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Carnosine as a potential therapeutic for the management of peripheral vascular disease

  • Author Footnotes
    1 These authors contributed equally to the preparation of this manuscript.
    Jack Feehan
    Footnotes
    1 These authors contributed equally to the preparation of this manuscript.
    Affiliations
    Institute for Health and Sport, Victoria University, Footscray, VIC, Australia
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  • Author Footnotes
    1 These authors contributed equally to the preparation of this manuscript.
    Rohit Hariharan
    Footnotes
    1 These authors contributed equally to the preparation of this manuscript.
    Affiliations
    Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton VIC, Australia
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  • Timothy Buckenham
    Affiliations
    Christchurch Clinical School of Medicine University of Otago and Christchurch Hospital, Christchurch, New Zealand
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  • Charles Handley
    Affiliations
    Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton VIC, Australia
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  • Aruni Bhatnagar
    Affiliations
    Diabetes and Obesity Center, Christina Lee Brown Environment Institute, University of Louisville, Louisville, KY, USA
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  • Shahid Pervez Baba
    Affiliations
    Diabetes and Obesity Center, Christina Lee Brown Environment Institute, University of Louisville, Louisville, KY, USA
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  • Barbora de Courten
    Correspondence
    Corresponding author. Department of Medicine, School of Clinical Sciences, 246 Clayton Road, Clayton, VIC 3168, Australia.
    Affiliations
    Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton VIC, Australia

    School of Health and Biomedical Sciences, RMIT, Bundoora
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  • Author Footnotes
    1 These authors contributed equally to the preparation of this manuscript.

      Highlights

      • Carnosine is an endogenous antioxidant, with potential for significant benefit in peripheral vascular disease.
      • Carnosine may prevent atherosclerotic plaque formation via antiglycating, anti-inflammatory actions.
      • Carnosine is safe, with few reported side effects, making it suitable for use in vulnerable populations.

      Abstract

      Aims

      To evaluate the potential role of carnosine in the management of peripheral vascular disease.

      Data synthesis

      Peripheral vascular disease is growing in its burden and impact; however it is currently under researched, and there are a lack of strong, non-invasive therapeutic options for the clinicians. Carnosine is a dipeptide stored particularly in muscle and brain tissue, which exhibits a wide range of physiological activities, which may be beneficial as an adjunct treatment for peripheral vascular disease. Carnosine's strong anti-inflammatory, antioxidant and antiglycating actions may aid in the prevention of plaque formation, through protective actions on the vascular endothelium, and the inhibition of foam cells. Carnosine may also improve angiogenesis, exercise performance and vasodilatory response, while protecting from ischemic tissue injury.

      Conclusions

      Carnosine may have a role as an adjunct treatment for peripheral vascular disease alongside typical exercise and surgical interventions, and may be used in high risk individuals to aid in the prevention of atherogenesis.

      Clinical recommendation

      This review identifies a beneficial role for carnosine supplementation in the management of patients with peripheral vascular disease, in conjunction with exercise and revascularization. Carnosine as a supplement is safe, and associated with a host of beneficial effects in peripheral vascular disease and its key risk factors.

