Gregorio Brevetti
email: brevetti@unina.it

Massimo Vittorio Schiano

Massimo Chiariello

Cellular adhesion molecules and peripheral arterial disease

Keywords: atherosclerosis, cellular adhesion molecules, intermittent claudication, peripheral arterial disease

come

Cellular adhesion molecules and peripheral arterial disease

Introduction

Over the past decade, enormous advances have occurred in the understanding of the cellular and molecular mechanisms involved in atherogenesis. In particular, it is now clear that one of the major pathogenetic mechanisms of atherosclerosis is the recruitment of circulating leukocytes to the blood vessel wall, and their subsequent migration into the subendothelial spaces. [START_REF] Price | Cellular adhesion molecules and atherogenesis[END_REF] This process is predominantly mediated by cellular adhesion molecules (CAMs), which are expressed on both the vascular endothelium and the leukocytes in response to several atherogenic stimuli. [START_REF] Price | Cellular adhesion molecules and atherogenesis[END_REF][START_REF] Li | An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium[END_REF][START_REF] Tummala | Angiotensin II induces vascular cell adhesion molecule-1 expression in rat vasculature: a potential link between the renin-angiotensin system and atherosclerosis[END_REF][START_REF] Albaugh | Nicotine induces mononuclear leukocyte adhesion and expression of adhesion molecules, VCAM and ICAM, in endothelial cells in vitro[END_REF] The contention that CAMs are causally related to the development of atherosclerosis is supported by the finding that knock-out mice deficient in E-selectin, P-selectin or intracellular adhesion molecule-1 (ICAM-1) have a reduced response to atherosclerotic stimuli. [START_REF] Dong | The combined role of P-and E-selectins in atherosclerosis[END_REF][START_REF] Collins | P-selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice[END_REF] Furthermore, increased expression of adhesion molecules have been found in human atherosclerotic plaques. [START_REF] Tenaglia | Levels of expression of P-selectin, E-selectin, and intercellular adhesion molecule-1 in coronary atherectomy specimens from patients with stable and unstable angina pectoris[END_REF][START_REF] O'brien | Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content[END_REF] Thus, evaluation of CAMs' expression may importantly contribute to improve our knowledge about the mechanisms responsible for the initiation, progression and clinical manifestations of atherosclerosis. Unfortunately, adhesion molecules are difficult to measure in vivo, but their soluble forms, probably resulting from proteolytic shedding, 9,10 may be detected in serum or plasma, and are increased in a variety of conditions in which their blood levels parallel the amount expressed on the cell surface. [START_REF] Adams | Detection of circulating intercellular adhesion molecule-1 after liver transplantation: evidence of local release within the liver during graft rejection[END_REF][START_REF] Nakai | Concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) correlated with expression of VCAM-1 mRNA in the human atherosclerotic aorta[END_REF] On the basis of this information, numerous studies have investigated the relationships between the soluble forms of the adhesion molecules and the various aspects of the atherosclerotic disease. The scope of this article is to summarize the findings of studies which investigated the role of soluble CAMs in peripheral arterial disease (PAD), which represents a major manifestation of atherosclerosis. ligands. Three families of CAMs play a crucial role in the pathogenesis of atherosclerosis: the selectins, the integrins and the immunoglobulin superfamily (Table 1). These different types of CAMs interact with CAM chemoattractant/activator molecules in a programmed, sequential manner (Figure 1) to form the so-called leukocyte-endothelial cell adhesion cascade. [START_REF] Albelda | Adhesion molecules and inflammatory injury[END_REF]

Selectins

The initial phase of the adhesion cascade (ie leukocyte tethering and rolling along the endothelium surface) is mediated by members of the selectin family. The three members of this family are named according to the cell type on which each of them was originally identified: E-selectin (endothelium), P-selectin (platelets) and L-selectin (lymphocytes), but they are not exclusively located on these cells.

Although the selectins are closely related in structure and function, their patterns of expression are quite different. The expression of E-selectin appears to be largely restricted to activated endothelial cells, as this molecule is almost absent from resting endothelium. [START_REF] Bevilacqua | Selectins[END_REF] Once transcriptionally induced following cytokine stimulation, E-selectin, by interacting with their counter-receptors, participates in the adhesion of neutrophils, monocytes, and of a subpopulation of memory T lymphocytes to the endothelium. [START_REF] Bevilacqua | Selectins[END_REF][START_REF] Graber | T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1[END_REF] Eosinophils and basophils may also bind to endothelium through E-selectin. [START_REF] Weller | Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1[END_REF][START_REF] Bochner | Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules[END_REF] P-selectin is stored in the ␣-granules of platelets and in Weibel-Palade bodies of the endothelium. From these intracellular pools, after activation by thrombin and other mediators, P-selectin can be rapidly mobilized to the cell surface, where it binds neutrophils and monocytes, [START_REF] Mayadas | Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice[END_REF] interacting with the P-selectin glycoprotein ligand (PSGL-1). Thus, P-selectin mediates platelet-leukocyte and endothelial cell-leukocyte adhesion. Of the three selectins, only L-selectin is constitutively expressed at the cell surface. [START_REF] Bevilacqua | Selectins[END_REF] It is present in almost all leukocytes, and is shed from the cell surface within minutes after leukocyte activation. Soluble forms of E-selectin, P-selectin and L-selectin may be detected in serum and plasma.

Integrins

Although rolling is a prerequisite for the eventual firm adherence to blood vessels, the selectin-dependent adhesion of leukocytes does not lead to firm adhesion and transmigration unless another set of adhesion molecules is engaged. For neutrophils, firm adhesion requires activation of the integrin family, resulting in binding to one of the adhesion molecules on the surface of the endothelial cells.

The integrins comprise a superfamily of heterodimer transmembrane proteins composed of noncovalently associated ␣and ␤-subunits. [START_REF] Luscinskas | Integrins as dynamic regulators of vascular function[END_REF] Different cell types assemble and express different ␣␤ complexes. Beta-integrins are distributed in various kinds of cells, and mediate cell adhesion to proteins found in extracellular matrix. Among the ␤1 subunits, the ␣ 4 ␤ 1 integrin, [START_REF] Rose | The affinity of integrin alpha(4)beta(1) governs lymphocyte migration[END_REF] expressed on resting lymphocytes, may be the most relevant to atherosclerosis, as it has been shown to modulate the evolution of ischemic stroke. [START_REF] Becker | Antibody to the alpha4 integrin decreases infarct size in transient focal cerebral ischemia in rats[END_REF] Beta-2 integrins are strictly expressed in leukocytes and interact with immunoglobulin superfamily members (ICAM-1, ICAM-2, and ICAM-3) to induce a firm arrest of the white cells at the surface of the activated endothelium. The ␤3 subfamily of integrins are expressed in platelets. The ␣ 2 b/␤ 3 integrin, also known as glycoprotein (GP) IIb/IIIa, is specific for this cell type and its activation by thrombin and other agonists represents the final common pathway for platelet aggregation. Furthermore, GP IIb/IIIa also binds fibronectin and von Willebrand factor, thus mediating platelet adhesion to the endothelium and subendothelial matrix, and enabling the formation of a platelet thrombus with attachment to the vessel wall. [START_REF] Price | Cellular adhesion molecules and atherogenesis[END_REF] Different from GP IIb/IIIa, ␣V␤ 3 integrin is more widely expressed and is involved in cell migration and proliferation. [START_REF] Schwartz | Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation. Involvement of the alphaV integrins[END_REF]

Immunoglobulin superfamily

Transmigration of leukocytes, ie the final step of the leukocyte-endothelial cell cascade (Figure 1), is mediated by certain members of the immunoglobulin superfamily. [START_REF] Hogg | Structure and function of intercellular adhesion molecule-1[END_REF][START_REF] Muller | PECAM-1 is required for transendothelial migration of leukocytes[END_REF] These molecules are membrane glycoprotein receptors containing a variable number of extracellular immunoglobulin-like domains. [START_REF] Price | Cellular adhesion molecules and atherogenesis[END_REF] The ICAM family comprises five members, but ICAM-4 and ICAM-5, which are expressed in erythroid cells and in the brain, respectively, are out of the scope of this review. ICAM-1 and ICAM-2 are present constitutively on endothelial cells and leukocytes; however, the first one is upregulated by proinflammatory cytokines, whereas constitutive expression of ICAM-2 is relatively stable. [START_REF] Albelda | Adhesion molecules and inflammatory injury[END_REF] ICAM-3 is found in endothelial cells and leukocytes, and it is the only ICAM present on neutrophils. Each ICAM may bind more than one ligand. Unlike ICAMs, vascular cell adhesion molecule-1 (VCAM-1) is mainly restricted to the endothelial cells, although it may be present in several non-vascular cell types. [START_REF] Simmons | Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells[END_REF] Through an interaction with the integrin ␣ 4 ␤ 1 , endothelial VCAM-1 mediates the adhesion of lymphocytes and monocytes. Adhesion of eosinophils and basophils to activated endothelium also appears to involve VCAM-1, which, in addition, participates in the process of leukocyte migration. [START_REF] Matheny | Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase[END_REF] Another important member of the immunoglobulin superfamily is the platelet-endothelial cell adhesion molecule-1 (PECAM-1), which is expressed at relatively low levels on the surface of leukocytes and platelets, but at higher levels on the endothelium. [START_REF] Albelda | Adhesion molecules and inflammatory injury[END_REF] Its location at the junctions between endothelial cells has suggested a role in transendothelial migration of leukocytes. [START_REF] Muller | PECAM-1 is required for transendothelial migration of leukocytes[END_REF] In addition, PECAM-1 participates in homophilic binding between adjacent cells. [START_REF] Newton | Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31[END_REF] Soluble forms of ICAM-1, ICAM-3, VCAM-1 and PECAM-1 may be measured in serum and plasma.

Soluble adhesion molecules in PAD

Although the origin and function of the soluble forms of CAMs remain to be clarified, elevated serum and plasma (circulating) levels of these molecules have been found to be associated with the atherosclerotic burden, severity of PAD, exercise-induced claudication, restenosis after percutaneous transluminal angioplasty and increased cardiovascular risk.

