Peter Danielsson
email: peter.danielsson@skane.se

L Truedsson

K-F Eriksson

L Norgren

Inflammatory markers and IL-6 polymorphism in peripheral arterial disease with and without diabetes mellitus

Keywords: diabetes mellitus, inflammation, interleukin-6, peripheral arterial disease, polymorphism

published or not. The documents may come

Inflammatory markers and IL-6 polymorphism in peripheral arterial disease with and without diabetes mellitus

Introduction

Peripheral arterial disease (PAD) is a manifestation of atherosclerosis, recognized as a chronic inflammatory disease of the vessel wall, [START_REF] Virchow | Phloge und Trombose im Gefässystem[END_REF][START_REF] Ross | Atherosclerosis -an inflammatory disease[END_REF] involving endothelial cells, white blood cells (WBC), platelets and inflammatory mediators. The symptomatic manifestation of PAD is characterized either by intermittent claudication (IC) or critical limb ischemia (CLI).

Parameters reflecting the inflammatory, response in PAD, although not extensively investigated, are increased compared with controls [START_REF] Danielsson | Response of inflammatory markers to balloon angioplasty in peripheral arterial occlusive disease[END_REF][START_REF] Fiotti | Long term prognosis in patients with peripheral arterial disease treated with antiplatelet agents[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] but also compared with patients suffering from coronary artery disease. [START_REF] Mazzone | Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation[END_REF] To study the inflammatory response, abundant markers are available, but proinflammatory cytokines (interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)␣, adhesion molecules on WBC (CD11b/ CD18) interacting with endothelial cell markers (ICAM, VCAM, selectins) are relevant and well described. [START_REF] Menger | Adhesion molecules as determinants of disease: from molecular biology to surgical research[END_REF] Elevated levels of cytokines in the circulation have been found in several situations where inflammation is an important part of the pathology (e.g. elevated IL-6 levels have been associated with increased cardiovascular risk [START_REF] Ridker | Plasma concentration of interleukin-6 and the risk of future Markers in peripheral arterial disease with and without diabetes 197 Vascular Medicine 2005; 10: 191-198 myocardial infarction among apparently healthy men[END_REF] ). IL-6 is a multifunctional proinflammatory cytokine that mediates many functions in host defence. It is also an important stimulus to the production of C-reactive protein (CRP), [START_REF] Yudkin | Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link?[END_REF] which is a relatively well established predictor for cardiovascular disease. [START_REF] Ridker | C-reactive protein, the metabolic syndrome, and risk of incident cardiovascular events: an 8-year follow-up of 14 719 initially healthy American women[END_REF] The concentration of IL-6 is likely to be influenced by several environmental and genetic factors. Such a genetic factor may be the polymorphic site (Ϫ174G/C) in the promoter region of the IL-6 gene. [START_REF] Fishman | The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis[END_REF] However, the significance of this polymorphism and its relationship to cardiovascular risk is unclear. In previous studies, the G allele has been reported to be associated with PAD [START_REF] Flex | The Ϫ174G/C polymorphism of the interleukin-6 gene promoter is associated with peripheral artery occlusive disease[END_REF] and the C allele with increased IL-6 production and high cardiovascular mortality. [START_REF] Jones | Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms[END_REF] In PAD, the clinical situation is often complicated and several sources of inflammatory response can be distinguished besides the atherosclerosis per se, such as ulcers, gangrene, infections, ischemic stress or the effect of diabetes mellitus (DM). [START_REF] Pradhan | C-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus[END_REF][START_REF] Dandona | Endothelium, inflammation, and diabetes[END_REF] Patients suffering from DM usually present an aggressive form of atherosclerosis with early and severe PAD. The increased metabolic stress is suggested to be responsible for the accelerated atherosclerotic process, [START_REF] Dandona | A rational approach to pathogenesis and treatment of type 2 diabetes mellitus, insulin resistance, inflammation, and atherosclerosis[END_REF] however this is not predictable, since the mechanisms are still unknown.

The aim of this study was to characterize the inflammatory response in different stages of PAD in populations with and without DM by studying some of the most important inflammatory mediators and also to investigate a possible relationship to polymorphism in the promoter region of the IL-6 gene.

Patients and methods

Patients

Demographic data are presented in Table 1. Five groups of 20 patients each and one control group, also of 20 patients, were included. All groups had a comparable age range. The group size was chosen before the start of the study. Recruitment was from patients referred to the Department of Vascular Diseases because of vascular symptoms; diabetic patients and male controls were participants in a health control study [START_REF] Eriksson | No excess 12-year mortality in men with impaired glucose tolerance who participated in the Malmo Preventive Trial with diet and exercise[END_REF] and the female controls were from a health control program at the hospital. Samples were collected between November 2001 and February 2003. In addition, DNA samples from 200 healthy blood donors were available.

The study was approved by the Ethics Committee of Lund University and the participants gave their informed consent.

Blood sampling

All blood sampling was performed in the morning with patients and controls fasting. The samples were collected in pyrogen-free tubes with and without ethylene diamine tetra-acetic acid (EDTA). EDTAblood was prepared for cell and DNA analysis and serum was separated from coagulated blood and frozen at Ϫ20°C within 4 hours of collection.

Routine blood analysis

Hemoglobin, high sensitive CRP (hsCRP), cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), glycated hemoglobin (HbA1c) and creatinine were analysed. CD18 expression. The different cell populations were identified using their morphological characteristics (forward versus side scatter). The cell concentration was determined by adding a known volume and concentration of Flow-Count Fluorospheres (Beckman Coulter, Brea, CA, USA) to the blood sample prior to flow cytometry analysis. CD11b and CD18 adhesion molecules were measured on the surface of granulocytes and monocytes using monoclonal r-phycoerythrin-conjugated anti-CD11b and fluorescein isothiocyanate-conjugated anti-CD18 (Dako A/S, Glostrup, Denmark). The analyses were made by a Coulter Epics XL-MCL (Coulter Electronics, Hialeah, FL, USA) at 488 nm. To keep a constant signal level, the fluorescence intensity was adjusted before analysis according to results obtained using Flow-Set Fluorospheres (Beckman Coulter). The mean channel fluorescence values for CD11b and CD18, respectively, were recorded.

Analysis of cell activation

Analysis of cytokines and soluble adhesion molecules and receptors

The serum concentrations of IL-6, IL-8 and TNF␣ were measured using an automated immunoassay system, Immulite ® (DPC, Los Angeles, CA, USA). The concentrations of the soluble adhesion molecules sE-selectin, sP-selectin, vascular cell adhesion molecule (sVCAM-1), intercellular adhesion molecule (sICAM-1) and the soluble TNF␣ receptors sTNF␣-R1 and sTNF␣-R2 were measured by enzyme-linked immunosorbent assay using kits from R&D Systems (Minneapolis, MN, USA).

Analysis of the Ϫ Ϫ174 G/C IL-6 gene promoter polymorphism

Extraction of genomic DNA from peripheral blood cells was performed according to the procedure described by Miller et al. [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] A nested polymerase chain reaction (PCR) was used to amplify the 5Ј-flanking region of the IL-6 gene, identifying the Ϫ174G/C polymorphism. Primers used for the outer PCR were 5Ј-CAG AAG AAC TCA GAT GAC TGG-3Ј and 5Ј-GCT GGG CTC CTG GAG GGG-3Ј amplifying a 611 bp fragment. [START_REF] Fishman | The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis[END_REF] Denaturation at 95°C for 5 minutes was carried out before the cycling program consisting of 25 cycles with denaturation at 95°C for 1 minute, annealing at 63°C for 1 minute, and extension at 72°C for 2 minutes. The program ended with a prolonged extension at 72°C for 10 minutes. The inner PCR amplified a 196 bp fragment using 5Ј-CGC TTC TTA GCG CTA GCC TC-3Ј and biotinylated 5Ј-GAG ACT CTA ATA TTG AGA CTC-3Ј primers. The amplification started with denaturation at 95°C for 5 minutes before the cycling program, which consisted of 50 cycles of denaturation at 95°C for 1 minute, then annealing using a touchdown program starting at 57ЊC for 2 minutes for 10 cycles, and then at 54ЊC for 40 cycles and 72°C of extension for 1 minute, and ending with an extension at 72°C for 5 minutes. The PCR reactions were performed in 25 l volumes in a Gene Amp PCR system 2400 thermocycler (Applied Biosystems, Foster City, CA, USA). The outer reaction mixture contained 0.5 g of genomic DNA and the inner 0.4 l of the product from the outer PCR reaction. Both PCR mixes contained 1.5 mM MgCl 2 , 0.2 mM dNTP (Amersham Biosystem, NJ, USA), 0.5 M of each primer (MVG Biotech, Ebersberg, Germany) and 0.5 units of AmpliTaq Gold™ (Applied Biosystems, Foster City, CA, USA). The Ϫ174G/C polymorphism was detected using the pyrosequencing technique. [START_REF] Ahmadian | Singlenucleotide polymorphism analysis by pyrosequencing[END_REF] The primer used for sequencing was 5Ј-CCC TAG TTG TGT CTT GC-3Ј and the reaction was carried out as described by the manufacturer on a PSQ™96MA System (Pyrosequencing AB, Uppsala, Sweden).

Statistics

The Kruskall-Wallis test was used to compare the tested variables between the six groups. However, the groups differed with respect to potential confounders, namely sex, age and smoking habits (Table 1). Moreover, the Kruskall-Wallis test cannot separate the effect of PAD from the effect of DM on the tested variables. Therefore, a multivariate linear regression analysis was also conducted, with age, gender and smoking habits as covariates in order better to assess relationships between the clinical stage of PAD, DM and the tested variables. The dependent variables were log-transformed in order to obtain regression residuals that conformed more closely to the normal distribution. The primary predictors in the regression analysis were binary markers of DM, IC and CLI. The dataset was considered too small to allow for interaction between clinical stage of PAD and DM in the regression modelling. The regression analysis permitted comparison of the tested variables among patients with and without DM, while controlling for differences in age, sex and PAD. It was also used to compare patients with IC and CLI with those without PAD, respectively, while controlling for differences in age, sex and DM. The allele distribution between the groups was analyzed by a chi-squared test, using an exact method for p-value calculation available in Stat-Xact-5 (Cytel Software Corporation, Cambridge, MA, USA). All other statistical analyses were performed using SPSS for Windows, release 11.5.1 (SPSS Inc., Chicago, IL, USA). P-values less than 0.05 were considered significant.

