koenig

. Persistent inflammatory residual risk despite aggressive cholesterol-lowering therapy: what is next? Curr Opin Cardiol 2021; 36: 776–783.

47.

Liu

Wang

Xiang

, et al. Association of residual inflammatory risk with stroke recurrence in patients with acute ischaemic stroke or transient ischaemic attack. Eur J Neurol 2022; 29: 2258–2268.

48.

Guedeney

Claessen

Kalkman

, et al. Residual inflammatory risk in patients with low LDL cholesterol levels undergoing percutaneous coronary intervention. J Am Coll Cardiol 2019; 73: 2401–2409.

49.

Ridker

Bhatt

Pradhan

, et al. Inflammation and cholesterol as predictors of cardiovascular events among patients receiving statin therapy: a collaborative analysis of three randomised trials. J Lancet 2023; 401: 1293–1301.

50.

Ridker

Lei

Ray

, et al. Effects of bempedoic acid on CRP, IL-6, fibrinogen and lipoprotein(a) in patients with residual inflammatory risk: a secondary analysis of the CLEAR harmony trial. J Clin Lipidol 2023; 17: 297–302.

51.

Adamstein

Cornel

Davidson

, et al. Association of interleukin 6 inhibition with ziltivekimab and the neutrophil-lymphocyte ratio: a secondary analysis of the RESCUE clinical trial. JAMA Cardiol 2023; 8: 177–181.

52.

Everett

MacFadyen

Thuren

, et al. Inhibition of interleukin-1β and reduction in atherothrombotic cardiovascular events in the CANTOS trial. J Am Coll Cardiol 2020; 76: 1660–1670.

53.

Hilvo

Wallentin

Ghukasyan Lakic

, et al. Prediction of residual risk by ceramide-phospholipid score in patients with stable coronary heart disease on optimal medical therapy. J Am Heart Assoc 2020; 9: e015258.

54.

Ridker

MacFadyen

Glynn

, et al. Comparison of interleukin-6, C-reactive protein, and low-density lipoprotein cholesterol as biomarkers of residual risk in contemporary practice: secondary analyses from the Cardiovascular Inflammation Reduction Trial. Eur Heart J 2020; 41: 2952–2961.

55.

Bohula

Giugliano

Leiter

, et al. Inflammatory and cholesterol risk in the FOURIER trial. Circulation 2018; 138: 131–140.

56.

Napoli

de Nigris

Williams-Ignarro

, et al. Nitric oxide and atherosclerosis: an update. Nitric Oxide 2006; 15: 265–279.

57.

Förstermann

. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000; 190: 244–254.

58.

Cannon

3rd . Role of nitric oxide in cardiovascular disease: focus on the endothelium. Clin Chem 1998; 44: 1809–1819.

59.

Rudic

Sessa

. Nitric oxide in endothelial dysfunction and vascular remodeling: clinical correlates and experimental links. Am J Hum Genet 1999; 64: 673–677.

60.

Loscalzo

. Nitric oxide insufficiency, platelet activation, and arterial thrombosis. Circ Res 2001; 88: 756–762.

61.

Stamler

Loh

Roddy

, et al. Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation 1994; 89: 2035–2040.

62.

Tousoulis

Kampoli

Tentolouris

, et al. The role of nitric oxide on endothelial function. Curr Vasc Pharmacol 2012; 10: 4–18.

63.

Wilkinson

Franklin

Cockcroft

. Nitric oxide and the regulation of large artery stiffness: from physiology to pharmacology. Hypertension 2004; 44: 112–116.

64.

Tousoulis

Oikonomou

Economou

, et al. Inflammatory cytokines in atherosclerosis: current therapeutic approaches. Eur Heart J 2016; 37: 1723–1732.

65.

Ridker

Rifai

Pfeffer

, et al. Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction. Circulation 2000; 101: 2149–2153.

66.

Yuan

Carter

Bruzelius

, et al. Effects of tumour necrosis factor on cardiovascular disease and cancer: a two-sample Mendelian randomization study. EBioMedicine 2020; 59: 102956.

67.

Lindmark

Diderholm

Wallentin

, et al. Relationship between interleukin 6 and mortality in patients with unstable coronary artery disease: effects of an early invasive or noninvasive strategy. JAMA 2001; 286: 2107–2113.

68.

Abbate

Trankle

Buckley

, et al. Interleukin-1 blockade inhibits the acute inflammatory response in patients with ST-segment-elevation myocardial infarction. J Am Heart Assoc 2020; 9: e014941.

69.

Guan

Liang

, et al. Therapeutic potential of MCC950, a specific inhibitor of NLRP3 inflammasome. Eur J Pharmacol 2022; 928: 175091.

70.

