Bayrische Thomas Thum
email: thum_t@klinik.uni-wuerzburg.de

Johann Bauersachs

Spotlight on endothelial progenitor cell inhibitors: short review

Keywords: drug therapy, endothelial progenitor cells, endothelium, nitric oxide, reactive oxygen species

published or not. The documents may come

Introduction

Endothelial progenitor cells (EPCs) are bone-marrowderived cells that may enter the systemic circulation to replace diseased mature endothelial cells in blood vessels. [START_REF] Asahara | Isolation of putative progenitor endothelial cells for angiogenesis[END_REF] Endothelial cell injury is the main stimulus for the development of atherosclerotic plaques. [START_REF] Ross | Atherosclerosis -an inflammatory disease[END_REF] EPCs contribute significantly to endothelial function and may represent a circulating repair pool of endothelial cells. [START_REF] Cho | Mobilized endothelial progenitor cells by granulocyte-macrophage colony-stimulating factor accelerate reendothelialization and reduce vascular inflammation after intravascular radiation[END_REF][START_REF] Hill | Circulating endothelial progenitor cells, vascular function, and cardiovascular risk[END_REF][START_REF] Isner | Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair[END_REF] Suppression of this pool is considered to have detrimental effects on the cardiovascular system. Patients with reduced numbers of EPCs are at increased risk for endothelial injury and, in general, for atherosclerotic plaque formation. [START_REF] Hill | Circulating endothelial progenitor cells, vascular function, and cardiovascular risk[END_REF] Moreover, inadequate coronary collateral development in patients with coronary heart disease is associated with reduced numbers of circulating EPCs. [START_REF] Lambiase | Circulating humoral factors and endothelial progenitor cells in patients with differing coronary collateral support[END_REF] Both endothelial function and damage appear to be the result of an altered balance between endothelial injury and the capacity for repair (Figure 1). In a mathematical model it was demonstrated that only minor elevations in the amount of circulating EPCs could significantly delay endothelial layer defects over time. [START_REF] Op Den Buijs | Mathematical modeling of vascular endothelial layer maintenance: the role of endothelial cell division, progenitor cell homing and telomere shortening[END_REF] This is, however, only a theoretical model and does not account for EPC functional capacity, which, besides the number of EPCs, appears to be a major determinant of EPCmediated beneficial effects. [START_REF] Heeschen | Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease[END_REF] In general, it is assumed that increasing the amount and enhancing the function of EPCs will improve cardiovascular outcome.

Thus, much attention has been given to drugs that enhance the peripheral EPC pool, such as granulocytemacropage colony-stimulating factor, [START_REF] Takahashi | Ischemia-and cytokineinduced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[END_REF] various HMG-CoA (3-hydroxyl-3-methylglutaryl coenzyme A) reductase inhibitors, [START_REF] Dimmeler | HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway[END_REF][START_REF] Vasa | Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease[END_REF] erythropoietin [START_REF] Bahlmann | Erythropoietin regulates endothelial progenitor cells[END_REF] and, recently, angiotension-converting enzyme (ACE) inhibitors. [START_REF] Min | Improvement in endothelial progenitor cells from peripheral blood by ramipril therapy in patients with stable coronary artery disease[END_REF] Drug-mediated improvement of EPC mobilization and function was shown to limit atherosclerosis and restenotic processes in coronary and carotid arteries, and to enhance re-endothelialization and neovascularization after myocardial infarction, although long-term effects need to be identified. [START_REF] Landmesser | Statininduced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase[END_REF][START_REF] Vasa | Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[END_REF][START_REF] Kong | Cytokine-induced mobilization of endothelial progenitor cells enhances repair of injured arteries[END_REF] Moreover, recent research is additionally strongly focused on the transplantation of progenitor cells [START_REF] Wollert | Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial[END_REF][START_REF] Schachinger | Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOP-CARE-AMI Trial[END_REF] and, although early results are convincing, long-term outcomes and potential adverse side effects of this invasive procedure are unclear. [START_REF] Yoon | Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction[END_REF] There is uncertainty about potential inhibitors of EPCs, although their therapeutic reduction could be an interesting way of enhancing the beneficial effects of EPCs. Understanding the molecular mechanisms of EPC mobilization, differentiation, homing, adhesion and function is a first step to developing useful drugs that may normalize disturbed EPC biology.

Endogenous inhibitors of EPCs

In general, the number of EPCs is tightly correlated with cardiac risk factors and the Framingham risk score. [START_REF] Cho | Mobilized endothelial progenitor cells by granulocyte-macrophage colony-stimulating factor accelerate reendothelialization and reduce vascular inflammation after intravascular radiation[END_REF][START_REF] Vasa | Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[END_REF] Certain endogenous mediators of vascular dysfunction and injury may not only adversely affect the mature endothelium of the vessel wall but diminish the number of circulating progenitor cells.

Nitric oxide inhibitors and reactive oxygen species

One of the most important functions of endothelial cells is the production of nitric oxide (NO) by the endothelial NO synthase (eNOS). [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF] Besides its wellknown protective impact on vascular tone and wall homeostasis, NO is essentially involved in the mobilization and function of vascular progenitor cells. Indeed, Dimmler's group was the first to demonstrate that eNOS knockout mice suffer from decreased EPC mobilization. [START_REF] Aicher | Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells[END_REF] Moreover, a variety of EPC mobilizing agents such as statins act, at least in part, via NOmediated effects. [START_REF] Landmesser | Statininduced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase[END_REF] Our group recently demonstrated that bone marrow-derived NO is correlated with enhanced EPC mobilization to the circulation after myocardial infarction (T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript). Particular attention should therefore be given to potential endogenous inhibitors of NO availability that may adversely affect EPC mobilization and/or function. One interesting candidate could be the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA), which induces endothelial dysfunction [START_REF] Boger | LDL cholesterol upregulates synthesis of asymmetric dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases[END_REF] and impairs angiogenesis in vivo. [START_REF] Jang | Angiogenesis is impaired by hypercholesterolemia -role of asymmetric dimethylarginine[END_REF] Furthermore, ADMA levels are closely correlated with coronary artery disease and acute coronary events. [START_REF] Boger | Asymmetrical methylarginine (ADMA) as a cardiovascular risk factor: epidemiological and prospective data[END_REF][START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF] Preliminary data suggest that ADMA plasma levels are inversely correlated with circulating progenitor cells. [START_REF] Thum | Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine[END_REF] The effects of ADMA on EPCs are currently under intense investigation. The treatment of cells with ADMA leads to increased intracellular production of reactive oxygen species (ROS). [START_REF] Scalera | Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence[END_REF] ROS, in turn, may hamper the function of EPCs, although EPCs from healthy controls are equipped with anti-oxidative enzymes such as manganese superoxide dismutase. [START_REF] Dernbach | Anti-oxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress[END_REF] Recently, it has been shown that the endogenous release of ROS is higher in the EPCs of patients with coronary artery disease compared with healthy controls. [START_REF] Fischer-Rasokat | Increased endogenous reactive oxygen species generation in endothelial progenitor cells of patients with coronary artery disease are associated with functional impairment[END_REF] Likewise, antioxidative enzymes were repressed in patients with coronary artery disease and showed markedly repressed migratory capacity. [START_REF] Fischer-Rasokat | Increased endogenous reactive oxygen species generation in endothelial progenitor cells of patients with coronary artery disease are associated with functional impairment[END_REF] ROS should therefore be considered as an important endogenous inhibitor of EPCs. [START_REF] Thum | Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine[END_REF]

Oxidized lipoproteins

The direct toxic effects of oxidized low-density lipoproteins (oxLDL) on endothelium are well known. [START_REF] Li | Antisense to LOX-1 inhibits oxidized LDLmediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells[END_REF][START_REF] Thum | Mechanistic role of cytochrome P450 monooxygenases in oxidized low-density lipoprotein-induced vascular injury -therapy through LOX-1 receptor antagonism?[END_REF] Recent evidence also suggests adverse effects of oxLDL on EPCs. [START_REF] Imanishi | Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation[END_REF] Treatment of cultured peripheral blood mononuclear cells with oxLDL reduces their differentiation into EPCs. [START_REF] Imanishi | Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation[END_REF] Likewise, proliferation, migration and in vitro vasculogenesis were reduced in a concentration-dependent manner, demonstrating negative quantitative and qualitative effects on EPCs. [START_REF] Imanishi | Oxidized lowdensity lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction[END_REF] Direct toxic effects of oxLDL include exaggerated production of ROS, enhanced expression of adhesion molecules, and downregulation of eNOS, leading to endothelial dysfunction. [START_REF] Li | Antisense to LOX-1 inhibits oxidized LDLmediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells[END_REF][START_REF] Thum | Mechanistic role of cytochrome P450 monooxygenases in oxidized low-density lipoprotein-induced vascular injury -therapy through LOX-1 receptor antagonism?[END_REF] Indirectly, by impairing EPC differentiation and function, oxLDL probably reduces the endothelial repair pool, contributing to its pro-atherogenic effects.