No significant differences was found in cholesterol or LDL concentrations. In the regression analyses, adjusted for age, gender and smoking habits, hsCRP, creatinine and TG were significantly higher and hemoglobin and HDL significantly lower in CLI patients than in patients without PAD (Table 3). In IC patients compared with patients without PAD, the concentration of creatinine was significantly higher and HDL significantly lower. In the same analyses, DM was significantly associated with increased levels of TG and HbA1c. 4 and5) Significant differences between the six groups were recorded for IL-6, TNF␣, sTNF␣-R1, sTNF␣-R2, sVCAM-1, sICAM-1, WBC count, and granulocyte and monocyte counts (Table 4). By contrast, no statistically significant differences between the six groups were discerned with respect to IL-8, sE-selectin, sP-selectin, and CD11b and CD18 on monocytes or granulocytes. In the regression analyses adjusted for age, gender and smoking habits, patients with advanced PAD (CLI) had, compared with those without PAD, significantly higher concentrations of IL-6, TNF␣, sVCAM-1, sICAM-1, sTNF␣-R1, sTNF␣-R2, and of WBC, monocyte and granulocyte counts and CD11b on granulocytes (Table 5). The results were not dependent on the presence of ulcers among the CLI patients (p Ͼ 0.12 for all tested variables; Kruskall-Wallis test comparing CLI patients with ulcers (n ϭ 25) with CLI patients without ulcers (n ϭ 15)). Significantly elevated levels of sVCAM-1, monocyte and granulocyte counts were also recorded in IC patients. No clear association between diabetes and any of these markers was found in the regression analyses.

Cell activation and cytokines (Tables

IL-6 polymorphism (Table 6)

No significant differences in the frequency distribution of any of three genotypes (GG,CC,GC) were found between the six groups (p Ͼ 0.3; chi-squared test for a 3 ϫ 6 cross-table). Neither was any significant difference found when CLI and IC patients (groups 3-6 collated into a single group) were compared with a control group of 200 healthy blood donors (p Ͼ 0.3; chi-squared test for a 3 ϫ 2 crosstable ).

Discussion

The main finding in this study was a significant interrelationship between advanced PAD and increased inflammatory activity. By contrast, DM was not found to be associated with increased levels of any of the studied inflammatory markers, indicating that DM alone, if well regulated as it was in these patients, does not cause significant endothelial or inflammatory cell activation. In less advanced PAD, represented by patients with IC (groups 3 and 4), associations were found with increased levels of sVCAM-1 and monocyte and granulocyte counts. This response may depend on the inflammatory activity in atherosclerotic plaques, a pattern demonstrated in several studies. [START_REF] Peter | Soluble vascular cell adhesion molecule-1 (VCAM-1) as potential marker of atherosclerosis[END_REF][START_REF] Brevetti | High levels of adhesion molecules are associated with impaired endotheliumdependent vasodilation in patients with peripheral arterial disease[END_REF][START_REF] Blankenberg | Adhesion molecules and atherosclerosis[END_REF] Such association, but also high levels of IL-6, were found in patients with advanced PAD but no relationship to polymorphism in the promoter region of the IL-6 gene was observed in any group.

It may be argued that the statistical results are hampered by the fact that normality of the regression residuals cannot be checked properly, given the large number of parameters (n ϭ 6) in the regression model in relation to the total number of observations (n ϭ 120). However, it should be noted that the results of the regression analyses (Tables 3 and5) and the Kruskall-Wallis test (Tables 2 and4), with one exception (CD11b on granulocytes), are entirely consistent with respect to variables that differ significantly between the groups. The inconsistency for CD11b and granulocytes is most likely due to the fact that regression analysis is more sensitive than the Kruskall-Wallis test in situations when only few groups differ markedly (see CLI patients, p ϭ 0.008, Table 5) from the others. Several markers have been analysed in this study and the elevated risk of false positive results must be considered, especially for a marker such as CD11b on granulocytes, where the Kruskall-Wallis test failed to reveal differences between the groups. However, overall, the results were consistent and not likely to be explained by chance alone.

In group 2 (DM) all the patients were men, which could be a confounding factor but, in groups 4 and 6, there were seven and five women with DM respectively.

Regarding the effect of renal insufficiency, small molecules such as IL-6 are partly eliminated by glomerular filtration (Ͻ70 KDa) and renal failure may therefore influence the results. In the present series, however, there were no patients on dialysis and creatinine levels ranged from 221 to 644 mol/l among the six patients classified as having renal failure. Most studies on inflammatory markers and endothelial cell activation have focused on coronary artery disease. There is, however, a considerable difference between coronary artery disease and PAD patients. The latter are usually older and more commonly have DM. A major difference is probably the more advanced and generalized atherosclerosis occurring in PAD, indicated by higher levels of WBC activation and expression of endothelial cell markers. [START_REF] Mazzone | Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation[END_REF] Only a few studies have explored inflammatory markers in PAD but the results demonstrate a correlation between the clinical stage of PAD and WBC/endothelial cell activation, which is in line with our results. [START_REF] Mazzone | Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation[END_REF][START_REF] Fiotti | Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease[END_REF][START_REF] Cimminiello | PAD: epidemiology and pathophysiology[END_REF][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][START_REF] Tsakiris | Circulating cell adhesion molecules and endothelial markers before and after transluminal angioplasty in peripheral arterial occlusive disease[END_REF][START_REF] Blann | Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis[END_REF] No elevated levels of pro-inflammatory cytokines have been described in PAD patients, but raised baseline levels of CRP [START_REF] Ridker | Plasma concentration of C-reactive protein and risk of developing peripheral vascular disease[END_REF] and inflammatory markers (soluble TNF and IL-6 receptors, alpha1-antitrypsin and haptoglobin) suggest increased inflammatory activity and activation of a regulatory feedback system. [START_REF] Fiotti | Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease[END_REF] In this study, advanced-stage PAD (groups 5 and 6) was related to increased expression of the majority of the tested inflammatory markers. Possible sources for the elevated inflammatory response in more advanced PAD could be the atherosclerosis per se, tissue ischemia or activity from existing ulcers. When the critically ischemic patients with and without ulcers were compared, it could be seen that the results did not depend on the presence of ulcers. In another study, yet to be published, we have demonstrated that revascularization in CLI did not significantly influence the inflammatory activity at 4 weeks after successful surgery. These findings suggest that the increased expression of inflammatory markers in CLI depends on advanced and active atherosclerosis and not on tissue ischemia or ulcers. Previous studies have also shown a correlation between inflammatory activity and the stage of PAD. [START_REF] Danielsson | Response of inflammatory markers to balloon angioplasty in peripheral arterial occlusive disease[END_REF][START_REF] Mazzone | Increased expression of CD11b/CD18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation[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] In the current study, this trend was most obvious for sVCAM-1 and granulocyte and monocyte counts. Atherosclerotic lesions can be very quiescent, with low inflammatory activity, for long periods but if there is plaque rupture or thrombosis formation the inflammatory response will increase. Such activity is more likely to be found in the most advanced stages of PAD, but high activity in atherosclerotic lesions can also be expected in patients with IC.

Other research has revealed elevated levels of soluble endothelial markers and increased activation of WBC in patients with DM [START_REF] Dandona | Inflammation: the link between insulin resistance, obesity and diabetes[END_REF] and macrovascular complications. [START_REF] Caimi | Polymorphonuclear leukocyte integrin pattern, at baseline and after activation, in type 2 diabetic subjects with macrovascular complications[END_REF] The response is, however, of low intensity and related to metabolic control. [START_REF] Aso | Plasma interleukin-6 is associated with coagulation in poorly controlled patients with type 2 diabetes[END_REF][START_REF] Aggarwal | Comparison of inflammatory markers in patients with diabetes mellitus versus those without before and after coronary arterial stenting[END_REF] The interaction of WBC and endothelial cells is probably required for the accelerated atherogenesis observed in DM, but the coexistence of other mechanisms is most likely. Impaired endothelial cell regulation by nitric oxide [START_REF] Hsueh | Role of endothelial dysfunction in insulin resistance[END_REF] and endothelin-1 [START_REF] Lam | Role of endothelin in diabetic vascular complications[END_REF] and the modification of LDL by glycosylation [START_REF] Matsunaga | Glycated highdensity lipoprotein regulates reactive oxygen species and reactive nitrogen species in endothelial cells[END_REF] are other features of DM that are responsible for accelerated atherogenesis.

In this study, no increased expression of WBC or endothelial cell markers was found as an indication of increased WBC/endothelial cell interaction in DM. The lack of response in the DM patients in this study could be explained by reasonably good metabolic control (HbA1c: 6.10-6.75%) and low cholesterol levels. In turn this may be an effect of previous participation in a lifestyle and cardiovascular risk factor modification program. [START_REF] Eriksson | No excess 12-year mortality in men with impaired glucose tolerance who participated in the Malmo Preventive Trial with diet and exercise[END_REF] Variations in the IL-6 response, suggested to cause variations in cardiovascular risk, have been postulated to be related to a polymorphism at position Ϫ174 in the promoter region of the IL-6 gene. The significance of this polymorphism is not unambiguous and may be of other importance in different vascular areas. In the UK small aneurysm study, [START_REF] Jones | Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms[END_REF] the CC genotype was associated with the highest IL-6 levels and highest mortality risk. In a recent study by Flex and coworkers, [START_REF] Flex | The Ϫ174G/C polymorphism of the interleukin-6 gene promoter is associated with peripheral artery occlusive disease[END_REF] the GG genotype was noted to be significantly more common in PAD patients compared with controls.

In this study, high levels of IL-6 were associated with CLI, reflecting augmented inflammatory activity in advanced atherosclerosis. However, no difference in the IL-6 genotype was found in these patients compared with the controls (Table 6). The results are based on a relatively small population, but they contradict a strong association between genotype and PAD.

The high IL-6 levels in advanced PAD patients probably depend more on other stimuli than the genotype of the IL-6 gene.

Conclusions

In conclusion, the results of this study confirm the correlation between advanced PAD, but not DM with good metabolic control, and an increased inflammatory response and may suggest a relationship depending on advanced atherosclerosis but not on the presence of ulcers. High IL-6 levels correlated with advanced PAD, but the Ϫ174 IL-6 gene promoter polymorphism was not found to be associated with PAD. The clinical implication, based on these findings, is that it is of the greatest importance to address the general atherosclerotic process in PAD patients by prevention and treatment options, in addition to treating the reduced blood flow that is causing the PAD symptoms.

  194 P Danielsson et al Vascular Medicine 2005; 10: 191-198

  196 P Danielsson et al Vascular Medicine 2005; 10: 191-198

10: 191-198 Table 1 Clinical characteristics of the participants stratified into six groups, each comprising 20 individuals; a median values and ranges.