Circulating levels of soluble CAMs in PAD patients vs controls

Circulating levels of sE-selectin, sP-selectin, sLselectin, sICAM-1 and sVCAM-1 have all been shown to be increased in PAD, although the findings of different studies are not consistent (Table 2). Elevated levels of sE-selectin have been detected in PAD patients with diabetes mellitus, [START_REF] Boulbou | Increased soluble E-selectin levels in type 2 diabetic patients with peripheral arterial disease[END_REF][START_REF] Elhadd | Elevated soluble cell adhesion molecules E-selectin and intercellular cell adhesion molecule-1 in type-2 diabetic patients with and without asymptomatic peripheral arterial disease[END_REF] but conflicting results were reported in non-diabetics. [START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFalpha), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Blann | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF] Similarly, although Pselectin has been consistently found to be overexpressed in platelets of PAD patients, [START_REF] Zeiger | P-selectin expression, platelet aggregates, and platelet-derived microparticle formation are increased in peripheral arterial disease[END_REF][START_REF] Koksch | Coagulation, fibrinolysis and platelet P-selectin expression in peripheral vascular disease[END_REF][START_REF] Cassar | Platelet activation is increased in peripheral arterial disease[END_REF] increased levels of the soluble form of this molecule were observed in some studies [START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFalpha), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Blann | Soluble L-selectin in peripheral arterial disease: relationship with soluble E-selectin and soluble P-selectin[END_REF] but not in others. [START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Kirkpatrick | Repeated exercise induces release of soluble P-selectin in patients with intermittent claudication[END_REF] With respect to the members of the immunoglobulin superfamily, sICAM-1 has been reported to be increased in PAD [START_REF] Elhadd | Elevated soluble cell adhesion molecules E-selectin and intercellular cell adhesion molecule-1 in type-2 diabetic patients with and without asymptomatic peripheral arterial disease[END_REF][START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFalpha), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Blann | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF][START_REF] Blann | Circulating ICAM-1 and VCAM-1 in peripheral artery disease and hypercholesterolaemia: relationship to the location of atherosclerotic disease, smoking, and in the prediction of adverse events[END_REF][START_REF] Brevetti | High levels of adhesion molecules are associated with impaired endotheliumdependent vasodilation in patients with peripheral arterial disease[END_REF] with one exception. [START_REF] Boulbou | Increased soluble E-selectin levels in type 2 diabetic patients with peripheral arterial disease[END_REF] For sVCAM-1, the findings are less consistent. The explanation for these heterogeneous results remains obscure. Increased circulating levels of the adhesion molecules are due to a variety of molecular and cellular factors, including the increased transcription of genes, alteration of mRNA stability, changes in translation, production of alternatively spliced forms and enhanced proteolytic cleavage from the cell surface. [START_REF] Blankenberg | Adhesion molecules and atherosclerosis[END_REF] None of these factors was investigated in the studies reported in Table 2. In addition, little is known about the clearance and catabolism of soluble CAMs. This lack of knowledge of the mechanisms determining the blood levels of these molecules is obviously a barrier in understanding between study discrepancies, which may be due also to differences in study populations and diagnostic criteria. [START_REF] Boulbou | Increased soluble E-selectin levels in type 2 diabetic patients with peripheral arterial disease[END_REF][START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Blann | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF][START_REF] Peter | Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin[END_REF] Soluble CAMs and atherosclerotic burden in PAD Given the systemic nature of atherosclerosis, individuals with PAD are often affected by concomitant coronary and/or carotid artery disease. [START_REF] Eberhardt | Cardiovascular morbidity and mortality in peripheral arterial disease[END_REF] A recent study [START_REF] Brevetti | Increased inflammatory status and higher prevalence of three-vessel coronary artery disease in patients with concomitant coronary and peripheral atherosclerosis[END_REF] has shown that patients with both peripheral and coronary artery disease have plasma levels of sICAM-1 and sVCAM-1 similar to those affected only by coronary artery disease. Similarly, in two separate studies, Blann et al found that, compared with patients with arterial disease at one site, those with disease at two or more sites had similar circulating levels of sPselectin [START_REF] Blann | Soluble P selectin in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease and its risk factors[END_REF] and sE-selectin. [START_REF] Blann | Circulating endothelial cell markers in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease[END_REF] These findings indicate that soluble forms of the adhesion molecules do not differentiate the extent of atherosclerosis. However, in 52 PAD patients, Peter et al [START_REF] Peter | Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin[END_REF] observed that serum levels of sVCAM-1 significantly correlated with the extent of atherosclerosis angiographically detected in infrarenal aorta and in the arteries of the lower limbs. Therefore, although the association of the adhesion molecules with the atherogenic process is well established, the associations between the circulating levels of the soluble forms and the atherosclerotic burden are less robust. This may in part be explained by the complex interaction of effects on protein synthesis and rate of shedding, which could be responsible for differences in associations between the plasma levels of different adhesion molecules and the extent of atherosclerosis.

Soluble CAMs and PAD severity

From a clinical point of view, PAD patients are classically divided into four stages according to the Fontaine's classification: stage I, asymptomatic PAD; stage II, intermittent claudication; stage III, pain at rest; stage IV, trophic lesions. Interestingly, Peter et al [START_REF] Peter | Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin[END_REF] found that serum levels of sVCAM-1, although showing a high correlation with leg atherosclerosis, did not reflect clinical stages of PAD. Similarly, a more recent study reported that plasma levels of both sVCAM-1 and sICAM-1 did not differentiate stage II from stage I patients. [START_REF] Silvestro | Inflammatory status and endothelial function in asymptomatic and symptomatic peripheral arterial disease[END_REF] However, when stage I and stage II patients were considered collectively, plasma levels of these two adhesion molecules showed a significant, albeit weak, negative relationship with the ankle/brachial index (ABI), [START_REF] Silvestro | Inflammatory status and endothelial function in asymptomatic and symptomatic peripheral arterial disease[END_REF] which is a marker of PAD severity. [START_REF] Fowkes | Validity of duplex scanning in the detection of peripheral arterial disease in the general population[END_REF] With respect to the relationship between selectins and PAD severity, patients with ischemic rest pain (stage III) have been found to have similar levels of sP-selectin than those less severely affected. [START_REF] Makin | Thrombogenesis and endothelial damage/dysfunction in peripheral artery disease. Relationship to ethnicity and disease severity[END_REF] Taken together, these findings suggest that plasma levels of sCAMs, while not reflecting clinical stages, correlate with more objective measures of PAD severity, such as the ABI and the extent of atherosclerosis in the arteries of the lower limbs.

Adhesion molecules and exercise-induced claudication

Although in PAD the primary determinant of inadequate blood supply to the affected limb during exercise is a flow-limiting lesion of a conduit artery, increased leukocyte adhesivity and platelet aggregation, by altering blood rheology, may contribute to the pathophysiology of intermittent claudication. [START_REF] Brevetti | Microcirculation and tissue metabolism in peripheral arterial disease[END_REF] Indeed, acute exercise affects various components of the immune system, including the function and number of leukocytes, [START_REF] Nieman | Immune response to heavy exertion[END_REF] and the surface expression of adhesion molecules may importantly contribute to such responses by changing patterns of cell trafficking. [START_REF] Shephard | Adhesion molecules, catecholamines and leucocyte redistribution during and following exercise[END_REF] In PAD patients, in whom intermittent claudication is associated with neutrophil activation [START_REF] Neumann | Activation and decreased deformability of neutrophils after intermittent claudication[END_REF] and systemic endothelial dysfunction, [START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF] exercise has been shown to induce reduction in the number of leukocytes expressing the ␣M/␤ 2 and ␣X/␤ 2 integrins. [START_REF] Ciuffetti | Human leucocyte-endothelial interactions in peripheral arterial occlusive disease[END_REF] This indicates that leukocyte-endothelial interactions occur during intermittent claudication. In addition, plasma levels of both sICAM-1 (the endothelial counter-receptor of ␣M/␤ 2 integrin) and sVCAM-1 have been shown to increase after maximal exercise in claudicants, but not in control subjects. [START_REF] Brevetti | Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication[END_REF][START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFalpha), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF] A more recent study [START_REF] Silvestro | Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication[END_REF] reported that the increase in sICAM-1 levels paralleled an increase in thiobarbituric acid-reactive substances (TBARS), and was prevented by intravenous administration of antioxidant vitamin C. Furthermore, such an increase was not observed after a submaximal exercise not heavy enough to induce TBARS elevation.

Collectively, these findings indicate that changes in sICAM-1 observed in claudicants performing a maximal exercise are presumably due to increased oxidative stress. Also P-selectin levels have been found to increase with exercise, [START_REF] Signorelli | High circulating levels of cytokines (IL-6 and TNFalpha), adhesion molecules (VCAM-1 and ICAM-1) and selectins in patients with peripheral arterial disease at rest and after a treadmill test[END_REF][START_REF] Kirkpatrick | Repeated exercise induces release of soluble P-selectin in patients with intermittent claudication[END_REF] thus indicating that platelet activation occurs during claudication.

Adhesion molecules, percutaneous transluminal intervention and restenosis

Because percutaneous transluminal angioplasty (PTA) has low procedural risk, PAD patients with lifestylelimiting claudication are often considered for this therapeutic approach. However, the long-term benefit of PTA continues to be compromised by the recurrence of stenotic lesion.