Glucose and diabetes mellitus

Chronically elevated glucose levels in diabetic patients correlate with endothelial dysfunction and atherosclerosis. [START_REF] Haffner | Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction[END_REF][START_REF] Hurst | Increased incidence of coronary atherosclerosis in type 2 diabetes mellitus: mechanisms and management[END_REF] Tepper et al were the first to demonstrate that human EPCs from patients with type 2 diabetes exhibit impaired proliferation, adhesion and incorporation into vascular structures. [START_REF] Tepper | Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures[END_REF] Likewise, Loomans and colleagues determined levels of circulating EPCs in type 1 diabetes. [START_REF] Loomans | Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes[END_REF] They found that EPC numbers were reduced by 40-50%, which could explain the impaired wound angiogenesis and healing in diabetes, as well as reduced collateral formation in ischemia. Although these studies suggest increased glucose levels as an underlying cause, there is no conclusive evidence. Glucose-stimulated ROS production within EPCs may be involved. Indeed, high glucose levels stimulate ROS production via a protein kinase C-dependent activation of NADPH oxidase in endothelial cells. [START_REF] Inoguchi | Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase [Review][END_REF][START_REF] Hink | Mechanisms underlying endothelial dysfunction in diabetes mellitus[END_REF] Moreover, eNOS exposure to oxidants causes increased enzymatic uncoupling and generation of oxygen superoxides in diabetes. [START_REF] Zou | Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite[END_REF] In addition, some of the vasculotoxic effects are mediated by advanced glycation end-products. [START_REF] Xu | Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products[END_REF] Thus, more studies are required to determine the mechanism by which diabetes mellitus impairs EPC function.

Endostatin

Endostatin is an endogenous anti-angiogenic molecule that reduces the number and clonogenic potential of circulating EPCs. [START_REF] Capillo | Continuous infusion of endostatin inhibits differentiation, mobilization, and clonogenic potential of endothelial cell progenitors[END_REF] Cellular stress upregulates endothelial endostatin release [START_REF] Deininger | Endothelial endostatin release is induced by general cell stress and modulated by the nitric oxide/cGMP pathway[END_REF] and therefore enhanced endostatin levels may also result in impaired EPC mobilization and function, as well as progression of atherosclerosis in certain patients. Indeed, endostatin is enhanced in the pericardial fluid of patients with coronary artery disease and reduced formation of collaterals. [START_REF] Panchal | Reduced pericardial levels of endostatin correlate with collateral development in patients with ischemic heart disease[END_REF]

C-reactive protein

As elevated levels of C-reactive protein (CRP) emerged as powerful predictors of cardiovascular disease, [START_REF] Willerson | Inflammation as a cardiovascular risk factor[END_REF][START_REF] Ridker | Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events[END_REF] their role in the biology of EPC may also be of importance. Indeed, human recombinant CRP directly inhibits EPC differentiation, survival, function, angiogenesis and the response to chronic ischemia. [START_REF] Verma | C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease[END_REF] These effects were attributed to a reduction of eNOS expression in EPCs and could be prevented by pretreatment of EPCs with the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone. [START_REF] Verma | C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease[END_REF] Moreover, CRP additionally decreases secretion of arteriogenic chemokines in EPCs and may impair their vascular regenerative capacity. [START_REF] Suh | C-reactive protein impairs angiogenic functions and decreases the secretion of arteriogenic chemo-cytokines in human endothelial progenitor cells[END_REF] Thus, elevated levels of CRP in patients with coronary artery disease [START_REF] Ridker | Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events[END_REF] could be mechanistically correlated with diminished EPC function in such patients, as reported previously. [START_REF] Heeschen | Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease[END_REF] It is important to note that statins are able to reduce CRP levels, [START_REF] Albert | Effect of statin therapy on C-reactive protein levels: the pravastatin inflammation/CRP evaluation (PRINCE): a randomized trial and cohort study[END_REF] which may contribute to their well-known EPC-inducing properties. 11

Angiotensin II

Angiotensin II is the effector peptide of the reninangiotensin system and has been implicated in the pathogenesis of atherosclerosis and coronary artery disease. [START_REF] Schmidt-Ott | The multiple actions of angiotensin II in atherosclerosis[END_REF] Angiotensin II potentiates vascular endothelial growth factor (VEGF)-induced human EPC proliferation and network formation through upregulation of the VEGF receptor kinase domain-containing receptor after 24 hours of treatment. [START_REF] Imanishi | Angiotensin II potentiates vascular endothelial growth factor-induced proliferation and network formation of endothelial progenitor cells[END_REF] However, after longer periods (Ͼ7 days), angiotensin II has a significant inhibitory effect on the mitogenic activity of EPCs. [START_REF] Imanishi | Biphasic effects of angiotensin II on the proliferative activity in endothelial progenitor cells[END_REF] Moreover, angiotensin II accelerates the rate of senescence by diminishing telomerase activity in EPCs. [START_REF] Ferrara | Clinical applications of angiogenic growth factors and their inhibitors[END_REF] Thus, increased angiotensin II levels in various cardiovascular diseases may increase atherosclerotic processes by enhanced senescence of circulating EPCs. Moreover, it has been reported that the treatment of patients with stable coronary artery disease with ACE inhibitors resulted in an increase of circulating EPCs. [START_REF] Min | Improvement in endothelial progenitor cells from peripheral blood by ramipril therapy in patients with stable coronary artery disease[END_REF] Furthermore, in rats with myocardial infarction, we were able to demonstrate that ACE inhibition resulted in an improved matrix metalloproteinase 9-mediated mobilization of EPCs from bone marrow to the circulation (T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript).

Exogenous inhibitors of EPCs

Pharmaceutical drugs or other xenobiotics that reduce the circulating EPC pool or cause EPC dysfunction can theoretically impair endothelial function and promote atherosclerosis.

Although not investigated in this context so far, inhibitors of the VEGF protein could be such candidates. VEGF inhibitors were originally designed for anti-angiogenic cancer treatment and indeed early results are promising. [START_REF] Ziche | Development of new drugs in angiogenesis[END_REF] However, VEGF also plays a role in the NO-mediated mobilization of progenitor cells [START_REF] Dimmeler | HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway[END_REF][START_REF] Landmesser | Statininduced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular function, and survival after experimental myocardial infarction requires endothelial nitric oxide synthase[END_REF] (and T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript) and systemic inhibition may lead to impaired endothelial repair mechanisms.

Moreover, exogenous substances that affect the bioavailability of NO may additionally affect EPCs. Indeed, infusion with the NO synthase inhibitor N-nitro-L-arginine methyl ester or with ADMA resulted in a decrease of effective renal plasma flow, and an increase in renovascular resistance and blood pressure. [START_REF] Kielstein | Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans[END_REF] It is likely that this treatment effectively reduces the eNOS activity of blood vessels and bone marrow, thus also leading to a reduction in EPC mobilization and function.

In addition, cigarette smoke was identified as an exogenous inhibitor of EPCs. [START_REF] Kondo | Smoking cessation rapidly increases circulating progenitor cells in peripheral blood in chronic smokers[END_REF] Indeed, the number of circulating EPCs was reduced in chronic smokers and smoking cessation led to a rapid restoration of EPC levels. [START_REF] Wang | Effects of nicotine on the number and activity of circulating endothelial progenitor cells[END_REF] This may be explained in part by the fact that smoking inhibits the release of physiological amounts of NO, which is important for EPC mobilization. [START_REF] Aicher | Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells[END_REF] Nicotine was found to increase the number and function of EPCs in vitro, [START_REF] Wang | Effects of nicotine on the number and activity of circulating endothelial progenitor cells[END_REF] as well as angiogenesis, [START_REF] Heeschen | Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis[END_REF] so the responsible cigarette smoke constituents causing impairment of EPCs are still not known and need to be identified.