	Full and differential WBC counts were performed.
	Activation of granulocytes and monocytes was assessed
	by flow cytometry measurement of CD11b and
	Group 2: DM 74 (73-76) 20 0 2 14 20 (4) 14 6 1 2 2 1 0 0 84 (63-107) Blood pressure (mmHg) a 130 (120-160) 145 (110-180) 145 (120-180) 150 (120-170) 140 (100-180) 165 (110-190) Group 3: Group 4: Group 5: Group 6: Characteristic controls IC DM ϩ IC CLI CLI ϩ DM Age (years) a 75 (69-87) 67.5 (40-86) 73 (59-82) 77 (60-94) 71.5 (61-91) Men 12 11 13 12 15 Women 8 9 7 8 5 Current smoker 1 7 5 3 3 Former smoker 6 11 14 13 13 DM (insulin dependent) 0 (0) 0 (0) 20 (7) 0 (0) 20 (10) Hypertension 5 13 11 10 14 Angina pectoris 1 4 6 5 4 Heart failure 0 1 5 3 3 Stenosis of a carotid 0 1 1 2 0 artery Cerebrovascular disease 2 2 6 1 1 Lung disease 0 0 2 0 0 Renal failure 0 0 1 1 4 Ulcer/gangrene 0 0 0 9 16 Weight (kg) a 72 (44-83) 75 (59-107) 78 (60-113) 71 (50-95) 82.5 (44-112) Vascular Medicine 2005; Group 1: Ankle pressure (mmHg) a 135 (120-160) 150 (110-160) 110 (50-150) 90 (50-140) 75 (0-150) 55 (0 Ն 200)

DM, diabetes mellitus; IC, Intermittent claudication; CLI, critical limb ischemia.

Table 2

 2 Variation of standard laboratory parameters; median values, ranges and p-values for testing the hypothesis of no difference between the six groups (Kruskall-Wallis test) are presented; n ϭ 20 in each group.

		Group 1:	Group 2:	Group 3:	Group 4:	Group 5:	Group 6
	Parameter	controls	DM	IC	DM ϩ IC	CLI	CLI ϩ DM	p-value
	Hb (g/l)	136.5 (94-143) 136.5 (112-156) 144 (130-165) 132.5 (100-148) 120 (78-146)	127.5 (96-152)	Ͻ0.001
	hsCRP (mg/l)	4.5 (0.3-246)	0.9 (0.3-2.1)	4.8 (0.5-14.9)	5.1 (0.5-43.7) 6.0 (0.3-97.6)	11.5 (1.1-114) Ͻ0.001
	Creatinine	79 (11-157)	91 (79-174) 81.5 (58-241)	104 (54-221) 105 (58-344)	108 (60-644)	0.02
	(mol/l)
	Cholesterol	5.3 (3.7-7.6)	4.8 (3.7-6.6) 4.93 (3.6-8.4)	5.3 (3.0-7.4) 5.2 (3.7-7.4)	5.25 (3.3-7.35)	0.79
	(mmol/l)
	LDL (mmol/l) 3.35 (1.9-4.8)	2.9 (1.7-4.6)	3.1 (1.6-6.7)	3.5 (0.1-4.2) 3.1 (1.8-5.7)	2.8 (1.6-5.1)	0.82
	HDL (nmol/l)	1.46 (0.6-2.7)	1.2 (0.5-2.5) 1.04 (0.7-2.5)	1.1 (0.4-3.5) 1.3 (0.65-2.1)	0.97 (0.49-1.85) 0.01
	TG (mmol/l)	0.95 (0.5-1.9)	1.22 (0.4-4.6)	1.3 (0.6-3.0)	1.4 (0.8-8.2) 1.2 (0.6-3.4)	2.1 (0.6-5.0)	0.02
	HbA1c (%)	4.65 (4.0-4.9)	6.75 (5.1-8.7)	4.6 (4.1-5.6)	6.1 (4.3-9.1) 4.6 (3.8-5.3)	6.5 (4.6-10.9) Ͻ0.001
	DM, diabetes mellitus; IC, intermittent claudication; CLI, critical limb ischemia; Hb, hemoglobin; hsCRP, high sensitive
	C-reactive protein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; HbA1c, glycated
	hemoglobin..

Table 3

 3 Standard laboratory parameters: p-values for the effect of DM compared with all patients without DM and the effect of IC and CLI, respectively, compared with patients without PAD in a multiple regression analysis adjusted for age, gender and smoking habits.

	Parameter	DM	IC	CLI
	Hb	Ͼ0.30	0.15	Ͻ0.001
	hsCRP	Ͼ0.30	0.093	Ͻ0.001
	Creatinine	Ͼ0.30	0.027	Ͻ0.001
	Cholesterol	Ͼ0.30	Ͼ0.30	Ͼ0.30
	HDL	0.21	0.020	0.016
	LDL	0.29	Ͼ0.30	Ͼ0.30
	TG	0.011	0.055	0.006
	HbA1c	Ͻ0.001	Ͼ0.30	Ͼ0.30
	Abbreviations: refer to

Table 2 .

 2 Markers in peripheral arterial disease with and without diabetes 195 Variation of laboratory parameters reflecting leukocyte and endothelial cell activation; median values, ranges and

	p-values for testing the hypothesis of

Vascular Medicine 2005; 10: 191-198 Table 4

Table 6

 6 Distribution of the Ϫ174G/C IL-6 promoter genotypes in the controls, patient groups and 200 healthy blood donors; n (%) for each group.

		Blood	Group 1:	Group 2:	Group 3: a	Group 4: a	Group 5: a	Group 6: a
	Genotype	donors	controls	DM	IC	IC ϩ DM	CLI	CLI ϩ DM
	GG	53 (26)	5 (25)	2 (10)	7 (37)	4 (21)	5 (26)	4 (22)
	CC	48 (24)	6 (30)	6 (30)	5 (26)	1 (5)	4 (21)	5 (28)
	GC	99 (50)	9 (45)	12 (60)	7 (37)	14 (74)	10 (53)	9 (50)

a DNA was not available from all patients in these groups.

Table 5

 5 Laboratory parameters reflecting leukocyte and endothelial cell activation: p-values for the effect of DM compared with all patients without DM and the effect of IC and CLI, respectively, compared with patients without PAD in a multiple regression analysis adjusted for age, gender and smoking habits.

	Parameter	DM	IC	CLI
	IL-6	0.12	Ͼ0.30	0.022
	IL-8	0.29	Ͼ0.30	0.30
	TNF␣	Ͼ 0.30	Ͼ0.30	0.003
	sTNF␣-RI	0.06	Ͼ0.30	0.002
	sTNF␣-R2	0.11	Ͼ0.30	0.007
	sVCAM-1	Ͼ0.30	0.026	Ͻ0.001
	sICAM-1	Ͼ0.30	Ͼ0.30	0.001
	sE-selectin	0.12	Ͼ0.30	0.15
	sP-selectin	Ͼ0.30	Ͼ0.30	0.08
	WBC count	Ͼ0.30	Ͼ0.30	0.010
	Granulocyte count	0.22	0.006	Ͻ0.001
	Monocyte count	Ͼ0.30	0.027	0.011
	CD18, granulocytes	Ͼ0.30	Ͼ0.30	Ͼ0.30
	CD11b, granulocytes	Ͼ0.30	Ͼ0.30	0.008
	CD18, monocytes	0.13	Ͼ0.30	0.26
	CD11b, monocytes	Ͼ0.30	Ͼ0.30	0.11
	Abbreviations: refer to Table 4.

Vascular Medicine 2005; 10: 191-198

Acknowledgements

The invaluable help with the statistical analysis given by Jonas Björk, PhD, is gratefully acknowledged. We also thank Mrs Camilla Nilsson for organizing the sampling, and Mrs Annica Andreasson and Mrs Birgitta Gullstrand for their skilful work with flow cytometry analysis and IL-6 genotyping, respectively.


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

Jens Rainer Allenberg

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Renal artery thrombosis in acute type B aortic

dissection: what do you do?

A 68-year-old man with an acute onset of chest pain was admitted to hospital. No signs of myocardial ischemia were identified on electrocardiography or by blood tests. Physical examination was unremarkable and chest radiography was normal. A computed tomographic (CT) scan confirmed acute aortic dissection type B. The dissecting membrane had extended from the false lumen into the origin of the left renal artery (Figure 1A andB). Distally, appositional thrombus formation was seen in the left proximal renal artery (Figure 2). Contrast enhancement of the left kidney was reduced. The current creatinine level was 1.3 mg/dl. The patient underwent immediate uncomplicated renal artery prosthetic bypass reconstruction using a retroperitoneal approach (Figure 3). Postoperative magnetic resonance angiography imaging demonstrated bypass patency and segmental renal infarction (Figure 4A andB). Creatinine and urea levels showed no significant postoperative elevation. The patient was transferred back to the intensive care unit for aggressive antihypertensive treatment.

Figure 1A

 1A Figure 1A The arrow indicates dissection of the left renal artery. Note minor contrast enhancement of the left kidney. Computed tomographic angiography: axial view.

Figure 1B

 1B Figure 1B Dissecting membrane causing renal artery occlusion.

Figure 2

 2 Figure 2 Arrow indicates a thrombus in the left renal artery distal to the dissecting membrane. Magnetic resonance angiography: maximal intensity projection.

Figure 3

 3 Figure 3 Intraoperative view of the iliac-renal bypass graft. Upward pointing arrow: iliac anastomosis; downward pointing arrow: renal anastomosis.

Figure 4A

 4A Figure 4A Magnetic resonance angiography: postoperative maximal intensity projection.

Figure 4B

 4B Figure 4B Note left upper segmental kidney infarction. Magnetic resonance angiography.


