Hrs Girn
email: hrsgirn@aol.com

N M Orsi

S Homer-Vanniasinkam

Hardev Ramandeep

Singh Girn

An overview of cytokine interactions in atherosclerosis and implications for peripheral arterial disease

Keywords: atherosclerosis, biomarkers, cytokines, interleukins, peripheral arterial disease

Over the last three decades, a surge in research into the inflammatory pathophysiology of atherosclerosis has highlighted an array of cytokines and other inflammatory mediators associated with underlying inflammatory burden. The ability to identify and simultaneously measure multiple cytokines in peripheral blood highlights their potential as biomarkers of atherosclerosis. This has prompted much research in vascular medicine to identify the 'at-risk' groups for atherostenotic or atheroaneurysmal disease. This review is compiled with similar intentions and aims to discern the relevant evidence for cytokine profiling in peripheral arterial disease (PAD), where such information is lacking, while providing a holistic overview of cytokine interactions in atherosclerosis. This is pertinent given that cytokine profiles from coronary artery disease and aortic aneurysm studies cannot be directly extrapolated to PAD due to differences in inflammatory environments that exist in these conditions. Whilst plaque morphology and blood rheology play an important role in the cardiac manifestations of atherosclerosis, tissue thrombogenecity is very important in PAD. Further, cytokines act in concert rather than in isolation in a disease process, and no single cytokine in a cross-sectional model is able to serve as an absolute screening marker. Thus, it is essential to understand the regulation of cytokine production in atherosclerosis prior to evaluating the viability and merits of a multimarker approach for clinical risk stratification in PAD.

Introduction

Atherosclerosis remains the leading cause of morbidity and mortality in the developed world. Chronic inflammation is the hallmark of atherosclerosis. It affects every step of the atherosclerotic process, from emergence of the fatty streak [START_REF] Stary | A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association[END_REF] -the first identifiable lesion of atherosclerosis -to endothelial activation and subsequent vascular dysfunction and vessel occlusion. More specifically, its clinical manifestations in the distal aorta and arteries in the legs are referred to as peripheral arterial disease (PAD) and affects about 4.5% of the population above 40 years of age, increasing to 14.5% above 70 years of age. [START_REF] Selvin | Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999-2000[END_REF] The PARTNERS study, which was a multi-centre, cross-sectional study conducted in 25 cities and 350 primary care practices throughout the United States, reported a PAD prevalence rate of 29%. [START_REF] Hirsch | Peripheral arterial disease detection, awareness, and treatment in primary care[END_REF] Although PAD is not directly life threatening, affected patients have a higher risk of morbidity and mortality due to generalized atherosclerosis. It remains largely under-diagnosed, [START_REF] Hirsch | Peripheral arterial disease detection, awareness, and treatment in primary care[END_REF] with an estimated six asymptomatic patients for every known symptomatic patient. [START_REF] Dormandy | Management of peripheral arterial disease (PAD). TASC working group. TransAtlantic Intersociety consensus (TASC)[END_REF] The inflammatory phenomenon related to atherosclerosis has been extensively studied over the past two decades and a variety of inflammatory and immunological mediators have been identified which positively correlate with the underlying disease load. They regulate every stage of the inflammatory cascade, from endothelial activation to the adhesion of inflammatory cells and platelets, and subsequent remodelling of the internal vascular environment.

Cytokines are key regulatory glycoproteins allied to inflammatory/immunological processes which modulate all aspects of vascular inflammation by altering the proliferation, differentiation and function of an extensive array of cell types. They are intimately associated with atherogenesis and modulate plaque morphology and stabilization. Atheromatous plaque consists of a lipid core covered by a fibrotic cap on the luminal surface. Integrity of the fibrous cap depends on the balance between the smooth muscle cell (SMC) proliferation and migration, and matrix metalloproteinase (MMP)-induced degradation of the extracellular matrix (ECM). The local balance between the pro-and anti-inflammatory cytokines is instrumental in determining structure of the fibrous cap and SMC degradation, thereby influencing plaque stability. Cytokines were initially discovered about 50 years ago through their mitogenic and chemotactic effects on lymphocyte and monocyte populations. Although initial research focused more on pro-inflammatory cytokines [T-helper cell (Th) 1 ] in relation to disease progression, it is the imbalance between pro-and antiinflammatory (Th 2 cytokines) that is emerging to be pivotal to this process. Th 2 cytokines, which were initially considered to be protective, have now been found to be strongly associated with human abdominal aortic aneurysm (AAA) development. By comparison, stenotic atherosclerotic lesions are associated with Th 1 responses. [START_REF] Schonbeck | T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm[END_REF] Cytokines act by binding to specific receptors, an interaction which has clarified their involvement in atherosclerosis and highlighted potential new avenues of therapeutic intervention. There are over 200 cytokines cloned to date and various classification systems have been used for the purpose of their categorization, although no single system has proven sufficiently comprehensive to be considered as a standard format. The difficulty in categorizing cytokines has largely related to the complexity and overlap of their biological roles. Structurally, cytokines can be classified into nine different families (Table 1): the interleukin (IL)-1, IL-2/IL-4, IL-6/IL-12, interferon (IFN), tumour necrosis factor (TNF), chemokines, IL-10, transforming growth factor ␤ (TGF-␤) and IL-17 families. This system will be used for discussing representative cytokines from each family in the context of atherosclerosis and PAD for the purpose of this review. In this respect, IL-17 is potentially pro-atherogenic because of its role in augmenting the expression of pro-inflammatory cytokines such as IL-6 from macrophages. Although it has recently been found to be associated with unstable angina, [START_REF] Hashmi | Role of interleukin-17 and interleukin-17induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease[END_REF] there is no direct evidence implicating it with atherosclerosis and its family will therefore not be discussed as a part of this review.

The interleukin (IL)-1 family

IL -1 and IL -1 receptor antagonist

The IL-1 cytokine family comprises four main members: IL-1␣, IL-1␤, IL-1 receptor antagonist (IL-1ra) and IL-18. Although IL-1 can upregulate host defences and act as an immunoadjuvant, the family is primarily considered to be pro-inflammatory. [START_REF] Dinarello | Interleukin-1[END_REF] IL-1 was one of the first cytokines to be implicated in vessel wall inflammation in atherosclerosis, and in facilitating early lesion formation. Its effect on leukocyte recruitment and transmigration allows it to maintain a proinflammatory milieu once the inflammation has been initiated. Increased IL-1 levels have also been associated with an increased tendency for plaque rupture. [START_REF] Libby | Cytokines regulate vascular functions related to stability of the atherosclerotic plaque[END_REF] The only member of this family with paradoxical properties is IL-1ra, a naturally occurring cytokine antagonist, which plays an anti-inflammatory role in regulating IL-1 function. In this respect, IL-1ra injections in rheumatoid arthritis patients can reduce inflammatory joint destruction. [START_REF] Bresnihan | Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist[END_REF] However, no similar therapeutic approaches have been determined for this cytokine in cardiovascular medicine. IL-1ra has been shown to be up-regulated in atherosclerosis and to correlate with the clinical stage of the disease in a small cohort of PAD patients in a number of studies. [START_REF] Fiotti | Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease[END_REF][START_REF] Mcdermott | Patterns of inflammation associated with peripheral arterial disease: the InCHIANTI study[END_REF]