A number of animal studies suggest that adhesion molecules may play a role in the pathophysiology of peripheral vessel restenosis. In experimental models, the absence of ␣M/␤ 2 integrin and blockade of both VCAM-1 and its ligand ␣ 4 ␤ 1 reduced intimal hyperplasia after ballon-induced carotid artery injury. [START_REF] Simon | Decreased neointimal formation in Mac-1 Ϫ/Ϫ mice reveals a role for inflammation in vascular repair after angioplasty[END_REF][START_REF] Oguchi | Monoclonal antibody against vascular cell adhesion molecule-1 inhibits neointimal formation after periadventitial carotid artery injury in genetically hypercholesterolemic mice[END_REF][START_REF] Lumsden | Anti-VLA-4 antibody reduces intimal hyperplasia in the endarterectomized carotid artery in nonhuman primates[END_REF] Similarly, P-selectin-deficient mice were protected from neointimal hyperplasia after transluminal endothelial injury of the femoral artery. [START_REF] Smyth | Beta(3)-integrin-deficient mice but not P-selectin-deficient mice develop intimal hyperplasia after vascular injury: correlation with leukocyte recruitment to adherent platelets 1 hour after injury[END_REF] Furthermore, in rabbit models of balloon-induced injury of the iliac artery, antibody blockade of the ␣M/␤ 2 integrin, and treatment with the oligosaccharide SLe x , which blocks the selectin effects, resulted in a lower intimal/media ratio and a smaller area of stenosis. [START_REF] Rogers | A mAb to the beta2leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits[END_REF][START_REF] Barron | Intimal hyperplasia after balloon injury is attenuated by blocking selectins[END_REF] In humans, only two studies reported an association between adhesion molecules and restenosis in PAD. [START_REF] Tsakiris | Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease[END_REF][START_REF] Belch | The white blood cell adhesion molecule E-selectin predicts restenosis in patients with intermittent claudication undergoing percutaneous transluminal angioplasty[END_REF] Tsakiris et al found that those patients who developed restenosis 6 months after PTA had higher circulating levels of sP-selectin and sVCAM-1. [START_REF] Tsakiris | Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease[END_REF] Similarly, in 42 patients with intermittent claudication, increased plasma levels of sE-selectin were found to be predictive of restenosis at 1 year. [START_REF] Belch | The white blood cell adhesion molecule E-selectin predicts restenosis in patients with intermittent claudication undergoing percutaneous transluminal angioplasty[END_REF] Taken together, animal and human studies indicate that all three families of CAMs may be involved in the process of restenosis after PTA, and suggest that the blockade of some of them may be a potential therapeutic strategy to decrease restenosis. In this regard it is noteworthy that cilostazol, by inhibiting leukocyte ␣M/␤ 2 integrin, the transcardiac expression of which is increased during percutaneous coronary intervention, [START_REF] Inoue | Stent-induced expression and activation of the leukocyte integrin Mac-1 is associated with neointimal thickening and restenosis[END_REF] leads to a potential reduction in restenosis after coronary stent implantation. [START_REF] Inoue | Cilostazol inhibits leukocyte integrin Mac-1, leading to a potential reduction in restenosis after coronary stent implantation[END_REF] Unfortunately, although cilostazol is effectively used in PAD to improve claudication, [START_REF] Regensteiner | Effect of cilostazol on treadmill walking, community-based walking ability, and health-related quality of life in patients with intermittent claudication due to peripheral arterial disease: meta-analysis of six randomized controlled trials[END_REF] no study has investigated its ability to prevent post-PTA restenosis in these patients.

sCAMs and risk prediction

Although the mechanisms that modulate the circulating levels of sCAMs remain unclear, the value of these molecules for the prediction of vascular risk has been assessed in apparently healthy subjects [START_REF] Ridker | Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men[END_REF][START_REF] Ridker | C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women[END_REF][START_REF] Hwang | Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study[END_REF][START_REF] Ridker | Soluble P-selectin and the risk of future cardiovascular events[END_REF] and in patients with CAD. [START_REF] Blankenberg | Circulating cell adhesion molecules and death in patients with coronary artery disease[END_REF][START_REF] Mulvihill | Risk stratification in unstable angina and non-Q wave myocardial infarction using soluble cell adhesion molecules[END_REF] With respect to PAD, the Physician's Health Study, conducted among middle-aged healthy men, showed that increased plasma levels of sICAM-1 but not sVCAM-1 were associated with a threefold higher risk of developing symptomatic atherosclerosis in the lower limbs. [START_REF] Pradhan | Soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, and the development of symptomatic peripheral arterial disease in men[END_REF] Noteworthy, this predictive power was maintained after adjustment for classic risk factors and C-reactive protein. The finding of the Physician's Health Study seems to be consistent with the observation that polymorphism of the EE genotype of the ICAM-1 gene significantly increases the risk of PAD. [START_REF] Gaetani | The K469E polymorphism of the ICAM-1 gene is a risk factor for peripheral arterial occlusive disease[END_REF] Furthermore, a recent 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] has shown that sICAM-1 is a significant predictor of lower-extremity atherosclerotic progression measured by ABI over 12 years of follow-up independently of cardiovascular risk factors. In the setting of secondary prevention, Blann et al found that increased circulating levels of sICAM-1 were a significant predictor of adverse cardiovascular events in a group of patients with ultrasound-proven atherosclerosis (stenosis Ͼ 70%) of carotid, iliac or femoral arteries. [START_REF] Blann | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF] Different results were observed in another study. [START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] In a well-defined population of patients with obliterative atherosclerosis of the lower limbs, plasma levels of sVCAM-1 were 618 Ϯ 258 ng/ml in those with and 496 Ϯ 164 ng/ml in those without an event ( p ϭ 0.016). The corresponding sICAM-1 values were 344 Ϯ 239 ng/ml and 275 Ϯ 99 ng/ml ( p ϭ 0.079). Thus, an increased cardiovascular risk was associated with increased plasma levels of sVCAM-1 (relative risk ϭ 4.06; 95% confidence intervals ϭ 1.53-10.74), but not of sICAM-1. [START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] The discrepancy between the two studies is difficult to explain. The expression of ICAM-1 is strongly upregulated by inflammatory cytokines in endothelial cells, fibroblasts, epithelial cells and multiple cells of hematopoietic lineage. [START_REF] Blankenberg | Adhesion molecules and atherosclerosis[END_REF] By contrast, VCAM-1 is more anatomically restricted to activated endothelial cells in lesion-prone areas, [START_REF] Li | An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium[END_REF] and is more prominent on intimal neovasculature than on the arterial luminal surface of complex lesions. [START_REF] O'brien | Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content[END_REF] Thus, although ICAM-1 and VCAM-1 are structurally and functionally similar, plasma levels of sICAM-1 may be considered a marker of a more generalized inflammatory process, whereas VCAM-1 seems to be an indicator of plaque burden or activity. Probably as a consequence of these differences, sVCAM-1 emerges as a risk predictor in patients with advanced atherosclerosis, [START_REF] Blankenberg | Circulating cell adhesion molecules and death in patients with coronary artery disease[END_REF][START_REF] Mulvihill | Risk stratification in unstable angina and non-Q wave myocardial infarction using soluble cell adhesion molecules[END_REF][START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] whereas sICAM-1 is a better prognostic indicator in healthy individuals. [START_REF] Ridker | Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men[END_REF][START_REF] Ridker | C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women[END_REF] It is noteworthy that, in PAD, the predictive value of sVCAM-1 is independent of classic risk factors, previous myocardial infarction and stroke, [START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] which are conditions associated with increased circulating levels of adhesion molecules. [START_REF] Gearing | Circulating adhesion molecules in disease[END_REF][START_REF] Ohta | Soluble vascular celladhesion molecule-1 and soluble intercellular adhesion molecule-1 correlate with lipid and apolipoprotein risk factors for coronary artery disease in children[END_REF] Even more important, sVCAM-1 measurement adds to the predictive value of ABI (Figure 2), which is to date the most powerful prognostic risk indicator in PAD. [START_REF] Brevetti | Intermittent claudication and risk of cardiovascular event[END_REF][START_REF] Mckenna | The ratio of ankle and arm arterial pressure as an independent predictor of mortality[END_REF] In particular, among patients with an ABI Ͻ median (ie patients at higher cardiovascular risk), those with sVCAM-1 Ͼ median had a 3.4-fold increased risk of feature events as compared to patients with sVCAM-1 Ͻ median. A similar additive prognostic value has been reported for reduced brachial artery flowmediated dilation. [START_REF] Brevetti | Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle-brachial pressure index[END_REF] Collectively, these findings indicate that markers of endothelium activation/dysfunction are strong predictors of cardiovascular risk in PAD.

Potential significance of adhesion molecules in clinical practice

As our knowledge of the atherosclerotic process improves, so will our working concepts of the strategies for defining and reducing the cardiovascular risk. Therefore, studies of the adhesion molecules, by providing new insights into the pathophysiology of PAD and its consequences, have potential clinical implications.

First, circulating levels of sCAMs could be used to gauge the cardiovascular risk of PAD patients. However, at the present, sCAMs' assay does not fulfill all the conditions required because a diagnostic test enters into clinical practice. [START_REF] Pearson | Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association[END_REF] Nevertheless, the finding that in PAD, sVCAM-1 measurement improves the stratification of prognosis provided by a well-established risk factor such as the ABI, [START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] encourages further efforts in this field.

Second, the observation that increased preprocedural levels of sVCAM-1, sP-selectin and sE-selectin are associated with restenosis after PTA [START_REF] Tsakiris | Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease[END_REF][START_REF] Belch | The white blood cell adhesion molecule E-selectin predicts restenosis in patients with intermittent claudication undergoing percutaneous transluminal angioplasty[END_REF] suggests that these markers may be of some value in the selection of patients for PTA and could constitute a new therapeutic target. Indeed, the phosphodiesterase III inhibitor cilostazol prevents restenosis after percutaneous coronary angioplasty, [START_REF] Inoue | Cilostazol inhibits leukocyte integrin Mac-1, leading to a potential reduction in restenosis after coronary stent implantation[END_REF][START_REF] Douglas | Rationale and design of the randomized, multicenter, cilostazol for RESTenosis (CREST) trial[END_REF] probably by inhibiting leukocyte ␣M/␤ 2 integrin. [START_REF] Inoue | Cilostazol inhibits leukocyte integrin Mac-1, leading to a potential reduction in restenosis after coronary stent implantation[END_REF] Future studies will clarify whether cilostazol is effective also in preventing peripheral vessel restenosis after PTA.

Finally, for sICAM-1 and sVCAM-1, which are considered important markers of inflammation and endothelial dysfunction, another potential use that might be considered is that of monitoring therapies aimed at reducing the inflammatory status and improving endothelial function. If plasma levels of these molecules do not fall, treatment might be intensified. Large clinical trials are needed to verify whether PAD patients with elevated levels of circulated CAMs would benefit from any specific therapy.