Guo

. Effects of tranilast on inflammasome and macrophage phenotype in a mouse model of myocardial infarction. J Interferon Cytokine Res 2021; 41: 102–110.

71.

Holzer

Hoke

Sabeti-Sandor

, et al. Disparate effects of adalimumab and fumaric acid esters on cardiovascular risk factors in psoriasis patients: results from a prospective, randomized, observer-blinded head-to-head trial. J Eur Acad Dermatol Venereol 2021; 35: 441–449.

72.

Broch

Anstensrud

Woxholt

, et al. Randomized trial of interleukin-6 receptor inhibition in patients with acute ST-segment elevation myocardial infarction. J Am Coll Cardiol 2021; 77: 1845–1855.

73.

Chen

Cao

Guo

, et al. Maraviroc, an inhibitor of chemokine receptor type 5, alleviates neuroinflammatory response after cerebral ischemia/reperfusion injury via regulating MAPK/NF-κB signaling. Int Immunopharmacol 2022; 108: 108755.

74.

Gilbert

Lekstrom-Himes

Donaldson

, et al. Effect of CC chemokine receptor 2 CCR2 blockade on serum C-reactive protein in individuals at atherosclerotic risk and with a single nucleotide polymorphism of the monocyte chemoattractant protein-1 promoter region. Am J Cardiol 2011; 107: 906–911.

75.

Zhao

Kostapanos

Griffiths

, et al. Low-dose interleukin-2 in patients with stable ischaemic heart disease and acute coronary syndromes (LILACS): protocol and study rationale for a randomised, double-blind, placebo-controlled, phase I/II clinical trial. BMJ Open 2018; 8: e022452.

76.

Kim

Lee

Seo

, et al. Rituximab protects against development of atherosclerotic cardiovascular disease after kidney transplantation: a propensity-matched study. Sci Rep 2019; 9: 16475–20191111.

77.

Joensuu

Aaltonen

Aronen

, et al. Cost-effectiveness of biologic compared with conventional synthetic disease-modifying anti-rheumatic drugs in patients with rheumatoid arthritis: a Register study. Rheumatology (Oxford, England) 2016; 55: 1803–1811.

78.

Yazdany

Dudley

Chen

, et al. Coverage for high-cost specialty drugs for rheumatoid arthritis in Medicare Part D. Arthritis Rheumatol 2015; 67: 1474–1480.

79.

Pereira

Faria

Lago

, et al. Infection and malignancy risk in patients treated with TNF inhibitors for immune-mediated inflammatory diseases. Curr Drug Saf 2017; 12: 162–170.

80.

Winthrop

Mariette

Silva

, et al. ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (soluble immune effector molecules [II]: agents targeting interleukins, immunoglobulins and complement factors). Clin Microbiol Infect 2018; 24 (Suppl. 2): S21–S40.

81.

Buckley

Abbate

. Interleukin-1 blockade in cardiovascular diseases: a clinical update. Eur Heart J 2018; 39: 2063–2069.

82.

Beinsberger

Heemskerk

Cosemans

. Chronic arthritis and cardiovascular disease: altered blood parameters give rise to a prothrombotic propensity. Semin Arthritis Rheum 2014; 44: 345–352.

83.

Jones

Panova

. New insights and long-term safety of tocilizumab in rheumatoid arthritis. Ther Adv Musculoskelet Dis 2018; 10: 195–199.

84.

Deftereos

Beerkens

Shah

, et al. Colchicine in cardiovascular disease: in-depth review. Circulation 2022; 145: 61–78.

85.

Imazio

Nidorf

. Colchicine and the heart. Eur Heart J 2021; 42: 2745–2760.

86.

Benedek

. Methotrexate: from its introduction to non-oncologic therapeutics to anti-TNF-α. Clin Exp Rheumatol 2010; 28: S3–S8.

87.

Cipriani

Ruscitti

Carubbi

, et al. Methotrexate: an old new drug in autoimmune disease. Expert Rev Clin Immunol 2014; 10: 1519–1530.

88.

Pincus

Gibson

Castrejón

. Update on methotrexate as the anchor drug for rheumatoid arthritis. Bull Hosp Jt Dis 2013; 71(Suppl. 1): S9–19.

89.

van Huizen

Menting

Gyulai

. International eDelphi study to reach consensus on the methotrexate dosing regimen in patients with psoriasis. JAMA Dermatol 2022; 158: 561–572.

90.

Damiani

Amerio

Bardazzi

, et al. Real-world experience of methotrexate in the treatment of skin diseases: an Italian Delphi Consensus. Dermatol Ther 2023; 13: 1219–1241.

91.

Hoekstra

Haagsma

Neef

, et al. Bxotrexate comparing oral and subcutaneous administration in patients with rheumatoid arthritis. J Rheumatol 2004; 31: 645–648.

92.