IL-1␤ ␤

IL-1␤ plays a significant role in inflammation, as demonstrated by studies on IL-1␤-deficient mice: these animals failed to exhibit an acute phase response, remained apyrexic and exhibited no rise in circulating IL-6 levels when receiving an inflammatory stimulus. [START_REF] Zheng | Resistance to fever induction and impaired acute-phase response in interleukin-1 betadeficient mice[END_REF] IL-1␤ has been implicated in enhancing expression of cell adhesion molecules on the endothelial surface and has consequently been deemed to be pro-atherogenic. [START_REF] Dinarello | IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family[END_REF] Taken together, these findings highlight the potential role of IL-1␤ in PAD through its eliciting of IL-6 production, an independent predictor of PAD progression in the Edinburgh artery study. [START_REF] Tzoulaki | C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study[END_REF] However, while IL-1␤ levels have been found to be increased in the pericardial fluid of patients with unstable angina, [START_REF] Oyama | Elevated interleukin-1beta in pericardial fluid of patients with ischemic heart disease[END_REF] plasma levels of IL-1␤ have not been found to be indicative of PAD. [START_REF] Fiotti | Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease[END_REF][START_REF] Mcdermott | Patterns of inflammation associated with peripheral arterial disease: the InCHIANTI study[END_REF] IL-18 IL-18 is a more recently identified member of the IL-1 family, initially identified for its role in inducing IFN-␥ production. IL-18 is an immunomodulator and does not fall neatly into the typical Th 1 :Th 2 dichotomy, inasmuch as it can induce either of these responses depending on the prevailing immunological environment. Like many cytokines, it acts synergistically with other similar proteins and mediators in order to potentiate its effects. Experimental evidence from studies using apo E -/-mice confirms IL-18 to be atherogenic. While exogenous administration of IL-18 in these mice enhances atherosclerotic lesions, [START_REF] Whitman | Interleukin-18 enhances atherosclerosis in apolipoprotein E(-/-) mice through release of interferon-gamma[END_REF] there is conversely a reduction in atherosclerosis by inhibiting IL-18. [START_REF] Mallat | Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability[END_REF][START_REF] Elhage | Reduced atherosclerosis in interleukin-18 deficient apolipoprotein E-knockout mice[END_REF] IL-18 has a bearing on the progression of human atherosclerotic plaques, particularly in relation to their stability. [START_REF] Mallat | Expression of interleukin-18 in human atherosclerotic plaques and relation to plaque instability[END_REF] Furthermore, IL-18 genetic polymorphisms have been associated with clinical outcome in coronary artery disease. [START_REF] Tiret | Genetic analysis of the interleukin-18 system highlights the role of the interleukin-18 gene in cardiovascular disease[END_REF] Although IL-18 is considered to be a predictor of future cardiovascular risk, [START_REF] Gilardini | Adiponectin is a candidate marker of metabolic syndrome in obese children and adolescents[END_REF] there are currently no data to indicate that IL-18 levels are elevated in association with PAD.

The IL-2 family

IL-2

The IL-2 family comprises of four representative members: IL-2, IL-4, IL-5 and granulocyte-monocyte colony-stimulating factor (GM-CSF). The most noteworthy action of IL-2 relates to its mitogenic effects on T lymphocytes in response to antigenic stimulation, including the generation of both cytotoxic and suppressor T cells. However, despite extensive attempts to fully understand the actions and signalling pathways of IL-2, the nature of its pleiotropic effects on an array of cell types remains elusive. Although IL-2 is detected in atheromatous lesions, its precise role in vascular plaque formation remains poorly understood. Indeed, although IL-2 levels are elevated in the blood and in the aortic tissue of patients with aortic aneurysm disease, the rate of aneurysm expansion appears to depend on the overall Th 1 :Th 2 cytokine imbalance [START_REF] Lindholt | Chronic inflammation, immune response, and infection in abdominal aortic aneurysms[END_REF] in favour of Th 2 cytokines. Although studies to date have failed to address the role of IL-2 in PAD, this cytokine is nevertheless integral to vascular inflammatory processes and promotes the maintenance and function of naturally occurring regulatory T cells, mostly at the transcriptional levels both in vivo and in vitro. [START_REF] Wan | The roles for cytokines in the generation and maintenance of regulatory T cells[END_REF]

IL-4

Since IL-4 is produced by Th 2 cells, it has been proposed to play a protective role in atherosclerosis. This is supported by phenomena such as IL-4-induced suppression of pro-atherogenic cytokines (e.g. IL-12) and inhibition of SMC proliferation and macrophage adhesiveness. [START_REF] Vadiveloo | Interleukin-4 inhibits human smooth muscle cell proliferation[END_REF] However, IL-4 has been found to cause an increased expression of P-selectin, [START_REF] Khew-Goodall | Stat6 activation is essential for interleukin-4 induction of P-selectin transcription in human umbilical vein endothelial cells[END_REF] vascular cell adhesion molecule-1 (VCAM-1), and MMP-1 and 12, which are implicated in aortic aneurysm formation. IL-4 responses have also been found to predominate in aortic aneurysms both in animal models and human studies. [START_REF] Shimizu | Local cytokine environments drive aneurysm formation in allografted aortas[END_REF] More recent studies also indicate that IL-4 causes differential IL-12 expression, [START_REF] Morita | Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis[END_REF] rather than a distinct inhibitory effect, as was previously noted. Although there is some evidence that IL-4 levels are elevated in acute coronary syndrome in comparison to stable disease, [START_REF] Szodoray | TH1/TH2 imbalance, measured by circulating and intracytoplasmic inflammatory cytokines -immunological alterations in acute coronary syndrome and stable coronary artery disease[END_REF] no definitive involvement of IL-4 has been implicated in the severity of manifestation of PAD to date; small cross-sectional studies reflect non-significant variations in IL-4 levels in association with PAD (which are very low overall). This may also be explained by a shift in the Th 1 :Th 2 balance in favour of a Th 1 milieu in atherostenotic disease, as detailed above.

GM-CSF

GM-CSF stimulates the proliferation, differentiation and function of multiple myeloid cells including neutrophils, monocytes/macrophages, eosinophils and dendritic cells. Its main clinical use is in mobilizing peripheral blood stem cells in myelosuppressive disorders, drug-induced agranulocytosis and immunodeficiency syndromes. It has been implicated in atherosclerosis because a decrease in plasma cholesterol levels has been observed in patients receiving GM-CSF for haematological disorders. These findings are mirrored by GM-CSF treatment of Watanabe heritable hyperlipidaemic rabbits, which also exhibit a significant decrease in total cholesterol levels for up to 2 weeks following cessation of recombinant GM-CSF administration. [START_REF] Ishibashi | Potent cholesterollowering effect by human granulocyte-macrophage colonystimulating factor in rabbits. Possible implications of enhancement of macrophage functions and an increase in mRNA for VLDL receptor[END_REF] Related leporine studies [START_REF] Shindo | Granulocytemacrophage colony-stimulating factor prevents the progression of atherosclerosis via changes in the cellular and extracellular composition of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits[END_REF] have also indicated that GM-CSF can prevent the progression of atherosclerotic plaques. After 7.5 months of GM-CSF treatment, a 45% reduction in the atherosclerotic lesion of the aortic arch was noted along with a reduction in intimal thickening compared to untreated animals.

GM-CSF has traditionally been considered a proinflammatory cytokine based on in vivo and in vitro studies using lipopolysaccharide challenge. [START_REF] Hamilton | Stimulation of macrophage plasminogen activator activity by colony-stimulating factors[END_REF] However, this distinction becomes blurred in atherosclerosis, [START_REF] Hamilton | GM-CSF biology[END_REF] where GM-CSF has been shown to confer some protective effects. [START_REF] Hamilton | GM-CSF biology[END_REF] GM-CSF is also a potential marker of chronic and severe inflammation (as opposed to acute responses) as its levels become more resistant to suppression by inhibitors such as glucocorticoids in this situation. [START_REF] Cosio | Histone acetylase and deacetylase activity in alveolar macrophages and blood monocytes in asthma[END_REF] Recently, there has been renewed interest in the role of GM-CSF in atherosclerosis because of its angiogenic properties. Following pioneering studies in rodents, subcutaneous GM-CSF administration has been shown to promote collateral flow in the coronary circulation, with a decrease in ECG signs of myocardial ischaemia. [START_REF] Seiler | Promotion of collateral growth by granulocyte-macrophage colony-stimulating factor in patients with coronary artery disease: a randomized, doubleblind, placebo-controlled study[END_REF] Unfortunately, these results have not been reproduced in PAD patients [START_REF] Van Royen | START Trial: a pilot study on STimulation of ARTeriogenesis using subcutaneous application of granulocyte-macrophage colony-stimulating factor as a new treatment for peripheral vascular disease[END_REF] (START trial), despite beneficial effects being seen in porcine models of PAD. [START_REF] Grundmann | Granulocytemacrophage colony-stimulating factor stimulates arteriogenesis Potential cytokine biomarkers in PAD 307 Vascular Medicine 2007; 12: 299-309 in a pig model of peripheral artery disease using clinically applicable infusion pumps[END_REF] Although there are currently no data supporting the role of GM-CSF as a cytokine signature of PAD, our preliminary studies in this area have nevertheless indicated that there is a trend for GM-CSF levels to be marginally higher in claudicants (present authors, unpublished).