Conclusions

Studies of adhesion molecules provide a substantial insight into the pathophysiology of PAD. However, the clinical utility of measuring the circulating forms of these markers remains uncertain. First, to have a clinical role, the assay of these molecules should be less expensive. Second, there must be a greater number of prospective studies demonstrating consistently that adhesion molecules, in PAD, add substantially to our ability to predict risk beyond that achievable by use of the ABI and traditional risk factors. Finally, of critical need are a large number of studies showing that a reduction in the plasma levels of adhesion molecules, obtained through specific interventions, results in a reduction of cardiovascular risk. Therefore, although extraordinarily valuable as research tools, we must await the completion of prospective evaluations of various panels of these molecules before introducing their use into daily clinical practice. (Adapted from ref. [START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] ).

Figure 1

 1 Figure 1 Schematic representation of the leukocyte tethering/rolling, adhesion and transmigration across the endothelium, and the cellular adhesion molecules involved in these processes.

Figure 2

 2 Figure 2Kaplan-Meier analysis of event (cardiac ϩ cerebrovascular)-free survival curves refers to a subgroup of patients categorized as being above or below the median values for sVCAM-1 and ABI. The combination of sVCAM-1 and ABI below the median was associated with the highest cardiovascular event incidence. (Adapted from ref.[START_REF] Silvestro | Adhesion molecules and cardiovascular risk in peripheral arterial disease. Soluble vascular cell adhesion molecule-1 improves risk stratification[END_REF] ).

Table 1

 1 Adhesion molecules involved in atherosclerosis, their cellular function and their biological and pathological implications.

			Biological
	Adhesion	Cellular	and pathological
	molecules	function	implications
	Selectins	Leukocyte-	Leukocyte
		endothelium	rolling and
		interaction	tethering
		Leukocyte-
		platelet
		interaction
	Integrins	Leukocyte-	Firm adhesion,
		endothelium	spreading,
		interaction	homing to
		Platelet-	inflamed tissue
		endothelium
		interaction
	Immunoglobulins	Leukocyte-	Firm adhesion,
		leukocyte	leukocyte
		interaction	extravasation,
		Leukocyte-	inflammatory
		endothelium	and immune
		interaction	responses

Table 2

 2 Studies showing an increase (YES) or not (NO) in plasma levels of adhesion molecules in PAD patients versus controls.

		sE-selectin	sP-selectin	sL-selectin	sICAM-1	sVCAM-1
	Boulbou et al 28	YES (d)	-	-	NO (d)	NO (d)
	Elhadd et al 29	YES (d)	-	-	YES (d)	-
	Brevetti et al 30	NO	NO	-	YES	NO
	Signorelli et al 31	YES	YES	YES	YES	YES
	Blann and McCollum et al 32	NO	-	-	YES	NO
	Blann et al 36	NO	YES	NO	-	-
	Kirkpatrick et al 37	-	YES	-	-	-
	Blann et al 38	-	-	-	YES	YES
	Brevetti et al 39	-	-	-	YES	NO
	Peter et al 40	-	-	-	-	YES

(d), patients with diabetes mellitus.

Vascular Medicine 2006; 11: 39-47


Oliver K Mohrs
email: o.mohrs@gmx.de

Markus K Heinemann

Dieter Eckhardt

Bernd Nowak

Hans-Ulrich Kauczor

Thomas Voigtlaender

Mark A Creager

Images in vascular medicine

Panel A Panel B

A 17-year-old woman had suffered from dyspnoea and cyanosis since childhood. Physical examination revealed central and peripheral cyanosis. Auscultation of the heart and lungs was unremarkable. Arterial blood gas analysis demonstrated an oxygenation saturation of 83%. This did not improve during inhalation of oxygen. Echocardiography showed good left ventricular function; the ventricles and atria were not enlarged. Intracardiac shunts, patent ductus arteriosus and valve disease were excluded. Intravenous administration of D-galactose-microbubbles caused an enhancement of the left atrium and ventricle, consistent with an intracardiac or extracardiac shunt. A chest radiograph showed diffuse opacities in the periphery of the right lung (Panel A, arrows). Owing to the differential diagnosis of abnormal pulmonary vasculature, the patient was referred for cardiovascular MRI.

Panel C

Using contrast-enhanced MR angiography we found multiple arteriovenous malformations mainly in the upper and middle lobe of the right lung (Panel B, arrows; PA ϭ pulmonary artery, PV ϭ pulmonary vein). These were not configured in a typical way as nodular structures with feeding (Panel C, dotted arrow) and draining vessels (Panel C, curved arrow), but showed a reticular pattern.

Velocity-encoded MRI-based flow measurement excluded a left-to-right shunt comparing flow volumes of the main pulmonary arteries and ascending aorta.

In order to quantify the intrapulmonary shunt volume we performed further flow measurements in the segmental pulmonary veins draining the three main malformations. Over 30% of the pulmonary blood flow shunted through the arteriovenous malformations, explaining decreased blood oxygen saturation, the dyspnoea and the abnormal findings of contrast-enhanced echocardiography.

In our report, we present the potential of MRI to detect even very rare malformations of the pulmonary vasculature and to allow for functional assessment of haemodynamic parameters.


Stefan Ockert
email: stefan_ockert@med.uni-heidelberg.de

Dittmar Böckler

Marika Ganten

Hardy Schumacher

Jens-Rainer Allenberg

Images in vascular medicine

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

  year-old patient was treated for a symptomatic inflammatory aortic aneurysm of 8 cm in diameter in November 2002. He underwent an uncomplicated open aortic repair with implantation of an 18 ϫ 9 mm bifurcated Dacron prosthesis. Fifteen months after the operation ultrasound examination detected a persistent aneurysm without shrinking. For further investigation a 16-row multidetector computed tomography was performed. The analysis of the CTA showed an expanded aneurysm sac around the perfused prosthesis (Panel A, transverse CTA: arrow shows the sac; Panel, coronal CTA: arrow shows persistent inflammation). Contrast medium extravasation into the sac as an indication of active bleeding could not be detected. The aortic wall demonstrated a thickening of 3-5 mm as a sign of persistent inflammation. A 3D CTA volume rendering reconstruction shows the expanded aneurysm sac with intense calcification of the aortic wall (Panel C, lateral view: arrow shows calcification of the aneurysm sac; Panel D, anterior-posterior view).


Rafal Poreba

Karol Poczatek

Arkadiusz Derkacz

Ryszard Andrzejak

Andrzej Szuba

Images in vascular medicine

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A 67-year-old woman with a 30-year history of hypertension was referred to our department for poorly controlled blood pressure and unstable angina (CCS II/III). A physical examination revealed no significant abnormalities. The systolic arterial pressure on the left arm was 15 mmHg lower than that measured on the right arm. The patient underwent coronary angiography in our institution. Cardiac catheterization from femoral access was impossible due to an inability to cross the aortic arch. Successful catheterization was achieved through the right radial artery. Coronary angiography revealed disseminated atherosclerotic lesions with multiple stenoses up to 50%. Simultaneous aortography demonstrated an atypical long narrowing of the aortic arch. An angio-CT revealed the normal proximal part of the ascending aorta. Behind the origin of the brachiocephalic trunk, atypical constriction of the arch of aorta with winding course and numerous angular bends was seen (Panels A and B). The constriction involved the whole length of the arch behind the take-off of the brachiocephalic trunk. The Botall duct was not visualized. Post-stenotic dilatation of up to 3.5 cm of descending aorta was present (Panel A). The classic pattern of collateral circulation was not visible. The patient did not consent to surgical reconstruction.

Coarctation of the aorta may be present in about 0.2% of cases of hypertension. Atypical aortic coarctation is very rare and found in 0. Williams or Turner's syndromes is unlikely (lack of cutaneous neurofibromas and normal physical examination). Atherosclerotic disease of the aorta can be a cause of aortic coarctation; however, published reports usually describe atherosclerotic coarctation being due to heavily calcified, 'coral reef'-type lesions. Morphological features of fibromuscular dysplasia in CT angiography (beads on a string appearance, a long-segment tubular stenosis), absence of clinical signs and symptoms of inflammatory process, lack of aortic calcifications and smooth lumen of the aorta may support the diagnosis of fibromuscular dysplasia rather then aortitis (eg Takayasu disease) or atherosclerosis. However, without histopathological verification we cannot rule out atherosclerotic disease, aortitis or developmental abnormality.

a'

  5-2% of patients with aortic coarctation. Causes of atypical coarctation of the aorta include developmental vascular abnormalities, genetic disorders (Williams syndrome, neurofibromatosis type I, Turner's syndrome), fibromuscular dysplasia, large vessel vasculitides (especially Takayasu disease) and atherosclerosis. Diagnosis of neurofibromatosis type I, Vascular Medicine 2006; 11: Departments of Internal, Occupational Diseases and Hypertension, and b Cardiology, Wroclaw University of Medicine, Wroclaw, Poland Address for correspondence: Andrzej Szuba, Associate Professor of Medicine, Department of Internal Medicine, Occupational Diseases and Hypertension, Wroclaw Medical University, Pasteura 4 Street; 50-367 Wroclaw, Poland. E-mail: szubaa@yahoo.com 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.


Dittmar Böckler
email: dittmar_boeckler@med.uni-heidelberg.de

Hardy Schumacher Stefan Ockert

Jens-Rainer Allenberg

Undiagnosed fatal mesenteric ischemia in acute type B aortic dissection

A 48-year-old man with a 2-day history of acute onset of upper back discomfort was admitted to the hospital. Physical examination was unremarkable and a chest radiograph was normal. A computed tomographic (CT) scan confirmed acute aortic dissection type B without ischemic complications (Panel A, white arrows show dissection membrane). The patient was treated with aggressive blood-pressure control in the intensive care unit. Measurement of serum lactate, GOT

'

  oxalat-transaminase), GPT (glutamat-pyruvattransaminase), LDH (laktatdehydrogenase), creatinine and urea confirmed highly elevated levels 20 hours later. The patient did not have abdominal pain. A CT scan was repeated (Panel B), suggesting hemodynamic impairment of the superior mesenteric artery (SMA) originating from the collapsed true lumen (TLC; arrows) due to low contrast enhancement. Selective angiography suggested compromised perfusion of the SMA due to delayed contrast inflow (Panel C, movie clip). The patient underwent endovascular repair. An acute abdomen developed 12 hours after endograft implantation. At immediate surgery the colon was found to be ischemic and a bowel resection was performed (Panel D). The patient died of multiorgan failure on day 3 after surgery. Medial dissection of the aortic wall with fragmentation of elastic lamellae, suspicious for connective tissue disorder, is seen on histological sections (van Gieson-elastic stain, 100ϫ) (Panel E, arrows indicate tears of elastic fibers). Mesenteric ischemia remains a very difficult diagnostic and therapeutic problem. The role of endovascular treatment of acute asymptomatic type B dissection in the early onset needs to be evaluated in the future. 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.