Tanaka

. Subcutaneous injection of methotrexate: advantages in the treatment of rheumatoid arthritis. Mod Rheumatol 2023; 33: 633–639.

93.

Herman

Veng-Pedersen

Hoffman

, et al. Pharmacokinetics of low-dose methotrexate in rheumatoid arthritis patients. J Pharm Sci 1989; 78: 165–171.

94.

Seideman

Beck

Eksborg

, et al. The pharmacokinetics of methotrexate and its 7-hydroxy metabolite in patients with rheumatoid arthritis. Br J Clin Pharmacol 1993; 35: 409–412.

95.

Gao

Wang

Wei

. The effects of drug transporters on the efficacy of methotrexate in the treatment of rheumatoid arthritis. Life Sci 2021; 268: 118907.

96.

Dalrymple

Stamp

O’Donnell

, et al. Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis. Arthritis Rheum 2008; 58: 3299–3308.

97.

Inoue

Yuasa

. Molecular basis for pharmacokinetics and pharmacodynamics of methotrexate in rheumatoid arthritis therapy. Drug Metab Pharmacokinet 2014; 29: 12–19.

98.

Howard

McCormick

Pui

, et al. Preventing and managing toxicities of high-dose methotrexate. Oncologist 2016; 21: 1471–1482.

99.

Romão

Lima

Bernardes

, et al. Three decades of low-dose methotrexate in rheumatoid arthritis: can we predict toxicity? Immunol Res 2014; 60: 289–310.

100.

Kinder

Hassell

Brand

, et al. The treatment of inflammatory arthritis with methotrexate in clinical practice: treatment duration and incidence of adverse drug reactions. Rheumatology (Oxford, England) 2005; 44: 61–66.

101.

Verschueren

De Cock

Corluy

, et al. Effectiveness of methotrexate with step-down glucocorticoid remission induction (COBRA Slim) versus other intensive treatment strategies for early rheumatoid arthritis in a treat-to-target approach: 1-year results of CareRA, a randomised pragmatic open-label superiority trial. Ann Rheum Dis 2017; 76: 511–520.

102.

Krijbolder

Verstappen

van Dijk

, et al. Intervention with methotrexate in patients with arthralgia at risk of rheumatoid arthritis to reduce the development of persistent arthritis and its disease burden (TREAT EARLIER): a randomised, double-blind, placebo-controlled, proof-of-concept trial. J Lancet 2022; 400: 283–294.

103.

Erre

Cadoni

Meloni

, et al. Methotrexate therapy is not associated with increased liver stiffness and significant liver fibrosis in rheumatoid arthritis patients: a cross-sectional controlled study with real-time two-dimensional shear wave elastography. Eur J Intern Med 2019; 69: 57–63.

104.

Erre

Castagna

Sauchella

, et al. Prevalence and risk factors of moderate to severe hepatic steatosis in patients with rheumatoid arthritis: an ultrasonography cross-sectional case-control study. Ther Adv Musculoskelet Dis 2021; 13: 1759720X211042739.

105.

Solomon

Glynn

Karlson

, et al. Adverse effects of low-dose methotrexate: a randomized trial. Ann Intern Med 2020; 172: 369–380.

106.

Wright

Jr. Williamson

Whelton

, et al. A randomized trial of intensive versus standard blood-pressure control. New Engl J Med 2015; 373: 2103–2116.

107.

ONTARGET

Investigators

Yusuf

Teo

, et al. Telmisartan, ramipril, or both in patients at high risk for vascular events. New Engl J Med 2008; 358: 1547–1559.

108.

Conrad

Misra

Verbakel

, et al. Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK. J Lancet 2023; 401: 1878–1890.

109.

Hitchon

El-Gabalawy

. Oxidation in rheumatoid arthritis. Arthritis Res Ther 2004; 6: 265–278.

110.

Sparks

. Rheumatoid arthritis. Ann Intern Med 2019; 170: ITC1–ITC16.

111.

Erre

Paliogiannis

Castagna

, et al. Meta-analysis of neutrophil-to-lymphocyte and platelet-to-lymphocyte ratio in rheumatoid arthritis. Eur J Clin Invest 2019; 49: e13037.

112.

Bassu

Zinellu

Sotgia

, et al. Oxidative stress biomarkers and peripheral endothelial dysfunction in rheumatoid arthritis: a monocentric cross-sectional case-control study. Molecules 2020; 25: 3855.

113.

Erre

Bassu

Giordo

, et al. Association between paraoxonase/arylesterase activity of serum PON-1 enzyme and rheumatoid arthritis: a systematic review and meta-analysis. Antioxidants (Basel, Switzerland) 2022; 11: 20221123.

114.

Erre

Cacciapaglia

Sakellariou

, et al. C-reactive protein and 10-year cardiovascular risk in rheumatoid arthritis. Eur J Intern Med 2022; 104: 49–54.

