Circulating CD40 ligand is elevated only in patients with more advanced symptomatic peripheral arterial diseases

Thrombosis Research (2006) 118, 619 — 626

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Circulating CD40 ligand is elevated only in patients with more advanced symptomatic peripheral arterial diseases Wen-Jane Lee a, Wayne Huey-Herng Sheu a,b, Ying-Tsung Chen b,c, Tsun-Jui Liu d,e, Kae-Woei Liang d,e, Chih-Tai Ting d,f, Wen-Lieng Lee d,f,* a

Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan Department of Medicine, Chung Shan Medical University, Taichung, Taiwan c Department of Medicine, Taichung Veterans General Hospital, Taiwan d Cardiovascular Center, Taichung Veterans General Hospital, Taiwan e Institute of Clinical Medicine, National Yang Ming University School of Medicine, Taipei, Taiwan f Department of Medicine, National Yang Ming University School of Medicine, Taiwan b

Received 10 June 2005; received in revised form 26 October 2005; accepted 27 October 2005 Available online 13 December 2005

KEYWORDS Peripheral arterial disease; CD40 ligand; hs-CRP; Monocyte chemotactic protein 1

Abstract Introduction: CD40 and its ligand participate in atherosclerosis formation, progression and destabilization. Increased soluble CD40 ligand (sCD40L) was observed in hypercholesterolemia, unstable angina and conditions with platelet activation. To date, there is no report on the association of sCD40L with angiographic peripheral artery disease (PAD) disease severity. Materials and methods: From March 1999 to April 2004, consecutive patients having angiographically documented PAD and given consents for pre-procedural serum sample use were recruited into this study. The key PAD lesions should be z 70% diameter stenotic at the lower limbs and patients were dichotomized into two groups depending on total PAD lengths. Peer angiographic control subjects were those free of coronary disease, PAD and major medical diseases. The serum samples were thawed and analyzed for sCD40L, monocyte chemotactic protein 1 (MCP-1) and hs-CRP in a single batch.

Abbreviations: PAD, peripheral artery disease; sCD40L, soluble CD40 ligand; MCP-1, monocyte chemotactic protein 1; hs-CRP, high-sensitivity C-reactive protein; CAD, coronary artery disease. * Corresponding author. Cardiovascular Center, Taichung Veterans General Hospital, 160, Sec. 3, Chung-Kang Road, Taichung 407, Taiwan. Tel.: +886 4 2359 2525x3124; fax: +886 4 2359 9257. E-mail address: [email protected] (W.-L. Lee). 0049-3848/$ - see front matter D 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.10.012

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W.-J. Lee et al. Results: A total of 63 well-defined lower-limb PAD patients and 30 control subjects were studied. Patients with PAD lengths N5 cm (N = 38) presented higher sCD40L than those with lesions V 5 cm (N = 25) (5430 F 459 vs. 3889 F 507 pg/ml, p = 0.037) and control subjects (5430 F 459 vs. 3973 F 551 pg/ml, p = 0.037). However, there was no significant difference in circulating MCP-1 (375 F 49 vs. 310 F 49 pg/ml and 297 F 24 pg/ml, respectively, p = 0.371) or hs-CRP (0.64 F 0.16 vs. 0.51 F 0.15 mg/ml and 0.46 F 0.15 mg/dl, respectively, p = 0.682) across three groups. PAD patients with associated coronary lesions did not differ in circulating CD40L, MCP-1 or hs-CRP from without and control subjects. Conclusions: Soluble CD40L was significantly elevated in patients with more advanced symptomatic PAD and might be an indicator for disease extent stratification. The distribution of sCD40L in PAD was not affected by coronary involvement or not. D 2005 Elsevier Ltd. All rights reserved.

Introduction Today, multiple lines of evidence suggest that atherosclerosis is a chronic inflammatory disease and implicates components of the immune system in atherogenesis [1—3]. Recently, research work found the participations of potent immune mediator CD40 and its counterpart CD40 ligand (CD40L or CD154) in atherosclerosis formation, progression and destabilization [4]. CD40L and soluble CD40L (sCD40L) have structural domains that allow them to have several important functions: ligation to CD40, signaling when bound to receptors, and binding to platelet glycoprotein IIbIIIa (stabilizing thrombus and stimulating platelets) [5]. Ligation of CD40 on atheroma-associated cells initiates expression of molecules involved in atherogenesis and subsequent complications [4—11]. More than 95% of the circulating CD40L exists in platelets [5], and sCD40L is thought to chiefly derive from shedding of the surface-expressed proteins from the stimulated platelets [12,13]. Increased sCD40L was observed in patients with hypercholesterolemia and unstable angina, and had strong independent prognostic value in healthy individuals [12—14]. One study found that elevated sCD40L in acute coronary syndrome is associated with increased risk of death or non-fatal myocardial infarction [15]. As platelets are subject to be activated in PAD with associated endothelial dysfunction, particularly those with advanced diseases, and the released CD40L is pro-atherogenetic, it is reasonable to speculate increased sCD40L in PAD. To date there are only rare reports on the sCD40L in PAD [16,17], and no one addressing the relationship between sCD40L and angiographic PAD lesion severity. This study was aimed to investigate the circulating levels of CD40L and monocyte chemotactic protein 1 (MCP-1), one of the major signaling mediators of

CD40L, in patients with symptomatic angiographically documented PAD, and to explore their possible relationships to PAD disease severity.