Russell Stein

Ingrid Hriljac

Jonathan L Halperin

Susan M Gustavson

Victoria Teodorescu

Jeffrey W Olin
email: jeffrey.olin@msnyuhealth.org

Dr Thom W Rooke

Limitation of the resting ankle-brachial index in symptomatic patients with peripheral arterial disease

Keywords: claudication, peripheral arterial disease, vascular laboratory

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Peripheral arterial disease (PAD) affects 5 to 10 million adults in the USA. [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][START_REF] Criqui | Peripheral arterial disease -epidemiological aspects[END_REF] Patients with PAD have a 6.6-fold greater risk of death from coronary disease compared to patients without PAD and a 3.1-fold greater mortality from any cause. [START_REF] Criqui | Mortality over a period of 10 years in patients with peripheral arterial disease[END_REF] In the PARTNERS study of 6979 primary care patients, 29% of those who were either older than 70 years or between the ages of 50 and 69 years with a history of tobacco smoking or diabetes mellitus had PAD. [START_REF] Hirsch | Peripheral arterial disease detection, awareness, and treatment in primary care[END_REF] Despite its known association with coronary artery disease, PAD was typically unrecognized or under-treated because many patients with PAD are asymptomatic or have atypical symptoms, and findings on physical examination may be subtle or misleading. [START_REF] Mcdermott | Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general medicine practice[END_REF][START_REF] Criqui | The sensitivity, specificity, and predictive value of traditional clinical evaluation of peripheral arterial disease: results from noninvasive testing in a defined population[END_REF] The lower the ankle-brachial index (ABI), the higher the cardiovascular mortality. [START_REF] Hirsch | Peripheral arterial disease detection, awareness, and treatment in primary care[END_REF][START_REF] Mcdermott | Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general medicine practice[END_REF][START_REF] Papamichael | Ankle-brachial index as a predictor of the extent of coronary atherosclerosis and cardiovascular events in patients with coronary artery disease[END_REF] It is recommended that the ABI be measured in high-risk patients to better identify patients who may benefit from more stringent cardiovascular risk factor modifications. [START_REF] Mcdermott | Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general medicine practice[END_REF] When taken at rest, the ABI is highly specific (99%) and sensitive (94-97%) for the detection of high-grade stenosis as assessed by angiography. [START_REF] Carter | Clinical measurement of systolic pressures in limbs with arterial occlusive disease[END_REF][START_REF] Ouriel | Doppler ankle pressure: an evaluation of three methods of expression[END_REF][START_REF] Yao | Ankle systolic pressure measurements in arterial disease affecting the lower extremities[END_REF][START_REF] Carter | Indirect systolic pressures and pulse waves in arterial occlusive diseases of the lower extremities[END_REF] The ABI may underestimate the severity of PAD in patients with non-compressible vessels due to arterial calcification; [START_REF] Quigley | A comparison of Doppler ankle pressures and skin perfusion pressure in subjects with and without diabetes[END_REF] however, most commonly in the presence of diabetes mellitus, end-stage renal disease or advanced age. [START_REF] Taguchi | Pipe-stem" brachial arteries: a cause of pseudohypertension[END_REF] In patients with arterial calcification, pulse volume recording (PVR) in the vascular laboratory overcomes this limitation of ABI measurement, with greater sensitivity for the diagnosis of PAD. [START_REF] Ouriel | Doppler ankle pressure: an evaluation of three methods of expression[END_REF][START_REF] Kempczinski | Segmental volume plethysmography in the diagnosis of lower extremity arterial disease[END_REF][START_REF] Darling | Quantitative segmental pulse volume recorder: a clinical tool[END_REF] Additionally, falsely negative results may arise in some patients with PAD who have normal ABI at rest but a fall in the ABI after limb exercise. [START_REF] Carter | Response of ankle systolic pressure to leg exercise in mild or questionable arterial disease[END_REF][START_REF] Ouriel | Critical evaluation of stress testing in the diagnosis of peripheral vascular disease[END_REF] Spurred by the results of the PARTNERS study and other initiatives, it is recommended that practitioners measure the ABI in the office in patients at risk of PAD, raising questions about the proper role of the vascular laboratory in diagnosis. The aims of this study were to determine the diagnostic utility of measuring the ABI at rest in patients referred to the vascular laboratory for evaluation of suspected PAD, and to assess the added value of PVR and post-exercise studies in patients with a normal ABI. The results may help guide more appropriate outpatient use of vascular diagnostic laboratory testing by general physicians.

Methods

In this retrospective and observational study, the vascular diagnostic laboratory database was queried for symptomatic outpatients referred for measurement of segmental blood pressures, ABI and PVRs by physicians not specialized in the evaluation and management of patients with peripheral vascular disease. All vascular studies were performed at the Marie-Josée and Henry R Kravis Vascular Diagnostic Laboratory in the Zena and Michael A Wiener Cardiovascular Institute of the Mount Sinai School of Medicine. The laboratory is accredited by the Intersocietal Commission for the Accreditation of Vascular Laboratories and all of the technologists and medical directors (VT, JWO) are Registered Vascular Technologists. Of 707 patients undergoing outpatient physiologic arterial evaluations between February 1, 2003 and July 31, 2004, 396 met these inclusion criteria. Vascular specialists were defined as physicians who predominantly practiced vascular surgery, vascular medicine, cardiovascular medicine with an emphasis on peripheral vascular diseases or interventional radiology. Indications for testing included intermittent claudication, cutaneous ulceration, rest pain, decreased pulses or cold feet.

The ABI was measured in both legs by sphygmomanometry, using an 8 MHz continuous-wave Doppler ultrasound transducer. The higher of the posterior tibial (PT) or dorsalis pedis (DP) artery systolic blood pressure values was taken as the numerator for each leg and the higher of the right and left brachial systolic blood pressure readings as the denominator. An ABI Յ 0.90 in either leg was considered abnormal. Arterial non-compressibility was diagnosed when the Doppler systolic signal was not obliterated at ankle cuff inflation pressures Ն250 mmHg.

Exercise evaluations were performed at the request of referring physicians. Ankle and brachial arterial pressure measurements were made at rest and repeated immediately after walking for 5 minutes on a treadmill at 12% grade and 2.0 mph (3.2 k/h) or until symptoms forced the patient to stop. A post-exercise ABI Յ 0.90 was considered abnormal. Segmental PVR was measured in the lower limbs using a standard protocol. [START_REF] Sumner | Volume plethysmography in vascular disease: an overview[END_REF] Any prolongation or attenuation of the pulse volume waveforms were considered abnormal.

Results

The study population (n ϭ 396) consisted of equal numbers of men and women (mean age 69 years, range 19-100 years). Among 396 studies, resting ABI values were normal in 183 (46.2%), abnormal in 159 (40.2%) and non-compressible in 54 (13.6%). The resting ABI values based on indications for testing in each referral group are shown in Figure 1.

Some patients with normal ABI did not undergo exercise testing because of their inability to exercise, severe cardiac disease, or physician or patient refusal. Of the 138 patients who underwent exercise testing, 84 had normal ABI readings at rest. Most of those with normal resting ABI values who underwent exercise tests (71.4%) had been referred because of intermittent claudication (Figure 2). In the 84 patients who had a normal ABI at rest and underwent exercise testing, the ABI fell below 0.9 after exercise in 26 (31%).

Arterial non-compressibility was detected in 54 (13.6%) patients, whose average age was 67 years. Thirteen (24%) of those with non-compressible vessels had abnormal PVR results, compared to five with normal resting ABI who had abnormal PVR findings (2.7%).

Discussion

Although PAD is prevalent, there are no exerciseinduced leg symptoms in approximately half of the cases and the diagnosis is often overlooked in clinical practice. [START_REF] Criqui | The sensitivity, specificity, and predictive value of traditional clinical evaluation of peripheral arterial disease: results from noninvasive testing in a defined population[END_REF] The Rose criteria [START_REF] Rose | The diagnosis of ischaemic heart pain and intermittent claudication in field surveys[END_REF] for intermittent claudication (exertional pain involving the calf that impedes walking, resolves within 10 minutes of rest, and neither begins at rest nor resolves on walking) are satisfied in fewer than 10% of patients with PAD [START_REF] Mcdermott | Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general medicine practice[END_REF] and the femoral pulse is preserved in about 87%. [START_REF] Criqui | The sensitivity, specificity, and predictive value of traditional clinical evaluation of peripheral arterial disease: results from noninvasive testing in a defined population[END_REF] The difficulty in using clinical symptoms to diagnose PAD is demonstrated by the fact that, among the study population, 47% of those with symptoms suspicious for claudication (51% of patients, with rest pain and 44% referred for ulceraction) had normal ABI values. The patients referred for claudication with normal resting ABI likely had either lesser degrees of stenosis that were not detected with resting ABI alone or symptoms simulating claudication but due to some other cause.

However, the normal resting ABI values in patients with rest pain or ulceration in all likelihood reflect errors in diagnosis by the referring non-vascular specialist.