115.

Zinellu

Mangoni

. Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratio and disease activity in rheumatoid arthritis: a systematic review and meta-analysis. Eur J Clin Invest 2023; 53: e13877.

116.

Bordy

Totoson

Prati

, et al. Microvascular endothelial dysfunction in rheumatoid arthritis. Nat Rev Rheumatol 2018; 14: 404–420.

117.

Mangoni

Tommasi

Sotgia

, et al. Asymmetric dimethylarginine: a key player in the pathophysiology of endothelial dysfunction, vascular inflammation and atherosclerosis in rheumatoid arthritis? Curr Pharm Des 2021; 27: 2131–2140.

118.

Erre

Buscetta

Paliogiannis

, et al. Coronary flow reserve in systemic rheumatic diseases: a systematic review and meta-analysis. Rheumatol Int 2018; 38: 1179–1190.

119.

Erre

Piga

Fedele

, et al. Prevalence and determinants of peripheral microvascular endothelial dysfunction in rheumatoid arthritis patients: a multicenter cross-sectional study. Mediators Inflamm 2018; 2018: 6548715.

120.

Mangoni

Baghdadi

Shanahan

, et al. Methotrexate, blood pressure and markers of arterial function in patients with rheumatoid arthritis: a repeated cross-sectional study. Ther Adv Musculoskelet Dis 2017; 9: 213–229.

121.

Ambrosino

Tasso

Lupoli

, et al. Non-invasive assessment of arterial stiffness in patients with rheumatoid arthritis: a systematic review and meta-analysis of literature studies. Ann Med 2015; 47: 457–467.

122.

Arosio

De Marchi

Rigoni

, et al. Forearm haemodynamics, arterial stiffness and microcirculatory reactivity in rheumatoid arthritis. J Hypertens 2007; 25: 1273–1278.

123.

Karakulak

Sahiner

Maharjan

, et al. Evaluation of the ambulatory arterial stiffness index in patients with rheumatoid arthritis. Blood Press Monit 2015; 20: 254–259.

124.

Woodman

Baghdadi

Shanahan

, et al. The temporal relationship between arterial stiffening and blood pressure is modified by methotrexate treatment in patients with rheumatoid arthritis. Front Physiol 2017; 8: 593.

125.

Jafri

Bartels

Shin

, et al. Incidence and management of cardiovascular risk factors in psoriatic arthritis and rheumatoid arthritis: a population-based study. Arthritis Care Res 2017; 69: 51–57.

126.

Chen

Fernandez

, et al. Temporal relationship between elevated blood pressure and arterial stiffening among middle-aged black and white adults: the Bogalusa Heart Study. Am J Epidemiol 2016; 183: 599–608.

127.

Feldmann

Brennan

Maini

. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996; 14: 397–440.

128.

Tetta

Camussi

Modena

, et al. Tumour necrosis factor in serum and synovial fluid of patients with active and severe rheumatoid arthritis. Ann Rheum Dis 1990; 49: 665–667.

129.

Kay

Calabrese

. The role of interleukin-1 in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford, England) 2004; 43(Suppl. 3): iii2–iii9.

130.

Srirangan

Choy

. The role of interleukin 6 in the pathophysiology of rheumatoid arthritis. Ther Adv Musculoskelet Dis 2010; 2: 247–256.

131.

Restivo

Candiloro

Daidone

, et al. Systematic review and meta-analysis of cardiovascular risk in rheumatological disease: symptomatic and non-symptomatic events in rheumatoid arthritis and systemic lupus erythematosus. Autoimmun Rev 2022; 21: 102925.

132.

Baghdadi

Woodman

Shanahan

, et al. The impact of traditional cardiovascular risk factors on cardiovascular outcomes in patients with rheumatoid arthritis: a systematic review and meta-analysis. PLoS One 2015; 10: e0117952.

133.

Santos-Moreno

Rodríguez-Vargas

Martínez

, et al. Metabolic abnormalities, cardiovascular disease, and metabolic syndrome in adult rheumatoid arthritis patients: current perspectives and clinical implications. Open Access Rheumatol 2022; 14: 255–267.

134.

Xiao

Zhu

, et al. Methotrexate use reduces mortality risk in rheumatoid arthritis: a systematic review and meta-analysis of cohort studies. Semin Arthritis Rheum 2022; 55: 152031.

135.

Sun

Liu

, et al. Methotrexate can prevent cardiovascular events in patients with rheumatoid arthritis: an updated meta-analysis. Medicine (Baltimore) 2021; 100: e24579.

136.

Baoqi

Dan

Xingxing

, et al. Effect of anti-rheumatic drugs on cardiovascular disease events in rheumatoid arthritis. Front Cardiovasc Med 2021; 8: 812631.