Matthias Frick

Hannes F Alber

Alexander Rinner

Alois Suessenbacher

Hanno Ulmer

Severin P Schwarzacher

Otmar Pachinger

Franz Weidinger
email: f.weidinger@uibk.ac.at

Relationship of sonographic wall components of the brachial artery to hypertension and coronary atherosclerosis

Keywords: atherosclerosis, brachial artery, coronary artery disease, hypertension, medial thickness, ultrasound

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

Introduction

With high-resolution ultrasound, the noninvasive assessment of early atherosclerotic changes in peripheral arteries has become possible. [START_REF] Salonen | Ultrasonographic manifestations of common carotid atherosclerosis in elderly eastern Finnish men. Prevalence and associations with cardiovascular diseases and risk factors[END_REF][START_REF] Burke | Arterial wall thickness is associated with prevalent cardiovascular disease in middleaged adults. The Atherosclerosis Risk in Communities (ARIC) Study[END_REF][START_REF] Gnasso | Carotid intimamedia thickness and coronary heart disease risk factors[END_REF][START_REF] Gariepy | Evidence for in vivo carotid and femoral wall thickening in human hypertension. Groupe de Prevention Cardio-vasculaire en Medecine du Travail[END_REF][START_REF] Lemne | Carotid intima-media thickness and plaque in borderline hypertension[END_REF][START_REF] Roman | Parallel cardiac and vascular adaptation in hypertension[END_REF] Previous studies have revealed that sonographically measured wall thickness (WT; i.e. intima plus media thickness) of the carotid artery is increased in patients with coronary artery disease (CAD) compared with controls. [START_REF] Salonen | Ultrasonographic manifestations of common carotid atherosclerosis in elderly eastern Finnish men. Prevalence and associations with cardiovascular diseases and risk factors[END_REF][START_REF] Burke | Arterial wall thickness is associated with prevalent cardiovascular disease in middleaged adults. The Atherosclerosis Risk in Communities (ARIC) Study[END_REF] More importantly, carotid artery WT is also increased in patients with vascular risk factors but without symptomatic atherosclerotic disease. [START_REF] Salonen | Ultrasonographic manifestations of common carotid atherosclerosis in elderly eastern Finnish men. Prevalence and associations with cardiovascular diseases and risk factors[END_REF][START_REF] Gnasso | Carotid intimamedia thickness and coronary heart disease risk factors[END_REF][START_REF] Gariepy | Evidence for in vivo carotid and femoral wall thickening in human hypertension. Groupe de Prevention Cardio-vasculaire en Medecine du Travail[END_REF][START_REF] Lemne | Carotid intima-media thickness and plaque in borderline hypertension[END_REF][START_REF] Roman | Parallel cardiac and vascular adaptation in hypertension[END_REF] Furthermore, patients with arterial hypertension (HT) in particular have been found to show increased WT. [START_REF] Gariepy | Evidence for in vivo carotid and femoral wall thickening in human hypertension. Groupe de Prevention Cardio-vasculaire en Medecine du Travail[END_REF][START_REF] Lemne | Carotid intima-media thickness and plaque in borderline hypertension[END_REF][START_REF] Roman | Parallel cardiac and vascular adaptation in hypertension[END_REF] Whether this reflects adaptation to a higher blood pressure or early atherosclerotic changes is unknown. [START_REF] Bots | Increased common carotid intima-media thickness. Adaptive response or a reflection of atherosclerosis? Findings from the Rotterdam Study[END_REF] In our previous studies we have shown that brachial artery (BA) WT is increased in patients with CAD. [START_REF] Weidinger | Association of wall thickness of the brachial artery measured with high-resolution ultrasound with risk factors and coronary artery disease[END_REF][START_REF] Frick | Morphological rather than functional or mechanical sonographic parameters of the brachial artery are related to angiographically evident coronary atherosclerosis[END_REF] It is interesting that, in particular, patients with HT but without CAD have been shown to have an increased WT. [START_REF] Weidinger | Association of wall thickness of the brachial artery measured with high-resolution ultrasound with risk factors and coronary artery disease[END_REF] Whether or not this is mainly due to medial hypertrophy, which has been suggested in experimental HT, [START_REF] Simon | Detection of preclinical atherosclerosis may optimize the management of hypertension[END_REF][START_REF] Lever | Essential hypertension: a disorder of growth with origins in childhood?[END_REF] still needs to be clarified.

The use of 13 MHz high-resolution ultrasound gives the opportunity to visualize different wall components of superficially located peripheral arteries such as the BA. The aim of this study was therefore to investigate whether sonographically assessed echodense (ED; 'intimal') or echolucent (EL; 'medial') thickening of the BA is associated with HT and/or CAD.

Methods

Patients

Written informed consent was obtained from all participants. A total of 201 men (mean age 54 Ϯ 10 years, range 19-73) in whom coronary angiography was performed because of chest pain were consecutively enrolled into the study. Patients with unstable angina pectoris (Ͻ7 days before inclusion), a left ventricular ejection fraction Ͻ40%, significant valvular disease and/or other severe disease such as cancer or infection were excluded from the study. According to the presence or absence of CAD (defined as a visually estimated diameter stenosis Ն30% in Ն1 major vessel) and/or HT (systolic blood pressure Ն140 mmHg and/or diastolic pressure Ն90 mmHg: i.e. hypertension stage I according to the definition in the fifth report of the Joint National Committee, [START_REF]The fifth report of the Joint National Committee on detection, evaluation and treatment of high blood pressure (JNC V)[END_REF] based on the average of two or more readings taken on each of two or more different days or as current use of antihypertensive drugs), the participants were divided into four groups: group 1 consisted of patients without either HT or CAD (n ϭ 26); group 2 included CAD patients without HT (n ϭ 63); 34 patients with HT but no CAD comprised group 3; and group 4 patients (n ϭ 78) had both HT and CAD.

Vascular risk factors were assessed as follows: smokers were defined as people who had smoked at least one cigarette a day for Ն1 year within the previous 5 years; HT was defined as described above; patients with a plasma low-density lipoprotein cholesterol Ͼ130 mg/dl or who were currently receiving cholesterol lowering therapy were classified as being hypercholesterolemic; [START_REF]Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)[END_REF] patients were considered to be diabetic if they were receiving treatment with insulin or oral hypoglycemic agents, or if the fasting blood glucose level exceeded 140 mg/dl; and a family history of CAD was sought. In addition, fasting blood samples were obtained for the measurement of plasma total, high-density and low-density lipoprotein cholesterol. The investigations conformed to the principles outlined in the Declaration of Helsinki.

Sonographic assessment of the brachial artery

High-resolution ultrasound (13 MHz, Acuson Sequoia C 256, Siemens, Mountain View, CA, USA) and a regional expansion system were used to evaluate BA WT, wall components (ED and EL) and BA endothelial function. The ultrasound examination was always performed between 10 a.m. and noon by an observer blinded to patients' diagnosis. All vasoactive drugs were withdrawn 18-24 hours before examination. The patients were instructed not to smoke and to remain fasting prior to the ultrasound examination. After a rest period of at least 10 minutes in the supine position the right BA was scanned longitudinally above the antecubital fossa. After optimizing gain settings and the transducer position, thus yielding a clear image of the BA as a vessel arch, images were ECG-triggered to the peak of the T wave and stored on hard disk for offline measurement. The offline assessments of the combined wall thickness and the intimal and medial thicknesses were carried out on separate days several weeks apart.

Measurement of the combined wall thickness

Combined WT was assessed as described in previous publications. [START_REF] Weidinger | Association of wall thickness of the brachial artery measured with high-resolution ultrasound with risk factors and coronary artery disease[END_REF][START_REF] Frick | Morphological rather than functional or mechanical sonographic parameters of the brachial artery are related to angiographically evident coronary atherosclerosis[END_REF] Briefly, the combined intimal and medial thickness of the far wall was measured (using electronic calipers) as the distance between the lumen-intima and media-adventitia borders. Measurements were made at two sites per image on four different images per patient. The mean of the eight measurements was defined as the BA WT.

Assessment of wall components

In addition to the combined WT, the ED (intimal) and EL (medial) wall components were assessed at different times several weeks apart by an observer blinded to the diagnosis and the combined WT. The ED thickness was measured as the distance between the lumenintima interface and the intima-media interface, and the EL thickness was assessed as the distance between the intima-media interface and the media-adventitia interface at the peak of the vessel arch on different days several weeks apart. Measurements were carried out at four sites per image on four different images per patient. The four sites per image were defined as being at or close to 1 mm of the 'peak' of the vessel arch over a length of approximately 10 mm. This peak is usually the location that gives the clearest image. The mean of the 16 measurements was defined as the ED (intimal) or the EL (medial) thickness, respectively.

Interobserver variability of brachial artery measurements

The interobserver variability in our laboratory was determined for 10 patients by two observers working independently of each other. The mean difference in ED thickness between the observers was 0.006 Ϯ 0.01 mm (r ϭ 0.85; p ϭ 0.02), that for EL thickness was 0.009 Ϯ 0.02 mm (r ϭ 0.98; p Ͻ 0.01), and for the combined WT it was 0.005 Ϯ 0.04 mm (r ϭ 0.91; p Ͻ 0.01).

Evaluation of peripheral endothelial function

Changes in vessel diameter after reactive hyperemia (flow-mediated, endothelium-dependent vasodilation, FMD) and after sublingual nitroglycerine (endotheliumindependent vasodilation, NMD) were examined according to previously described methods. [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF][START_REF] Frick | Short and long term changes of flow-mediated vasodilation in patients under statin therapy[END_REF] In brief, the resting diameter was defined as the mean of three lumen diameter measurements. After suprasystolic compression of the right upper arm at 260 mmHg for 4.5 minutes, the cuff was deflated and serial posthyperemia scans were stored on the hard disk. The mean of the three widest diameters was taken as the post-hyperemia diameter. When the BA diameter had returned to baseline, 0.8 mg of nitroglycerine was given sublingually and the mean of the three widest diameters during the next 10 minutes was recorded. Vasodilation (FMD, NMD) was calculated as the percentage change in diameter compared with baseline.

Statistical analysis

The data are expressed as means Ϯ standard deviations (range) or as frequencies (percentages). Patient characteristics were compared by an unpaired t-test or the Mann-Whitney U-test for continuous variables and the chi-squared test or Fisher's exact test for categorical variables, as appropriate. Pearson's, Spearman's and eta correlation coefficients were determined to assess the association of the combined WT and wall components with clinical characteristics. After log transformation, linear regression analyses for these parameters were performed. One-way analysis of variance followed by the least significant difference test or the Kruskal-Wallis-H test followed by the Mann-Whitney U-test was used for comparison of combined WT and wall components as well as FMD and NMD between groups, as appropriate. Logistic regression analyses were performed to determine wall components as independent predictors of the presence of HT or CAD, adjusting for major risk factors. p-values of Ͻ0.05 were considered to be statistically significant. All analyses were conducted using statistical software (SPSS for Windows, version 7.5.2G).

Results

Patient characteristics

As already noted, group 1 patients had neither HT nor CAD (n ϭ 26), group 2 comprised CAD patients without HT (n ϭ 63), group 3 patients had HT but no CAD (n ϭ 34), and group 4 had both HT and CAD (n ϭ 78). The clinical characteristics of the patients are shown in Table 1. Age, number of risk factors, total and low-density lipoprotein cholesterol, pulse pressure, body mass index, and prior statin and antihypertensive therapy showed differences between groups.