An ABI value Ͻ0.9 correlates with angiographic evidence of obstructive PAD; sensitivity approaches 95% and specificity is almost perfect for the exclusion of patients without the disease. [START_REF] Carter | Clinical measurement of systolic pressures in limbs with arterial occlusive disease[END_REF][START_REF] Ouriel | Doppler ankle pressure: an evaluation of three methods of expression[END_REF][START_REF] Yao | Ankle systolic pressure measurements in arterial disease affecting the lower extremities[END_REF][START_REF] Carter | Indirect systolic pressures and pulse waves in arterial occlusive diseases of the lower extremities[END_REF][START_REF] Belch | Prevention of Atherothrombotic Disease Network. Critical issues in peripheral arterial disease detection and management: a call to action[END_REF] Few contemporary studies have evaluated the accuracy of the ABI for diagnosis of PAD in the broad spectrum of patients encountered in general clinical practice. Older studies were limited to cases with relatively advanced PAD. In a study of 146 limbs with angiographically documented atherosclerosis, for example, 112 (77%) had complete arterial occlusion; systolic blood pressure at the ankle was below normal in all cases. [START_REF] Carter | Indirect systolic pressures and pulse waves in arterial occlusive diseases of the lower extremities[END_REF] In the 85 control limbs without angiographically apparent stenosis, the ABI was Ն0.99. The ABI was less accurate across the middle part of the spectrum in 34 limbs with mild to severe arterial stenosis; abnormal in 44% of limbs with mild stenosis and 76% of those with severe stenosis. [START_REF] Carter | Indirect systolic pressures and pulse waves in arterial occlusive diseases of the lower extremities[END_REF] The predictive value of reduced ABI measurements for the detection of PAD of lesser severity is limited, with no significant difference in ABI between asymptomatic patients with angiographically normal-appearing arteries and those with nonobstructive PAD. [START_REF] Ouriel | Doppler ankle pressure: an evaluation of three methods of expression[END_REF] Exercise testing may enhance the detection of PAD in patients with non-obstructive PAD who have (falsely) normal ABI readings. [START_REF] Yao | Ankle systolic pressure measurements in arterial disease affecting the lower extremities[END_REF] The inability of resting ABI measurement to detect mild arterial stenosis results from preservation of perfusion pressure as described by Poisseuille's law governing flow across a zone of luminal narrowing in proportion to the 4th power of the radius. [START_REF] May | Critical arterial stenosis[END_REF][START_REF] May | Hemodynamic effects of arterial stenosis[END_REF] During exercise, flow cannot increase beyond the zone of significant stenosis despite the reduction of resistance resulting from arteriolar dilatation, so blood pressure drops distally (Ohm's law). [START_REF] Cappelen | The effect of obstructive arterial disease on the peripheral arterial blood pressure. Registrations from the dorsalis pedis artery of normal persons and patients before and after operation[END_REF] A fall in systolic pressure after exercise is the most sensitive test for detection of hemodynamically significant stenosis; thus, measurement of the ABI after exercise is most useful for the detection of PAD when stenosis is not severe enough to compromise perfusion at rest.

Of the patients who had normal ABI measurements at rest, 31% had abnormal readings after exercise. While angiographic documentation of the presence (and severity) or absence of PAD was not obtained, previous studies in patients with clinically suspected PAD and normal resting ABI readings have yielded conflicting results with respect to the utility of stress testing. In a study of 218 patients with PAD, stress testing increased the diagnostic yield by only 1.6% compared to ABI measurements at rest, and stress testing increased the yield by only 6% when rest and post-exercise ABI measurements were compared in the contralateral, asymptomatic limbs. The incremental yield rose to 22% when patients with intermittent claudication and normal resting ABI values were subjected to exercise testing. [START_REF] Ouriel | Critical evaluation of stress testing in the diagnosis of peripheral vascular disease[END_REF] Taken together with our findings, available data support the value of exercise assessment in patients with exertional limb symptoms who have normal ABI at rest. Many patients with normal ABI did not undergo exercise testing. The reasons for this included an inability to exercise on a treadmill, pre-existing cardiac disease, or at the request of the referring physician.

An alternative to treadmill exercise testing is active pedal plantar flexion (heel raises). Heel raising exercises are carried out to a maximum height achievable by a patient at a maximum speed possible for 30 seconds. Heel raising exercises produce changes in ankle pressure that correlate well with treadmill exercise. [START_REF] Amirhamzeh | A comparative study of treadmill tests and heel raising exercise for peripheral arterial disease[END_REF] A relatively high proportion of patients (13.6%) had arterial non-compressibility that produced falsely elevated ABI readings. In a previous study of over 600 limbs, Carter found the incidence of non-compressibility to be only 1%. [START_REF] Carter | Indirect systolic pressures and pulse waves in arterial occlusive diseases of the lower extremities[END_REF] The Strong Heart Study demonstrated the value of identifying subjects with either abnormally high or low ABI measurements; both cardiovascular and all-cause mortality were lowest in patients with ABI values between 1.0 and 1.39. Risk-adjusted odds ratios for all-cause mortality were 1.69 (1.34-2.14) and 1.77 (1.48-2.13) for those with an ABI Ͻ 0.90 and Ͼ1.40, respectively. Similar risk ratios for cardiovascular mortality were 2.52 for those in the low and 2.09 for the high ABI categories. [START_REF] Resnick | Relationship of high and low ankle brachial index to all-cause and cardiovascular disease mortality: the Strong Heart Study[END_REF]

Limitations

This study of outpatients referred to the vascular laboratory has inherent limitations linked to its retrospective design. Eligibility was limited to patients referred by physicians not specially trained in the care of patients with vascular disease, and the potential for referral bias makes it difficult to generalize the findings to other populations. Only limited information was available about other clinical features such as diabetes or manifestations of atherosclerotic disease in vascular beds other than the lower extremities. Perhaps most importantly is the lack of angiographic confirmation of the presence or severity of PAD by which to assess the overall accuracy of the noninvasive laboratory evaluation.

Conclusions

This study demonstrated that nearly half of the patients referred by non-vascular specialists to the outpatient vascular laboratory because of suspected arterial disease had a normal ABI. Even patients referred because of rest pain and/or ulceration had a normal ABI 51% and 44% of the time, respectively. Of the 84 patients with normal ABI readings, exercise testing disclosed an abnormality consistent with PAD in almost a third. While it is recommended that the ABI be measured at rest in patients at risk of PAD in primary care practice, [START_REF] Hirsch | Peripheral arterial disease detection, awareness, and treatment in primary care[END_REF] these findings suggest that patients with symptoms of PAD should be more completely evaluated in a vascular laboratory. Furthermore, when the ABI is normal at rest in patients with symptoms of claudication, exercise testing is recommended to enhance the sensitivity for the detection of PAD.

This study also demonstrated arterial noncompressibility in 13.6% of patients referred for ABI. PVRs were abnormal in 24% of this patient subgroup and thus it is a valuable diagnostic test, especially in patients with calcified vessels.

Figure 1

 1 Figure 1 Resting ankle-brachial index values in relation to the indication for study.

Figure 2

 2 Figure 2 Indications for exercise testing in patients with a normal resting ankle-brachial index.

Vascular Medicine 2006; 11: 29-33


Philippe Lacroix
email: philippe.lacroix@unilim.fr

Victor Aboyans

Mh H Criqui

F Bertin

T Bouhamed

F Archambeaud

M Laskar

Type-2 diabetes and carotid stenosis: a proposal for a screening strategy in asymptomatic patients

Keywords: carotid artery disease, screening, type 2 diabetes, ultrasonography

come

Introduction

Diabetes is a well-known risk factor for cardiovascular diseases. The prevalence of diabetes is dramatically increasing in western populations; currently 20 million individuals have diabetes in the USA, of whom more than 5 million remain undiagnosed. [START_REF] Winer | Epidemiology of diabetes[END_REF] The risk of coronary disorders or stroke in diabetic patients is three times that in individuals without this condition. [START_REF] Stamler | Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial[END_REF][START_REF] Weinberger | Factors contributing to stroke in patients with atherosclerotic disease of the great vessels: the role of diabetes[END_REF] Stroke is the third leading cause of death in western countries and is associated with a high level of disability and causes a tremendous financial burden. Carotid artery stenosis is one of the main causes of stroke. Duplex scanning allows efficient screening for carotid artery disease, but a trained physician or technologist is required and the equipment is still expensive. In most of the guidelines regarding diabetes, general screening for carotid disease is not recommended, mainly due to the cost and the low return when prevalence of carotid disease is low. [START_REF]Prevention Conference VI: Diabetes and Cardiovascular Disease: Writing Group III. Risk assessment in persons with diabetes[END_REF]5 Focused screening is recommended where there is a history of neurological symptoms or cervical bruit. 5 On the other hand, the Asymptomatic Carotid Atherosclerosis Study (ACAS) 6 and the Asymptomatic Carotid Surgery Trial (MRC ACST) [START_REF] Mrc Asymptomatic | Prevention of disabling and fatal strokes by successful carotid endarterectomy in patients without recent neurological symptoms: randomised controlled trial[END_REF] demonstrated a positive effect of carotid endarterectomy in asymptomatic patients with carotid stenosis Ն60%, with a prerequisite surgical complications rate under 3%. In daily practice, many physicians extend screening to a larger subgroup of diabetics.

The objectives of this study were (1) to determine the prevalence of high-grade carotid stenosis in patients with type-2 diabetes without any history of cerebrovascular disease and (2) to increase the efficiency of duplex screening by identifying a high-risk subgroup with a predictive model. were systematically included. These patients were referred to our department of internal medicine which takes care of diabetic patients as a university hospital center. They were either hospitalized for an accurate equilibration of their disease or seen in the outpatient clinic for a biennial check-up. In addition to type-2 diabetes, the inclusion criteria were age Ͼ18 years, life expectancy Ͼ12 months and informed consent agreement. All the patients with a previous history of recent (Ͻ6 weeks) stroke or transient ischemic attack, a history of carotid surgery or cervical radiotherapy were excluded from this study. Personal and family history and medications were recorded.