137.

Rodríguez-Vargas

Santos-Moreno

Rubio-Rubio

, et al. Vascular age, metabolic panel, cardiovascular risk and inflammaging in patients with rheumatoid arthritis compared with patients with osteoarthritis. Front Cardiovasc Med 2022; 9: 894577.

138.

Roubille

Richer

Starnino

, et al. The effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis. Ann Rheum Dis 2015; 74: 480–489.

139.

Xie

Chen

Yun

, et al. Benefits of methotrexate use on cardiovascular disease risk among rheumatoid arthritis patients initiating biologic disease-modifying antirheumatic drugs. J Rheumatol 2021; 48: 804–812.

140.

Wei

Galaviz

Kowalski

, et al. Comparison of cardiovascular events among users of different classes of antihypertension medications: a systematic review and network meta-analysis. JAMA Netw Open 2020; 3: e1921618.

141.

Naci

Brugts

Fleurence

, et al. Comparative benefits of statins in the primary and secondary prevention of major coronary events and all-cause mortality: a network meta-analysis of placebo-controlled and active-comparator trials. Eur J Prev Cardiol 2013; 20: 641–657.

142.

Mangoni

Woodman

Piga

, et al. Patterns of anti-inflammatory and immunomodulating drug usage and microvascular endothelial function in rheumatoid arthritis. Front Cardiovasc Med 2021; 8: 681327.

143.

D’Andrea

Desai

, et al. Cardiovascular risks of hydroxychloroquine vs methotrexate in patients with rheumatoid arthritis. J Am Coll Cardiol 2022; 80: 36–46.

144.

Ridker

Everett

Pradhan

, et al. Low-dose methotrexate for the prevention of atherosclerotic events. New Engl J Med 2019; 380: 752–762.

145.

Panja

Khatua

Halder

. Simultaneous binding of folic acid and methotrexate to human serum albumin: insights into the structural changes of protein and the location and competitive displacement of drugs. ACS Omega 2018; 3: 246–253.

146.

Goldman

Lichtenstein

Oliverio

. Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell. J Biol Chem 1968; 243: 5007–5017.

147.

Morgan

Baggott

Vaughn

, et al. Supplementation with folic acid during methotrexate therapy for rheumatoid arthritis. A double-blind, placebo-controlled trial. Ann Intern Med 1994; 121: 833–841.

148.

Mangoni

Jackson

. Homocysteine and cardiovascular disease: current evidence and future prospects. Am J Med 2002; 112: 556–565.

149.

Mangoni

Sherwood

Asonganyi

, et al. Short-term oral folic acid supplementation enhances endothelial function in patients with type 2 diabetes. Am J Hypertens 2005; 18: 220–226.

150.

Mangoni

Ouldred

Swif

, et al. Vascular and blood pressure effects of folic acid in older patients with cardiovascular disease. J Am Geriatr Soc 2001; 49: 1003–1004.

151.

Mangoni

Sherwood

Swift

, et al. Folic acid enhances endothelial function and reduces blood pressure in smokers: a randomized controlled trial. J Intern Med 2002; 252: 497–503.

152.

Huo

Qin

, et al. Efficacy of folic acid therapy in primary prevention of stroke among adults with hypertension in China: the CSPPT randomized clinical trial. JAMA 2015; 313: 1325–1335.

153.

Gomes

FLT

Maranhão

Tavares

, et al. Regression of atherosclerotic plaques of cholesterol-fed rabbits by combined chemotherapy with paclitaxel and methotrexate carried in lipid core nanoparticles. J Cardiovasc Pharmacol Ther 2018; 23: 561–569.

154.

Mašić

Stengaard-Pedersen

Løgstrup

, et al. Similar lipid level changes in early rheumatoid arthritis patients following 1-year treat-to-target strategy with adalimumab plus methotrexate versus placebo plus methotrexate: secondary analyses from the randomised controlled OPERA trial. Rheumatol Int 2021; 41: 543–549.

155.

Enrich

Rentero

de Muga

, et al. Annexin A6-linking Ca(2+) signaling with cholesterol transport. Biochim Biophys Acta 2011; 1813: 935–947.

156.

Zhang

Spencer

Voruganti

, et al. Association of functional polymorphism rs2231142 (Q141K) in the ABCG2 gene with serum uric acid and gout in 4 US populations: the PAGE Study. Am J Epidemiol 2013; 177: 923–932.

157.

Wang

Deng

, et al. The difference of lipid profiles between psoriasis with arthritis and psoriasis without arthritis and sex-specific downregulation of methotrexate on the apolipoprotein B/apolipoprotein A-1 ratio. Arthritis Res Ther 2022; 24: 17.

158.