      Keywords

      1. Introduction

      Peripheral vascular disease (PVD) is a growing concern for healthcare globally, in the face of an aging population and, with rising prevalence of cardiometabolic risk factors. PVD, also known as peripheral artery disease, is characterized by progressive occlusion of arteries due to atherosclerosis, leading to reduced blood flow in the limbs. This causes pain with physical activity, known as intermittent claudication, and progressive limb ischemia. The prevalence of PVD is challenging to assess and is therefore underreported, with more than 50% of cases being asymptomatic [
      • Shu J.
      • Santulli G.
      Update on peripheral artery disease: epidemiology and evidence-based facts.
      ]. The rates vary drastically across region and socioeconomic groups due to differences in prevalence rates of cardiometabolic risk factors [
      • Song P.
      • Rudan D.
      • Zhu Y.
      • Fowkes F.J.I.
      • Rahimi K.
      • Fowkes F.G.R.
      • et al.
      Global, regional, and national prevalence and risk factors for peripheral artery disease in 2015: an updated systematic review and analysis.
      ]. The global prevalence of PVD in those aged 25 and older is 5.56%, however it rises sharply with age affecting more than 18% of those over 85 [
      • Fowkes F.G.R.
      • Rudan D.
      • Rudan I.
      • Aboyans V.
      • Denenberg J.O.
      • McDermott M.M.
      • et al.
      Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis.
      ]. Regardless of the presence of symptoms, PVD is associated with increased mortality, significant disability, and reduced quality of life [
      • Sampson U.K.A.
      • Fowkes F.G.R.
      • McDermott M.M.
      • Criqui M.H.
      • Aboyans V.
      • Norman P.E.
      • et al.
      Global and regional burden of death and disability from peripheral artery disease: 21 world regions, 1990 to 2010.
      ,
      • Criqui M.H.
      • Aboyans V.
      Epidemiology of peripheral artery disease.
      ]. Despite this significant burden, PVD receives significantly less research and healthcare attention than other cardiovascular diseases.
      Presently very few effective treatments are available for PVD. Currently, two drugs, pentoxifyline and cilostazol, are approved for treating PVD. However, the effectiveness of these drugs is limited, hence they are not routinely used in clinical practice and are not recommended for use in Australia. Structured exercise programs, such as treadmill walking are effective in alleviating PVD symptoms, increase walking endurance, and improve a number of cardiometabolic risk factors [
      • Lane R.
      • Harwood A.
      • Watson L.
      • Leng G.C.
      Exercise for intermittent claudication.
      ]. However, exercise is limited by the intermittent claudication associated with physical activity [
      • Mazari F.A.
      • Khan J.A.
      • Samuel N.
      • Smith G.
      • Carradice D.
      • McCollum P.C.
      • et al.
      Long-term outcomes of a randomized clinical trial of supervised exercise, percutaneous transluminal angioplasty or combined treatment for patients with intermittent claudication due to femoropopliteal disease.
      ]. Open surgical revascularization is the most invasive option, and is also effective in reducing symptoms and increasing exercise tolerance [
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American heart association.
      ], particularly when combined with exercise [
      • Greenhalgh R.
      The adjuvant benefit of angioplasty in patients with mild-to-moderate intermittent claudication (MIMIC) managed by supervised exercise, smoking cessation advice and best medical therapy: results from two randomised trials for occlusive femoropopliteal and aortoiliac occlusive arterial disease.
      ]. Operative approaches are limited by cost, type of disease (not appropriate for diffuse disease), invasiveness and general surgical risk, and therefore are considered inappropriate in the early stages of the disease. Endovascular revascularisation, such as angioplasty, is a minimally invasive alternative to open surgery which is now performed more commonly than open bypass for peripheral vascular disease [
      • Goodney P.P.
      • Beck A.W.
      • Nagle J.
      • Welch H.G.
      • Zwolak R.M.
      National trends in lower extremity bypass surgery, endovascular interventions, and major amputations.
      ]. Given the negative cycle of PVD limiting physical activity, which subsequently worsens the disease, new treatments are required to help manage symptoms and prevent disease progression.
      Carnosine is a naturally occurring dipeptide, with potent antioxidant, antiglycating and geroprotective effects [
      • Hipkiss A.R.
      Carnosine and its possible roles in nutrition and health.
      ,
      • Baye E.
      • Ukropcova B.
      • Ukropec J.
      • Hipkiss A.
      • Aldini G.
      • de Courten B.
      Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease.
      ]. This dipeptide is synthesised in vivo from the amino acids, β-alanine and histidine by the enzyme carnosine synthase (CARNS) [
      • Drozak J.
      • Veiga-da-Cunha M.
      • Vertommen D.
      • Stroobant V.
      • Van Schaftingen E.
      Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1).
      ]. Carnosine is present in the meat and in particular red meat, is a rich dietary source of carnosine [
      • Ghodsi R.
      • Kheirouri S.
      Carnosine and advanced glycation end products: a systematic review.
      ]. β-alanine is the precursor amino-acid, which is not incorporated into proteins. Numerous studies show that carnosine concentrations can be increased through supplementation of β-alanine alone [
      • Hill C.A.
      • Harris R.C.
      • Kim H.J.
      • Harris B.D.
      • Sale C.
      • Boobis L.H.
      • et al.
      Influence of β-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity.
      ,
      • Derave W.
      • Özdemir M.S.
      • Harris R.C.
      • Pottier A.
      • Reyngoudt H.
      • Koppo K.
      • et al.
      β-Alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters.
      ], increasing its concentrations in skeletal muscle, cardiac and brain tissues [
      • Hipkiss A.R.
      Carnosine and its possible roles in nutrition and health.
      ,
      • Boldyrev A.A.
      • Aldini G.
      • Derave W.
      Physiology and pathophysiology of carnosine.
      ]. The chemical nature of carnosine offers significant physiological activity (Fig. 1). The imidazole ring derived from histidine imparts a high proton buffering capacity, while its nucleophilic structure helps it to form conjugates with reactive aldehydes, such as acrolien [
      • Carini M.
      • Aldini G.
      • Beretta G.
      • Arlandini E.
      • Facino R.M.
      Acrolein-sequestering ability of endogenous dipeptides: characterization of carnosine and homocarnosine/acrolein adducts by electrospray ionization tandem mass spectrometry.
      ,
      • Regazzoni L.
      • de Courten B.
      • Garzon D.
      • Altomare A.
      • Marinello C.
      • Jakubova M.
      • et al.
      A carnosine intervention study in overweight human volunteers: bioavailability and reactive carbonyl species sequestering effect.
      ]. Reactive aldehydes are the downstream effectors of reactive oxygen species, which are formed due to decreased antioxidant ability in the face of oxidative stress [
      • Jaganjac M.
      • Tirosh O.
      • Cohen G.
      • Sasson S.
      • Zarkovic N.
      Reactive aldehydes--second messengers of free radicals in diabetes mellitus.
      ]. They are generated by cytotoxic conversion of macromolecules to form advanced glycation end products (AGEs), such as methylglyoxal and advanced lipoxidation end products (ALEs), such as 4 hydroxynonenal. These cytotoxic products of oxidative stress cause changes to carbohydrate and lipid metabolism, acting as secondary signaling messengers of cytotoxic metabolic events [
      • Baye E.
      • Ukropcova B.
      • Ukropec J.
      • Hipkiss A.
      • Aldini G.
      • de Courten B.
      Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease.
      ,
      • Jaganjac M.
      • Tirosh O.
      • Cohen G.
      • Sasson S.
      • Zarkovic N.
      Reactive aldehydes--second messengers of free radicals in diabetes mellitus.
      ]. The single nitrogen of the histidine also forms complexes with divalent metal ions allowing chelation [
      • Bertinaria M.
      • Rolando B.
      • Giorgis M.
      • Montanaro G.
      • Guglielmo S.
      • Buonsanti M.F.
      • et al.
      Synthesis, physicochemical characterization, and biological activities of new carnosine derivatives stable in human serum as potential neuroprotective agents.
      ], and the synergistic effect of the imidazole ring and the free amino group of β-alanine aid in inhibiting formation of harmful advanced glycation end products [
      • Reddy V.P.
      • Garrett M.R.
      • Perry G.
      • Smith M.A.
      Carnosine: a versatile antioxidant and antiglycating agent.
      ]. While carnosine is yet to be widely evaluated in atherosclerotic disease, these mechanisms have a naturally synergistic potential as an intervention in PVD, targeting a number of key processes underpinning PVD development, progression and symptoms.
      Figure 1
      Figure 1Proposed therapeutic mechanisms for carnosine in PVD. okLDL: oxidized low density lipoprotein, NO: Nitric oxide.