IL-7

IL-7 was first discovered almost two decades ago for its role in promoting the growth of B-cell precursors in bone marrow culture systems. [START_REF] Namen | Stimulation of B-cell progenitors by cloned murine interleukin-7[END_REF] This cytokine is discussed under the IL-2 family because its receptor shares the common ␥ chain with other IL-2 family members. IL-7 is now an established regulator of T-cell homeostasis in both thymic and extra-thymic tissues, [START_REF] Jiang | Cell biology of IL-7, a key lymphotrophin[END_REF] and has more recently been implicated as a proinflammatory factor in atherosclerosis, with higher levels detected in patients with unstable angina, compared to stable angina and controls. [START_REF] Damas | Interleukin-7-mediated inflammation in unstable angina: possible role of chemokines and platelets[END_REF] This study also revealed that aspirin causes a significant decline in both spontaneous and stimulated IL-7 release from platelets in vitro. However, only one study has investigated IL-7 levels in relation to PAD (as a subgroup of an atherosclerotic cohort), and revealed that IL-7 was undetectable in these patients. [START_REF] Prunet | Multiplexed flow cytometric analyses of pro-and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients[END_REF] The IL-6/IL-12 family

IL-6

This pro-inflammatory cytokine is probably the most consistent biomarker of PAD identified thus far (Table 2). The Edinburgh artery, 14 InCHIANTI 11 and MESA [START_REF] Allison | The effect of novel cardiovascular risk factors on the ethnic-specific odds for peripheral arterial disease in the Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF] studies have all individually established its role as an independent predictor of PAD in community screening, irrespective of ethnicity. Furthermore, IL-6 has been found to be associated with PAD severity [START_REF] Nylaende | Markers of vascular inflammation are associated with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease[END_REF] and its genetic polymorphisms have been implicated in increasing susceptibility of type 2 diabetics to develop PAD. [START_REF] Libra | Analysis of G(-174)C IL-6 polymorphism and plasma concentrations of inflammatory markers in patients with type 2 diabetes and peripheral arterial disease[END_REF] IL-6 enhances cell adhesion molecule (CAM) expression by endothelial and smooth muscle cells, and enhances the production of acute phase reactants such as C-reactive protein (CRP) and TNF-␣ by the hepatocytes. As such, plasma IL-6 concentrations in healthy populations correlate with metabolic and inflammatory factors such as TNF-␣ and CRP, which are considered relevant to atherosclerotic progression, [START_REF] Haddy | IL-6, TNF-alpha and atherosclerosis risk indicators in a healthy family population: the STANISLAS cohort[END_REF] and in the case of the latter, associated with increased risk of death from cardiovascular pathology. More specifically, IL-6 has a positive feedback interaction with intimal cells and macrophages to further stimulate its own production, along with that of IL-8 and monocyte chemoattractant protein (MCP)-1. IL-6 also increases leucocyte recruitment into the atherosclerotic plaque by stimulating chemokine release from the endothelium and by enhancing expression of intercellular adhesion molecule-1 (ICAM-1) by the smooth muscle cells. [START_REF] Der Thusen | Interleukins in atherosclerosis: molecular pathways and therapeutic potential[END_REF] It may also stimulate smooth muscle cells to evolve into foam cells, [START_REF] Klouche | Enzymatically degraded, nonoxidized LDL induces human vascular smooth muscle cell activation, foam cell transformation, and proliferation[END_REF] thereby adversely effecting plaque morphology.

IL-12

IL-12 (p70) is a heterodimeric protein, composed of a heavy chain (p40) and a covalently associated light chain (p35). The expression of each of these subunits is independently regulated, but biologically active IL-12 can only be produced by cells with the capacity to produce both light and heavy chains. The primary function of IL-12, as proven in both animal models and humans, is the induction and sustenance of Th 1 responses to infection/antigen exposure by facilitating the secretion of cytokines such as IL-2, IFN-␥, IL-18 and an array of chemokines. While IL-12 has been implicated in the progression of atherosclerosis, functional blockade of endogenous IL-12 production can reduce atherogenesis in LDL receptor-deficient mice and improve plaque stability. [START_REF] Hauer | Blockade of interleukin-12 function by protein vaccination attenuates atherosclerosis[END_REF] Immunohistochemical studies have also revealed an increased presence of IL-12 (p70) in human atherosclerotic plaques compared to normal arteries, [START_REF] Uyemura | Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis[END_REF] thereby supporting its role in promoting plaque inflammation in human coronary arteries by enhancing local T-cell recruitment. [START_REF] Zhang | Interleukin 12 induces T-cell recruitment into the atherosclerotic plaque[END_REF] While raised IL-12 levels in the coronary arteries in vivo are a feature of unstable angina, [START_REF] Baidya | T helper cell related interleukins and the angiographic morphology in unstable angina[END_REF][START_REF] Steppich | Cytokine profiles and T cell function in acute coronary syndromes[END_REF] no evidence is available regarding IL-12 profiles in PAD to date.

IL-12 acts synergistically with IL-18 to elicit Th 1 responses, as well as facilitating the release of IFN-␥ by Th 1 cells. IFN-␥ and IL-12 act in combination to promote Th 1 function too, with IFN-␥ further up-regulating IL-12 production. Paradoxically, IL-12 also regulates Th 2 responses by inducing the production of IL-10 and IL-13 while inhibiting IL-4 and IL-5 production. [START_REF] Morita | Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis[END_REF] This is part of a protective negative feedback mechanism, which comes into play in chronic inflammatory states in particular. IL-10 causes feedback inhibition of IL-12 [START_REF] Uyemura | Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis[END_REF] while IL-4 has a similar effect on IFN-␥. The interaction between IL-4 and IL-12 is more complex: although initial reports indicated that IL-4 inhibited IL-12, recent evidence suggests that IL-4 has more subtle effects by inducing differential IL-12 expression rather than overall inhibition. [START_REF] Hochrein | Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells[END_REF] Thus, although IL-12 enhances IFN-␥ production, it also concomitantly enhances production of anti-inflammatory cytokines that limit IFN-␥ production, highlighting the complex network system in which cytokines operate. Understanding these interactions is pivotal to clarifying the inflammatory pathophysiology allied to atherosclerotic progression and revealing the prognostic potential of cytokines in PAD (Figure 1).

The interferon (IFN) family

IFN-␥ ␥

IFN-␥ is a pleiotropic pro-inflammatory cytokine primarily secreted by T lymphocytes and natural killer cells following activation by immune and inflammatory stimuli. It induces expression of ICAM-1, VCAM-1 and selectins by endothelial and smooth muscle cells, and promotes leucocyte adhesion at the site of the vascular lesion. [START_REF] Chung | Statin inhibits interferongamma-induced expression of intercellular adhesion molecule-1 (ICAM-1) in vascular endothelial and smooth muscle cells[END_REF] As outlined, it acts in concert with IL-12 and IL-18 to promote atherosclerosis. [START_REF] Tedgui | Cytokines in atherosclerosis: pathogenic and regulatory pathways[END_REF] More specifically, a reduction in atherosclerosis was also noted in cholesterol-diet fed mice in the absence of IFN-␥. [START_REF] Buono | Influence of interferon-gamma on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse[END_REF] By contrast, IFN-␥ has also been associated with a reduction in atherosclerotic lesion size in apo E -/-mice. [START_REF] Xiong | Key roles of CD4ϩ T cells and IFN-gamma in the development of abdominal aortic aneurysms in a murine model[END_REF] IFN-␥ is intimately related to plaque morphology, with enhanced expression reported in unstable plaques. [START_REF] Leon | Gamma interferon: a central mediator in atherosclerosis[END_REF] This relationship with atherosclerotic lesions has been consistently reproduced in clinical samples as well as rodent atherosclerosis models. There is also an associated rise in pro-inflammatory cytokines such as MCP-1 and macrophage inflammatory protein (MIP)-1␣ and ␤ within the plaque. IFN-␥ potentially destabilizes the fibrous cap by inhibiting SMC proliferation as well as IL-1␤ and TGF-␤-mediated collagen synthesis. However, there is an ambivalent element to its otherwise pro-atherogenic role through its attenuation of constitutive and inducible MMP expression within the plaque. [START_REF] Young | Cytokines in the pathogenesis of atherosclerosis[END_REF] In addition, it regulates foam cell apoptosis within the lipid core of the plaque. [START_REF] Harvey | Interferon-gamma and atherosclerosis: pro-or anti-atherogenic?[END_REF] It is, therefore, difficult to determine whether IFN-␥ forms unstable plaque from the outset or if there is a shift in the plaque modulation mechanism of IFN-␥ with advanced atherosclerosis, causing plaque instability at a later stage. Although IFN-␥ has not been investigated in the context of PAD, its profiles should be studied in relation to IL-12, IL-4 and IL-10 levels given what has been discussed above (Figure 1B).