Krahel

Baran

Kamiński

, et al. Methotrexate decreases the level of PCSK9-A novel indicator of the risk of proatherogenic lipid profile in psoriasis. The preliminary data. J Clin Med 2020; 9: 20200326.

159.

Artenstein

Opal

. Proprotein convertases in health and disease. New Engl J Med 2011; 365: 2507–2518.

160.

Coppinger

Movahed

Azemawah

, et al. A comprehensive review of PCSK9 inhibitors. J Cardiovasc Pharmacol Ther 2022; 27: 10742484221100107.

161.

Baghdadi

. Effect of methotrexate use on the development of type 2 diabetes in rheumatoid arthritis patients: a systematic review and meta-analysis. PLoS One 2020; 15: e0235637.

162.

Xie

Yang

, et al. Incident diabetes associated with hydroxychloroquine, methotrexate, biologics and glucocorticoids in rheumatoid arthritis: a systematic review and meta-analysis. Semin Arthritis Rheum 2020; 50: 598–607.

163.

Nam

Kim

, et al. Risk of new-onset diabetes mellitus associated with antirheumatic drugs in patients with rheumatoid arthritis: a nationwide population study. J Clin Med 2022; 11: 20220410.

164.

Baker

Sauer

Teng

, et al. Initiation of disease-modifying therapies in rheumatoid arthritis is associated with changes in blood pressure. J Clin Rheumatol 2018; 24: 203–209.

165.

Toms

Panoulas

John

, et al. Methotrexate therapy associates with reduced prevalence of the metabolic syndrome in rheumatoid arthritis patients over the age of 60- more than just an anti-inflammatory effect? A cross sectional study. Arthritis Res Ther 2009; 11: R110.

166.

Bilecik

Tuna

Samancı

, et al. Prevalence of metabolic syndrome in women with rheumatoid arthritis and effective factors. Int J Clin Exp Med 2014; 7: 2258–2265.

167.

Costa

Caso

Atteno

, et al. Impact of 24-month treatment with etanercept, adalimumab, or methotrexate on metabolic syndrome components in a cohort of 210 psoriatic arthritis patients. Clin Rheumatol 2014; 33: 833–839.

168.

Seitz

Zwicker

Loetscher

. Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes. Arthritis Rheum 1998; 41: 2032–2038.

169.

Edwards

3rd Bendele

Reznikov

, et al. Soluble human p55 and p75 tumor necrosis factor receptors reverse spontaneous arthritis in transgenic mice expressing transmembrane tumor necrosis factor alpha. Arthritis Rheum 2006; 54: 2872–2885.

170.

Yamasaki

Soma

Kawa

, et al. Methotrexate inhibits proliferation and regulation of the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by cultured human umbilical vein endothelial cells. Br J Dermatol 2003; 149: 30–38.

171.

Quan

Pan

Singh

, et al. Cardiovascular inflammation is reduced with methotrexate in diabetes. Mol Cell Biochem 2017; 432: 159–167.

172.

Shao

, et al. Methotrexate improves perivascular adipose tissue/endothelial dysfunction via activation of AMPK/eNOS pathway. Mol Med Rep 2017; 15: 2353–2359.

173.

Municio

Dominguez-Soto

Fuentelsaz-Romero

, et al. Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism. Ann Rheum Dis 2018; 77: 752–759.

174.

Stigliano

Ramirez

Singh

, et al. Methotrexate-loaded hybrid nanoconstructs target vascular lesions and inhibit atherosclerosis progression in ApoE(-/-) mice. Adv Healthcare Mater 2017; 6: 1601286.

175.

Mangoni

Zinellu

Sotgia

, et al. Protective effects of methotrexate against proatherosclerotic cytokines: a review of the evidence. Mediators Inflamm 2017; 2017: 9632846.

176.

Owczarczyk-Saczonek

Drozdowski

Maciejewska-Radomska

, et al. The effect of subcutaneous methotrexate on markers of metabolic syndrome in psoriatic patients – preliminary report. Postepy Dermatol Alergol 2018; 35: 53–59.

177.

Gong

Zhang

Sun

, et al. The nonspecific anti-inflammatory therapy with methotrexate for patients with chronic heart failure. Am Heart J 2006; 151: 62–68.

178.

Zhang

Chen

Zhang

, et al. Effects of methotrexate in a rabbit model of in-stent neoatherosclerosis: an optical coherence tomography study. Sci Rep 2016; 6: 33657.

179.

Fiorelli

Lourenço-Filho

Tavares

, et al. Methotrexate associated to lipid core nanoparticles improves cardiac allograft vasculopathy and the inflammatory profile in a rabbit heart graft model. Braz J Med Biol Res 2017; 50: e6225.

180.

Zhang

Zhao

, et al. Effects of methotrexate on plasma cytokines and cardiac remodeling and function in postmyocarditis rats. Mediators Inflamm 2009; 2009: 389720.