Conclusion

Endogenous and exogenous inhibitors of EPCs are likely to contribute to endothelial dysfunction and atherosclerosis progression by impairment of the self-renewal capacity of endothelium. The identification of such substances is therefore of clinical importance. Therapeutic reduction of EPC inhibitors may be a promising approach for the future treatment of atherosclerosis.

Figure 1

 1 Figure 1 Schematic overview of the potential role of EPC inhibitors on the mobilization, differentiation, homing and function of EPCs. Impairment of EPC-mediated vascular repair and direct toxic endothelial effects are likely to contribute to the development of endothelial dysfunction and atherosclerosis. ADMA, asymmetric dimethylarginine; AGE, advanced glycation end-products; EPC, endothelial progenitor cell; L-NAME, N(G)-nitro-L-arginine methyl ester; NO, nitric oxide; oxLDL, oxidized low-density lipoprotein; VEGF, vascular endothelial growth factor.

Acknowledgment

This work was supported in part by the Ernst und Berta Grimmke-Stiftung (TT).


Rainer H Böger
email: boeger@uke.uni-hamburg.de

Edzard Schwedhelm

Renke Maas

Sabine Quispe-Bravo

Cord Skamira

Prof. Dr med H Rainer

Böger

ADMA and oxidative stress may relate to the progression of renal disease: rationale and design of the VIVALDI study

Keywords: endothelium, 8-iso-prostaglandin F 2, nitric oxide, risk marker, telmisartan

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 and background

Despite improved treatment of hypertension, urinary tract obstruction and infection, many forms of renal disease associated with permanent nephron loss still progress inexorably to chronic end-stage renal failure. Although this may be caused by a variety of kidney diseases, in the USA and western Europe this population group is dominated by patients with diabetes or hypertension. According to the United States Renal Data System registry data for the year 2002, diabetes was the attributed cause of end-stage renal failure in 35.7% of patients who started dialysis. Hypertension was responsible for 24% of cases. Glomerulonephritis accounts for about 15.6% of cases and cystic disease, hereditary causes and urological diseases together constitute another 6.4%. The cause is unknown in 18.3% of these patients. [START_REF]United States Renal Data System. Annual data report: atlas of end-stage renal disease in the United States[END_REF] Since the early 1980s the endothelium has been recognized as a major regulator of vascular homeostasis. Endothelial cells, as the inner lining of blood vessels, are strategically located between circulating blood and the vascular smooth muscle. In a person with a body weight of 70 kg, the endothelium covers an area of approximately 700 m 2 and weighs about 1-1.5 kg. [START_REF] Lüscher | The endothelium. Target and promoter of hypertension?[END_REF] Functional integrity of the endothelium is crucial for the maintenance of blood flow and antithrombotic capacity because the endothelium releases humoral factors that control relaxation and contraction, thrombogenesis and fibrinolysis, and platelet activation and inhibition. Thus, the endothelium contributes to blood pressure control, blood flow and vessel patency. It is now clear that impaired endothelial function contributes substantially to cardiovascular disorders such as atherosclerosis, hypertension and heart failure, which lead to hypoperfusion, vascular occlusion and end-organ damage.

Endothelial dysfunction is also a common feature in patients who are diabetic. [START_REF] Van De Ree | Impaired endothelium-dependent vasodilation in type 2 diabetes mellitus and the lack of effect of simvastatin[END_REF] It is considered a major step in atherosclerosis and acute atherothrombotic events. Futhermore, it predicts cardiovascular mortality. [START_REF] Heitzer | Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease[END_REF] Endothelial dysfunction has also been documented in a large proportion of patients with type 2 diabetes mellitus. [START_REF] Calles-Escandon | Diabetes and endothelial dysfunction: a clinical perspective[END_REF] The most probable explanation for this observation is a disturbed oxidative balance in the vasculature, resulting in endothelial dysfunction. [START_REF] Giugliano | Oxidative stress and diabetic vascular complications[END_REF] A major pathophysiological alteration of type 2 diabetes is insulin resistance. Studies carried out in people with type 2 diabetes mellitus show an inverse correlation between measures of oxidative stress and insulin resistance. [START_REF] Paolisso | Evidence for a relationship between oxidative stress and insulin action in non-insulindependent (type II) diabetic patients[END_REF][START_REF] Sano | Oxidative stress measurement by in vivo electron spin resonance spectroscopy in rats with streptozotocin-induced diabetes[END_REF] Both, diabetes mellitus [START_REF] Haffner | Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction[END_REF] and renal dysfunction 10 are now considered as major cardiovascular risk factors and are associated with greatly elevated cardiovascular morbidity and mortality.

Endothelial dysfunction: the role of ADMA and oxidative stress

Endothelial dysfunction is mainly characterized by impaired production of the key endogenous vasodilator, nitric oxide (NO). In the endothelium, nitric oxide is formed from L-arginine by the endothelial isoform of nitric oxide synthase (eNOS). In humans NO synthesis may be impaired by asymmetrical dimethylarginine (ADMA), an endogenously formed compound that inhibits NOS activity by displacing L-arginine from the substrate binding site. [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] Infusion of ADMA impairs endothelium-mediated vasodilation. [START_REF] Calver | Effect of local intra-arterial asymmetric dimethylarginine (ADMA) on the forearm arteriolar bed of healthy volunteers[END_REF][START_REF] Achan | Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolised by dimethylarginine dimethylaminohydolase[END_REF] Elevated ADMA plasma levels are associated not only with endothelial dysfunction [START_REF] Böger | Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease[END_REF][START_REF] Böger | ADMA: a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia[END_REF] but also with increased oxidative stress, [START_REF] Böger | An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes[END_REF] thereby linking endothelial dysfunction and redox mechanisms in vascular disease. Elevated plasma ADMA concentrations have also been found in patients with diabetes. [START_REF] Abbasi | Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus[END_REF][START_REF] Stühlinger | Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor[END_REF] Functional impairment or decreased expression of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH) has been suggested as one possible mechanism for elevated ADMA concentrations in diabetes. [START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF] This is brought about at least in part by redox-mediated dysregulation of DDAH activity. [START_REF] Ito | Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF] Furthermore, several investigators recently demonstrated that an elevated plasma ADMA concentration is an independent risk factor for cardiovascular mortality in patients with cardiovascular disease and in patients with chronic renal failure. [START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF] Several groups of drugs have been studied with respect to their ability to lower ADMA plasma concentration and to improve the related impairment in NO-mediated vasodilation. Until now, only metformin, inhibitors of the angiotensin converting enzyme, and AT 1 receptor blockers have been found, in small clinical studies, to lower plasma ADMA concentrations. [START_REF] Asagami | Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes[END_REF][START_REF] Chen | Long-term angiotensin-converting enzyme inhibition reduces plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X[END_REF][START_REF] Delles | Angiotensin converting enzyme inhibition and angiotensin II AT1-receptor blockade reduce the levels of asymmetrical N(G),N(G)-dimethylarginine in human essential hypertension[END_REF] The strong relationship between elevated ADMA concentration, cardiovascular disease and mortality has made ADMA a goal of primary interest for pharmacotherapeutic intervention in large, controlled clinical trials.