      2. Current therapeutic approaches for PVD

      The current paradigm of medical management for PVD is centred on three key approaches: exercise prescription, pharmacotherapy, and revascularization surgery. While combinations of these treatments are effective in reducing the symptoms associated with PVD, however each of the interventions have limitations to their use.
      Therapeutic exercise prescription is typically recommended as a first line treatment for symptomatic PVD [
      • Hamburg N.M.
      • Balady G.J.
      Exercise rehabilitation in peripheral artery disease: functional impact and mechanisms of benefits.
      ]. As exercise both directly improves peripheral muscular and vascular function, as well as important risk factors such as obesity, hyperglycaemia and hyperlipidaemia, it plays a central role in management of the disease. The likely mechanisms underpinning these effects are increased capillary density in the lower leg, decreased inflammation, and improved vasodilatory response with exercise training [
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American heart association.
      ]. The majority of research on exercise prescription for PVD is centred on supervised treadmill walking programs, with the most recent evidence confirming beneficial effects on aerobic capacity, plantar flexor muscle strength, peak walking distance and initial claudication time [
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American heart association.
      ,
      • Parmenter B.J.
      • Raymond J.
      • Fiatarone Singh M.A.
      The effect of exercise on fitness and performance-based tests of function in intermittent claudication: a systematic review.
      ]. A smaller number of studies have evaluated resistance training as an interventions, which has been shown to have similar outcomes in muscular strength, walking capacity and claudication time [
      • Parmenter B.J.
      • Raymond J.
      • Fiatarone Singh M.A.
      The effect of exercise on fitness and performance-based tests of function in intermittent claudication: a systematic review.
      ]. While exercise has a number of benefits for these patients, there are significant challenges to its effective utilization clinically. The characteristic claudication of PVD causes significant pain on exercise, leading to challenges with compliance, as well as difficulty in acquiring sufficient training volume for benefit. Patients are commonly unable to sustain walking effort for longer durations, particularly if they are older, and more deconditioned due to co-morbidity and inactivity. General compliance to exercise programs is often poor, while supervised exercise is more expensive, and may be inaccessible to many patients. PVD also commonly co-exists with a host of other factors, which may limit exercise participation, such as diabetes, cardiovascular disease, musculoskeletal disease and obesity, and while these are all likewise improved by exercise, they also reduce compliance and the ability to perform exercise effectively.
      Pharmacological treatments for PVD rely on two major approaches, antiplatelet and hemorheological therapies. There are two approved medications for the specific treatment of PVD, the antiplatelet medication Cilostazol, and the hemorheological medication Pentoxifylline. Other drugs such as aspirin are also used in combination with these drugs [
      • Duprez D.A.
      Pharmacological interventions for peripheral artery disease.
      ]. While these approaches are targeted specifically at management of PVD, patients are also likely to be treated with statins, anti-hypertensive and hypoglycaemic agents, to limit progression and secondary events. Antiplatelet therapies are widely used, and are effective in reducing systemic thrombotic events, however their efficacy in reducing acute limb ischemia, amputation or symptoms of claudication are limited, particularly in the case of aspirin [
      • Bonaca M.P.
      • Creager M.A.
      Pharmacological treatment and current management of peripheral artery disease.
      ]. Cilostazol has been shown to cause modest improvements in walking distance [
      • Dawson D.L.
      • Cutler B.S.
      • Hiatt W.R.
      • Hobson II, R.W.
      • Martin J.D.
      • Bortey E.B.
      • et al.
      A comparison of cilostazol and pentoxifylline for treating intermittent claudication.
      ], however it does not improve all cause, or cardiovascular mortality [
      • Hiatt W.R.
      • Money S.R.
      • Brass E.P.
      Long-term safety of cilostazol in patients with peripheral artery disease: the CASTLE study (Cilostazol: a Study in Long-term Effects).
      ]. Additionally, while Cilostazol is broadly considered safe, it is not suitable for use in patients with heart failure, or reduced ejection fraction (which constitute a large proportion of patients), and has a number of side-effects, making durability a potential issue [
      • Bonaca M.P.
      • Creager M.A.
      Pharmacological treatment and current management of peripheral artery disease.
      ]. Pentoxifylline has been shown to improve perfusion in patients with PVD, through reduced blood viscosity and increased cellular deformability [
      • Rao K.M.K.
      • Simel D.L.
      • Cohen H.J.
      • Crawford J.
      • Currie M.S.
      Effects of pentoxifylline administration on blood viscosity and leukocyte cytoskeletal function in patients with intermittent claudication.
      ]. However, the clinical benefits of Pentoxifylline are small, making it rarely prescribed, typically only used in those who cannot tolerate other agents [
      • Bonaca M.P.
      • Creager M.A.
      Pharmacological treatment and current management of peripheral artery disease.