Materials and methods Study design and subjects The Windows 2000-based cardiac catheterization report databank has recently been established in this institute, using the hospital-information-system data stored in the mainframe, and contains the entire angiographic report data over past 12 years. In addition, the serum databank consisting of all patients who underwent different sorts of cardiac catheterization and were willing to donate their blood samples for research uses is in operation since March 1999. This study is part of the programs taking these two advantages to look into challenging cardiovascular issues. All patients who had undergone lower limb angiographic studies for suspected PAD or percutaneous transluminal angioplasty of documented PAD since March 1999 were reviewed. Patients who had angiographically documented symptomatic PAD involving major lower-limb vessels (iliac and femoral arteries) were screened for study enrollment. The key PAD lesion should be z 70% diameter stenotic at the common/external iliac, or common/superficial femoral arteries, and have clinically relevant presentations (claudications or weakened pulses). One hundred and forty-seven patients who met the angiographic inclusion criteria were subsequently queried in the serum databank. Of these, eightyfive subjects had pre-procedural serum samples at the time of angiography and were screened for study recruitment. Only patients with conclusive and well-defined lower-limb angiograms which

CD40 ligand in peripheral arterial disease were available for QCA measurements were considered eligible for recruitment. The relevant clinical demographic data were retrieved from the database and completed by reviewing the medical chart records thoroughly. The complete cine records were reviewed and angiographic measurements (reference vessel size, minimal lumen diameter, diameter stenosis) were made on a viewing workstation with software for quantitative analysis of angiograms (QCA) (Philips Inturis Suite, R2.2). All lower-limb PAD lesions that were z 50% diameter-stenotic were counted, and PAD patients were dichotomized into two groups depending on total PAD lengths V 5 or N 5 centimeters (cm). The associated major systemic arterial involvements of coronary, extra-cranial (carotid, vertebral, subclavian) and renal arteries, if corresponding angiograms were completed, were counted if the lesions were at least 50% diameter-stenotic. Patients presenting with acute myocardial infarction or acute coronary syndrome leading to index angiographic studies were excluded. Patients with pre-existing rheumatic mitral stenosis or emergent PAD were also considered ineligible. PAD due to below-knee involvement were not to be considered in this study as most angiographic studies were focused on above-knee lesions and below-knee lesions were a particular PAD subset. A half-numbered peer coronary angiographic control subjects were enrolled for comparison. Their coronary lesion was excluded by coronary angiography, whereas PAD and major medical diseases were ruled out by detailed physical examination and history-taking during the admission for elective coronary angiographic study for chest pain.

Measurement of circulating CD40 ligand, MCP-1 and hs-CRP All serum samples were already stored in 70 8C freezers till use. These blood samples were drawn from the antecubital vein at 7:00 AM—8:00 AM after an overnight fasting before angiographic procedures. The adequately sized aliquots of serum samples of all enrolled patients were thawed and analyzed for circulating CD40 ligand, MCP-1 and hsCRP in a single batch. Serum CD40 ligand and MCP-1 were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Minneapolis, MN, USA). The intraand inter-assay coefficients of variation for CD40ligand were 5.00% and 6.20% with a sensitivity of b 10.0 pg/mL. The intra- and inter-assay coefficients of variation for MCP-1 were 5.80% and 5.70%

621 with a sensitivity of b5.0 pg/mL. Serum hs-CRP was determined by particle-enhanced immunoturbidimetry (Latex microparticles sensitized with duck anti-CRP IgY kit, provided by Good Biotech Corp., Taichung, Taiwan). The intra-assay and inter-assay coefficients of variance were 1.4% and 1.42%, respectively.

General biochemistry analyses The frozen plasma and serum were also thawed for determination of plasma glucose, serum cholesterol and triglyceride by commercially available kits. Plasma glucose was measured by the glucose oxidase—peroxidase method (Wako Diagnostics, Tokyo, Japan). Fasting serum triglyceride and cholesterol concentrations were assayed by an enzymatic method using commercial kits (WAKO, Tokyo, Japan). The high-density lipoprotein (HDL) cholesterol level was determined in the supernatant of plasma after magnesium chloride—phosphotungstic precipitation of apolipoprotein B-containing lipoproteins. The LDL cholesterol concentration was estimated by the formula of Friedewald et al.