Subjects were considered to have hypercholesterolemia if they had a total blood cholesterol Ͼ240 mg/dl or were taking lipid-lowering drugs. They were considered as hypertensive if they reported a history of hypertension or were taking antihypertensive drugs. Patients were categorized as 'non-smokers' or 'ever smokers'. Subjects were considered to have coronary heart disease (CHD) if they had a history of myocardial infarction, angina pectoris, ischemic disorders on the electrocardiogram, or were taking antiangina drugs. Subjects were considered to have peripheral arterial disease (PAD) if they had a history of claudication or vascular surgery or an abnormal ankle-brachial index (ABI). The normal ABI range in this study was determined as 0.85-1.5. Albuminuria was measured on 24-hour urine and microalbuminuria was considered for albumin excretion Ն30 mg/24 hour. All the subjects underwent fundus examination by an ophthalmologist and were classified as 'diabetic retinopathy' (either proliferative or non-proliferative) or 'no diabetic retinopathy'.

For each leg, the ABI was determined by dividing the mean of the posterior tibial and dorsalis pedis systolic pressures by the mean of the systolic pressures of both arms, except when the difference between both arms exceeded 15 mmHg. In this latter case, only the highest of both values was taken for the denominator. The validation of the protocol for the ABI measurement has been extensively published elsewhere. [START_REF] Aboyans | The intra-and interobserver variability of ankle-arm blood pressure index according to its mode of calculation[END_REF] The ultrasound screening method for cervical vascular disorders included a continuous wave Doppler followed by a duplex study (ATL HDI 3000 or ATL HDI 5000, Bothell, WA, USA) of both the carotid and vertebral arteries. All the investigations were performed by experienced vascular physicians. The duplex stenosis determination in our laboratory has previously been validated versus surgical pieces after endarterectomy, and already published elsewhere. [START_REF] Aboyans | A predictive model for screening cerebrovascular disease in patients undergoing coronary artery bypass grafting[END_REF] Briefly, in none of the 50 cases of carotid endarterectomy was our Duplex estimation of stenosis overestimated. Stenosis was considered Ն60% when the maximal velocity within the lesion was Ͼ2.6 m/s and the end-diastolic velocity Ͼ0.7 m/s. [START_REF] Moneta | Screening for asymptomatic internal carotid artery stenosis: duplex criteria for discriminating 60% to 99% stenosis[END_REF] The artery was considered as occluded in the absence of any Doppler signal, either by pulsed or color Doppler. According to the results of the carotid ultrasound screening in this study, subjects were classified as normal (no atherosclerotic lesions), stenosis Ͻ60% (small plaques up to Ͻ60% stenosis), and stenosis Ն60% (including occlusion).

Statistical analysis

Comparisons were performed with Student's t-test for continuous and the chi-squared analysis for discrete variables. All the factors presented in Table 1 were included in the univariate analysis. Multivariate analysis was performed using multiple logistic regression. All the factors with a p-value Ͻ0.25 were included in the multivariate analysis. A p-value Ͻ0.05 was considered statistically significant. Age was first analyzed as a continuous variable then as a discrete one with a cut-off point at 70 years. Two analyses were performed: first, we compared subjects with normal cervical arteries versus subjects with carotid lesions (plaque or stenosis); the second analysis compared subjects with no or low-grade stenosis (Ͻ60%) versus those with high-grade stenosis (Ն60%). Finally, we studied the value of screening limited to subgroups selected according to the predictive factors of Ն60% stenosis.

Results

Patient demographic data

A total of 300 consecutive subjects were included in the analysis. The mean age was 61.7 Ϯ 11.5 years (range: 33-88) in the male population and 63.5 Ϯ 13.7 years (range: 20-91) in the female population. The mean The main population data are presented in Table 1.

Overall cervical screening results

Out of the 300 subjects who underwent the duplex scan investigation, 205 (68.3%) were abnormal; 191 (63.6%) had Ͻ60% stenosis and 14 (4.7%) presented with Ն 60% stenosis (seven with 60-99% stenosis and seven occlusions). Figure 1 presents the results according to age categories.

Relationship between risk factors and presence of carotid plaque or stenosis

The results of the univariate analysis are presented in Table 2. Ten factors were identified. In the multivariate analysis, six factors appeared to be independently predictive (Table 3). An age Ͼ70 years, a personal history of hypertension, CHD, diabetic retinopathy, or familial history of diabetes were predictive of a higher risk of carotid artery lesions, whereas a BMI Ͼ27 kg/m 2 seemed to be protective.

Risk factors for the presence of carotid stenosis Ն Ն60% or occlusion

Fourteen patients had an internal carotid artery stenosis Ն60%. All the subjects except one were male. Among the 16 factors included in the univariate analysis, seven were significant (Table 4). The presence of diabetic retinopathy, an ABI Ͻ0.85 and a personal history of neurological disorders or bruit remained predictive of stenosis or occlusion (Table 5). Female sex was a protective factor. As the female prevalence of Ն60% carotid stenosis was limited we focused our analysis on the male population. The independent predictors of stenosis or occlusion were a personal history of coronary disease (OR: 3.34; 95%CI: 1.01-11.01, p ϭ 0.046) and an ABI Ͻ0.85 (OR: 3.66; 95% CI: 1.04-12.84, p ϭ 0.042). Seventy-nine males (47.6%) presented one of these two criteria. The distribution of male individuals was studied in order to determine the diagnostic characteristics of a strategy where the presence of either criteria would be a prerequisite to the performance of cervical duplex (Table 6). The sensitivity of this mode of selection prior to cervical duplex was 77%, the specificity at 54.9%, the positive predictive value at 12.7% and the negative predictive value at 96.6%. Males without at least one of these two factors had a very low probability of high-grade carotid stenosis or occlusion (3.4% vs 12.7% in males with one or both of these factors, p Ͻ 0.05).

Discussion

Previous studies have demonstrated the efficiency of screening for symptomatic carotid artery disease in order to prevent stroke. Conversely, controversy remains on the appropriateness of screening asymptomatic subjects. The positive predictive value of screening depends on the prevalence of carotid artery stenosis in the population. In the general population the prevalence of carotid artery stenosis Ͼ50% ranges between 2% in Italy 11 and 7% for women and 9% for men in the Framingham study. [START_REF] Fine-Edelstein | Precursors of extracranial carotid atherosclerosis in the Framingham study[END_REF] In our study, limited to a diabetic population managed in a university hospital, we found a 4.7% prevalence of carotid artery stenosis Ն60%, which is close to the general population. Mass screening for carotid artery stenosis is not recommended. In a study estimating the prevalence of carotid artery stenosis Ͼ50% up to 8%, Hill [START_REF] Hill | Symposium: controversies in cerebrovascular disease 2. Should patients be screened for asymptomatic carotid artery stenosis?[END_REF] considered that it was necessary to screen up to 850-1500 patients in order to prevent one stroke. As the prevalence of high-grade stenosis in our population was even lower, we may conclude that systematic screening in the diabetic population is not cost-effective. Diabetes was not predictive of carotid artery atherosclerosis in the Framingham [START_REF] Fine-Edelstein | Precursors of extracranial carotid atherosclerosis in the Framingham study[END_REF] and Troms 14 studies. In the Troms study, male gender was an independent predictor of carotid atherosclerosis. [START_REF] Mathiesen | Prevalence of risk factors associated with carotid artery stenosis: the Troms study[END_REF] was a protective factor for high-grade stenosis. More surprisingly, our data suggest a lower prevalence of carotid plaque in obese subjects. A similar trend has already been reported in post-menopausal women [START_REF] Lassila | Prevalence and determinants of carotid atherosclerosis in healthy postmenopausal women[END_REF] as well as in a Mexican-American population. [START_REF] Hunt | Genetic basis of variation in carotid artery plaque in the San Antonio Family Heart Study[END_REF] In contrast, BMI was positively correlated with the probability of carotid plaque in another general population study. [START_REF] Sun | Carotid atherosclerosis, intima media thickness and risk factors -an analysis of 1781 asymptomatic subjects in Taiwan[END_REF] We cannot exclude a systematic bias, based on the mode of selection of our patients. In addition, the relationship between obesity and carotid atherosclerosis seems to be mainly related to central obesity and visceral fat. [START_REF] Lakka | Abdominal obesity is associated with accelerated progression of carotid atherosclerosis in men[END_REF] We do not have these data in our study. Further investigations are needed to elucidate this relationship.

The main goal of our study was to define the subgroup of diabetics who may benefit from screening. Similarly, other authors have focused their research on carotid screening in a selected population. In a population study, Rockman et al [START_REF] Rockman | Focused screening for occult carotid artery disease: patients with known heart disease are at high risk[END_REF] found a 18.2% prevalence of asymptomatic carotid artery stenosis in cases of heart disease; patients with both hypertension and heart disease had a 22.1% prevalence. Regarding PAD patients, the overall prevalence of asymptomatic carotid artery stenosis was 14% in the SMART study. [START_REF] Simons | Carotid artery stenosis in patients with peripheral arterial disease: the SMART study[END_REF] Three factors, higher age, lower weight and higher diastolic blood pressure, were independently associated with stenosis. The prevalence increased from 8% in those with one risk factor to 50% in those with three. In our present study, a personal history of CHD and symptomatic or asymptomatic PAD were associated with an increased risk of carotid stenosis in the male population. Screening therefore seems inappropriate in other subgroups due to a very low probability of carotid stenosis.

The clinical consequences of screening asymptomatic carotid stenosis are limited. In the Asymptomatic Carotid Atherosclerosis Study, the 5-year absolute risk reduction for death and stroke associated with surgery was only 5.8%. 6 The relative risk reduction for stroke and death in men treated with surgery was at 66% in men but only 17% in women. However, this difference was not statistically significant. Whitty et al [START_REF] Whitty | Investigating individual subjects and screening populations for asymptomatic carotid stenosis can be harmful[END_REF] estimated that screening for asymptomatic carotid artery stenosis below a 20% prevalence cannot be recommended, and may be harmful. Even at 20% prevalence, the number of strokes prevented per 10 000 subjects screened was 112. In the present study, the prevalence of carotid stenosis was 12.7% in the male high-risk group (with an abnormal ABI and/or history of CHD). Thus, as the indication of asymptomatic carotid revascularization keeps on generating debates, screening for asymptomatic carotid stenosis is similarly controversial. [START_REF] Hill | Symposium: controversies in cerebrovascular disease 2. Should patients be screened for asymptomatic carotid artery stenosis?[END_REF][START_REF] Whitty | Investigating individual subjects and screening populations for asymptomatic carotid stenosis can be harmful[END_REF][START_REF] Perry | Consensus against both endarterectomy and routine screening for asymptomatic carotid artery stenosis[END_REF] Additionally, in a recent national audit on complications after carotid endarterectomy, [START_REF] Kragsterman | Risk factors for complications after carotid endarterectomy. A population-based study[END_REF] diabetes was associated with a higher risk of peri-operative complication.