The tumour necrosis factor (TNF) family

TNF-␣ ␣ TNF-␣ is a pro-inflammatory prototype member of this family. Initially identified as an anti-tumour agent, it is now recognized as a pleiotropic cytokine with multiple, dose-dependent actions on both local and systemic inflammation. TNF-␣ is involved in atherosclerotic progression from the initial stages of intimal thickening to the subsequent vessel occlusion. [START_REF] Barath | Detection and localization of tumor necrosis factor in human atheroma[END_REF] It stimulates selectin and adhesion molecule expression by endothelial cells, SMCs and macrophages, promoting interaction between endothelium, leucocytes and platelets, thus creating a pro-inflammatory and prothrombotic local environment. These changes magnify the impact of the local vascular injury and enhance plaque formation. TNF-␣ has also been implicated in modulating plaque morphology and is associated with plaque rupture. It stimulates production of MMP-1-9,11 and 13 production in the endothelium, SMCs and macrophages. [START_REF] Young | Cytokines in the pathogenesis of atherosclerosis[END_REF][START_REF] Galis | Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions[END_REF] Locally, within the atheroma, it increases expression of tissue factor, a potent thrombogenic protein, by macrophage-derived foam cells and the endothelial cells. [START_REF] Taubman | Tissue factor in the pathogenesis of atherosclerosis[END_REF] There is ample evidence implicating TNF-␣ in PAD progression (Table 2). [START_REF] Fiotti | Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease[END_REF][START_REF] Depalma | Statins and biomarkers in claudicants with peripheral arterial disease: cross-sectional study[END_REF][START_REF] Danielsson | Inflammatory markers and IL-6 polymorphism in peripheral arterial disease with and without diabetes mellitus[END_REF][START_REF] Depalma | Cytokine signatures in atherosclerotic claudicants[END_REF] Osteoprotegerin, another member of the TNF family, has also been associated with PAD severity. [START_REF] Ziegler | Osteoprotegerin plasma concentrations correlate with severity of peripheral artery disease[END_REF] Along with CRP and IL-6, TNF-␣ production has also been found to have a significant correlation in atherosclerotic parents and their offspring (STANISLAS cohort), [START_REF] Haddy | IL-6, TNF-alpha and atherosclerosis risk indicators in a healthy family population: the STANISLAS cohort[END_REF] implicating it as an atherosclerotic risk indicator in healthy families. The impact of TNF-␣ on vascular injury in both acute and chronic inflammatory conditions has made it an important therapeutic target. Unfortunately, results allied to TNF neutralization therapies have been inconsistent thus far. Trials using TNF-␣ antagonist, such as infliximab (ATTACH trial) [START_REF] Chung | Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-alpha, in patients with moderate-to-severe heart failure: results of the anti-TNF Therapy Against Congestive Heart Failure (ATTACH) trial[END_REF] and etanercept (RENNAISANCE and RECOVER trials), 73 failed to show any improvement in cardiac failure, while the ATTACH trial was associated with an increased allcause mortality. Although there have been no studies focusing on anti-atherogenic therapeutic interventions in PAD, a recent study by DePalma and co-workers, [START_REF] Depalma | Statins and biomarkers in claudicants with peripheral arterial disease: cross-sectional study[END_REF] indicated that there were no marked differences in TNF-␣ levels in PAD patients receiving/not receiving statin therapy.

The chemokine family

Chemokines are a large superfamily of small proteins that direct leucocytes to sites of inflammation and can be broadly classified into three distinct groups on the basis of structure and function: CC (e.g. monocyte chemoattractant protein-1), CXC (e.g. IL-8) and CX3C (e.g. fractalkine) chemokine families. MCP-1 and IL-8 are both pro-inflammatory cytokines and contribute to atherogenesis by mediating monocyte/ neutrophil-endothelial interactions.

MCP-1

MCP-1 is the most widely studied chemokine in PAD and it is consistently expressed in both animal and human atherosclerotic plaques. This atherogenic cytokine plays a crucial role in monocyte recruitment into the vascular lesion, which is one of the earliest steps in atherogenesis. While sub-endothelial migration of monocytes is facilitated by engagement of this cytokine with its receptor (CCR2), this interaction is only necessary to allow entry of monocytes into the intima and does not play a significant role in the subsequent macrophage action. [START_REF] Boisvert | Modulation of atherogenesis by chemokines[END_REF] There is a plethora of data from mouse studies clearly demonstrating the role of MCP-1 and its receptor in modulation of the atherosclerotic process. [START_REF] Shin | The role of chemokines in human cardiovascular pathology: enhanced biological insights[END_REF] MCP-1 has also been found to enhance SMC proliferation by stimulating binding activity of activator protein-1 and promotes expression of integrins and tissue factor on SMCs, generating a pro-inflammatory and a pro-thrombotic environment.

Recently, the ARIC study [START_REF] Hoogeveen | Plasma MCP-1 level and risk for peripheral arterial disease and incident coronary heart disease: Atherosclerosis Risk in Communities study[END_REF] found MCP-1 to be an independent risk factor for PAD and reported significantly higher levels of MCP-1 in the PAD population. Incidental coronary heart disease was also found to increase significantly in association with MCP-1 levels. Another study correlated MCP-1 levels with the severity of PAD, as assessed by angiographic scores and maximal treadmill walking distance. [START_REF] Nylaende | Markers of vascular inflammation are associated with the extent of atherosclerosis assessed as angiographic score and treadmill walking distances in patients with peripheral arterial occlusive disease[END_REF] Consequently, MCP-1 has been suggested as a novel potential target for atherosclerotic therapy. Indeed, statins have been found to have an independent effect on reducing MCP-1 levels. [START_REF] Ballantyne | Markers of inflammation and their clinical significance[END_REF] In this respect, administration of 20 mg simvastatin/day can reduce MCP-1 levels by 7% from baseline in hypercholesterolaemic patients. [START_REF] Leu | Fluvastatin reduces oxidative stress, decreases serum monocyte chemotactic protein-1 level and improves endothelial function in patients with hypercholesterolemia[END_REF]

IL-8 (CXCL8)

First described in 1987, IL-8 is probably one of the best characterized neutrophil chemoattractants consistently found in macrophage-rich atherosclerotic plaques. [START_REF] Charo | Chemokines in the pathogenesis of vascular disease[END_REF] It enhances all aspects of endothelial-neutrophil interaction and migration, facilitating neutrophil recruitment at the site of the vascular inflammation. IL-8 also triggers firm adhesion of rolling monocytes to vascular endothelium and infiltration of the vessel wall, and is thus implicated in early atherosclerotic progression. [START_REF] Ito | Inflammatory cytokines and cardiovascular disease[END_REF] Mice deficient in IL-8 receptor show decreased macrophage clustering in the plaque and smaller atherosclerotic lesions. [START_REF] Boisvert | A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice[END_REF] A Japanese study involving tissue sampling from human abdominal aortic aneurysms and carotid artery stenoses showed significantly increased expression of IL-8 and its receptor (CXCR2), implicating this chemokine in both aneurysmal and atheroocclusive disease processes. [START_REF] Yamagishi | Sustained upregulation of inflammatory chemokine and its receptor in aneurysmal and occlusive atherosclerotic disease: results from tissue analysis with cDNA macroarray and real-time reverse transcriptional polymerase chain reaction methods[END_REF] The role of IL-8 in destabilizing the atherosclerotic plaque has been a subject of close scrutiny because of its inhibitory effect on tissue inhibitor of metalloproteinase (TIMP)-1 expression in macrophages, which leads to a consequent increase in MMP activity. [START_REF] Moreau | Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterolloaded human macrophages: relevance to stability of atherosclerotic plaque[END_REF] Furthermore, the potentially detrimental effects of IL-8 may be compounded by its effects on SMC proliferation. While IL-8 has been associated with unstable angina and acute myocardial infarction, [START_REF] Martins | Risk factor analysis of plasma cytokines in patients with coronary artery disease by a multiplexed fluorescent immunoassay[END_REF] there are some studies addressing its role in PAD. Kirk et al. [START_REF] Kirk | Interleukin-8 (IL-8) may contribute to the activation of neutrophils in patients with peripheral arterial occlusive disease (PAOD)[END_REF] indicated that significantly higher levels of IL-8 were detectable in PAD patients compared with controls, both at rest and post-exercise. However, this observation was not reproduced in the study of Danielsson et al. [START_REF] Danielsson | Inflammatory markers and IL-6 polymorphism in peripheral arterial disease with and without diabetes mellitus[END_REF] which compared the cytokine profiles of PAD patients with/without diabetes and controls.