      ]. The small benefits, limitations, and side-effects of these pharmacological agents, makes their widespread use and impact minimal, particularly in significant outcomes such as mortality. In fact, neither Cilostazol nor Pentoxifylline are approved for management of PVD in Australia, paving the way for novel agents to reduce the clinical burden of PVD.
      While these medications are hampered by poor efficacy and adverse events, other commonly used cardiometabolic medications are also used in the management of PVD. The angiotensin converting enzyme (ACE) inhibitors and statins are widely used to manage the hypertension and dyslipidaemia, respectively, commonly seen alongside PVD. ACE inhibitors have been shown to have antiatherogenic, antiproliferative and antimigratory effects on mononuclear cells and protect against rupture of atherosclerotic plaques [
      • Lonn E.M.
      • Yusuf S.
      • Jha P.
      • Montague T.J.
      • Teo K.K.
      • Benedict C.R.
      • et al.
      Emerging role of angiotensin-converting enzyme inhibitors in cardiac and vascular protection.
      ]. ACE inhibitors also have the ability to improve vascularisation by boosting endogenous fibrinolysis [
      • Lonn E.M.
      • Yusuf S.
      • Jha P.
      • Montague T.J.
      • Teo K.K.
      • Benedict C.R.
      • et al.
      Emerging role of angiotensin-converting enzyme inhibitors in cardiac and vascular protection.
      ]. ACE inhibitors primarily exert their activity by inhibiting production of Angiotensin II in the kidney, which results in vasodilation of renal and peripheral arteries. The statins improve plasma hypercholesterolemia via inhibiting the action of HMG-CoA reductase, a crucial enzyme in the production of plasma LDL [
      • Laws P.E.
      • Spark J.I.
      • Cowled P.A.
      • Fitridge R.A.
      The role of statins in vascular disease.
      ]. Statins have been shown to improve endothelial dysfunction and also provide anti-inflammatory, anti-proliferative and anti-thrombogenic effects [
      • Laws P.E.
      • Spark J.I.
      • Cowled P.A.
      • Fitridge R.A.
      The role of statins in vascular disease.
      ], which are all relevant in the context of PVD. Similar to carnosine, statins can also stabilise atherosclerotic plaque formation and inactivate NFkB, a potent activator of inflammatory cascades [
      • Laws P.E.
      • Spark J.I.
      • Cowled P.A.
      • Fitridge R.A.
      The role of statins in vascular disease.
      ], providing further potential for utility in PVD. However, unlike carnosine, ACE inhibitors and statins have side effects, which can prevent their use.
      The last line of therapeutic intervention is revascularization, which can be performed via endovascular or open surgical approach, depending upon location and extent of atherosclerosis. Revascularization is generally effective in restoring effective perfusion to an area, and reducing symptoms of the disease [
      • Devine E.B.
      • Alfonso-Cristancho R.
      • Yanez N.D.
      • Edwards T.C.
      • Patrick D.L.
      • Armstrong C.A.L.
      • et al.
      Effectiveness of a medical vs revascularization intervention for intermittent leg claudication based on patient-reported outcomes.
      ]. It is shown to be more effective than exercise interventions, however the best outcomes are achieved through a combined approach of surgical and supervised walking [
      • Treat-Jacobson D.
      • McDermott M.M.
      • Bronas U.G.
      • Campia U.
      • Collins T.C.
      • Criqui M.H.
      • et al.
      Optimal exercise programs for patients with peripheral artery disease: a scientific statement from the American heart association.
      ]. Endovascular revascularization has been associated with improved long-term amputation free survival compared with open surgery [
      • Wiseman J.T.
      • Fernandes-Taylor S.
      • Saha S.
      • Havlena J.
      • Rathouz P.J.
      • Smith M.A.
      • et al.
      Endovascular versus open revascularization for peripheral arterial disease.
      ]. A recent Cochrane review suggests combination therapy, such as endovascular revascularization with either supervised exercise or drug therapy offers greater clinical improvement than either supervised exercise therapy or drug therapy alone [
      • Fakhry F.
      • Fokkenrood H.J.
      • Spronk S.
      • Teijink J.A.
      • Rouwet E.V.
      • Hunink M.G.M.
      Endovascular revascularisation versus conservative management for intermittent claudication.
      ]. However, it is typically reserved for patients with chronic critical limb ischemia (CCLI), characterized by extreme circulation limitation and pain at rest, or with severe claudication, due to risks associated with the procedures due to risk of adverse limb, or cardiovascular events [
      • Vartanian S.M.
      • Conte M.S.
      Surgical intervention for peripheral arterial disease.
      ]. Additionally, it is commonly not recommended for those with key risk factors for critical events, such as in smokers, type 2 diabetics, limiting its applicability [
      • Biscetti F.
      • Nardella E.
      • Rando M.M.
      • Cecchini A.L.
      • Gasbarrini A.
      • Massetti M.
      • et al.
      Outcomes of lower extremity endovascular revascularization: potential predictors and prevention strategies.
      ]. Importantly, given that even asymptomatic and undiagnosed PVD is associated with negative outcomes, revascularization is unable to be applied to many patients.
      While therapeutic interventions are available for PVD, they are limited by compliance, limited efficacy, or inability for broad application to patients. To combat this, novel, safe and cost-effective approaches are urgently needed to reduce the burden of PVD more broadly.