The results of this study also indicate that the prevalence of carotid atherosclerosis is high in diabetic subjects as more than 68% had lesions. De Angelis et al [START_REF] De Angelis | Prevalence of carotid stenosis in type 2 diabetic patients asymptomatic for cerebrovascular disease[END_REF] had previously described a higher prevalence of carotid atherosclerosis in type-2 diabetic subjects compared with non-diabetic subjects. Carotid atherosclerosis was identified as a risk factor for CHD in the Cardiovascular Health Study. [START_REF] O'leary | Carotid-artery intima thickness as a risk factor for myocardial infarction and stroke in older adults[END_REF] In the Insulin Resistance Atherosclerosis Study, diabetic subjects without CHD had similar carotid intima-media thickness compared with non-diabetic subjects with a history of CHD. [START_REF] Haffner | Carotid artery atherosclerosis in type-2 diabetic and nondiabetic subjects with and without symptomatic CHD (the Insulin Resistance Atherosclerosis Study)[END_REF] These data suggest that all diabetic subjects may be treated the same as secondary prevention for CHD with statins and antiplatelet drugs. In the Medical Research Council / Heart Protection Study, [START_REF] Heart | MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial[END_REF] simvastatin reduced the rate of first occurrence of major vascular events by 22% regardless of the baseline LDL cholesterol or history of coronary disease. Recently, the American Diabetes Association recommended the use of aspirin for primary prevention in type-2 diabetics at increased cardiovascular risk (age Ͼ40, family history of cardiovascular disease, hypertension, smoking, dyslipidemia, albuminuria). [START_REF] Diabetes | Aspirin therapy in diabetes[END_REF] Taking into account all these data, the diagnosis of carotid plaques (Ͻ60% stenosis) has a non-significant effect on the optimal management of diabetic patients, and future studies in this field should, as in our first aim, focus on the determination of criteria predictive of high-grade (Ն60%) carotid stenosis, which could then potentially be considered for additional specific treatment, namely a carotid surgery, or alternatively a carotid angioplasty.

Our study has some limitations. First, the diabetic patients studied were all referred to a medical department specifically managing these patients, and therefore the results obtained here cannot be extended to all diabetic subjects. However, it is reasonable to consider these patients at a higher level of cardiovascular risk compared to overall diabetic subjects, and consequently the prevalence of high-grade carotid stenosis could be even lower in an extended population of diabetic subjects. Conversely, as we excluded symptomatic patients as well as those with carotid surgery history, we depleted to some extent our diabetic group of high-risk patients. We cannot determine the effect of this selection criteria on our results. However, we attempted to focus our study on those diabetic subjects who had no reason for referral to a vascular laboratory other than being diabetic per se. Finally, other ABI thresholds could be suggested for this study (ie 0.9 for the lower threshold and 1.4 for the higher threshold). Presently, there is no consensus on the actual thresholds of ABI, [START_REF] Aboyans | L'index de pression systolique: élément incontournable dans le dépistage, le diagnostic et la prise en charge de l'artériopathie oblitérante des membres inférieurs[END_REF] and our thresholds correspond to another report on the probability of carotid stenosis according to ABI values in a different population. [START_REF] Aboyans | A predictive model for screening cerebrovascular disease in patients undergoing coronary artery bypass grafting[END_REF] As the prevalence of carotid disease is proportional to PAD severity, [START_REF] Aboyans | A predictive model for screening cerebrovascular disease in patients undergoing coronary artery bypass grafting[END_REF][START_REF] Long | The correlation between the severity of peripheral arterial disease and carotid occlusive disease[END_REF] it is plausible that by choosing a more lenient criteria we would weaken the predictive value of ABI to determine Ն60% carotid stenosis.

In conclusion, in diabetic patients, the field for screening carotid disease is narrow. Among patients with type-2 diabetes mellitus, the probability of finding Ն60% stenosis is highest among men with a history of CHD or an ABI Ͻ0.85.

  duration of diabetes was 11 Ϯ 9.2 years (range: 1-40).

  Screening carotid artery stenosis in diabetics 97Vascular Medicine 2006; 11: 93-99

Table 1

 1 Demographic characteristics of the population (total 300).

	Demographic factors	No. (%)
	Age, years: mean (ϮSEM) (range)	62.5 (Ϯ12.6)
		(20-91)
	Male sex	166 (55.3)
	Ever smokers	146 (48.7)
	Hypercholesterolemia	191 (63.7)
	Hypertension	200 (66.7)
	BMI, kg/m 2 : mean (ϮSEM) (range)	29.1 (Ϯ5.2)
		(18.9-46.7)
	Personal history of CHD	62 (20.6)
	Personal history of PAD	58 (19.3)
	Abnormal ABI a	94 (31.4)
	Personal history of ischemic	24 (8)
	neurological disorder or
	cervical bruit
	Microalbuminuria	50 (16.7)
	Diabetic retinopathy	62 (20.7)
	Family history of cardiovascular	91 (30.3)
	disease
	Family history of diabetes	194 (64.7)

94 P Lacroix et al Vascular Medicine 2006; 11: 93-99 a Abnormal ABI: Ͻ0.85 or Ͼ1.5, including symptomatic and asymptomatic PAD.

Table 2

 2 Relationship between variables and positive screening test (whatever the lesion): univariate analysis.

		No lesion	Lesion
		n ϭ 95(%)	n ϭ 205(%)	p
	Age Ͼ 70	10 (10.5)	114 (55.6)	Ͻ 0.0001
	Male sex	44 (46.3)	122 (59.5)	0.044
	Ever smokers	54 (56.8)	105 (51.2)	ns
	Hypercholesterolemia	66 (69.5)	125 (60.9)	ns
	Hypertension	52 (54.7)	148 (72.1)	0.004
	BMI (kg/m 2 )	74 (77.8)	121 (59.0)	0.002
	History of diabetes Ͼ10 years	37 (38.9)	120 (58.5)	0.002
	Personal history of CHD	9 (9.4)	53 (25.8)	0.001
	Personal history of PAD	8 (8.4)	50 (24.3)	0.001
	Abnormal ABI a	18 (18.9)	76 (37.0)	0.001
	Personal history of ischemic	6 (6.3)	18 (8.7)	ns
	neurological disorder or
	cervical bruit
	Microalbuminuria	11 (11.5)	39 (19.0)	ns
	Diabetic retinopathy	10 (10.5)	52 (25.3)	0.005
	Family history of diabetes	53 (55.7)	141 (68.7)	0.039

a Abnormal ABI: Ͻ0.85 or Ͼ1.5, including symptomatic and asymptomatic PAD.

Table 3

 3 Characteristics independently associated with carotid lesion in diabetic patients.

		Odds
		ratio	95% IC	p
	Age Ͼ70	6.93	3.21-14.98	Ͻ 0.0001
	Hypertension	2.18	1.21-3.95	0.009
	BMI Ͼ27 (kg/m 2 )	0.35	0.18-0.67	0.001
	Diabetic retinopathy	2.38	1.06-5.33	0.03
	Personal history of	2.93	1.26-6.79	0.01
	CHD
	Family history of	2.79	1.53-5.11	0.0008
	diabetes

Table 4

 4 Relationship between variables and carotid stenosis Ն60% or occlusion on the univariate analysis.

		No lesion	Lesion
		n ϭ 286(%)	n ϭ14(%)	p
	Male sex	153 (53.5)	13 (92.9)	0.089
	Age Ͼ70 years	94 (32.8)	7 (50.0)	ns
	Ever smokers	135 (47.2)	11 (78.5)	0.04
	Hypercholesterolemia	181 (63.2)	10 (71.4)	ns
	Hypertension	190 (63.2)	10 (71.4)	ns
	BMI Ͼ27 kg/m 2	188 (66.4)	7 (50.0)	ns
	History of diabetes Ͼ 10 years	147 (51.3)	10 (71.4)	ns
	Personal history of CHD	55 (19.2)	7 (50.0)	0.01
	Personal history of PAD	51 (17.8)	7 (50.0)	0.008
	Abnormal ABI a	83 (29.0)	10 (71.4)	0.002
	ABI Ͻ 0.85	41 (14.3)	7 (50.0)	0.001
	Personal history of ischemic	20 (6.9)	4 (28.5)	0.01
	neurological disorder or
	cervical bruit
	Microalbuminuria	45 (15.7)	5 (35.7)	ns
	Diabetic retinopathy	54 (18.8)	8 (57.1)	0.001

a Abnormal ABI: Ͻ0.85 or Ͼ1.5, including symptomatic and asymptomatic PAD.

Table 5

 5 Characteristics independently associated with carotid stenosis Ͼ60% or occlusion in diabetic patients.

		Odds
		ratio	95% CI	p
	Female	0.09	0.01-0.78	0.028
	Abnormal ABI a	3.94	1.21-12.84	0.023
	Personal history of	4.54	1.16-17.81	0.029
	ischemic neurological
	disorder or
	cervical bruit
	Diabetic retinopathy	3.62	1.12-11.73	Ͻ 0.0001
	a Abnormal ABI: Ͻ0.85 or Ͼ1.5, including symptomatic
	and asymptomatic PAD.

Table 6

 6 Distribution of 166 diabetic males according to the presence or absence of prerequisite criteria a for a carotid duplex and the presence of Ն60% carotid stenosis.

		Carotid	Carotid
		stenosis	stenosis
		Ն60%	Ͻ60%
	Presence of at least one criteria	10	69
	Absence of both criteria	3	84

a History of CHD or an abnormal ABI Յ0.85 or Ͼ1.5.

Vascular Medicine 2006; 11: 93-99