The IL-10 family IL-10 IL-10 was initially described as a cytokine synthesis inhibitory factor because of its ability to directly inhibit Th 1 and Th 2 cytokine production. This role makes it one of the key regulators of both local and systemic inflammatory/immune responses, and it has been shown to confer a protective effect in atherosclerosis. This is illustrated by the study of Mallat and co-workers, who demonstrated that IL-10-deficient mice fed an atherogenic diet had a threefold increase in lipid accumulation compared with their wild-type counterparts. [START_REF] Mallat | Protective role of interleukin-10 in atherosclerosis[END_REF] The plaques in the study group were also more vulnerable and had increased thrombogenic tendencies. Furthermore, Namiki et al showed that intramuscular IL-10 gene transfer significantly reduced the atherosclerotic plaque area in such mice, with an associated decline in IL-12 and IFN-␥ mRNA expression in the spleens of the study group animals. [START_REF] Namiki | Intramuscular gene transfer of interleukin-10 cDNA reduces atherosclerosis in apolipoprotein E-knockout mice[END_REF] IL-10 is produced by a variety of inflammatory cells and is found in advanced atherosclerotic plaques where it has been associated with decreased apoptosis in the lipid central core, thereby reducing the likelihood of rupture. [START_REF] Mallat | Expression of interleukin-10 in advanced human atherosclerotic plaques: relation to inducible nitric oxide synthase expression and cell death[END_REF] Systemic and intralesional delivery of IL-10 has been shown to diminish atherosclerosis in a variety of experimental models. [START_REF] Terkeltaub | IL-10: An 'immunologic scalpel' for atherosclerosis?[END_REF] One of the reasons for this effect could be a reduction in inflammatory cell recruitment within the plaque: IL-10 has been shown to inhibit monocyte adherence to human aortic endothelial cells in vitro. [START_REF] Oslund | Interleukin-10 blocks atherosclerotic events in vitro and in vivo[END_REF] The ability of IL-10 to suppress certain CD40/CD40L ligand-mediated monocyte responses may account for some of its anti-atherogenic effects, [START_REF] Poe | IL-4 and IL-10 modulation of CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis[END_REF] while others may be mediated indirectly through a suppression of pro-inflammatory cytokine (e.g. MCP-1, IL-6, IL-12 and IFN-␥) secretion.

Data regarding the role of IL-10 in PAD in humans are surprisingly scant. It is difficult to determine whether higher or lower IL-10 levels are to be expected as these will vary according to the timing of onset of the disease. On one hand, lower IL-10 levels in PAD would favour disease progression. On the other, higher levels might be expected as a modulatory phenomenon to the associated increase in local inflammatory burden. This complex relationship has been illustrated in coronary artery disease patients, where lower IL-10 levels coupled with higher proinflammatory cytokine levels were observed in patients with unstable angina who subsequently developed a cardiovascular event. [START_REF] Anguera | Elevation of serum levels of the anti-inflammatory cytokine interleukin-10 and decreased risk of coronary events in patients with unstable angina[END_REF][START_REF] Smith | Serum levels of the antiinflammatory cytokine interleukin-10 are decreased in patients with unstable angina[END_REF] Conversely, higher IL-10 levels have been associated with a favourable prognosis in patients with acute coronary syndromes (CAPTURE trial). [START_REF] Heeschen | Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes[END_REF] As such, this suggests that IL-10 may have poor predictive merit for clinical outcome in atherosclerosis. In this respect, the ratio between IL-10 and various pro-inflammatory cytokines may prove more useful as they account for some of the dynamics of the inflammatory processes involved. While IL-10 levels have also been consistently found to be elevated in association with abdominal aortic aneurysms, [START_REF] Davis | Cytokine pattern in aneurysmal and occlusive disease of the aorta[END_REF] DePalma et al [START_REF] Depalma | Cytokine signatures in atherosclerotic claudicants[END_REF] found IL-10 to be significantly lower in PAD. These findings were resonated by the large InCHIANTI study, which pointed to a marked trend for circulatory IL-10 concentrations to be lower in patients with PAD. 11

The transforming growth factor (TGF) family

TGF-␤ ␤

The TGF family of growth, differentiation and morphogenic glycosylated preproteins comprises of over 50 members. TGF-␤ is both the prototype member of this superfamily as well as one of the most studied cytokines in vascular biology due to its role in atherosclerosis and neo-intimal hyperplasia. Following the work done by Metcalfe and Grainger in the mid-1990s, TGF-␤ is considered to be beneficial in atherosclerosis, leading to the so-called 'protective cytokine' hypothesis. [START_REF] Metcalfe | Transforming growth factor-beta and the protection from cardiovascular injury hypothesis[END_REF] Although this hypothesis was initially postulated based on the role of TGF-␤ in inhibiting vascular smooth muscle cell (VSMC) proliferation and migration, its other immunomodulatory effect on T-cell regulation both within and outside the plaque, [START_REF] Grainger | Transforming growth factor beta and atherosclerosis: so far, so good for the protective cytokine hypothesis[END_REF] and inhibition of pro-inflammatory cytokine (IL-6, granulocyte colony stimulating factor and MCP-1) gene expression [START_REF] Yamagami | Effects of TGFbeta2 on immune response-related gene expression profiles in the human corneal endothelium[END_REF] gave further momentum to this concept. Whilst inhibition of VSMC proliferation is consistent across studies, the effects of TGF-␤ on macrophages and cytokine inhibition is variable and governed by the local inflammatory environment. [START_REF] Ashcroft | Bidirectional regulation of macrophage function by TGF-beta[END_REF][START_REF] Turner | Transforming growth factor beta induces the production of interleukin 6 by human peripheral blood mononuclear cells[END_REF] As for many other cytokines, most of the initial evidence regarding the anti-atherogenic properties of TGF-␤ was drawn from studies using knockout mice. However, TGF-␤-deficient mice died shortly after birth due to systemic inflammation associated with leucocytic infiltration in virtually every organ system, [START_REF] Kulkarni | Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death[END_REF] highlighting the anti-inflammatory effects of TGF-␤ by down-regulation of CAM expression and the consequent reduction in leucocyte infiltration. Studies using heterozygous mouse models proved equally informative, as these animals presented with reduced rather than absent TGF-␤ profiles. Exposing these mice to a high-fat diet enhanced endothelial activation and arterial lipid deposition compared with controls. 100 In line with these observations, Mallat et al noted that inhibition of TGF-␤ signalling is also associated with accelerated atherosclerotic lesion development in apoE-deficient mice and favours the development of those with an increased inflammatory component and decreased collagen content. 101 A related study further showed that TGF-␤ has a definitive role in influencing plaque morphology, with more vulnerable plaques formed in the case of TGF-␤ deficiency due to decreased fibrosis, increased plaque inflammatory cell counts, greater areas of rupture and increased intraplaque haemorrhage. 102 The anti-atherosclerotic role of TGF-␤ is supported by the negative correlation between its plasma levels and coronary artery disease, 103 with similar trends also noted in association with PAD in the InCHIANTI study. [START_REF] Mcdermott | Patterns of inflammation associated with peripheral arterial disease: the InCHIANTI study[END_REF] These findings suggest that together with IL-10, TGF-␤ may have anti-atherogenic properties which may prove useful in a therapeutic setting.

Conclusion

There are multiple regulatory levels of vascular inflammation with direct or indirect involvement of cytokines at every step. Traditional risk factors such as smoking, hypertension, diabetes, obesity and high cholesterol levels -though useful in population studies -fail to explain the variable nature of disease progression between individuals. Although cytokines are clearly implicated in the pathophysiology of atherosclerosis, there is currently scant information of their role in PAD in particular. However, early evidence in human studies indicates that markers such as IL-6 are closely associated with disease manifestation. Given the intimate interrelationships between cytokines in physiological immune/inflammatory networks, this argues in favour of the case that other cytokines whose role has not yet been implicated in cardiovascular pathophysiology are very likely to also play a role (and likely above and beyond the conventional Th 1 :Th 2 dichotomy). This postulate is further supported by the animal studies reviewed herein.

The findings summarized in this review substantiate the role of cytokines in atherosclerosis and highlight the differences in cytokine responses in different subcatergorizations of atherostenotic or atheroaneurysmal disease processes. As proteins involved in the pathophysiological process per se, cytokines have the potential to provide useful information on disease progression on an individual basis and serve as valuable adjuncts to existing clinical screening and investigative tools in forming a composite picture. Also, the potential role of cytokines in gauging the response to the medical treatment (e.g. statins, aspirin) of atherosclerosis needs to be investigated through larger longitudinal clinical trials. Only then would we be in a position to assess the merit of cytokines as clinical risk stratification tools in cardiovascular medicine.

  Potential cytokine biomarkers in PAD 303Vascular Medicine 2007; 12: 299-309

Figure 1 (

 1 Figure 1 (A) Conventional Th 1 :Th 2 paradigm. Shift of balance in the favour of Th 1 responses promotes atherosclerotic plaque progression in the vessel wall. (B) Schematic representation of the negative and positive feedback interactions between Th 1 and Th 2 cytokines. IL-10 is the dominant anti-inflammatory cytokine that hinders expression of IL-12, IFN-␥ and IL-18. IL-12 enhances production of IFN-␥ and IL-18. It inhibits production of TGF-␤ and thereby contributes to plaque instability and fibrin cap degradation. Whilst promoting pro-atherogenic mechanisms, IL-12 also regulates atherogenic responses by enhancing production of IL-10.