Endothelial dysfunction and oxidative stress: the role of angiotensin II

A growing body of evidence is strengthening the view that angiotensin II is a major stimulus for vascular oxidative stress. Angiotensin II induces a mitogenic response in vascular smooth muscle cells through protein kinase C-extracellular signal-regulated kinasedependent and -independent pathways, [START_REF] Lu | Oleic acid and angiotensin II induce a synergistic mitogenic response in vascular smooth muscle cells[END_REF][START_REF] Benndorf | Angiotensin II type 2 receptor inhibits VEGF-induced migration and in vitro tube formation of human endothelial cells[END_REF] together with increased vascular NAD(P)H oxidase activity [START_REF] Mollnau | Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling[END_REF] and modulation of extracellular superoxide dismutase expression. [START_REF] Fukai | Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension[END_REF] The consequence is an aggravation of the oxidative burden associated with atherosclerosis and endothelial dysfunction. [START_REF] Warnholtz | Increased NADHoxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the reninangiotensin system[END_REF] Not surprisingly, AT 1 receptor blockade attenuates low-density lipoprotein oxidation and improves endothelial function in hypercholesterolemia, hypertension and type 2 diabetes. [START_REF] Rachmani | Losartan and lercanidipine attenuate low-density lipoprotein oxidation in patients with hypertension and type 2 diabetes mellitus: a randomized, prospective crossover study[END_REF][START_REF] Von Zur Mühlen | Treatment with irbesartan or atenolol improves endothelial function in essential hypertension[END_REF][START_REF] Wassmann | Angiotensin II type 1 receptor antagonism improves hypercholesterolemia-associated endothelial dysfunction[END_REF] Isoprostanes: markers and mediators of oxidative injury in the vascular system Markers of oxidative stress are various and data are often conflicting. [START_REF] Schwedhelm | Clinical pharmacokinetics of antioxidants and their impact on systemic oxidative stress[END_REF] This may mainly be attributed to the diversity of markers used and the heterogeneity of methods applied. In recent years new markers of lipid peroxidation have shown very promising results: F 2isoprostanes (reviewed by Morrow 36 ). They are formed endogenously by arachidonic acid oxidation in phospholipids. Subsequently, they are liberated from phospholipids by phospholipases and eliminated via the kidney. F 2 -isoprostanes are reliable markers and potentially significant mediators of oxidative stress in disease associated with the cardiovascular system. [START_REF] Schwedhelm | Urinary 8-isoprostaglandin F2a is a risk marker in patients with coronary heart disease: a matched case-control study[END_REF][START_REF] Cracowski | Isoprostanes, emerging biomarkers and potential mediators in cardiovascular diseases[END_REF] They have also been validated as reliable markers of oxidative stress in type 2 diabetes. [START_REF] Davi | In vivo formation of 8-iso-prostaglandin F2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation[END_REF][START_REF] Devaraj | Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes[END_REF][START_REF] Sampson | Plasma F2 isoprostanes: direct evidence of increased free radical damage during acute hyperglycemia in type 2 diabetes[END_REF][START_REF] Feillet-Coudray | Divergence in plasmatic and urinary isoprostane levels in type 2 diabetes[END_REF][START_REF] Helmersson | Association of type 2 diabetes with cyclooxygenase-mediated inflammation and oxidative stress in an elderly population[END_REF] Oxidative stress is involved in the microvascular and macrovascular complications seen in patients with diabetes. These pathophysiologic alterations are mainly attributed to atherosclerotic lesions in the vasculature. F 2isoprostanes have been detected in atherosclerotic lesions and they may be regarded as footprints left by the enhanced oxidative burden. However, it seems reasonable to assume that F 2 -isoprostanes are not only an index of oxidative injury but also contribute to the progression of atherosclerosis. [START_REF] Morrow | Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans[END_REF] They may be quantified by gas chromatography-mass spectrometry (GC-MS), GC-tandem MS, liquid chromatography-tandem mass spectrometry (LC-tandem MS), and immunoassays. Despite this variety of possible methods, reliable quantification of F 2 -isoprostanes is hindered by inappropriately performed enzyme immunoassays; this is subject to recent controversy. [START_REF] Tsikas | Handling of commercially available enzyme immunoassays for 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP) in clinical research and science: considerations from the analytical and review point of view[END_REF][START_REF] Schwedhelm | 8-isoprostaglandin F2alpha as a risk marker in patients with coronary heart disease -Response[END_REF] There does not appear to be a consensus concerning the best method, but chromatographic methods (GC-(tandem) MS, and LC-tandem MS) should be superior to immunoassays. [START_REF] Young | Oxidative stress and vascular disease: insights from isoprostane measurement[END_REF] Moreover, measurement of urinary F 2 -isoprostanes seems to be advantageous over plasma F 2 -isoprostanes, at least in type 2 diabetes. [START_REF] Feillet-Coudray | Divergence in plasmatic and urinary isoprostane levels in type 2 diabetes[END_REF][START_REF] Helmersson | Association of type 2 diabetes with cyclooxygenase-mediated inflammation and oxidative stress in an elderly population[END_REF] Telmisartan: an effective and long-lasting AT 1

receptor blocker

The discovery of a new pharmacological class of antihypertensives, the angiotensin II receptor antagonists (or sartans), has led to the clinical development and marketing of these new therapeutic agents since 1995, with the marketing approval of losartan by the Food and Drug Administration. Telmisartan belongs to the second generation of angiotensin II receptor antagonists. It was developed by Boehringer Ingelheim and was approved for use in hypertension by the Food and Drug Administration for North America in November 1998 and by the European Medicines Agency for Europe in December 1998. Telmisartan is an orally active nonpeptide type I angiotensin II receptor antagonist that lowers blood pressure with once daily dosing over a whole 24 hours dosing interval. The antihypertensive effect of once-daily dosing of telmisartan in patients with mild-to-moderate hypertension results in a significant reduction of sitting, supine and standing systolic and diastolic blood pressure, with usually little or no orthostatic change. The usual starting dose of telmisartan is 40 mg once daily, with the option to increase that dose to 80 mg daily. The 40 mg dosage reduces systolic and diastolic blood pressure by an average of 11.3/7.3 mmHg; with 80 mg this average is 13.7/8.1 mmHg. The antihypertensive activity occurs within 2 hours after single-dose administration and is maintained for the full 24-hour dosing interval. With ambulatory blood pressure monitoring, the 24-hour trough-to-peak ratio for telmisartan was determined to be at least 80% for both systolic and diastolic blood pressure. PRIME was the first clinical program to show beneficial effects in patients with hypertension and type 2 diabetes mellitus across the spectrum of early and late stage kidney disease. It consisted of two studies: IRMA 2 (irbesartan microalbuminuria type 2) and IDNT (irbesartan diabetic nephropathy trial).

IRMA 2, a 2-year multicenter, randomised, doubleblind, placebo-controlled study of 590 patients aged 30-70 years, assessed the effects of irbesartan compared with other proven antihypertensive treatments in slowing the progression of diabetic renal disease in hypertensive patients with type 2 diabetes and persistent microalbuminuria (20-200 g/min). [START_REF] Parving | for the Irbesartan in Patients with Type 2 Diabetes and Microalbuminuria Study Group. The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes[END_REF] In this study irbesartan (high dose, 300 mg) led to a 70% reduction in progression to later stage disease, overt diabetic nephropathy. It was therefore shown that irbesartan is renoprotective independently of its bloodpressure-lowering effect in patients with type 2 diabetes mellitus and microalbuminuria.

IDNT, the second study, was conducted in 1715 patients with proteinuria (overt nephropathy), hypertension and type 2 diabetes mellitus. [START_REF] Lewis | for the Collaborative Study Group. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes[END_REF] Irbesartan caused a 20% reduction in progression to doubling of serum creatinine, end-stage renal disease, or all-cause mortality compared with placebo, and a 23% reduction compared with amlodipine. Moreover, there was a 37% reduction in hospitalizations owing to congestive heart failure compared with amlodipine. The conclusion was that irbesartan is effective in protecting against the progression of nephropathy due to type 2 diabetes mellitus independently of the reduction in blood pressure.

In addition, in September 2001 the New England Journal of Medicine published the results of a similar trial to IDNT, the RENAAL study (Reduction of Endpoints in NIDDM with Angiotensin II Antagonist Losartan), examining the effects of losartan in patients with type 2 diabetes mellitus and overt nephropathy. The RENAAL study included 1513 patients from 29 countries. [START_REF] Brenner | for the RENAAL Study Investigators. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy[END_REF] There was a 28% reduction in the risk of developing end-stage renal disease when losartan was included in the antihypertensive treatment regimen, and hospitalization for congestive heart failure decreased by 32%.

In May 2001, data from the MARVAL study (MicroAlbuminuria Reduction with VALsartan) were presented at the 16th Annual Scientific Session of the American Society of Hypertension. MARVAL was a multicenter, double blind, randomized parallel study of 332 type 2 diabetic patients with microalbuminuria and adjusted blood pressure. [START_REF] Viberti | MicroAlbuminuria Reduction With VALsartan (MARVAL) Study Investigators. Microalbuminuria reduction with valsartan in patients with type 2 diabetes mellitus: a blood pressure-independent effect[END_REF] The patients were randomized to receive either valsartan or amlodipine once daily over 24 weeks. The study was designed to assess the blood-pressure-independent effects of valsartan versus amlodipine on urinary albumin excretion rates, a measure of microalbuminuria. The results showed that more patients returned to a normal albuminuric status after 24 weeks with valsartan (29.9%) compared with amlodipine (14.5%) (p ϭ 0.001). These two groups experienced similar rates of blood pressure reduction. The conclusion was that the angiotensin receptor blocker valsartan is more effective than the calcium channel blocker amlodipine in reducing microalbuminuria in type 2 diabetic patients with adjusted blood pressure.