181.

DeOliveira

Acedo

Gotardo

. Effects of methotrexate on inflammatory alterations induced by obesity: an in vivo and in vitro study. Mol Cell Endocrinol 2012; 361: 92–98.

182.

Saleem

Muhammad

Sharif

, et al. Methotrexate-loaded biodegradable nanoparticles exert anti-arthritic effect by downregulating pro-inflammatory cytokines in Freund’s complete adjuvant-induced arthritic rats. Inflammopharmacology 2022; 30: 1079–1091.

183.

Stähli

Scherler

Zappalà

, et al. In vitro activity of anti-rheumatic drugs on release of pro-inflammatory cytokines from oral cells in interaction with microorganisms. Front Oral Health 2022; 3: 960732.

184.

Zdanowska

Owczarczyk-Saczonek

Czerwińska

, et al. Adalimumab and methotrexate affect the concentrations of regulatory cytokines (interleukin-10, transforming growth factor-β1, and interleukin-35) in patients with plaque psoriasis. Dermatol Ther 2020; 33: e14153.

185.

Ridker

Everett

Thuren

, et al. Antiinflammatory therapy with canakinumab for atherosclerotic disease. New Engl J Med 2017; 377: 1119–1131.

186.

Iwamoto

Umemura

Toya

, et al. Identification of adenosine A2 receptor-camp system in human aortic endothelial cells. Biochem Biophys Res Commun 1994; 199: 905–910.

187.

Ansari

Nadeem

Talukder

, et al. Evidence for the involvement of nitric oxide in A2B receptor-mediated vasorelaxation of mouse aorta. Am J Physiol Heart Circ Physiol 2007; 292: H719–H725.

188.

Tang

Parker

Fei

, et al. Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney. Am J Physiol 1999; 277: F926–F933.

189.

Maimon

Titus

Sarelius

. Pre-exposure to adenosine, acting via A(2A) receptors on endothelial cells, alters the protein kinase A dependence of adenosine-induced dilation in skeletal muscle resistance arterioles. J Physiol 2014; 592: 2575–2590.

190.

Zou

Nithipatikom

, et al. Role of renal medullary adenosine in the control of blood flow and sodium excretion. Am J Physiol 1999; 276: R790–R798.

191.

Schindler

Karcz-Kubicha

Thorndike

, et al. Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists. Br J Pharmacol 2005; 144: 642–650.

192.

Mahmud

Feely

. Acute effect of caffeine on arterial stiffness and aortic pressure waveform. Hypertension 2001; 38: 227–231.

193.

Koupenova

Johnston-Cox

Ravid

. Regulation of atherosclerosis and associated risk factors by adenosine and adenosine receptors. Curr Atheroscler Rep 2012; 14: 460–468.

194.

Varani

Portaluppi

Merighi

, et al. Caffeine alters A2A adenosine receptors and their function in human platelets. Circulation 1999; 99: 2499–2502.

195.

Haskó

Pacher

. Regulation of macrophage function by adenosine. Arterioscler Thromb Vasc Biol 2012; 32: 865–869.

196.

Voloshyna

Reiss

. The ABC transporters in lipid flux and atherosclerosis. Prog Lipid Res 2011; 50: 213–224.

197.

Escher

Krozowski

Croft

, et al. Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux. J Biol Chem 2003; 278: 11015–11019.

198.

Meng

, et al. Novel insights: dynamic foam cells derived from the macrophage in atherosclerosis. J Cell Physiol 2021; 236: 6154–6167.

199.

Višnjić

Lalić

Dembitz

, et al. AICAr, a widely used AMPK activator with important AMPK-independent effects: a systematic review. Cells 2021; 10: 1095.

200.

Hardie

. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev 2011; 25: 1895–1908.

201.

Antonioli

Colucci

Pellegrini

, et al. The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders. Expert Opin Ther Targets 2016; 20: 179–191.

202.

Boin

Erre

Posadino

, et al. Oxidative stress-dependent activation of collagen synthesis is induced in human pulmonary smooth muscle cells by sera from patients with scleroderma-associated pulmonary hypertension. Orphanet J Rare Dis 2014; 9: 123.

203.

Igata

Motoshima

Tsuruzoe

, et al. Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression. Circ Res 2005; 97: 837–844.

204.

Wang

, et al. Pharmacological activation of AMPK prevents drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction. J Mol Cell Cardiol 2015; 86: 62–74.

205.

Chen

Peng

Sun

, et al. AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633. Circ Res 2009; 104: 496–505.

206.

Goirand

Solar

Athea

, et al. Activation of AMP kinase alpha1 subunit induces aortic vasorelaxation in mice. J Physiol 2007; 581: 1163–1171.

207.