      3. Prevent the plaque: carnosine as an inhibitor of atherogenesis

      Advanced glycation end products (AGEs) are well known to be associated with the development of atherosclerosis [
      • Alvarez E.
      • Paradela-Dobarro B.
      • González-Peteiro M.
      • González-Juanatey J.R.
      Impact of advanced glycation end products on endothelial function and their potential link to atherosclerosis.
      ] and type 2 diabetes [
      • Baye E.
      • Kiriakova V.
      • Uribarri J.
      • Moran L.J.
      • de Courten B.
      Consumption of diets with low advanced glycation end products improves cardiometabolic parameters: meta-analysis of randomised controlled trials.
      ]. AGEs cause significant oxidative stress, leading to inflammatory endothelial dysfunction, a critical precursor to formation of atherosclerotic plaques [
      • Kattoor A.J.
      • Pothineni N.V.K.
      • Palagiri D.
      • Mehta J.L.
      Oxidative stress in atherosclerosis.
      ]. Importantly, AGE formation is associated with adverse outcomes in patients with PVD, being strongly associated with disease progression and cardiovascular outcomes [
      • de Vos L.C.
      • Lefrandt J.D.
      • Dullaart R.P.F.
      • Zeebregts C.J.
      • Smit A.J.
      Advanced glycation end products: an emerging biomarker for adverse outcome in patients with peripheral artery disease.
      ].
      Carnosine strongly inhibits AGE formation [
      • Reddy V.P.
      • Garrett M.R.
      • Perry G.
      • Smith M.A.
      Carnosine: a versatile antioxidant and antiglycating agent.
      ], potentially reducing oxidative stress on the endothelium, and leading to decreased vascular injury and atherogenesis. Carnosine reduces the formation of pathogenic foam cells – cholesterol laden macrophages which contribute to the formation of plaques [
      • Rashid I.
      • van Reyk D.M.
      • Davies M.J.
      Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro.
      ]. It was proposed that this also occurred through a decrease in the glycation of LDL by reactive aldehydes, a key mediator of AGE formation [
      • Rashid I.
      • van Reyk D.M.
      • Davies M.J.
      Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro.
      ]. This effect on aldehyde reduction has also been indirectly inferred in humans [
      • Bispo V.S.
      • de Arruda Campos I.P.
      • Di Mascio P.
      • Medeiros M.H.
      Structural elucidation of a carnosine-acrolein adduct and its quantification in human urine samples.
      ]. Several animal models have identified that carnosine is able to protect against the development of atherosclerotic plaques in hypercholesteremic and diabetic mice, through its antiglycating actions [
      • Menini S.
      • Iacobini C.
      • Ricci C.
      • Fantauzzi C.B.
      • Pugliese G.
      Protection from diabetes-induced atherosclerosis and renal disease by D-carnosine-octylester: effects of early vs late inhibition of advanced glycation end-products in Apoe-null mice.
      ,
      • Menini S.
      • Iacobini C.
      • Ricci C.
      • Scipioni A.
      • Blasetti Fantauzzi C.
      • Giaccari A.
      • et al.
      D-Carnosine octylester attenuates atherosclerosis and renal disease in ApoE null mice fed a Western diet through reduction of carbonyl stress and inflammation.
      ,
      • Brown B.E.
      • Kim C.H.
      • Torpy F.R.
      • Bursill C.A.
      • McRobb L.S.
      • Heather A.K.
      • et al.
      Supplementation with carnosine decreases plasma triglycerides and modulates atherosclerotic plaque composition in diabetic apo E(-/-) mice.
      ,
      • Barski O.A.
      • Xie Z.
      • Baba S.P.
      • Sithu S.D.
      • Agarwal A.
      • Cai J.
      • et al.
      Dietary carnosine prevents early atherosclerotic lesion formation in apolipoprotein E-null mice.
      ]. In addition to the direct impact on plaque formation, carnosine has also been shown to protect against the harmful effects of obesity, ameliorating the associated dyslipidaemia in animal models [
      • Aldini G.
      • Orioli M.
      • Rossoni G.
      • Savi F.
      • Braidotti P.
      • Vistoli G.
      • et al.
      The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats.
      ].
      In addition to these direct pathogenic actions, carnosine influences a significant number of risk factors strongly associated with PVD. Animal studies show that carnosine reduces obesity, improves glucose metabolism, blood pressure, markers of chronic low-grade inflammation and oxidative stress, AGE/ALEs, lipid levels and peroxidation in a dose-dependent fashion - each of which could individually significantly affect the progression of PVD [
      • Baye E.
      • Ukropcova B.
      • Ukropec J.
      • Hipkiss A.
      • Aldini G.
      • de Courten B.
      Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease.
      ,
      • Brown B.E.
      • Kim C.H.
      • Torpy F.R.
      • Bursill C.A.
      • McRobb L.S.
      • Heather A.K.
      • et al.
      Supplementation with carnosine decreases plasma triglycerides and modulates atherosclerotic plaque composition in diabetic apo E(-/-) mice.
      ,
      • Aldini G.
      • Orioli M.
      • Rossoni G.
      • Savi F.
      • Braidotti P.
      • Vistoli G.
      • et al.
      The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats.
      ,
      • Sauerhofer S.
      • Yuan G.
      • Braun G.S.
      • Deinzer M.
      • Neumaier M.
      • Gretz N.
      • et al.
      L-carnosine, a substrate of carnosinase-1, influences glucose metabolism.
      ,
      • Lee Y.T.
      • Hsu C.C.
      • Lin M.H.
      • Liu K.S.
      • Yin M.C.
      Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation.
      ,
      • Nagai K.
      • Tanida M.
      • Niijima A.
      • Tsuruoka N.
      • Kiso Y.
      • Horii Y.
      • et al.
      Role of L-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by autonomic nerves in rats: involvement of the circadian clock and histamine.
      ,
      • Menon K.A.
      • Mousa A.Y.A.
      • Courten B.D.
      Effect of carnosine supplementation on cardiometabolic risk factors in obesity, prediabetes, and diabetes—a meta-analysis of randomized controlled trials.
      ].
      Taken together, these findings paint a compelling picture of carnosine as a means of preventing the onset of PVD as well as limiting its progression. However, carnosine may also have a role to play in protecting patients against the ischemic injury to peripheral tissues caused by the ongoing obstruction and hypoxia caused by PVD.