The effects of angiotensin converting enzyme inhibitors and angiotensin II AT 1 -receptor antagonists on mortality and renal outcomes in diabetic nephropathy have recently been reviewed. [START_REF] Strippoli | Effects of angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists on mortality and renal outcomes in diabetic nephropathy: systematic review[END_REF] Available data suggest that AT 1 receptor antagonists are beneficial for renal outcomes, with about a 22% reduction in risk of end stage renal disease and doubling of the serum creatinine concentration, around a 51% reduction in progression rates from microalbuminuria to macroalbuminuria, and about a 42% increase in regression from microalbuminuria to normoalbuminuria. [START_REF] Strippoli | Effects of angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists on mortality and renal outcomes in diabetic nephropathy: systematic review[END_REF]

Design of the VIVALDI trial

The VIVALDI trial has been designed as a prospective, multicenter, randomized, double-blind, doubledummy, parallel group trial to inVestigate the efficacy of telmIsartan versus VALsartan in hypertensive type 2 DIabetic patients with overt nephropathy. The primary goal is to show non-inferiority of telmisartan 80 mg with valsartan 160 mg in reducing proteinuria after 1 year of treatment. Furthermore, the treatment effects for telmisartan 80 mg versus valsartan 160 mg with regard to several renal function parameters, clinical end-points and parameters of endothelial function and oxidative stress after 1 year of treatment will be evaluated.

Between April 2003 and November 2004, 884 patients aged 30-80 years with type 2 diabetes and hypertension and overt nephropathy (defined by proteinuria in 24 h urine Ն900 mg and serum creatinine Յ265 mol/l or Յ3.0 mg/dl) were randomized in the study. Study medication is given while other antihypertensive therapy may be maintained, with the exception of angiotensin converting enzyme inhibitors and other angiotensin II receptor antagonists. If the goal blood pressure of 130/80 mmHg cannot be reached with the high dose of study medication, other antihypertensive medication may be given at any time during the study, starting 4 weeks after randomization. The sample size of the per protocol analysis population is set to include a total of 680 patients, 340 administered telmisartan 40 mg daily with mandatory titration after 2 weeks to 80 mg daily, and 340 patients taking valsartan 80 mg daily, with mandatory titration after 2 weeks to 160 mg daily.

The primary efficacy variable is the change from baseline in 24-hour proteinuria, after 1 year of treatment with telmisartan 80 mg versus valsartan 160 mg. Secondary end-points are the following: renal parameters (24-hour urinary albumin excretion rate, creatinine clearance, glomerular filtration rate, serum creatinine, 24-hour sodium excretion in urine); clinical end-points (composite of doubling of the serum creatinine concentration, end-stage renal disease or allcause death; composite of morbidity and mortality from cardiovascular causes); parameters characterizing endothelial dysfunction and oxidative stress (change from baseline in C-reactive protein, ADMA and 8-iso-PG F 2␣ levels); parameters characterizing insulin sensitivity (change from baseline in homeostasis model assessment index in patients not receiving insulin therapy; change from baseline in adiponectin).

Taken together, the VIVALDI trial will evaluate different aspects of the effects of the angiotensin II receptor blockers telmisartan and valsartan on renal function and the processes underlying the pathogenesis and facilitating the prevention of diabetic nephropathy.

ADMA and urinary 8-iso-prostaglandin F 2␣ as secondary end-points in the VIVALDI trial

As detailed above, serum levels of ADMA and urinary excretion rates of 8-iso-PGF 2␣ are novel markers of endothelial dysfunction and oxidative stress, respectively. For both, elevated levels have been reported in patients with type 2 diabetes. Moreover, both are associated with cardiovascular disease, although this association was more difficult to assess for 8-iso-PGF 2␣ because only in a few studies were sufficiently reliable urine collections carried out to allow the assessment of this marker in urine. However, in one study in which we collected urine samples carefully, a significant relationship between urinary excretion of 8-iso-PGF 2␣ and coronary heart disease was found. [START_REF] Schwedhelm | Urinary 8-isoprostaglandin F2a is a risk marker in patients with coronary heart disease: a matched case-control study[END_REF] In the case of ADMA, several prospective as well as numerous cross-sectional clinical studies have confirmed the predictive value of this marker for major cardiovascular adverse events and death. These data have been extensively reviewed elsewhere in this issue. [START_REF] Böger | Asymmetric dimethylarginine (ADMA) and cardiovascular disease: insights from prospective clinical trials[END_REF] The analysis of ADMA in a large cohort of type 2 diabetic patients in the VIVALDI trial will allow us to establish the relationship between ADMA levels and cardiovascular end-points in a prospective manner in the diabetic population. Moreover, it will also allow firm conclusions on the efficacy of the AT 1 receptor blocker telmisartan to lower ADMA levels. Only a few previous studies have been reported in which the effects of AT 1 blockers on ADMA were tested; these included very small numbers of participants (see the overview on therapeutic interventions by Maas in this issue [START_REF] Maas | Pharmacotherapies and their influence on asymmetric dimethylargine (ADMA)[END_REF] ). Moreover, they may have been flawed by the method of assessing ADMA, which has only recently been improved (see the overview by Schwedhelm, also in this issue [START_REF] Schwedhelm | Quantification of ADMA: analytical approaches[END_REF] ).

Although angiotensin II has long been viewed as a major determinant of oxidative stress in the vascular system, the effects of AT 1 receptor blockade on urinary F 2 -isoprostane excretion in type 2 diabetes mellitus have not yet been studied.

Thus, the VIVALDI study will give us detailed and high-quality information on the roles of these novel markers of endothelial dysfunction and oxidative stress to allow us to predict outcomes in diabetic patients. It will also allow us to study the interrelationship between ADMA, lipid peroxidation and angiotensin receptor blockade with telmisartan, and thus to find potential new clues to the mechanisms of action of this class of drugs in cardiovascular disease and diabetes mellitus.

47

 47

AT 1

 1 blockers in the prevention of renal failure in type 2 diabetes mellitus In September 2001 the New England Journal of Medicine published the results of PRIME (Program for Irbesartan Mortality and Morbidity Evaluation).

Vascular Medicine 2005; 10: S97-102

The VIVALDI study S99 Vascular Medicine 2005; 10: S97-102


Pär Wanby
email: wanby@telia.com

Petter Palmquist

Lars Brudin

Martin Carlsson

Genetic variation of the intestinal fatty acid-binding protein 2 gene in carotid atherosclerosis