Nagata

Takeda

Sata

, et al. AMP-activated protein kinase inhibits angiotensin II-stimulated vascular smooth muscle cell proliferation. Circulation 2004; 110: 444–451.

208.

Ford

Teschke

Reid

, et al. AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats. J Hypertens 2012; 30: 725–733.

209.

Buhl

Jessen

Pold

, et al. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes 2002; 51: 2199–2206.

210.

Samsonov

Podkuychenko

Khapchaev

, et al. AICAR protects vascular endothelial cells from oxidative injury induced by the long-term palmitate excess. Int J Mol Sci 2021; 23: 211.

211.

Pang

Wang

Song

, et al. AMPK upregulates K(Ca)2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice. Biochem Pharmacol 2021; 183: 114337.

212.

Angin

Beauloye

Horman

, et al. Regulation of carbohydrate metabolism, lipid metabolism, and protein metabolism by AMPK. Exp Suppl 2016; 107: 23–43.

213.

Steinberg

Hardie

. New insights into activation and function of the AMPK. Nat Rev Mol Cell Biol 2023; 24: 255–272.

214.

Heidary Moghaddam

Samimi

Asgary

, et al. Natural AMPK activators in cardiovascular disease prevention. Front Pharmacol 2021; 12: 738420.

215.

Cronstein

Naime

Ostad

. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. J Clin Investig 1993; 92: 2675–2682.

216.

Hess

Khasawneh

. Cancer metabolism and oxidative stress: insights into carcinogenesis and chemotherapy via the non-dihydrofolate reductase effects of methotrexate. BBA Clin 2015; 3: 152–161.

217.

Duryee

Klassen

Schaffert

, et al. Malondialdehyde-acetaldehyde adduct is the dominant epitope after MDA modification of proteins in atherosclerosis. Free Radic Biol Med 2010; 49: 1480–1486.

218.

Zimmerman

Clemens

Duryee

, et al. Direct antioxidant properties of methotrexate: inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging. Redox Biol 2017; 13: 588–593.

219.

Luna-López

Flores-González

Rivera-Ruz

, et al. Methotrexate induces an antioxidant hormetic response in primary rat astrocytes. Dose Response 2022; 20: 15593258221130752.

220.

Chowdhury

Khan

Heydon

, et al. Adherence to cardiovascular therapy: a meta-analysis of prevalence and clinical consequences. Eur Heart J 2013; 34: 2940–2948.

221.

Jekic

Maksimovic

Damnjanovic

. Methotrexate pharmacogenetics in the treatment of rheumatoid arthritis. Pharmacogenomics 2019; 20: 1235–1245.

222.

Campbell

Bateman

Stephenson

, et al. Methotrexate-induced toxicity pharmacogenetics: an umbrella review of systematic reviews and meta-analyses. Cancer Chemother Pharmacol 2016; 78: 27–39.

223.

Szostak

Machaj

Rosik

, et al. Using pharmacogenetics to predict methotrexate response in rheumatoid arthritis patients. Expert Opin Drug Metab Toxicol 2020; 16: 617–626.

224.

Baghdadi

Woodman

Shanahan

, et al. Genetic polymorphism of the methotrexate transporter ABCG2, blood pressure and markers of arterial function in patients with rheumatoid arthritis: repeated cross-sectional study. Pharmgenomics Pers Med 2018; 11: 205–210.

225.

Nidorf

Eikelboom

Budgeon

, et al. Low-dose colchicine for secondary prevention of cardiovascular disease. J Am Coll Cardiol 2013; 61: 404–410.

226.

Sharma

Wasko

Tang

, et al. Hydroxychloroquine use is associated with decreased incident cardiovascular events in rheumatoid arthritis patients. J Am Heart Assoc 2016; 5: e002867.

227.

Shapiro

Levy

. The association between hydroxychloroquine treatment and cardiovascular morbidity among rheumatoid arthritis patients. Oncotarget 2018; 9: 6615–6622.

Methotrexate and cardiovascular prevention: an appraisal of the current evidence

Abstract

Keywords

Introduction

Residual cardiovascular and inflammatory risk

Atheroprotective strategies targeting inflammation and immunity

Methotrexate pharmacology and role in cardiovascular risk in clinical studies

Pharmacology of methotrexate

Rheumatoid arthritis and cardiovascular disease

Methotrexate and cardiovascular risk

Effects of methotrexate on traditional cardiovascular risk factors

Lipid profile

Type 1 and type 2 diabetes

Arterial hypertension

Metabolic syndrome

Methotrexate and atheroprotection: Mechanistic insights

Cytokines

Adenosine and AMPK

Redox balance

Practical considerations for using methotrexate in cardiovascular prevention

Future research directions

Conclusion

Footnotes

Acknowledgements

Declarations

ORCID iD

References