      4. Limiting the ischemia: carnosine as an anti-ischemic agent

      Critical ischemia is the underlying cause of tissue loss in PVD patients [
      • Serrano Hernando F.J.
      • Martin Conejero A.
      [Peripheral artery disease: pathophysiology, diagnosis and treatment].
      ], and is the central mechanism underpinning it's characteristic symptoms of exercise intolerance. The ischemia of PVD is caused by progressive occlusion in peripheral vessels, and a failure of the circulation to generate collateral supply. Current studies show that post-natal vessel growth is regulated by the transcription factor, HIF-1α [
      • Vincent K.A.
      • Shyu K.G.
      • Luo Y.
      • Magner M.
      • Tio R.A.
      • Jiang C.
      • et al.
      Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor.
      ,
      • Ziello J.E.
      • Jovin I.S.
      • Huang Y.
      Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia.
      ]. Activation of HIF-1α leads to the expression of angiogenic genes such as VEGF [
      • Forsythe J.A.
      • Jiang B.H.
      • Iyer N.V.
      • Agani F.
      • Leung S.W.
      • Koos R.D.
      • et al.
      Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.
      ], essential in promoting the mobilization of pro-angiogenic endothelial progenitor cells (EPCs) [
      • Asahara T.
      • Takahashi T.
      • Masuda H.
      • Kalka C.
      • Chen D.
      • Iwaguro H.
      • et al.
      VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.
      ,
      • Aicher A.
      • Zeiher A.M.
      • Dimmeler S.
      Mobilizing endothelial progenitor cells.
      ,
      • Gerhardt H.
      • Golding M.
      • Fruttiger M.
      • Ruhrberg C.
      • Lundkvist A.
      • Abramsson A.
      • et al.
      VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.
      ] as well as promoting an inflammatory response, which is essential for collateral growth [
      • Silvestre J.S.
      • Mallat Z.
      • Tedgui A.
      • Levy B.I.
      Post-ischaemic neovascularization and inflammation.
      ,
      • Waeckel L.
      • Mallat Z.
      • Potteaux S.
      • Combadiere C.
      • Clergue M.
      • Duriez M.
      • et al.
      Impairment in postischemic neovascularization in mice lacking the CXC chemokine receptor 3.
      ]. Under normal conditions, HIF-1α is targeted for proteasomal degradation through the activity of prolyl hydroxylases (PHDs), which require iron for their activity. Recent studies show that metal chelators and pharmaceutical inhibitors of PHDs prevent HIF-1α proteasomal degradation and promote revascularization [
      • Wang G.L.
      • Semenza G.L.
      Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction.
      ]. However, the use of these chelators or inhibitors can cause toxicity. Similarly, clinical trials using HIF-1α and VEGF gene therapy in patients with chronic limb ischemia were largely negative or inconclusive because the effect of HIF-1α was localized at the site of injection [
      • Creager M.A.
      • Olin J.W.
      • Belch J.J.
      • Moneta G.L.
      • Henry T.D.
      • Rajagopalan S.
      • et al.
      Effect of hypoxia-inducible factor-1alpha gene therapy on walking performance in patients with intermittent claudication.
      ,
      • Annex B.H.
      Therapeutic angiogenesis for critical limb ischaemia.
      ]. Carnosine is an efficient chelator of metals [
      • Baran E.J.
      Metal complexes of carnosine.
      ], through which it imparts some of it's antioxidant effect. However, it may also be a non-toxic means of limiting HIF-1α degradation through iron sequestration, increasing its angiogenesis. This effect has been shown in mouse models of hind limb ischemia, in which carnosine administration led to increased expression of HIF-1α and VEGF, and enhanced perfusion [
      • Boakye A.A.
      • Zhang D.
      • Guo L.
      • Zheng Y.
      • Hoetker D.
      • Zhao J.
      • et al.
      Carnosine supplementation enhances post ischemic hind limb revascularization.
      ].
      While it is unclear whether atherosclerotic plaques can be reduced after onset, their impact can be mitigated through maintenance of circulation. Ongoing endothelial dysfunction commonly leads to altered function in endothelial nitric oxide synthase [
      • Varadharaj S.
      • Kelly O.J.
      • Khayat R.N.
      • Kumar P.S.
      • Ahmed N.
      • Zweier J.L.
      Role of dietary antioxidants in the preservation of vascular function and the modulation of health and disease.
      ]. This dysfunction leads to excess production of superoxide free radicals, and a subsequent decrease in the powerful vasodilator nitric oxide (NO) [
      • Varadharaj S.
      • Kelly O.J.
      • Khayat R.N.
      • Kumar P.S.
      • Ahmed N.
      • Zweier J.L.
      Role of dietary antioxidants in the preservation of vascular function and the modulation of health and disease.
      ]. This decrease in NO production leads to increased vasoconstriction, leading to progression or exacerbation of a number of cardiovascular conditions [
      • Daiber A.
      • Xia N.
      • Steven S.
      • Oelze M.
      • Hanf A.
      • Kröller-Schön S.
      • et al.
      New therapeutic implications of endothelial nitric oxide synthase (eNOS) function/dysfunction in cardiovascular disease.
      ]. Interestingly, this process is reversed through application of antioxidants, increasing the bioavailability of NO [
      • Lubos E.
      • Handy D.E.
      • Loscalzo J.
      Role of oxidative stress and nitric oxide in atherothrombosis.
      ]. Carnosine has been shown to improve vasodilation in animal models [
      • Ririe D.G.
      • Roberts P.R.
      • Shouse M.N.
      • Zaloga G.P.
      Vasodilatory actions of the dietary peptide carnosine.
      ], however the mechanism for this effect has yet to be identified. This could lead to increased tissue perfusion, and limit hypoxia for those living with PVD. In addition to improving perfusion, carnosine may be able to limit the injury associated with hypoxia.
      Carnosine has also been shown to protect against ischemia in a number of animal models of organ damage induced by ischemia-reperfusion [
      • Stvolinsky S.L.
      • Dobrota D.
      Anti-ischemic activity of carnosine.
      ,
      • Bokeriya L.A.B.A.
      • Movsesyan R.R.
      • Alikhanov S.A.
      • Arzumanyan E.S.
      • Nisnevich E.D.
      • Artyukhina T.V.
      • et al.
      Cardioprotective effect of histidine-containing dipeptides in pharmacological cold cardioplegia.
      ,
      • Davis C.K.
      • Laud P.J.
      • Bahor Z.
      • Rajanikant G.K.
      • Majid A.
      Systematic review and stratified meta-analysis of the efficacy of carnosine in animal models of ischemic stroke.
      ]. The mechanisms underlying these effects are not fully understood but are potentially due in part to its proton buffering effect. As cells breakdown due to hypoxic injury, protons are released from both breakdown of tissue, mitochondrial dysfunction and the action of innate immune cells [
      • Kalogeris T.
      • Baines C.P.
      • Krenz M.
      • Korthuis R.J.
      Cell biology of ischemia/reperfusion injury.
      ]. The decreased pH and resulting oxidative damage lead to ongoing tissue stress and injury. Carnosine has significant pH buffering activity [
      • Culbertson J.Y.
      • Kreider R.B.
      • Greenwood M.
      • Cooke M.
      Effects of beta-alanine on muscle carnosine and exercise performance: a review of the current literature.
      ], offering a protective benefit against this, as well as inhibiting the respiratory burst of innate immune cells [
      • Boldyrev A.
      • Abe H.
      • Stvolinsky S.
      • Tyulina O.
      Effects of carnosine and related compounds on generation of free oxygen species: a comparative study.
      ]. This is likely to protect against some level of the hypoxia induced injury caused by PVD.
      While increasing perfusion is likely to aid in ameliorating the symptoms associated with exercise induced hypoxia and claudication, carnosine has a number of physiological impacts at the skeletal muscle level, which are likely to improve exercise tolerance.