Keywords: carotid atherosclerosis, FABP2, genetics, polymorphism

Introduction

Internal carotid artery (ICA) stenosis is a sign of advanced atherosclerotic disease, and ICA stenosis is recognized as a major risk factor of stroke. Conversely, carotid endarterectomy is effective in the prevention of stroke secondary to severe ICA stenosis. [START_REF] Rothwell | Analysis of pooled data from the randomised controlled trials of endarterectomy for symptomatic carotid stenosis[END_REF] Fatty acid-binding protein 2 (FABP2) is an abundant cytosolic protein expressed exclusively in the enterocytes of the proximal small intestine, and FABP2 is believed to be involved in fatty acid absorption and intracellular transport of dietary long-chain free fatty acids (FFAs). [START_REF] Zhang | Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit[END_REF][START_REF] Cohn | Use of transgenic mice to map cis-acting elements in the intestinal fatty acid binding protein gene (Fabpi) that control its cell lineage-specific and regional patterns of expression along the duodenal-colonic and crypt-villus axis of the gut epithelium[END_REF] A common gene polymorphism at codon 54 results in replacement of alanine (A) with threonine (T). [START_REF] Baier | An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance[END_REF] In vitro, the T-containing protein has a 2-fold greater affinity for long-chain FFAs. [START_REF] Baier | An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance[END_REF][START_REF] Baier | A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells[END_REF] This altered affinity for FFAs in the intestine is believed to affect the absorption, and consequently the fatty acid composition of serum lipids. In an earlier study using genotype-discordant sibling pairs we found that siblings with more T54 alleles had higher triglycerides and cholesterol concentrations than their siblings with more A54 alleles. [START_REF] Carlsson | The T 54 allele of the fatty acid-binding protein 2 is associated with a parental history of stroke[END_REF] In other studies the T54 allele has been associated with insulin resistance, [START_REF] Baier | An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance[END_REF][START_REF] Hegele | A review of intestinal fatty acid binding protein gene variation and the plasma lipoprotein response to dietary components[END_REF] obesity and serum lipid abnormalities, including higher fasting triglyceride concentrations [START_REF] Hegele | Genetic variation of intestinal fatty acid-binding protein associated with variation in body mass in aboriginal Canadians[END_REF] and elevated triglyceride [START_REF] Prately | Effects of an Ala54Thr polymorphism in the intestinal fatty acid-binding protein on responses to dietary fat in humans[END_REF] and FFA [START_REF] Ågren | Postprandial lipemic response is modified by the polymorphism at codon 54 of the fatty acid-binding protein 2 gene[END_REF] concentrations after an oral fat load. A few other case-control studies could not replicate these data. [START_REF] Vidgren | Threonine allele in codon 54 of the fatty acid binding protein 2 gene does not modify the fatty acid composition of serum lipids in obese subjects[END_REF][START_REF] Tahvanainen | Intestinal fatty acid binding protein polymorphism at codon 54 is not associated with postprandial responses to fat and glucose tolerance tests in healthy young Europeans. Results from EARS II participants[END_REF] Stroke has been associated with the metabolic syndrome and, in a recent study, [START_REF] Ninomiya | Association of the metabolic syndrome with history of myocardial infarction and stroke in the Third National Health and Nutrion Examination Survey[END_REF] with hypertriglyceridemia in particular. In addition, coronary restenosis after balloon angioplasty has been associated with a different polymorphism in the FABP2 gene. [START_REF] Horibe | Genetic risk for restenosis after coronary balloon angioplasty[END_REF] In our earlier study we suggested that the T54 allele might increase susceptibility to stroke, as we found a higher parental prevalence of stroke in TT and TA genotype carriers when compared with AA genotype carriers. [START_REF] Carlsson | The T 54 allele of the fatty acid-binding protein 2 is associated with a parental history of stroke[END_REF] Several reports have pointed out an association between increased levels of postprandial triglyceride-rich lipoproteins and the presence of or development of coronary artery disease. [START_REF] Hyson | Postprandial lipemia and cardiovascular disease[END_REF][START_REF] Simons | Chylomicrons and chylomicron remnants in coronary artery disease: a case-control study[END_REF][START_REF] Groot | Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease[END_REF] Since other earlier studies have shown that the FABP2 T54 allele is associated with postprandial lipemia, [START_REF] Hegele | A review of intestinal fatty acid binding protein gene variation and the plasma lipoprotein response to dietary components[END_REF][START_REF] Prately | Effects of an Ala54Thr polymorphism in the intestinal fatty acid-binding protein on responses to dietary fat in humans[END_REF][START_REF] Ågren | Postprandial lipemic response is modified by the polymorphism at codon 54 of the fatty acid-binding protein 2 gene[END_REF] and since postprandial lipemia is associated with carotid atherosclerosis, [START_REF] Sharrett | Association of postprandial triglyceride and retinyl palmitate responses with asymptomatic carotid artery atherosclerosis in middle-aged men and women[END_REF][START_REF] Ryu | Postprandial triglyceridemia and carotid atherosclerosis in middle-aged subjects[END_REF][START_REF] Boquist | Alimentary lipemia, postprandial triglyceride-rich lipoproteins, and common carotid intima-media thickness in healthy, middle-aged men[END_REF][START_REF] Karpe | Magnitude of alimentary lipemia is related to intima-media thickness of the common carotid artery in middle-aged men[END_REF] we raised the hypothesis that the FABP2 gene A54T polymorphism would be associated with an increased risk of ICA stenosis.

Simpler genetic effects probably underlie the development of ICA stenosis compared with stroke and different stroke subgroups. In this study we therefore chose to study the FABP2 gene polymorphism in relation to ICA stenosis instead of studying the effect on stroke itself.

Methods

Subjects

Between June 2000 and November 2002, 497 patients initially diagnosed with stroke or a transitory ischemic attack (TIA) were, upon their admission to the acute stroke unit, recruited to the Kalmar Stroke Study. Those who were investigated using ultrasound of the carotids were identified and included in the present study. Depending on the result of the ultrasound assessment, they were either classified as cases (patients with ICA stenosis, n ϭ 25) or as controls (patients without ICA stenosis, n ϭ 171). Of the study group (n ϭ 196), 133 (68%) had received a final diagnosis of ischemic cerebral infarction, 30 (15%) had suffered a cardio-embolic infarction, 29 (15%) had had a TIA, and the remaining four patients (2%) had been found not to have suffered a stroke or TIA.

As a reference of FABP2 genotype frequency in a healthy population we used 158 genotyped blood donors from the same hospital (age ϭ 55.4 Ϯ 3.9 years, 83 males, 75 females) whose internal carotid arteries had not been assessed with ultrasound. The blood donors were free from drugs and without a history of coronary, peripheral or cerebrovascular disease. Blood samples other than those used for DNA genotyping were not obtained. All participants gave informed consent to participate in the study, which received the approval of the Ethical Committee of Linköping University, Sweden.

The County Hospital of Kalmar is an acute-care hospital serving a population of approximately 150 000 in southeast Sweden. The population mainly consists of Swedish Caucasians with relatively few immigrants or subjects from other ethnic groups.

Clinical characterisation

The medical history of all subjects concerning diabetes, hypertension and current cigarette smoking was obtained from the patient or a relative following a written nurse-administrated standardized questionnaire. A history of hypertension was defined as the use of antihypertensive drugs and diabetes mellitus as the use of anti-diabetic drugs or diet treatment. Weight and height were measured with the subject in light clothing without shoes. Body mass index (BMI) was calculated as kilograms per m 2 . Blood pressure was measured with a mercury sphygmomanometer with the subject in the supine position.

Blood sampling procedures

At approximately 7 a.m. on the morning after admission to hospital, fasting venous blood samples were drawn to obtain DNA and for measurements of FFA, glucose, glycohaemoglobin (HbA1c), total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides. Blood samples were obtained in tubes without additives and kept on ice for 30 minutes prior to centrifugation. Serum was immediately separated and kept at Ϫ70ЊC until analysis. Fasting serum FFA concentrations were measured using an acyl-CoA oxidase-based colorimetric kit (Wako Chemicals Inc., Richmond, VA, USA), on a Cobas Mira (Roche, Basel, Switzerland). Fasting plasma glucose was measured using a glucose dehydrogenase method, and fasting plasma total cholesterol, HDLcholesterol, and triglyceride concentrations were analysed using commercially available kits using Cobas Integra 700 (Roche). Fasting plasma LDLcholesterol concentration was calculated with the use of Friedewald's formula. HbA1c was measured using a high performance liquid chromatography (HPLC) method. [START_REF] Jeppsson | Measurement of hemoglobin A1c by a new liquid-chromatography assay; methodology, clinical utility, and relation to tolerance evaluated[END_REF][START_REF] Eckerbom | Simplified analysis for glycated hemoglobin by ion-exchange chromatography[END_REF] The assays were performed according to the manufacturer's protocols.

Genotyping

The G to A nucleotide substitution in exon 2 of the FABP2 gene (A54T) was genotyped using an earlier described PCR-RFLP method [START_REF] Baier | A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells[END_REF] with the following changes: exon 2 was PCR-amplified from 25 ng genomic DNA in a 20 L volume consisting of 1 ϫ PCR buffer (Applied Biosystems, Roche, Basel, Switzerland), 0.25 mmol/L of each deoxy-NTP, 10 pmol of each primer, 2.5 mmol/L MgCl 2 , and 0.5 U Taq polymerase (Applied Biosystems, Roche). PCR reactions were initiated by denaturation (30 s at 94ЊC) followed by amplification for denaturation (30 s at 94ЊC), annealing (30 s at 55ЊC), and extension step (10 min at 72ЊC). The amplified 180 bp product was digested with Hin6I according to the manufacturer's instructions (Tamro Med-lab, Hisings Kärra, Sweden) and separated on a 3% agarose gel. The T54 allele lacking the Hin6I site migrated as a 180-bp fragment, whereas the A54 allele was cleaved and appeared as 99-bp and 81-bp fragments.