Wall components

The EL (medial) thickness was significantly greater in patients with CAD and/or HT compared with those without CAD and HT (Figure 1). In addition, a greater EL thickness was seen in groups 3 and 4 compared with group 2 (0.12 Ϯ 0.04 versus 0.15 Ϯ 0.05 versus 0.19 Ϯ 0.08 versus 0.20 Ϯ 0.06 mm; p Ͻ 0.001). Table 2 shows the univariate and multivariate associations of EL thickness with clinical characteristics. On linear regression analysis, age and HT were significantly correlated with EL thickness.

The ED (intimal) thickness revealed no significant differences between groups (0.15 Ϯ 0.04 versus 0.15 Ϯ 0.03 versus 0.16 Ϯ 0.03 versus 0.16 Ϯ 0.03 mm; NS). On multivariate analysis, ED thickness correlated only with age and BA diameter (Table 2).

On logistic regression analyses only EL thickness remained significantly associated with CAD or HT after adjustment for other factors (Table 3).

Combined wall thickness of the brachial artery

In groups 2, 3 and 4 the combined WT of the BA was significantly greater compared with group 1 (Figure 2). In addition, groups 3 and 4 revealed a greater WT than group 2 (Figure 2). Univariate analysis demonstrated a close correlation between the combined WT and the summation of ED plus EL thickness (r ϭ 0.90; p Ͻ 0.001).

Flow-mediated and nitroglycerine-mediated vasodilation

Patients without HT and/or CAD showed greater FMD values compared with the other three groups (p ϭ 0.028; Figure 3). In contrast, NMD revealed no significant differences between groups (19.6 Ϯ 6.1 versus 17.6 Ϯ 7.6 versus 17.9 Ϯ 6.6 versus 15.8 Ϯ 7.1%; NS).

Discussion

This study shows that assessment of the specific wall components of the BA is feasible. Assuming that the ED layer largely represents the intima and the EL layer the media, [START_REF] Salonen | Ultrasound B-mode imaging in observational studies of atherosclerotic progression[END_REF] the medial, but not the intimal, thickness of the BA is associated with HT and coronary atherosclerosis.

In our previous studies we have shown that the BA WT is increased in CAD patients compared with non-CAD patients. [START_REF] Weidinger | Association of wall thickness of the brachial artery measured with high-resolution ultrasound with risk factors and coronary artery disease[END_REF][START_REF] Frick | Morphological rather than functional or mechanical sonographic parameters of the brachial artery are related to angiographically evident coronary atherosclerosis[END_REF] In addition, we have seen that the pattern of BA wall thickening in CAD patients, in accordance with the low prevalence of advanced atherosclerosis in this vessel, [START_REF] Sorensen | Atherosclerosis in the human brachial artery[END_REF] is diffuse rather than localized. [START_REF] Weidinger | Association of wall thickness of the brachial artery measured with high-resolution ultrasound with risk factors and coronary artery disease[END_REF] As atherosclerosis is thought preferentially to affect the intima, we hypothesized that the sonographic ED (intimal) thickness may be increased in patients with CAD compared with hypertensive patients and controls. It is interesting that ED thickness was not significantly different between the groups. One possible explanation could be the small dimensions of the intimal layer and the inability to [START_REF] Sorensen | Atherosclerosis in the human brachial artery[END_REF] who demonstrated that fatty streaks and intimal thickening, but not advanced lesions, are common in the BA and are significantly correlated with atherosclerotic changes in the coronary arteries. Thus, the pattern of atherosclerosis of the BA is likely to be different to that in the coronary arteries. However, the BA as an easily accessible vessel may contribute to the noninvasive estimation of atherosclerosis. Another possible explanation for the lack of difference in the ED layer between groups may be the ultrasound frequency. It is known that the ED thickness is dependent on the longitudinal resolution of the ultrasound system and may therefore not be related to the anatomical intima. To our knowledge, however, no study has so far investigated the association of sonographic measurements performed with 13 MHz and a regional expansion system with histologic data.

In the present study, the EL (medial) thickness was significantly greater in patients with HT compared with both control patients and those with CAD (Figure 1). Although HT seems to be a stronger stimulus for medial thickening compared with CAD (Figure 1; group 2 versus 3), the greatest EL thickness was seen in patients who had both HT and CAD, suggesting an additive effect of both conditions on wall changes in the BA. In addition, logistic regression analyses revealed that EL, but not ED, thickness was significantly associated with the presence of HT and/or CAD. Whether this increased EL thickness in patients with HT indicates early atherosclerosis or an adaptive change due to increased vascular pressure remains to be elucidated. Models of hypertension [START_REF] Simon | Detection of preclinical atherosclerosis may optimize the management of hypertension[END_REF][START_REF] Lever | Essential hypertension: a disorder of growth with origins in childhood?[END_REF] have demonstrated that medial thickness increases as compensation for rising intravasal pressure, suggesting an adaptive mechanism. A recent study [START_REF] Tracy | Medial thickenings of coronary artery and the aging risk factor for atherosclerosis[END_REF] has revealed that medial thickening is a component of atheromatous arteries and is associated with age as a cardiovascular risk factor.

It is interesting to note that endothelial function was also decreased in patients with HT, similar to patients with CAD. This result supports previous studies [START_REF] Muiesan | Flow-mediated dilatation of the brachial artery and left ventricular geometry in hypertensive patients[END_REF][START_REF] Barenbrock | Flow-mediated vasodilation and distensibility in relation to intima-media thickness of large arteries in mild essential hypertension[END_REF] showing endothelial dysfunction in hypertensive patients. Assuming endothelial dysfunction to be an early marker of atherosclerosis, [START_REF] Ross | The pathogenesis of atherosclerosis -an update[END_REF] our data suggest that increased medial and combined WT in patients with HT may be an early marker of atherosclerosis. We did not find a significant difference in FMD between patients with hypertension or CAD only. These data support the hypothesis that BA endothelial function more closely reflects risk factor burden than the presence of CAD. [START_REF] Frick | Endothelial function in a large community[END_REF] Regarding the prognostic value, some [START_REF] Gokce | Predictive value of noninvasively determined endothelial dysfunction for longterm cardiovascular events in patients with peripheral vascular disease[END_REF][START_REF] Modena | Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women[END_REF] but not all [START_REF] Fathi | The relative importance of vascular structure and function in predicting cardiovascular events[END_REF] previous studies have suggested that FMD may be associated with future cardiovascular events. In addition, we have shown that BA WT also predicts clinical outcome. [START_REF] Suessenbacher | Brachial artery intima-media thickness is closely related to long-term cardiovascular events [Abstract][END_REF] One could therefore speculate that, by using a combined assessment of endothelial function, WT and perhaps even arterial wall substructures, patients at high risk could be identified more accurately.

Limitations

From our data we cannot conclude that the EL layer corresponds to the media and the ED layer to the intima. This issue deserves experimental work. Although the media thickness is probably underestimated and the intima overestimated when using our method, the error is likely to be very small as evidenced by the close correlation of WT versus ED plus EL thickness. Pending a histologic correlation of sonographic and anatomic vessel wall components, the combined measurement of intima and media thickness remains the only validated method for the in vivo assessment of human arterial wall structure. We included only patients who had undergone coronary angiography. This may have introduced a selection bias towards those with more advanced disease. Finally, the number of patients studied is insufficient to draw definite conclusions regarding the clinical utility of BA scanning.

Conclusion

This study demonstrates that EL, but not ED, thickness of the BA is independently correlated with the presence of CAD and HT. High-resolution ultrasound examination of the BA wall, in combination with the assessment of endothelial function, may contribute to the noninvasive assessment of early atherosclerosis. Further studies are required to confirm these results in larger patient groups.

Figure 1

 1 Figure 1 Box plots showing the influence of CAD and HT on brachial artery medial (EL) thickness. * p Ͻ 0.05, ** p Ͻ 0.001; CAD, coronary artery disease; HT, arterial hypertension.

  188 M Frick et al Vascular Medicine 2005; 10: 185-190

  Relationship of sonographic brachial artery thickness with hypertension and coronary atherosclerosis 189Vascular Medicine 2005; 10:185-190

Figure 3

 3 Figure 3 Box plots showing FMD between the different groups (abbreviations as Figure 1; FMD, flow-mediated vasodilation).

Figure 2

 2 Figure 2 Box plots showing the influence of HT and CAD on combined brachial artery wall thickness (abbreviations as Figure 1).

Table 1

 1 Relationship of sonographic brachial artery thickness with hypertension and coronary atherosclerosis 187 Patient characteristics (data expressed as mean Ϯ standard deviation or as n (%)).

		Group 1	Group 2	Group 3	Group 4
	Characteristic	(n ϭ 26)	(n ϭ 63)	(n ϭ 34)	(n ϭ 78)	p
	Age (years)	50 Ϯ 10	52 Ϯ 10	55 Ϯ 10	57 Ϯ 8	Ͻ0.001
	Number of risk factors	1.2 Ϯ 0.8	1.7 Ϯ 0.9	2.1 Ϯ 0.7	2.4 Ϯ 0.8	Ͻ0.001
	Diabetes mellitus	0	3 (5)	2 (6)	7 (9)	0.371
	Positive family history	5 (19)	20 (32)	5 (15)	22 (28)	0.233
	Hypercholesterolemia	20 (77)	56 (89)	25 (74)	61 (78)	0.247
	Smoker	7 (27)	30 (48)	9 (26)	19 (24)	0.022
	Total cholesterol (mg/dl)	232 Ϯ 36	230 Ϯ 45	213 Ϯ 43	210 Ϯ 42	0.016
	LDL-C (mg/dl)	164 Ϯ 29	153 Ϯ 42	140 Ϯ 40	140 Ϯ 37	0.018
	HDL-C (mg/dl)	50 Ϯ 12	45 Ϯ 11	47 Ϯ 12	44 Ϯ 11	0.212
	Triglyceride (mg/dl)	148 Ϯ 71	184 Ϯ 119	171 Ϯ 74	165 Ϯ 81	0.392
	PP (mmHg)	52 Ϯ 14	50 Ϯ 12	59 Ϯ 19	59 Ϯ 16	0.001
	BMI (kg/m 2 )	2 6 Ϯ 3	2 6 Ϯ 3	2 8 Ϯ 3	2 8 Ϯ 3	0.003
	Statins	1 (4)	10 (16)	8 (24)	24 (31)	0.018
	Antihypertensive medication	0 (0)	0 (0)	19 (56)	43 (55)	Ͻ0.001

Vascular Medicine 2005; 10: 185-190 LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; PP, pulse pressure; BMI, body mass index.