      5. Improving exercise tolerance

      The major symptom of PVD is a lack of exercise tolerance, with onset of claudication during physical exertion. As obstructive disease progresses the capacity for exertion decreases, and lack of physical activity subsequently worsens cardiovascular health. Perhaps carnosine's most well-known benefit is an increase in muscular performance, leading to its use as an athletic supplement [
      • Culbertson J.Y.
      • Kreider R.B.
      • Greenwood M.
      • Cooke M.
      Effects of beta-alanine on muscle carnosine and exercise performance: a review of the current literature.
      ]. However, its powerful action in skeletal muscle physiology could also aid in ameliorating the symptoms of patients living with PVD, improving quality of life, and increasing exercise capacity. Carnosine reduces lactic acid formation due to its buffering capacity, which may on its own promote walking endurance in patients with PVD by preventing muscle acidosis [
      • Saunders B.
      • Elliott-Sale K.
      • Artioli G.G.
      • Swinton P.A.
      • Dolan E.
      • Roschel H.
      • et al.
      beta-alanine supplementation to improve exercise capacity and performance: a systematic review and meta-analysis.
      ], however this is yet to be tested in those with PVD induced claudication. Administration of carnosine to individuals with heart failure was able to improve 6-min walk test, health related quality of life and key VO2 measures, with non-significant (p = 0.07) improvement in systolic cardiac function, but muscular effects were not evaluated [
      • Lombardi C.
      • Carubelli V.
      • Lazzarini V.
      • Vizzardi E.
      • Bordonali T.
      • Ciccarese C.
      • et al.
      Effects of oral administration of orodispersible levo-carnosine on quality of life and exercise performance in patients with chronic heart failure.
      ]. There has also been some suggestion that carnosine can improve excitation coupling, and therefore muscular performance and cardiac function [
      • Swietach P.
      • Youm J.-B.
      • Saegusa N.
      • Leem C.-H.
      • Spitzer K.W.
      • Vaughan-Jones R.D.
      Coupled Ca2+/H+transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling.
      ], however this has not been directly observed [
      • Derave W.
      • Everaert I.
      • Beeckman S.
      • Baguet A.
      Muscle carnosine metabolism and beta-alanine supplementation in relation to exercise and training.
      ].
      Carnosine has been widely used to increase athletic performance, and while it may seem logical that it would improve outcomes for those with PVD, there is little data available in humans. High-quality randomised controlled trials studying the impact of carnosine supplementation on exercise endurance and performance are required to guide its recommendation.

      6. Why carnosine?

      While carnosine has been more extensively studied in chronic diseases, there may be some utility in the study of cheaper β-alanine rather than carnosine for managing the disease. As the key rate-limiting component of in vivo carnosine synthesis, supplementation with β-alanine effectively increases concentrations of carnosine in muscle [
      • Hill C.A.
      • Harris R.C.
      • Kim H.J.
      • Harris B.D.
      • Sale C.
      • Boobis L.H.
      • et al.
      Influence of β-alanine supplementation on skeletal muscle carnosine concentrations and high intensity cycling capacity.
      ]. However, to our knowledge, there are no studies which investigated β-alanine, in any setting of cardiometabolic disease, or compared β-alanine and carnosine. Indeed, the other amino acid component of carnosine, histidine, has also been shown to have physiological effects, improving metabolic syndrome, giving a potential dual mechanism for carnosine supplementation [
      • Moro J.
      • Tomé D.
      • Schmidely P.
      • Demersay T.-C.
      • Azzout-Marniche D.J.N.
      Histidine: a systematic review on metabolism and physiological effects in human and different animal species.
      ]. There are also a number of other histidine containing dipeptides which have physiological functions similar to carnosine, the most important of these being anserine and acetylcarnosine. These two molecules are known to have a range of shared functions with carnosine, including its metal chelating and anti-oxidative properties, however, their therapeutic effect has not been evaluated in cardiometabolic diseases. While there may be no evidence on these compounds in cardiovascular disease, they may offer advantages over carnosine in some settings. Anserine and acetylcarnosine are more stable in the circulation, and resistant to carnosinase mediated degradation [
      • Pegova A.
      • Abe H.
      • Boldyrev A.
      Hydrolysis of carnosine and related compounds by mammalian carnosinases.
      ], potentially allowing for more long-lasting effects. While, these are worthy candidates for future investigation, carnosine remains the front-running therapeutic candidate for clinical use at present.