Ultrasonography, ECG and CT-scan

Duplex sonography combining B-mode imaging, colour flow and pulsed Doppler spectrum analysis was used (Sonos 5000 HP ™ or Acuson Sequoia ™ 512). A consultant clinical physiologist (MD) performed examinations using an 8 MHz probe with the subject in supine position. The Doppler angle was chosen as close to 60Њ as possible. Doppler measurements were made with the sampling volume in the common carotid artery (CCA) proximal to the bifurcation, in the internal carotid artery (ICA; where the highest peak velocity was chosen) and in the external carotid artery (ECA). Maximum systolic peak ICA velocities above the normal limit 1.05 m/s [START_REF] Bluth | Carotid duplex sonography: A multicenter recommendation for standardized imaging and Doppler criteria[END_REF] were classified as stenosis of the ICA (ranging from 50% to 100%; degrees of stenosis less than 50% are not possible to detect with this method). Both the right and left carotid in each subject were investigated and the highest ICA-stenosis value of the two was used when the subjects were classified in either 1 ϭ normal (Ͻ50% stenosis irrespective of the characteristics of a possible plaque; controls) or 2 ϭ stenosis (Ն50%; cases). In addition, a 12-lead electrocardiogram (ECG) and a brain-computed tomography (CT) scan were made.

Data analysis and statistics

Variables not normally distributed (FFA, triglycerides and HbA1c) are presented as median Ϯ range, other continuous variables as means Ϯ SD, and categorical variables as percentages, calculated for each of the two groups (normal ICA velocities vs. ICA stenosis). Group differences were tested by Student's t-test (if normally distributed) or Mann-Whitney's U-test, or for frequencies, Fisher's exact test (2-tailed) using a commercially available computer program (STATIS-TICA version 6.0, StatSoft ® , Tulsa, USA). P values Յ 0.05 were considered statistically significant. Logistic regression analysis was used to investigate associations between the dependent variable, ICA stenosis (yes/no) and the independent variables, FABP2 genotype (AA vs. TA ϩ TT) together with possible risk factors for atherosclerosis, of which some were fixed in the model (see below). Both univariate and multivariate (stepwise, backwards) analysis were used (blood donors excluded). The following risk factors for atherosclerosis were fixed: Age (categorized in quartiles, for cut-off limits see Table 3), known diabetes (yes/no), measured systolic blood pressure levels (categorized below or above the median value 160 mmHg) and smoking (yes/no). Other independent variables were gender (male/ female), a history of hypertension (yes/no), diastolic blood pressure levels (below the median value of 84 mmHg or not), and use of oral lipid lowering medication (yes/no). Since our hypothesis is that atherosclerosis is partly caused by hyperlipidaemia, which in turn is associated with the FABP2 genotype, blood lipids were not included in the model.

Results

Differences between groups

Clinical characteristics of the patients are shown in Table 1 where they are classified according to ICA lumen. The group of patients with ICA stenosis were older, suffered from more diabetes and had lower concentrations of cholesterol and LDL-cholesterol than patients without ICA stenosis. There were no group differences in the proportions that smoked or had a history of earlier hypertension, nor were there any significant differences in systolic or diastolic blood pressure, BMI, fasting glucose, HbA1c, oral anti-diabetics or insulin.

Allele and genotype frequencies in patients with ICA stenosis did not deviate from those predicted by the Hardy-Weinberg equilibrium. Due to few patients with the TT genotype (only 2 patients with ICA-stenosis), the TT and TA genotypes were combined. Allele and genotype frequencies in the group of ICA stenosis (TT ϩ TA, 68.0%; AA, 32.0% ) did not differ significantly from the group of patients without ICA stenosis (TT ϩ TA, 49.7%; AA, 50.3%; p ϭ 0.09) (Table 2). We also compared the genotype frequencies of the group of patients with ICA stenosis with the genotype frequencies in apparently healthy blood donors (TT ϩ TA, 48.7%; AA, 51.3%). Neither in this group did genotype frequencies differ significantly from the group of patients with ICA stenosis (patients with ICA stenosis vs. blood donors, p ϭ 0.07).

Association between FABP2 polymorphism and clinical characteristics

No associations between the different genotypes of the FABP2 gene polymorphism and blood pressure, BMI, fasting glucose, HbA1c and lipid concentrations were seen.

Association between ICA stenosis and FABP2 polymorphism in univariate and multivariate analysis

There were univariate statistically significant correlations between ICA stenosis and age (p Ͻ 0.03), a history of diabetes (p Ͻ 0.01), but not with the FABP2 TT ϩ TA genotype (p ϭ 0.09), blood pressure (systolic p ϭ 0.09; diastolic p ϭ 0.29; both categorized) or smoking (p ϭ 0.78). Nevertheless, when known risk factors were fixed in the model (diabetes, blood pressure, age and smoking), the association between FABP2 genotype and carotid stenosis became statistically significant (p ϭ 0.04) (Table 3). This was also true when using only statistically significant parameters in the model (diabetes, systolic blood pressure and FABP2; p ϭ 0.03 for FABP2). Systolic and diastolic blood pressure (continuous or categorized) are strongly coupled and, therefore, only one of them could be incorporated in the model. Systolic blood pressure was chosen because it had a slightly stronger association with carotid stenosis than diastolic blood pressure. Although lipids (LDL, HDL or triglycerides) were not included in the regression model for conceptual reasons (see methods) they had no influence on the significance in the association between FABP2 genotype and carotid stenosis.

Discussion

Stroke is a highly heterogeneous disease and the molecular basis of inherited stroke risk has been ill defined since different stroke subtypes have different genetic bases. Various genetic polymorphisms may also exert differential effects across different ages, sexes and ethnic groups.

To partly circumvent this problem, instead of studying the risk of stroke, we studied a so-called endophenotype or intermediated phenotype, i.e. ICA stenosis, in a group of patients with acute cerebrovascular disease. Thereby, we might enable differences in genotype frequencies between cases (patients with ICA stenosis) and controls (patients without ICA stenosis) to be detected. Known risk factors for ICA stenosis include age, male gender, hypertension, diabetes, and cigarette smoking. [START_REF] Khaw | Epidemiology of stroke[END_REF] ICA stenosis has also been associated with elevated triglycerides, low HDL and hypercholesterolemia. [START_REF] Ritto | Worsening lipid profile is associated with progression of carotid artery stenosis[END_REF] Apart from the above-mentioned risk factors for carotid stenosis, variants in multiple genes have been associated with ICA stenosis. Among them are matrix metalloproteinase polymorphisms [START_REF] Ghilardi | Matrix metalloproteinase-1 and matrix metalloproteinase-3 gene promotor polymorphisms are associated with carotid artery stenosis[END_REF] as well as the Marburg I polymorphism of factor VII. [START_REF] Willeit | Marburg I polymorphism of factor VII-activating protease: prominent risk predictor of carotid stenosis[END_REF] In addition, data from the San Antonio Family Heart Study indicates a substantial genetic influence in carotid artery plaque. [START_REF] Hunt | Genetic basis of variation in carotid artery plaque in the San Antonio Family Heart Study[END_REF] To our knowledge, this is the first direct study of the FABP2 (A54T) gene polymorphism in ICA stenosis. In favour of our hypothesis that the FABP2 gene could be considered a candidate gene polymorphism for ICA stenosis, the gene polymorphism has been shown to affect the function of the protein in vitro. [START_REF] Baier | An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance[END_REF][START_REF] Baier | A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells[END_REF] In vivo, the T54 allele has been associated with elevated plasma lipid levels. In the present study we did not find an association between the FABP2 T54 allele and significant fasting dyslipidaemia. Actually the patients with ICA stenosis did have lower total and LDL-cholesterol concentrations despite the fact that there were less than 10% on lipid lowering medication (statins), evenly distributed between the two groups. However, it is reasonable to believe that differences in lipid plasma concentration between different FABP2 genotypes are apparent, for the most part, in the postprandial state, but not in the fasting state. [START_REF] Prately | Effects of an Ala54Thr polymorphism in the intestinal fatty acid-binding protein on responses to dietary fat in humans[END_REF][START_REF] Ågren | Postprandial lipemic response is modified by the polymorphism at codon 54 of the fatty acid-binding protein 2 gene[END_REF] The results of the study suggest that subjects with the T54 allele have more carotid atherosclerosis. Populations from different ethnic groups may have different genetic backgrounds and therefore different frequencies of genetic polymorphisms. A strength of the present study is the well-characterized study group, which came from a homogenous population (Caucasian), enrolled from only one hospital. Another strength is that we have studied a genetic variant that makes biological sense.