Table 1

 1 Cytokine classification and implication in atherosclerosis.

	Cytokines	Family	Implication in atherosclerosis
	IL-1␣, IL-1␤, IL-1ra, IL-18	IL-1 family	Pro-atherogenic except IL-1ra
	IL-2, IL-4, GM-CSF	IL-2 family	Pro-atherogenic. IL-4 levels elevated in aortic
			aneurysms but precise categorization of IL-4
			and GM-CSF as atherogenic or protective
			is not possible
	IL-6, IL-12	IL-6 family	Pro-atherogenic
	CXCL1-16, CXCL-28,	Chemokines	Pro-atherogenic
	CX3CL1/fractalkine,
	IL-8, MCP-1
	IL-10	IL-10 family	Protective
	IL-17	IL-17 family	Pro-atherogenic
	IFN-␥	Interferon family	Pro-atherogenic
	TGF-␤	TGF-␤	Protective
	TNF-␣, TNF-␤, LT-␤	Tumour necrosis factor family	Pro-atherogenic
	LT, lymphotoxins.
	Other abbreviations can be found in the text.

Table 2

 2 Baseline cytokine trends in PAD patients in vivo.

	Year	Study	PAD group size	Conclusions
	2006	Nylaende et al 41	127	MCP-1, CD40L and IL-6, significantly associated with
				extent of angiographically proven PAD and maximal
				treadmill walking
	2006	DePalma et al 42	100	IL-6, TNF-␣R1 and CRP were significantly elevated in PAD;
			(47) no statins	no significant differences with statin dose except for
			(53) on statins	IL-6 (higher in PAD patients on statins)
	2006	Armitage et al 43	60	Significantly elevated IL-6 in PAD; endothelial reactive
				antibodies promote PAD by elevating IL-6
	2006	Libra et al 44	290	IL-6, CRP, fibrinogen and VEGF higher in diabetic PAD;
			(146) diabetic	IL-6-174G/G phenotype promotes PAD in type 2
			(144) non-diabetic	diabetics through increased IL-6 release
	2006	MESA study 45	275	African-American ethnicity associated with 50% increase
		Allison et al	(6653) total cohort	in odds of PAD; increase in IL-6 associated with
				largest risk for PAD
	2005	Edinburgh Artery	273	CRP, IL-6 and ICAM-1 as molecular markers of peripheral
		Study 14 Tzoulaki et al	(46) symptomatic	atherosclerosis and its progression; IL-6 strongest
			(227) asymptomatic	predictive power
	2005	InCHIANTI study 11	107	PAD population features increased IL-6, IL-1ra, fibrinogen
		McDermott et al		and CRP; trends for lower TGF-␤ in PAD
	2005	ARIC study 46	209	Significant association of MCP-1 with PAD and incident
		Hoogeveen et al		coronary heart disease
	2005	Danielsson et al 47	100	CLI was significantly associated with elevated white
				cell count, hsCRP, IL-6 and sTNF-␣R1 and 2; no effect
				of IL-6 promoter polymorphism
	2005	Ziegler et al 48	66	OPG values correlate with PAD severity
	2003	DePalma et al 49	91	IL-10 significantly lower in PAD, while IL-6, CRP and
				TNF-␣ significantly elevated
	2003	Silvestro et al 50	38	IL-6, CRP, sICAM-1 and sVCAM-1 associated with poor
				prognosis in PAD
	2000	Testa et al 51	8	IL-1␤ and IL-6 gene expression upregulated in skeletal
				muscle of CLI patients
	1999	Fiotti et al 10	16	Significant increase in TNF-␣R1 and 2, IL-6 and
				IL-1ra in PAD
	1999	Kirk et al 52	26	Significantly elevated IL-8 at rest and post-exercise in PAD
	VEGF, vascular endothelial growth factor; CLI, critical limb ischaemia; hsCRP, high-sensitivity C-reactive protein;
	sTNF-␣R1 and 2, soluble TNF-␣ receptors 1 and 2; OPG, osteoprotegerin; sVCAM, soluble vascular cell adhesion
	molecule.
	Other abbreviations can be found in the text.

Vascular Medicine 2007; 12: 299-309


Claudio S Cinà

Sonia Anand

Applying the gender lens to abdominal aortic aneurysm screening

Four randomized controlled trials (RCT), [START_REF] Lindholt | Screening for abdominal aortic aneurysms: single centre randomised controlled trial[END_REF][START_REF] Ashton | The Multicentre Aneurysm Screening Study (MASS) into the effect of abdominal aortic aneurysm screening on mortality in men: a randomised controlled trial[END_REF][START_REF] Norman | Population based randomised controlled trial on impact of screening on mortality from abdominal aortic aneurysm[END_REF][START_REF] Scott | The long-term benefits of a single scan for abdominal aortic aneurysm (AAA) at age 65[END_REF] including 125,201 men, have shown that ultrasound screening to identify abdominal aortic aneurysms (AAA) Ͼ5 cm followed by surgery reduces cause-specific mortality among individuals older than 65 years. This benefit is not apparent among men older than 75 years, [START_REF] Mastracci | Screening for abdominal aortic aneurysm in Canada: review and position statement of the Canadian Society for Vascular Surgery[END_REF] and there is some controversy regarding the benefit of screening for AAA among women. No specific recommendations are given in the ACA/AHA task force guidelines for screening of AAA in women. [START_REF] Hirsch | ACC/AHA guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery[END_REF] The United States Preventative Services Task Force (USP-STF) recommends one-time screening for AAAs in men 65 to 75 years of age who have ever smoked and recommends against routine screening in women, [START_REF] Fleming | Screening for abdominal aortic aneurysm: a best-evidence systematic review for the US Preventive Services Task Force[END_REF] and the Screening Abdominal Aortic Aneurysms Very Efficiently (SAAAVE) Act 8 supports only a screening program for AAA in male ever-smokers when they turn 65 years old.

Only one RCT, [START_REF] Scott | The long-term benefits of a single scan for abdominal aortic aneurysm (AAA) at age 65[END_REF] the Chichester trial, included women (n ϭ 9342) aged 65-80 years old. In this trial, the prevalence of AAA Ͼ3 cm among women (1.3%) was substantially lower than in men (7.6%). The subgroup analysis addressing the effect of screening in women concluded that screening followed by surgery did not reduce mortality [START_REF] Scott | Randomized clinical trial of screening for abdominal aortic aneurysm in women[END_REF] : the relative risk (RR) of the screened versus not screened female population was 1.49 (95% CI 0.72-3.10), whereas it was 0.60 (95% CI 0.45-0.80) among men. [START_REF] Mastracci | Screening for abdominal aortic aneurysm in Canada: review and position statement of the Canadian Society for Vascular Surgery[END_REF] However, the confidence interval for the RR in women is wide and therefore this finding cannot be definitive. In women, the incidence of ruptured AAA was similar in the control and screening groups, and in general the incidence of death from ruptured aneurysm increased with age, since more than 70% of ruptures occurred among women Ͼ 80 years. On the basis of the low prevalence of AAA in women and the unfavorable RR, screening of women may not be beneficial or cost effective. [START_REF] Scott | Randomized clinical trial of screening for abdominal aortic aneurysm in women[END_REF] The evidence available from the Chichester trial regarding the effect of population screening in women should be considered with caution because of the possibility of confounding factors or biases. The gender analysis was a subgroup analysis and, as expected, the number of participating women was considerably lower than men. In this study, only 3052 women attended screening which, given the low prevalence of AAA in this population, may leave a trial not powered enough to demonstrate the benefit of the intervention as evidenced by the wide confidence interval around the RR (amplification of systematic error). In addition, patients were excluded by the referring physician, before randomization, if it was thought that the patient was not a candidate for surgery (sampling bias). Since the risk factors associated with increased risk for surgery are the same as those associated with increased incidence of AAAs, it is possible that many women were excluded, giving a falsely lower incidence of AAAs (ascertainment bias).