      7. Carnosine prescription for PVD: what is its role?

      As previously described, carnosine is particularly suited to managing the symptoms of PVD as part of a multimodal treatment. Its broad physiological action, targets a number of key mechanisms which may provide benefit to those with PVD. Importantly, carnosine is well tolerated, with few reports of serious side-effects, beyond occasional transient paraesthesia with high doses [
      • Décombaz J.
      • Beaumont M.
      • Vuichoud J.
      • Bouisset F.
      • Stellingwerff T.
      Effect of slow-release β-alanine tablets on absorption kinetics and paresthesia.
      ]. This, coupled with its widespread benefits on key cardiometabolic risk factors, makes it a strong candidate for use regardless of direct impact on PVD [
      • Baye E.
      • Ukropcova B.
      • Ukropec J.
      • Hipkiss A.
      • Aldini G.
      • de Courten B.
      Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease.
      ,
      • Menon K.A.
      • Mousa A.Y.A.
      • Courten B.D.
      Effect of carnosine supplementation on cardiometabolic risk factors in obesity, prediabetes, and diabetes—a meta-analysis of randomized controlled trials.
      ]. However, it is also likely to have a beneficial effect on lower limb tissue perfusion, prevent formation and progression of atherosclerosis, and increase exercise capacity in those living with PVD. Given the strong associations with even asymptomatic PVD, decreased quality of life and mortality, novel treatments for the disease are vital. It is likely that carnosine would play an adjunctive role alongside standard care such as supervised exercise training. Carnosine also has a potential synergistic effect when combined with endovascular revascularization. Importantly, while PVD is on its own, a significant health challenge, the multimorbid nature of atherosclerotic disease cannot be forgotten. Patients with PVD are also likely to have coronary heart disease, hypertension, metabolic syndrome, and kidney disease, however given the shared pathology, it is likely that carnosine will have a beneficial impact across the cardiovascular risk factor and disease spectrum. Indeed, it is known to have benefits in type 2 diabetes [
      • Baye E.
      • Ukropcova B.
      • Ukropec J.
      • Hipkiss A.
      • Aldini G.
      • de Courten B.
      Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease.
      ,
      • Brown B.E.
      • Kim C.H.
      • Torpy F.R.
      • Bursill C.A.
      • McRobb L.S.
      • Heather A.K.
      • et al.
      Supplementation with carnosine decreases plasma triglycerides and modulates atherosclerotic plaque composition in diabetic apo E(-/-) mice.
      ,
      • Sauerhofer S.
      • Yuan G.
      • Braun G.S.
      • Deinzer M.
      • Neumaier M.
      • Gretz N.
      • et al.
      L-carnosine, a substrate of carnosinase-1, influences glucose metabolism.
      ], renal disease [
      • Elbarbary N.S.
      • Ismail E.A.R.
      • El-Naggar A.R.
      • Hamouda M.H.
      • El-Hamamsy M.
      The effect of 12 weeks carnosine supplementation on renal functional integrity and oxidative stress in pediatric patients with diabetic nephropathy: a randomized placebo-controlled trial.
      ], and heart failure [
      • Lombardi C.
      • Carubelli V.
      • Lazzarini V.
      • Vizzardi E.
      • Bordonali T.
      • Ciccarese C.
      • et al.
      Effects of oral administration of orodispersible levo-carnosine on quality of life and exercise performance in patients with chronic heart failure.
      ], strengthening the case for its use in this population.
      While the preliminary evidence on the use of carnosine in PVD is supportive, however there is a critical lack of human studies in the field. High quality trials are needed evaluating supplementation of carnosine alongside standard care, such as exercise or pharmacological intervention. Additionally, dose finding studies, and long-term evaluation of its outcomes are required to guide recommendations for its use. Finally, thorough investigation into the mechanisms underpinning its effects is required to further inform its use, and identify any relevant contraindications or interactions.

      8. Conclusion

      As the burden of atherosclerotic disease such as PVD continues its steady increase, identification of novel therapeutic strategies for its prevention and management are of critical importance. Particularly, in the instance of PVD, where current interventions are of limited efficacy. Carnosine is a strong candidate for therapeutic evaluation in this setting, with a wide range of physiological effects on factors central to the pathology of PVD. Its metal chelating, antioxidant, anti-inflammatory, antiglycating and pH buffering effects prevent the development and progression of plaques, as well as potentially increase tissue perfusion, and improve exercise capacity. Future human studies will help evaluate the clinical outcomes of supplementation, and thus guide translation into this underserved cohort.

      Funding

      No funding was acquired for the preparation of this manuscript. BdC is supported by a Royal Australasian College of Physicians Fellows Career Development Fellowship .

      Author contributions

      Conceptualization: B.D.C., R.H.; Writing - original draft: J.F.; Visualization: J.F.; Writing - review & editing: J.F., R.H., T.B., C.H., A.B., S.P.B., B.D.C. All authors have read and agreed to the published version of the manuscript.

      Declaration of competing interest

      The authors declare no conflicts of interest.

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