The results should, however, be interpreted with caution. Adequate sample size is important in genetic association studies. A limitation of the present study is the relatively small number of patients with carotid stenosis. A further limitation is that only current smoking (and not smoking in terms of current/ former/never smoking) was assessed. In addition, the FABP2 genotype frequencies only reached statistical significance in the multivariate regression analysis, not in the univariate analysis.

Furthermore, the control group (patients without ICA stenosis) were slightly younger than the patients with ICA stenosis. If the FABP2 A54T polymorphism is associated with the development of atherosclerosis, the frequency of the T54 allele may be higher in the present control group selected from patients with stroke than in an age-matched control group chosen from a healthier population. In order to partly circumvent this problem, we therefore compared the patients with ICA stenosis with a second control group, blood donors. Here we found a similar association with a nonsignificant trend towards a lower frequency of the T54 allele in the blood donors compared with the patients with ICA stenosis. The group of blood donors had not been assessed with an ultrasound of the carotids and the group was younger than the patients with ICA stenosis, but it is reasonable to assume that the frequency of ICA stenosis in this group will still be relatively low when the group has reached the same age as the group of patients with ICA stenosis, strengthening our hypothesis. In order to decide whether our results are likely to represent a true effect rather than statistical noise, an independent replication study in another population is needed.

In summary, our findings suggest a possible involvement of the FABP2 T54 allele in the pathogenesis of ICA stenosis.

  The FABP2 gene polymorphism and carotid atherosclerosis 105 Vascular Medicine 2005; 10: 103-108

  106 P Wanby et al Vascular Medicine 2005; 10: 103-108

Table 1

 1 Clinical characteristics of patients with and without internal carotid artery stenosis.

	Without ICA stenosis	ICA stenosis	p value

Data are mean Ϯ SD, median and range or fraction (%).

Table 2

 2 Genotype and allele frequencies in patients without ICA stenosis, with ICA stenosis and in healthy blood donors.

		Without		Blood
		ICA stenosis ICA stenosis donors
	FABP2 genotypes
	n	171	25	158
	TT (n, %)	17 (9.9)	2 (8.0)	12 (7.6)
	TA (n, %)	68 (39.8)	15 (60.0)	65 (41.1)
	AA (n, %)	86 (50.3)	8 (32.0) a	81 (51.3) b
	FABP2 alleles
	n	342	50	316
	T (n, %)	102 (29.8)	19 (38.0)	89 (28.2)
	A (n, %)	240 (70.2)	31 (62.0) c	227 (71.8)
	a p ϭ 0.09, TT ϩ TA vs. AA in patients with and without
	stenosis.
	b p ϭ 0.07, TT ϩ TA vs. AA in patients with stenosis and
	blood donors.
	c p ϭ 0.25, T allele vs. A allele in patients with and with-
	out stenosis.

Table 3

 3 Multivariate logistic regression analysis for risk factors in ICA stenosis.

			95%
		Odds Confidence
		ratio	interval	p value
	Diabetes mellitus
	no	1.00
	yes	4.93	1.76-13.8	Ͻ0.01
	FABP2 genotype
	AA	1.00
	TT ϩ TA	2.88	1.08-7.70	0.04
	Systolic blood pressure
	Ͻ160 mmHg	1.00
	Ն160 mmHg	2.51	0.91-6.95	0.08
	Age
	Ͻ67 years	1.00
	67-74 years	1.57	0.98-2.52
	75-81 years	2.47	0.96-6.36
	Ͼ81 years	3.89	0.94-16.0	0.06
	Current smoker
	no	1.00
	yes	2.27	0.74-7.0	0.15

Vascular Medicine 2005; 10: 103-108

Acknowledgements

This work was supported by a grant from the Medical Research Council of Southeast Sweden (FORSS). We are also indebted to Ingvor Gardtman, Ingela Nilsson and Inger Gustafsson for excellent technical assistance.


L Sieverding

Visualization of peripheral pulmonary artery stenosis using high-resolution multidetector computed tomography

A 3-year and 9-month-old patient (7.1 kg) with pulmonary atresia, ventricular septal defect and multifocal lung perfusion with major aorto-pulmonary collateral arteries (MAPCA) had shunt implantation connecting the hypoplastic central pulmonary arteries to the aorta (Panel A; S ϭ Shunt, AAo ϭ ascending aorta, RPA ϭ right pulmonary artery, LPA ϭ left pulmonary artery, RLLB ϭ right lower lung branch). Postoperatively the right upper lung was hyperperfused due to MAPCA, which was occluded interventionally in the catheterization laboratory. Unfortunately the patient could not be weaned off the ventilator due to persistent hyperperfusion of the right upper lung and hypoperfusion of the right lower lung.

The arterial and venous vascular system was visualized with multidetector computed tomography (MDCT, Panels A and B). After venous injection of 17 ml of contrast agent (Imeron 400) the neck, thorax and the upper abdomen were imaged within 10 seconds with nearly isotropic voxels (Somatom Sensation, 16 detectors, collimation 0.75 mm, 80 kV, 98 eff. mAs, voxelsize 0.5 ϫ 0.5 ϫ 0.6 mm 3 ).

Images in vascular medicine

Visualization of peripheral pulmonary artery stenosis using high-resolution multidetector computed tomography Precise knowledge of the pulmonary vasculature is the basis for adequate treatment of patients with pulmonary atresia and ventricular septal defect. [START_REF] Van Praagh | The child with congenital heart disease after surgery[END_REF][START_REF] O'leary | Pulmonary atresia and ventricular septal defect[END_REF] So far MAPCA have been visualized by selective injections of contrast agents in the catheterization laboratory. [START_REF] O'leary | Pulmonary atresia and ventricular septal defect[END_REF][START_REF] Geva | Gadolinium-enhanced 3-dimensional magnetic resonance angiography of pulmonary blood supply in patients with complex pulmonary stenosis or atresia: comparison with x-ray angiography[END_REF] With MDCT submillimetre high-resolution images with nearly isotropic voxels can be obtained. MDCT has the potential for visualization of the pulmonary vascular tree in complex congenital heart disease in children.

Movie

  A stenotic vessel (Panels A-D, arrow) to the right lower lung was detected in the maximal intensity projection image (Panel B) and the surface rendered model (Panel A). Catheterization was performed confirming the MDCT results (Panel C). The stenosis was balloon dilated successfully (Panel D, arrow).

GF

  Greil a , A Kuettner b , M Schoebinger c , H-P Meinzer c , CD Claussen b , M Hofbeck a and L Sieverding a a Department of Pediatric Cardiology, University of Tuebingen, Germany; b Department of Radiology, University of Tuebingen, Germany; c Division of Medical and Biological Informatics, German Cancer Research Center, Heidelberg, Germany Address for correspondence: Gerald F Greil, Department of Pediatric Cardiology, Children's Hospital, University of Tuebingen, Hoppe-Seyler-Strasse 1, 72076 Tuebingen, Germany. Tel: ϩ49 7071 2987147; Fax: ϩ49 7071 295127; E-mail: gerald.greil@med.uni-tuebingen.de Video footage of this can be seen at http://www.vascularmed journal.com by clicking on the 'Additional material' button. Perfusion of the right lung improved and the patient could be weaned off the ventilator.

  1A A 360-degree spin of the 3D surface rendered model shows anatomical structures as indicated in Panel A. The ascending aorta (AAo), the aortopulmonary shunt (S), the right pulmonary artery (RPA) with the right upper and middle pulmonary lobe and the left pulmonary artery (LPA) are coloured red. The stenotic segment from the right pulmonary artery to the right lower pulmonary lobe (arrow) is also coloured red. The right lower lung branch (RLLB) is coloured blue. Movie 2A Selective injection of contrast agent in the right pulmonary artery (RPA) showing the stenotic right lower lung branch (arrow) before balloon dilation. Movie 2B Selective injection of contrast agent in the right pulmonary artery (RPA) showing the increased diameter of the right lower lung branch (arrow) after balloon dilation.

Panel C

Panel D