Before making a final decision on the effectiveness of AAA screening in women, a number of features unique to women should be considered. The lower prevalence of AAA in women is most likely due to their lower burden of risk factors compared with men. The evidence supports that like in men, for women the probability of AAAs is increased among smokers (odds ratio (OR) 3.8), those aged Ͼ70 years (OR 1.8), family history (OR 2.6), and pre-existing cerebrovascular disease (OR 3.20). [START_REF] Lederle | 11 Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants[END_REF] Since the ORs of these risk factors were derived from a multivariate logistic model, the presence of multiple risk factors in a woman likely confers a multiplicative risk of having an AAA. It is also reasonable to expect that, as women's lifestyle practices become more similar to men's (e.g. increased smoking), the incidence of AAA would be expected to increase. Like coronary heart disease, the increase in prevalence of AAA among women appears to occur approximately one decade after men. For example, in the Chichester trial, women were screened starting at age 65: the prevalence of AAA was 0 in the 65-year-old group, 1% for 66-70, 1.8% for 71-80, and unknown in women older than 80 years. Because of this 10-year delay in onset, and lower burden of AAAs likely due to the currently more favorable cardiovascular risk factor profile of women, the cost effectiveness of screening and repair of AAA to prevent death does not favor screening at present. However, we must also consider the observation that although women have a lower incidence of AAA,

Editorial

Applying the gender lens to abdominal aortic aneurysm screening when they are found to have an AAA Ͼ 3 cm the risk of rupture is greater than that of men, 11,[START_REF] Clouse | Improved prognosis of thoracic aortic aneurysms[END_REF] and mortality associated with surgery for ruptured aortic aneurysms is higher compared with that in men. [START_REF] Scott | Randomized clinical trial of screening for abdominal aortic aneurysm in women[END_REF] In a population-based study of patients with thoracic aortic aneurysms, the 5-year cumulative risk of rupture was 20% (95% CI 12-28) and was significantly higher in women than in men (30% [95% CI 19-47] vs 9% [95% CI 1-17]; p Ͻ 0.01; RR 6.8; 95% CI 2-20). [START_REF] Clouse | Improved prognosis of thoracic aortic aneurysms[END_REF] The United Kingdom Small Aneurysm Trial also found that female sex increased the risk of aneurysm rupture (hazard ratio 3.0 [95% CI 1.99-4.53]; p Ͻ 0.001). 11,[START_REF] Lobato | Predictive factors for rupture of thoracoabdominal aortic aneurysm[END_REF] This higher risk of rupture in women may be because the prevalence of the disease was defined as an aorta with a diameter Ͼ3 cm, which is the usual threshold used for men, and it does not take into account the smaller size of a normal aorta in women. Thus, an aneurysm of 5 cm in a woman may have a higher rupture rate because it is equivalent to an aneurysm of 6 cm in a man.

Given the state of the evidence, a number of outstanding issues should be considered for the screening of AAA in women. First, the evidence does not support population-based screening over 65 years of age, due to the low incidence of AAA. However, it would be reasonable to recommend targeted screening of 'higher risk women', including those of an older age, who are current or had a long history of smoking, as well as those with co-existing vascular disease. In addition, accumulating data to assess whether sex-specific criteria to define AAA and the equivalent risk of rupture are also needed. While existing data amassed in large population-based studies or randomized trials may help us determine if this is a sensible approach, future studies of AAA diagnosis and screening among women are needed.


Editorial

Endovascular repair of the ruptured abdominal aortic aneurysm

The endovascular technique (EVAR) for elective repair of abdominal aortic aneurysms (AAA) has gained broad acceptance, although great variation exists between the proportion of AAA undergoing EVAR in different hospitals. At present only selected hospitals perform this kind of treatment. Based on the fact that the technical development has been rapid, new devices appear frequently and modifications of existing ones are common, there is still a need to follow up EVAR-treated patients carefully to assess the long-term outcome. Although EVAR causes less trauma to the patient than open surgery, mortality rates of the latter are generally acceptable at approximately 4%. [START_REF] Arko | Aneurysm-related death: primary endpoint analysis for comparison of open and endovascular repair[END_REF] One may therefore conclude that open surgery of elective AAA is still a reasonable treatment option.

The ruptured AAA (rAAA) is quite another challenge to the vascular surgeon. About 40% of those rAAA patients reaching the operating room for open surgery will not survive surgery or the postoperative period. [START_REF] Noel | Ruptured abdominal aortic aneurysm: the excessive mortality rate of conventional repair[END_REF] The question then is to what extent EVAR is suitable for the rAAA, and whether this technique is able to reduce mortality. Ohki and Veith claimed that catheter-based techniques appear to improve outcome for patients with rAAA. [START_REF] Ohki | Endovascular grafts and other imageguided catheter-based adjuncts to improve the treatment of ruptured aortoiliac aneurysms[END_REF] A study from The Netherlands 4 concluded that EVAR is feasible in about 40% of rAAA cases. Persistent hypotension and aortic neck morphology were the most common reasons to avoid EVAR. An even lower proportion of feasible patients was found in a French study, [START_REF] Alsac | Emergency endovascular repair for ruptured abdominal aortic aneurysms: feasability and comparison of early results with conventional open repair[END_REF] with the same reasons for precluding EVAR. Reported series usually include selected patients for EVAR, those not circulatory unstable and those with favourable neck morphology. Under such circumstances, a 30-day mortality rate as low as 8% for EVAR compared to 53% for open repair has been reported. [START_REF] Brandt | Endovascular repair of ruptured abdominal aortic aneurysm: feasibility and impact on early outcome[END_REF] With an intentionto-treat all patients by EVAR, Arya, et al. [START_REF] Arya | An intention-to-treat by endovascular repair policy may reduce overall mortality in ruptured abdominal aortic aneurysm[END_REF] only found 17 out of 51 patients suitable. They reported a 20% reduction of mortality rate in the EVAR group compared to open surgery.

The better way to evaluate the role of EVAR in rAAA would be via a randomized trial that included all patients. Several arguments are used against this approach, raising problems such as unsuitable morphology or persistent hypotension. In a randomized trial by Hinchliffe, et al., [START_REF] Hinchliffe | A randomised trial of endovascular and open surgery for ruptured abdominal aortic aneurysmresults of a pilot study and lessons learned for future studies[END_REF] only 32 out of 103 admitted patients were recruited. Thirty-day mortality was the same for EVAR and open repair (58%). The importance of an established protocol, including a multidisciplinary approach, was pointed out by Mehta, et al. [START_REF] Mehta | Establishing a protocol for endovascular treatment of ruptured abdominal aortic aneurysms: outcomes of a prospective analysis[END_REF] In our first experience, 15 out of 41 patients with rAAA had an endovascular repair, initiated under local anesthesia and with an aortic occlusion balloon inserted through the femoral artery to stabilize hemodynamics. The 30-day mortality rate was 13% after EVAR, and 46% after open repair. [START_REF] Larzon | Endovascular treatment of ruptured abdominal aortic aneurysms: a shift of the paradigm[END_REF] In our most recent series, operated on during a 5-year period, 49 patients had an open repair and 44 had an EVAR procedure. [START_REF] Larzon | Endografting decreases surgical mortality in ruptured abdominal aortic aneurysms[END_REF] The proportion of patients with rAAA undergoing EVAR increased from 30% to 78% from the first to the last third of the period. The use of local anesthesia as the single method increased from 15% to 50%. Overall, the 30-day mortality was 43% after open surgery and 11% after EVAR. A mortality as low as 4% (1/24) was recorded during the last third of this time period. Age, sex or proportion of patients refused from surgery did not differ between the periods, neither did the proportion of hypotensive patients vary. [START_REF] Larzon | Endografting decreases surgical mortality in ruptured abdominal aortic aneurysms[END_REF] Some dedicated centers have published encouraging results after EVAR, but there are also centers where improvement has not been obvious. In a recently published Cochrane Review, [START_REF] Dillon | Endovascular treatment for ruptured aortic aneurysm[END_REF] it was made clear that high-level evidence does not yet exist, while prospective studies at least find as good an outcome after EVAR as after open repair for rAAA. A trend towards lowered mortality, less blood loss and reduced ICU stay was also found. An important issue is whether an abdominal compartment syndrome occurs more frequently after EVAR than after open repair of rAAA due to the amount of blood left in the abdomen and retroperitonally. [START_REF] Mehta | Factors associatd with abdominal compartment syndrome complicating endovascular repair of ruptured abdominal aortic aneurysms[END_REF] Careful monitoring is of utmost importance with readiness to evacuate blood, as this complication increases mortality.

Patient selection may be one important reason for the variation in outcome that has been published. Another factor might be differences in operative technique. The use of bifurcated or aorto-uni-iliac stentgrafts might have an impact on the results, as access technique differs and the need for general anesthesia is reduced.

Mortality will also be affected by the proportion of EVAR performed. Hospitals starting with EVAR of rAAA will have to establish new protocols and availability of a 24-hour service. Several challenges therefore remain before one can establish the use of EVAR for rAAA more generally.