Table 2

 2 Univariate and multivariate correlation of sonographic parameters.

			ED thickness		EL thickness		Wall thickness
				p			p			p
			Univariate Multivariate		Univariate Multivariate		Univariate Multivariate
	Age	0.30 Ͻ0.01	Ͻ0.01	0.48 Ͻ0.01	Ͻ0.01	0.50 Ͻ0.01	Ͻ0.01
	LDL-C	Ϫ0.14	0.05	0.32	Ϫ0.09	0.28	n.i.	Ϫ0.09	0.20	n.i.
	BA diameter	0.31 Ͻ0.01	0.02	0.16	0.03	0.51	0.28 Ͻ0.01	0.08
	FMD	Ϫ0.19 Ͻ0.01	0.91	Ϫ0.17	0.02	0.52	Ϫ0.22	0.02	0.41
	NMD	Ϫ0.27 Ͻ0.01	0.88	Ϫ0.17	0.03	0.53	Ϫ0.23	0.02	0.45
	HT	0.15	0.04	0.33	0.42 Ͻ0.01	Ͻ0.01	0.39 Ͻ0.01	Ͻ0.01
	Diabetes	0.06	0.42	n.i.	0.17	0.02	0.22	0.18	0.01	0.11
	CAD	0.08	0.26	n.i.	0.18	0.01	0.03	0.22	0.02	0.01
	PP	0.25 Ͻ0.01	0.07	0.31 Ͻ0.01	0.07	0.22 Ͻ0.01	0.24
	BMI	0.07	0.34	n.i.	0.23 Ͻ0.01	0.04	0.23 Ͻ0.01	0.07
	ED	n.i.	n.i.	n.i.	0.15	0.04	0.39	0.48 Ͻ0.01	n.i.
	EL	0.15	0.04	0.49	n.i.	n.i.	n.i.	0.81 Ͻ0.01	n.i.
	ED, echodense; EL, echolucent; LDL-C, low-density lipoprotein cholesterol; n.i., not included; BA, brachial artery; FMD,
	flow-mediated vasodilation; NMD, nitroglycerine-mediated vasodilation; HT, arterial hypertension; CAD, coronary
	artery disease; PP, pulse pressure; BMI, body mass index.

Table 3

 3 Logistic regression analyses for the presence of arterial hypertension or coronary artery disease.

	CAD	HT

Vascular Medicine 2005; 10: 185-190


Jozef Dulak

Severin P Schwarzacher

Ralf H Zwick

Hannes Alber

Gunda Millonig

Caecilia Weiss

Heike Hügel

Matthias Frick

Alicja Jozkowicz

Otmar Pachinger

Professor Franz Weidinger
email: f.weidinger@uibk.ac.at

Effects of local gene transfer of VEGF on neointima formation after balloon injury in hypercholesterolemic rabbits

Keywords: atherosclerosis, gene therapy, vascular endothelial growth factor

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Gene transfer of vascular endothelial growth factor (VEGF) has been shown to improve collateral vessel formation in ischemic limbs, [START_REF] Takeshita | Gene transfer of naked DNA encoding for three isoforms of vascular endothelial growth factor stimulates collateral development in vivo[END_REF][START_REF] Tsurumi | Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion[END_REF] to induce re-endothelialization after balloon angioplasty [START_REF] Asahara | Accelerated restitution of endothelial integrity and endothelium dependent function after phVEGF 165 gene transfer[END_REF] and to prevent the development of neointima formation. [START_REF] Asahara | Accelerated restitution of endothelial integrity and endothelium dependent function after phVEGF 165 gene transfer[END_REF][START_REF] Van Belle | Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening[END_REF][START_REF] Laitinen | VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries[END_REF] The proangiogenic activity of VEGF is dependent on nitric oxide (NO). [START_REF] Laitinen | VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries[END_REF] Accordingly, inhibition of NO generation abolished the VEGF-induced angiogenesis in rabbit cornea [START_REF] Ziche | Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not fibroblast growth factor-induced angiogenesis[END_REF] and targeted mutation of endothelial, constitutive nitric oxide synthase gene (eNOS)impaired ischemia-induced angiogenesis despite normal expression of VEGF in eNOS knockout animals. [START_REF] Murohara | Nitric oxide synthase modulates angiogenesis in response to tissue ischemia[END_REF] NO generation can be impaired in atherosclerotic vessels and it may be restored by local delivery of L-Arg, the substrate for NO generation, thus preventing neointima formation. [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF] In view of these results, both VEGF and NO were thought to be beneficial in the prevention of neointima formation after ballon injury in normocholesterolemic animals. However, recent studies have shown that, although intracoronary or intramuscular application of adenoviral and plasmid VEGF is safe and feasible in patients with coronary heart or peripheral artery disease, it does not improve exercise performance or quality of life. 9,10 Thus, it seems that VEGF alone may be not sufficient for prevention of neointima formation. We therefore sought to determine whether endovascular VEGF gene transfer in combination with the local application of L-Arg would lead to a more effective inhibition of neointima formation in the hypercholesterolemic rabbit than either therapy alone.

Laboratories (Sulzfeld, Germany). Animal experiments were performed according to the National Institutes of Health guidelines. The rabbits had unlimited access to food and water during the entire study.

Ten rabbits fed with normal chow were used for testing the feasibility of transfection (injury alone n ϭ 3, injury ϩ L-Arg n ϭ 3, transfection with pSV␤-gal plasmid n ϭ 4).

Twenty-four animals were fed a 1% cholesterol diet for 3 weeks before the procedure. After angioplasty of the iliac artery a Dispatch TM delivery catheter (Scimed, Boston Scientific Inc., Boston, MA, USA) was introduced to the site of the injury using the internal iliac artery as a landmark. The animals were treated by local delivery of: (1) pSG5VEGF 165 plasmid (1000 g) alone (group 1; n ϭ 6); (2) pSG5VEGF 165 plasmid (1000 g) with L-Arg (800 mg) (group 2; n ϭ 7); (3) L-Arg (800 mg) alone (group 3; n ϭ 6); or (4) L-Arg (800 mg) with naked pSV␤-gal plasmid (1000 g) (group 4; n ϭ 5). Blood was collected before, and 7 and 28 days after the procedure, after which the animals were sacrificed. The cholesterol diet was continued until the end of the study, when vessels were snap frozen in liquid nitrogen and cryostat sections were cut and used for morphometry or immunocytochemistry.

Balloon injury and local drug delivery

The succesful utilization of a local drug delivery catheter [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF] has already been demonstrated by our group. For the experiments, the rabbits were anesthetized with an intramuscular injection of ketamine (5 mg/kg) and xylazine (35 mg/kg). When necessary, additional anesthesia was given intravenously throughout the procedure. The right carotid artery was exposed, carefully incised and a Tygon sheath (1.67 mm in diameter) inserted under fluoroscopic control into the descending aorta. A coronary angioplasty catheter (3 mm diameter) was advanced consecutively into both external iliac arteries for balloon angioplasty. The internal iliac branch was used as an anatomical landmark. The balloon was inflated at 8 atm three times for 30 s. The last inflation was followed by a backwards and forwards movement of the balloon in order to rub off the endothelium fully. After withdrawing the ballon catheter a Dispatch delivery catheter (3 mm) was introduced to the site of the injury. The plasmid diluted in 12 ml sterile saline was delivered to the injured segment by a perfusion pump (Braun, Melsungen, Germany) for 15 minutes. For the first 10 minutes the speed of delivery was 12 ml/h, followed by 80 ml/h. The normocholesterolemic animals (n ϭ 10) used in the preliminary study were sacrificed 3 days after injury, the arteries were removed and processed for determination of ␤-galactosidase activity. 9,10 RNA was isolated from some vessels (see below). The hypercholesterolemic animals (n ϭ 24) were sacrificed 28 days after injury.

Preparation of plasmids

Two types of plasmids were used in the experiments. The control plasmid, pSV␤-gal (Promega, Madison, USA) contains the reporter, the ␤-galactosidase gene driven by the SV40 promoter. The pSG5VEGF 165 plasmid contains human VEGF 165 cDNA, driven by the same SV40 promoter. Our former in vitro [START_REF] Dulak | Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells[END_REF] and in vivo studies [START_REF] Dulak | Gene transfer of naked VEGF plasmid induces the formation of microvessels but not mature collaterals in ischemic limb muscles[END_REF] demonstrated that the pSG5VEGF 165 plasmid generates the angiogenically active protein that induces endothelial cell proliferation [START_REF] Dulak | Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells[END_REF] and increases the number of microvessels in ischemic rabbit adductor muscle. [START_REF] Dulak | Gene transfer of naked VEGF plasmid induces the formation of microvessels but not mature collaterals in ischemic limb muscles[END_REF] Plasmids were isolated from overnight bacterial culture using a Qiagen EndoFree Plasmid Kit (Qiagen, Hilden, Germany) and dissolved in sterile saline. The integrity of the plasmid was determined by restriction enzyme digestion followed by agarose electrophoresis.

Determination of VEGF in vascular tissue

The determination of VEGF in vascular tissue was performed via the reverse transcription-polymerase chain reaction (RT-PCR). RNA was isolated from the arterial segments with the RNA Total Isolation Kit (Promega, Madison, WI, USA). One pair of primers (sense primer: 5 Ј CTG CTG TCT TGG GTG CAT TG; antisense primer 5 Ј CAC CGC CTC GGC TTG TCA CAT) was used to detect VEGF mRNA. With this pair of primers a 563 bp product was obtained from the amplification of the VEGF 165 isoform. RT was carried out at 70°C for 40 min, with Tth polymerase (Promega), followed by 40 cycles of PCR: 94°C for 40 s, 64°C for 40 s, 70°C for 40 s.

In order to detect the plasmid DNA in arterial segments we used the primers amplifying a part of the ampicillin-resistance gene (Amp R ) present in both pSV␤-gal and pSG5VEGF 165 plasmid. The sequence of the upper primer was: 5Ј GCT GAA GAT CAG TTG GGT GC and the lower primer was: 5Ј-GAA GTA AGT TGG CCG CAG TG. The product length was 310 bp. PCR was performed under the conditions specified above, with omission of the RT step. The PCR products were detected by agarose-gel electrophoresis with ethidium bromide staining.