Vascular Medicine 2008; 13: 45-46 Vascular Medicine 2008; 13: 45-46


Thomas Kau
email: t.kau@gmx.at

Johann Gasser

Gerald Lesnik

Maria Sinzig

Klaus A Hausegger

Editor in Chief, Mark A Creager

Two exceptionally rare

  L'archive ouverte pluridisciplinaire

Images in vascular medicine

Two exceptionally rare branching patterns of the aortic arch Thomas Kau, Johann Gasser, Gerald Lesnik, Maria Sinzig and Klaus A Hausegger Magnetic resonance angiography of the thoracic aorta was performed in a 66-year-old man scheduled for a Bentall operation. Beside the known aneurysm of the ascending aorta, a rare variant branching sequence of the aortic arch was found (Panel A: view from posterior; R, right; ls, left subclavian artery; lv, left vertebral artery; lc, left common carotid artery; rv, right vertebral artery; rc, right common carotid artery; rs, right subclavian artery). Three-dimensional surface reconstructions depicted a common brachiocephalic trunk (asterisk), in which both common carotid arteries and the right subclavian artery arose from a single trunk off the arch. This so-called 'bovine trunk' (misnomer 1 ) was combined with a regular origin of the left subclavian artery and a very uncommon variant that may be called 'arteria vertebralis lusoria', in which the right vertebral artery (long arrow) is the last branch of the aortic arch and courses to the right behind the trachea (short arrow) and esophagus (Panel B: maximum intensity projection image; v, ventral; d, dorsal; T, trachea). [START_REF] Lemke | Anomalous origin of the right vertebral artery: review of the literature and case report of the right vertebral artery origin distal to the left subclavian artery[END_REF] Goray, et al. hypothesized that this variant developed from the C8 intercostal artery with secondary proximal migration. [START_REF] Goray | Aortic arch variation: a unique case with anomalous origin of both vertebral arteries as additional branches of the aortic arch distal to left subclavian artery[END_REF] Our patient was asymptomatic in this regard with no sign of a swallowing disorder.

Only 1 week later, a 38-year-old woman was referred for magnetic resonance angiography of the thoracic aorta with suspicion of dysphagia lusoria on the basis of barium swallow (Panel C: view from posterior; abbreviations as in Panel A). Three-dimensional imaging confirmed the diagnosis of an arteria lusoria with the right subclavian artery being the last branch of the aortic arch. [START_REF] Bayford | An account on a singular case of obstructed deglutition[END_REF] Further, a variant origin of the vertebral arteries was found. The right vertebral artery originated from the right common carotid artery, while the left vertebral artery directly arose from the aortic arch between the left common carotid subclavian arteries. [START_REF] Lemke | Anomalous origin of the right vertebral artery: review of the literature and case report of the right vertebral artery origin distal to the left subclavian artery[END_REF] The branching in this report represent exceptionally rare combinations of variant vascular anatomy. With the exception of arteria subclavia lusoria, anomalous branching of the aortic arch usually does not result in clinical symptoms. The true value of detecting an anomalous origin is in the diagnostic gain for planning aortic arch surgery or endovascular intervention. An aberrant origin of the vertebral arteries also represents a potential pitfall following trauma to the neck. [START_REF] Goray | Aortic arch variation: a unique case with anomalous origin of both vertebral arteries as additional branches of the aortic arch distal to left subclavian artery[END_REF]

Vascular Medicine 2008; 13: 89-90


Jerzy Wojciuk
email: dr.wojciuk@bfwhospitals.nhs.uk

Graham M Hoadley

Michael J Brack

published or not. The documents may come

Marfan syndrome -aortic dissection type A, visualized by computed tomography angiography

Images in vascular medicine

Marfan syndromeaortic dissection type A, visualized by computed tomography angiography Jerzy Wojciuk 1 , Graham M Hoadley 2 and Michael J Brack 1

A 19-year-old man with known Marfan syndrome presented with a 4-day history of neck pain. He has previously been diagnosed as having aortic root dilatation. Transthoracic echocardiography demonstrated that the ascending aorta was dilated (6.9 cm) with a definite flap, suggesting aortic dissection. Following urgent transfer to our tertiary cardiology centre, he underwent contrast enhanced computed tomography angiography studies. Three-dimensional volume rendered images ( RSCA, right subclavian artery) showed that the aortic root was grossly dilated to 6 cm in diameter. A dissection flap was demonstrated in the root and ascending aorta, but this did not reach the arch and was shown to stop approximately 6 cm short of the origin of the brachiocephalic artery. There was no dilatation of the arch or descending aorta and no evidence of arch or descending thoracic aortic dissection. The patient was referred for surgical repair of the Type A dissection. Increasingly, multislice CT has been used as the investigation of choice in suspected cases of aortic dissection. [START_REF] Sebastia | Aortic dissection: diagnosis and follow-up with helical CT[END_REF][START_REF] Batra | Pitfalls in the diagnosis of thoracic aortic dissection at CT angiography[END_REF][START_REF] Lawler | Multi-detector row CT of thoracic disease with emphasis on 3D volume rendering and CT angiography[END_REF] This modality is non-invasive and provides excellent three-dimensional visualization of aortic pathology, enabling accurate preoperative assessment.

  Panel A: view from the front; LINV, left innominate vein; LPA, left pulmonary artery; Panel B: view from the right; SVC, superior vena cava; PA, pulmonary artery; Panel C: view from the left; D Ao, descending aorta; Panel D: right anterior oblique view; RCCA, right common carotid artery;


Rolf Weidenhagen
email: rolf.weidenhagen@med.uni-muenchen.de

Karin Herrmann

Reinhard Kopp

Thrombus of an abdominal aortic aneurysm

come

Images in vascular medicine

Thrombus of an abdominal aortic aneurysm

Rolf Weidenhagen 1 , Karin Herrmann 2 and Reinhard Kopp 1 A 78-year-old man presented with acute onset of severe abdominal and lower back pain. Multislice computed tomography showed an abdominal aortic aneurysm (Panel A: arrow) with a diameter of 10.8 cm and extravasation of contrast medium into the thrombus. Threedimensional post-processing with volume rendering technique in coronal view with additional single-slice reconstruction in the mid-sagittal plane shows the mural thrombus surrounding the contrast-enhanced lumen. The patient was considered an emergency case and oper-ated on within the following 2 hours. Infrarenal open surgical repair was done using a tube graft (Gelsoft; Vascutek Ltd, Renfrewshire, Scotland, UK). After the surgical opening of the aneurysm sac the +solid onion skin-like thrombus was removed in one piece. Usually, thrombus material is too smooth to be removed in its entire integrity. Panel B shows the mural thrombus ex situ with its intra-aneurysm vascular channel and the large outer thrombus mass. The patient recovered uneventfully.

'Images in vascular medicine' is a regular feature of Vascular Medicine. Readers may submit original, unpublished images related to clinical vascular medicine to: Mark A Creager, Editor in Chief, Vascular Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.


August Ysa
email: augustysa@osakidetza.net

Amaia Arruabarrena

Maite R Bustabad

Eduardo Perez

Adolfo Del Campo

True aneurysm of the superficial temporal artery

teaching and research institutions in France or abroad, or from public or private research centers.

Images in vascular medicine

True aneurysm of the superficial temporal artery August Ysa, Amaia Arruabarrena, Maite R Bustabad, Eduardo Perez, Adolfo del Campo and Juan Garcia-Alonso A 59-year-old female with hypertension presented with an 8-month history of headache and an asymptomatic pulsatile mass on her left lateral forehead (Panel A). There was no prior history of trauma or sepsis. Computed tomographic angiography obtained by the family physician confirmed the presence of an aneurysm of the superficial temporal artery (STA) (Panel B, arrow). Laboratory studies (leukocyte count, VDRL, C-reactive protein, immunoelectrophoresis, anti-nuclear and anti-smooth muscle antibodies) were normal. Subsequent imaging did not reveal aneurysms in other regions. Ultrasonography showed a 1.5 × 1 cm saccular aneurysm of the STA containing mural thrombus. The aneurysm was resected under local anaesthesia (operative findings, Panel C). Operative cultures were negative. Histopathological examination confirmed a true aneurysm of the STA with atherosclerotic characteristics. The postoperative period was uneventful.

True aneurysms of the STA are extremely rare. [START_REF] Uchida | Atherosclerotic superficial temporal artery aneurysm: report of a case[END_REF] Most superficial temporal artery aneurysms are pseudoaneurysms commonly involving the anterior branch of the STA and related to prior blunt or penetrating trauma to the side of the head. [START_REF] Pipinos | Superficial temporal artery aneurysms[END_REF] Most individuals with STA present with an asymptomatic pulsatile mass, although it can occasionally present with a throbbing headache or ear discomfort. Pain, dizziness, bleeding and neurologic deficits have also been described. The diagnosis is easily established by physical examination and confirmatory imaging. Differential diagnosis includes lipoma, cyst, haematoma, abscess, inflamed lymph node, and neuroma of the supraorbital nerve. [START_REF] Evans | Traumatic pseudoaneurysm of the superficial temporal artery: two cases and review of the literature[END_REF] Once diagnosed, repair of these aneurysms is generally recommended, although precise criteria for surgery are not established. Ligation of the feeding vessels followed by excision is the recommended treatment, as the rich facial vascular supply makes reconstruction of vessels unnecessary. When sur-gical resection is likely to be difficult because of the depth of the artery or its proximity to the facial nerve and the parotid gland, selective catheter embolization is an alternative option.

Vascular Medicine 2008; 13: 295-296