Determination of VEGF plasma levels

Two different enzyme-linked immunosorbent assay (ELISA) kits were used in order to differentiate between exogenous human VEGF (Accucyte ELISA: CytImmune Sciences Inc., College Park, MD, USA) and endogenous rabbit VEGF (Human Quantkine ELISA: R&D Systems, Abingdon, UK). The CytImmune ELISA kit contains antihuman VEGF antibodies generated in rabbits; these antibodies do not cross-react with rabbit VEGF. The antibodies from the Quantkine ELISA kit recognize both human and rabbit VEGF. [START_REF] Dulak | Gene transfer of naked VEGF plasmid induces the formation of microvessels but not mature collaterals in ischemic limb muscles[END_REF]

Immunohistochemical analysis

Cryostat sections fixed in acetone were incubated with antihuman VEGF or antirat VEGF goat-derived antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1:150 in DAKO antibody diluent (Dako, Carpenteria, CA, USA) with a background reducing component. Both used anti-VEGF antibodies cross-reacting with rabbit VEGF protein (J Dulak, unpublished observations). Anti-CD31 mouse antibody (Dako) was used to visualize the extent of reendothelialization. Antibodies were applied to sections for 2 hours at room temperature. The cells were washed several times with phosphate buffered saline (PBS) and incubated for 1 hour at room temperature with monoclonal biotin-conjugated antigoat immunoglobulin G (IgG) (for VEGF staining) or anti-mouse IgG (for CD31) (both antibodies from Sigma, Munich, Germany) diluted 1:200 in DAKO antibody diluent. Afterwards, the cells were washed again with PBS, and extravidine conjugated with alkaline phosphatase (Sigma) was applied to the sections for 30 min. Then the NBT/BCIP chromogen was added and the reaction was monitored under the microscope. After 10-15 min the sections were washed with water and covered with a glass slide.

Morphometric analysis

Frozen sections stained with hematoxylin and eosin were used for histomorphometry. Measurements of intimal and medial cross-sectional area were performed by observers blinded to the treatment group. Histological cross-sections were scanned using 50 ϫ magnification and digitized using an Image Analyst program (Zeiss, Jena, Germany).

The following borders were highlighted with a trackball: external elastic lamina (EEL), internal elastic lamina (IEL) and lumen-intima border. Crosssectional areas of the respective vessel wall layers were then determined and an intima/media ratio (I/M) calculated (intima: IEL/lumen; media: IEL/EEL). The intima was defined as the area between the lumen and the IEL, and the media between the IEL and the EEL. Four cross-sections were measured for each vessel segment.

Statistical analysis

For the morphometric analysis, analysis of variance (ANOVA) for repeated measurements was used followed by a post-hoc test (least significant difference) to identify statistically significant differences among groups.

For comparisons between two groups the Mann-Whitney U-test was applied. A value of p Ͻ 0.05 was considered to be statistically significant. Data are shown as means Ϯ standard errors or as box plot graphs.

Results

Feasibility of transfection of naked DNA with a Dispatch balloon catheter

The feasibility of local plasmid DNA delivery with a Dispatch catheter was tested in vivo in 10 normocholesterolemic animals: three rabbits had injury alone, three had injury plus delivery of L-Arg, and four were transfected with pSV␤-gal plasmid.

The delivery of naked plasmid resulted in the expression of ␤-galactosidase in treated vessels (Figure 1). No ␤-galactosidase activity was detected in only injured vessels or in injured vessels to which L-Arg had been delivered (not shown). The transfection efficiency was low (Ͻ0.1% of cells), and cells exhibiting ␤-galactosidase activity in the cytoplasm were observed in only some areas of the treated segments, mostly in the media (Figure 1). VEGF 165 isoform mRNA was detected in the vessels using RT-PCR and there were no differences between the groups (data not shown).

Effects of pSG5VEGF 165 transfer on neointima formation

Immunocytochemical staining for CD31 showed near complete re-endothelialization in all vessels 4 weeks after balloon injury (data not shown), in accordance with previous data. [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF][START_REF] Weidinger | Persistent dysfunction of regenerated endothelium after balloon angioplasty of rabbit iliac artery[END_REF] The vessels of animals treated with pSG5VEGF 165 plasmid showed a significantly higher I/M ratio compared with pSG5VEGF 165 plus L-arg (p Ͻ 0.05; ANOVA ϭ 0.036), while no significant differences were observed among the other groups (Figure 2).

VEGF in plasma and the vascular wall

During the first 3 weeks of cholesterol feeding the endogenous rabbit VEGF plasma levels tended to increase from 12.59 Ϯ 2.53 pg/ml to 17.06 Ϯ 1.57 pg/ml (n ϭ 11 and n ϭ 24, respectively; p ϭ 0.061) (Figure 3). Injury itself caused a significant further increase in endogenous VEGF plasma levels in all treated animals to 23.01 Ϯ 1.92 pg/ml (n ϭ 24, p Ͻ 0.02 vs level before injury). VEGF at 28 days after injury remained at similar levels (28.46 Ϯ 5.24, n ϭ 7; p Ͻ 0.01) (Figure 3). However, when using the CytImmune ELISA kit with antibodies specific for only human VEGF we were not able to detect exogenous VEGF in the rabbit plasma.

Immunocytochemical staining showed very abundant VEGF expression in all rabbit iliac arteries, which was independent of the kind of treatment (Figure 4A). Numerous positive cells were observed in both the media and the neointima of vessels transfected with pSV␤gal or pSG5VEGF 165 plasmid (Figure 4B). VEGF was also strongly expressed in the noninjured vessels of hypercholesterolemic animals. Thus, the anti-VEGF antiobodies did not differentiate between rabbit and human VEGF.

Discussion

The salient findings of this study were (1) the delivery of VEGF plasmid to injured vascular segments failed to prevent intimal growth; (2) intimal growth was reduced by L-Arg delivery in the setting of hypercholesterolemia; and (3) VEGF plasma levels were increased by hypercholesterolemia alone as well as by vessel injury.

The feasibility of delivering naked plasmid into the vessel wall was confirmed by demonstrating the expression of a reporter gene. We assume therefore that the observed effect of VEGF may result from the local production of VEGF in the transfected vessels. However, we were not able to detect exogenous VEGF in the rabbit plasma nor to document a further increment of VEGF in the vessel wall induced by VEGF plasmid transfection. This may indicate that the amount of VEGF produced by the plasmid vector was very low. Other studies have also shown no systemic release of exogenous VEGF protein, even when more efficient adenoviral vectors were used. [START_REF] Laitinen | Cathetermediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty[END_REF][START_REF] Magovern | Regional angiogenesis induced in nonischemic tissue by an adenoviral vector expressing vascular endothelial growth factor[END_REF][START_REF] Hiltunen | Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta[END_REF] The inability to observe an increase in VEGF expression after plasmid transfection may be caused by the low specificity of antihuman VEGF antibodies, which cross-reacted with rabbit protein.

Previous studies have shown inhibition of neointima formation with the use of VEGF gene transfer, a specific mitogen for endothelial cells. Indeed, several groups [START_REF] Asahara | Accelerated restitution of endothelial integrity and endothelium dependent function after phVEGF 165 gene transfer[END_REF][START_REF] Van Belle | Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening[END_REF][START_REF] Laitinen | VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries[END_REF] have shown a positive effect on endothelial regrowth after injection of VEGF. Given the lack of data in diet-induced hypercholesterolemia combined with mechanical injury, we sought to determine VEGF effects in this setting, which better mimics the conditions expected in human atherosclerotic vessels. [START_REF] Inoue | Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis[END_REF] It is interesting, and contrary to our expectation, that the neointima in vessels treated with VEGF plasmid was massive and the I/M ratio was higher than in the VEGF plus L-Arg treatment group. To determine whether VEGF was expressed in injured and locally treated vessels, we performed immunohistochemical staining. This approach, however, demonstrated very abundant VEGF expression in the media and neointima of both treated and uninjured rabbit arteries.

One explanation for the findings that VEGF did not inhibit neointimal proliferation as suggested in previous studies could be that the majority of those experiments were performed in normocholesterolemic animals. It has been demonstrated that VEGF is more highly expressed in atherosclerotic vessels than in normal vessels. [START_REF] Inoue | Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis[END_REF][START_REF] Ramos | Induction of macrophage VEGF in response to oxidized LDL and VEGF accumulation in human atherosclerotic lesions[END_REF][START_REF] Inoue | Oxidized LDL regulates vascular endothelial growth factor expression in human macrophages and endothelial cells through activation of peroxisome proliferator-activated receptor-gamma[END_REF] Indeed, in this study we observed an increase in VEGF plasma levels in rabbits fed cholesterol-rich chow, suggesting that hypercholesterolemia may elevate VEGF levels in plasma. Therefore, whereas normocholesterolemic animals appear to benefit from VEGF overexpression after arterial injury, this may not be the case in hypercholesterolemia, when endogenous VEGF is already enhanced and NO bioavailability is reduced. Indeed, another recent study also showed no inhibition of neointima formation after VEGF-A delivery to rabbits fed a 0.25% cholesterol diet. [START_REF] Hiltunen | Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta[END_REF] Moreover, a single intraperitoneal injection of a small amount of VEGF protein potentiated atherosclerotic plaque formation in apoE/apoB100 knockout mice and in hypercholesterolemic rabbits. [START_REF] Celletti | Vascular endothelial growth factor enhances atherosclerotic plaque progression[END_REF][START_REF] Celletti | Effect of human recombinant vascular endothelial growth factor 165 on progression of atherosclerotic plaque[END_REF] It has also been shown that VEGF is necessary for the initial steps of atherogenesis in the setting of NO inhibition, [START_REF] Zhao | Vascular endothelial growth factor is necessary in the development of arteriosclerosis by recruiting/activating monocytes in a rat model of long-term inhibition of nitric oxide synthesis[END_REF] and in an earlier study [START_REF] Yonemitsu | Characterization of in vivo gene transfer into the arterial wall mediated by the Sendai virus (hemagglutinating virus of Japan) liposomes: an effective tool for the in vivo study of arterial diseases[END_REF] VEGF delivery enhanced neointima formation.

Moreover, while this article was under review, several others were published that demonstrated the complex effect of VEGF delivery. In a study by Zhao et al [START_REF] Zhao | Essential role of vascular endothelial growth factor and flt-1 signals in neointimal formation after periadventitial injury[END_REF] the expression of VEGF and its receptors markedly increased with the development of neointima formation in hypercholesterolemic mice after cuff-induced periarterial injury. The conclusion was that VEGF may promote neointimal formation by acting as a pro-inflammatory cytokine, the effect being dependent on VEGFR1 (flt-1). In agreement with those studies, blockade of VEGF by soluble VEGF receptor gene transfer was shown to attenuate neointimal formation after intra-arterial injury in rabbits, rats and mice. [START_REF] Ohtani | Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells[END_REF] Khurana et al demonstrated that the application of VEGF-165 to the adventitia of injured arteries induced a marked increase in neointimal thickening beyond that seen with injury alone. [START_REF] Khurana | Vascular endothelial growth factor gene transfer inhibits neointimal macrophage accumulation in hypercholsterolemic rabbits[END_REF] On the other hand, the same group showed in another study that VEGF gene transfer inhibits collar-inhibited intimal thickening, macrophage accumulation and VCAM-1 expression in hypercholesterolemic rabbits. [START_REF] Khurana | Angiogenesisdependent and independent phases of intimal hyperplasia[END_REF] Hutter et al [START_REF] Hutter | growth factor synthesis in vascular smooth muscle cells is enhanced by 7-ketocholesterol and lysophosphatidylcholine independently of their effect on nitric oxide production[END_REF] showed that VEGF accelerated endothelial repair and inhibited neointima formation after arterial injury in mice. Thus there is some discordance in the preclinical literature, which makes it difficult to state unequivocally what may be the effect of local VEGF delivery in cardiovascular patients.

In our study, the VEGF plasma levels were upregulated by cholesterol feeding as well as by injury over the period of the experiment. This observation is in agreement with other data demonstrating an enhancement of VEGF synthesis in atherosclerotic conditions. Recently, we 29 and others [START_REF] Ramos | Induction of macrophage VEGF in response to oxidized LDL and VEGF accumulation in human atherosclerotic lesions[END_REF][START_REF] Inoue | Oxidized LDL regulates vascular endothelial growth factor expression in human macrophages and endothelial cells through activation of peroxisome proliferator-activated receptor-gamma[END_REF] have also shown that modified low density lipoprotein can enhance VEGF production by vascular smooth muscle cells and macrophages. The induction of VEGF has also been observed in injured vessels. [START_REF] Wysocki | Vascular endothelial growth factor (VEGF) expression during arterial repair in the pig[END_REF][START_REF] Hausner | Vascular endothelial growth factor in porcine coronary arteries following balloon angioplasty[END_REF] However, several studies have demonstrated that the systemic or topical delivery of L-Arg can prevent restenosis and improve endothelial function in hypercholesterolemic animals. [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF][START_REF] Janero | Nitric oxide and postangioplasty restenosis: pathological correlates and therapeutic potential[END_REF][START_REF] Brandes | N(G)-nitro-L-arginineand indomethacin-resistant endothelium-dependent relaxation in the rabbit renal artery: effect of hypercholesterolemia[END_REF] Increased generation of NO as a result of L-Arg supplementation may have inhibited neointima formation by attenuating the proliferation of smooth muscle cells as demonstrated in earlier studies. [START_REF] Schwarzacher | Local intramural delivery of L-arginine enhances nitric oxide generation and inhibits lesion formation after balloon angioplasty[END_REF] The pharmacodynamics of locally applied L-Arg has been addressed in one previous study. Niebauer et al [START_REF] Niebauer | Local L-arginine delivery after balloon angioplasty reduces monocyte binding and induces apoptosis[END_REF] have shown by using 3 H-L-Arginine that local L-Arg remained elevated for 1 week after its intramural delivery, which was associated with a persistent increase in NO release, inhibition of monocyte binding, and an increased rate of apoptosis of vascular smooth muscle cells as well as macrophages. Similar mechanisms may also have operated under our settings. Moreover, a recent randomized controlled trial demonstrated that locally applied L-Arg, by means of a Dispatch catheter, attenuated neointima formation after stent deployment in humans. [START_REF] Suzuki | Effect of local delivery of L-arginine on in-stent restenosis in humans[END_REF] Supplementation with L-Arg may restore the production of NO by reversing the effect of asymmetric dimethylarginine, an endogenous inhibitor of NOS activity, which is supported by both experimental and clinical data. [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF][START_REF] Jang | Angiogenesis is impaired by hypercholesterolemia: role of asymmetric dimethylarginine[END_REF][START_REF] Lundman | Mild-to-moderate hypertriglyceridemia in young men is associated with endothelial dysfunction and increased plasma concentrations of asymmetric dimethylarginine[END_REF][START_REF] Stuhlinger | Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine[END_REF] In conclusion, our results add to those already published that suggest that VEGF may not inhibit neointima formation in the setting of hypercholesterolemia. Thus, VEGF gene therapy applied locally in atherosclerotic vessels may not be beneficial. Moreover, recent clinical trials did not provide a definitive conclusion concerning the potential beneficial effect of either VEGF protein [START_REF] Henry | The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis[END_REF] or gene therapy [START_REF] Hedman | Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT)[END_REF][START_REF] Rajagopalan | Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, doubleblind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication[END_REF][START_REF] Khan | Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia[END_REF][START_REF] Yla-Herttuala | Gene transfer as a tool to induce therapeutic vascular growth[END_REF] in patients with cardiovascular disease. Further studies are therefore necessary in order to establish the therapeutic efficacy of VEGF using a model combining ischemia and atherosclerosis. This model would approximate more closely to a clinical trial.

Figure 1

 1 Figure 1 Expression of ␤-galactosidase reporter gene in rabbit iliac artery after local infusion of pSV␤-gal plasmid. Positive cells are visible as dark-stained cells (arrows). L: lumen; Adv: adventitia.

Figure 2

 2 Figure 2 The effect of local delivery of VEGF plasmid on neointima formation. Morphometric analysis of I/M ratio. Delivery of pSG5VEGF 165 alone resulted in a higher I/M ratio than other treatments. *p Ͻ 0.05; ANOVA ϭ 0.036.

Figure 3

 3 Figure 3 Endogenous VEGF in rabbit plasma. Levels of rabbit VEGF slightly increased in plasma during 3 weeks of cholesterol feeding. Injury resulted in an additional significant increase of VEGF, independent of the type of treatment applied. *p Ͻ 0.02 vs value before injury; **p Ͻ 0.01 vs value before injury.

Figure 4

 4 Figure4 Immunocytochemical demonstration of VEGF expression in the injured rabbit iliac arteries. Strong VEGF expression was observed in the vessels of hypercholesterolemic rabbits, independent of the type of treatment. VEGF was particularly strongly expressed under the endothelium (A, arrows) and at the border between neointima and media (B, arrow). IEL: internal elastica lamina. No staining was observed when the primary antibody was omitted (not shown).

  Collaborative Research Grant, and grant number PBZ-KBN 096/P05/2004 from the Polish Ministry of Scientific Research and Information Technology. Dr Alicja Józkowicz is an International Senior Research Fellow of the Wellcome Trust. J Dulak, SP Schwarzacher and RH Zwick contributed equally to this study.

Vascular Medicine 2005; 10: 285-291

Acknowledgements

This work was supported by a fellowship grant from the Austrian Society of Cardiology, a Polish-Austrian


H Gutierrez

J Sanz

S Dellegrottaglie

Sanjay Rajagopalan
email: sanjay.rajagopalan@mssm.edu

Images in vascular medicine Giant coronary aneurysm due to Kawasaki's disease

come   L'archive ouverte pluridisciplinaire

A 24-year-old Caucasian woman presented to her physician for a follow-up evaluation of Kawasaki's disease. She had a prior diagnosis of Kawasaki's disease resulting in an aneurysm of the left anterior descending artery (LAD) diagnosed by invasive arteriography 6 years previously but was otherwise asymptomatic. Multidetector computed tomography (MDCT) coronary angiography was performed using the following parameters: 80 ml of contrast followed by 50 ml of saline (rate 3. MDCT is a useful tool in the follow up of patients with coronary aneurysms due to Kawasaki's disease. It enables accurate measurement of the diameter of the aneurysms and assessment of the serial changes for determination of optimal timing for surgery.

  5 cc/s), rotation time ϭ 0.42 s, collimation ϭ 16 ϫ 0.75 mm, tube current ϭ 400 mAs, voltage ϭ 120 kV, pitch ϭ 0.31. MDCT revealed a giant coronary aneurysm (19 ϫ 9 mm) in the proximal segment of the LAD, involving the origin of a diagonal branch (arrow in Panel A, which shows a 3-dimensional volume rendered reconstruction; PA: pulmonary artery; LV: left ventricle). A small filling defect was noted in the anteromedial inner aspect of the aneurysm in maximum intensity projection (MIP) images, a finding compatible with mural thrombosis (arrow in Panel B). Three other serial aneurysms were seen in the proximal and middle segments of the right coronary artery (8.6 ϫ 4.5 mm, 14 ϫ 6.5 mm and 14 ϫ 10 mm, from proximal to distal, respectively) as shown by the arrows in Panel C (3-dimensional volume Vascular Medicine 2006; 11: 49-50 rendered image. RV: right ventricle; Ao: aorta) and Panel D (MIP reconstruction. LV: left ventricle; Ao: aorta). No other coronary abnormalities were noted. The patient was referred for coronary artery bypass artery grafting of the LAD and right coronary artery (RCA).

Acknowledgements

Dr Gutierrez is a Cardiology Fellow and his work is supported by a grant from the Spanish Society of Cardiology. Dr Sanz is a Post-Doctoral Research Fellow and his work is supported by a research grant from the Spanish Society of Cardiology ('Beca para la Formación en Investigación Post-Residencia'). Dr Dellegrottaglie is a Post-Doctoral Research Fellow and his work is supported by a grant from the Italian Society of Cardiology.


Nenad S Ilijevski
email: ilijevskidr@yubc.net

Sandra Radak

Bozidar Novakovic

Ana Miholjcic

Djordje Radak

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 55-year-old man presented with an enlarging painless mass in the base of the left side of the neck. His left thyroid lobe had been resected for goiter 16 years ago.

Ultrasonography revealed an aneurysm in the shape of a triangle in the transverse plane and ovoid shape in the saggital plane, 42 ϫ 34 ϫ 21 mm in size, closely attached to the lateral aspect of the internal jugular vein (IJV) (Panels A and B). At the junction of the middle and the distal third of this aneurysm, a communication with the IJV was seen, 6-7 mm in diameter. High-velocity blood flow was registered within the central part of the aneurysm, and much slower flow near the aneurysmal wall, causing a 'pseudothrombosis' effect. The flow through the aneurysmal neck was bidirectional and respiratory phase-dependent (Panel B).

Surgery was performed with resection of the aneurysm and lateral suture of the jugular vein. No thrombus was found within the aneurysm, and there was no reaction in the surrounding tissue to connect the previous neck surgery with the aneurysm formation. Pathologic examination showed all layers of a normal vein, with endothelial continuity and fibroblastic proliferation and some mixed muscle layers. Jugular vein aneurysms are rarely seen, 1,2 and most frequently have been described in children. They appear to have a benign natural history, and should be excised only if symptomatic, enlarging or disfiguring. [START_REF] Hopsu | Jugular vein aneurysm or phlebectasia[END_REF] Duplex 3D ultrasonography was a reliable technique for diagnosis and sufficient for surgical treatment of the problem.