Statistics All data of continuous variables are expressed as mean F SEM. Between-group differences in means were analyzed by one-way ANOVA and post hoc pairwise multiple comparisons by LSD (least significant difference) tests if differences existed among the means. Differences in categorical variables were measured by chi-square test with Yate’s correction. All statistics are performed by SPSS for Windows, release 10.0.1 (SPSS inc. Chicago). A two-tailed p-value b0.05 is considered statistically significant.

Results Demographic and angiographic characteristics From March 1999 to April 2004, a total of 63 welldefined lower-limb PAD patients met the enrollment criteria and were studied, of whom 38 had total PAD lengths N5 cm. In the same period, another 30 subjects who were free of coronary and peripheral arterial disease and major systemic diseases were recruited into the peer angiographic control group. The demographic characteristics of these three groups are presented in Table 1.

622 Table 1

W.-J. Lee et al. Demographic characteristics of PAD patients and control subjects

Age (years) Sex (M/F) Height (cm) Weight (kg) Hypertension (N) (%) Diabetes (N) (%) Smoking (N) (%) Hyperlipidemia (N) (%) Serum creatinine (mg/dl) Cholesterol (mg/dl) Triglyceride (mg/dl) Associated CAD (N/CAGs) LM (N) (%) SVD (N) (%) DVD (N) (%) TVD (N) (%) Aspirin (N) (%) Plavix/ticlopidine (N) (%) Any antiplatelet (N) (%)

PAD, lengths V5 cm

PAD, lengths N5 cm

Control subjects

p-value (ANOVA)

N = 25 (A)

N = 38 (B)

N = 30 (C)

72.1 F 2.1 23/2 162 F 1 58.4 F 1.5 16 (64%) 7 (28 %) 9 (36%) 10 (40%) 1.4 F 0.1 197 F 8 153 F 19 14/22 (64%) 4 (29%) 1 (7 %) 6 (43%) 7 (50%) 13 (52%) 3 (12%) 14 (56%)

73.3 F 1.2 29/9 160 F 1 65.7 F 2.2 24 (63%) 9 (24%) 10 (26%) 11 (29%) 1.9 F 0.2 196 F 8 157 F 17 28/35 (80%) 3 (11%) 6 (22%) 11 (39%) 11 (39%) 20 (53%) 2 (5%) 20 (53%)

67.4 F 2.04 23/7 159 F 1 61.8 F 2.0 26 (87%) 6 (20%) 14 (47%) 7 (23%) 1.7 F 0.4 190 F 10 139 F 18 0/30 (0%)

0.037 0.243 0.599 0.058 0.071 0.785 0.219 0.398 0.506 0.840 0.758

12 (40%) 0 12 (40%)

0.535 0.145 0.322

4 p = 0.03, control subjects vs. PAD patients with lengths N5 cm.

Subjects in three groups did not differ in sex, weight, height, serum lipid profiles, serum creatinine, diabetes or hypertension. However, PAD patients with total lengths N 5 cm were significantly older than control subjects (73.3 F 1.2 vs. 67.4 F 2.0 years, p = 0.013). Fifty-seven PAD patients had coronary angiograms and 42 of them (74%) had concurrent coronary artery disease (CAD). The incidence of CAD was 80% and 64% ( p = 0.172) in patients with PAD lengths N5 and V 5 cm, respectively. Left main coronary artery was involved in three and four patients in two groups, respectively. The incidences of single-vessel (22% vs. 7%), doublevessel (39% vs. 43%) and triple-vessel (39% vs. 50%) coronary diseases were similar in two PAD groups. There was no difference in use of aspirin (PAD lengths N5 cm vs. PAD lengths V 5 cm and control groups, 53% vs. 52% and 40%, p = 0.535), ticlopidine/ clopidogrel (12% vs. 5% and 0%, p = 0.145) or any Table 2

antiplatelet agent (56% vs. 52% and 40%, p = 0.322) across three groups at the time of blood sampling. The lesion distribution and angiographic characteristics (site, side, lesion length, vessel diameter and stenosis) of all major diseased peripheral arteries (42 common iliac, 31 external iliac and 36 superficial femoral arterial lesions), and associated systemic arteries (13 carotid, 5 subclavian, 6 vertebral and 19 renal artery involvements) in these PAD patients are shown in Table 2. These PAD lesions were evenly distributed on both sides of the body and at the three sites. The occlusion rate for common iliac, external iliac and superficial femoral lesions was 7 / 42 (17%), 6 / 31 (19%) and 17 / 36 (47%), respectively. Most (97%) of the common iliac lesions were less than 5 cm in lengths, whereas 36% of the external iliac and whereas 70% of the superficial femoral lesions were longer than 5 cm. Regarding the major (lower limb plus systemic)

Angiographic characteristics of peripheral arterial and associated systemic arterial lesions in PAD patients Peripheral arterial disease

Side (R/L) (N) Lesion length (N) (%) b1 cm 1—2 cm N2—5 cm N5—10 cm N10—20 cm N20 cm Mean diameter (mm) Mean stenosis (%) Total occlusions (N) (%)

Associated systemic arterial disease

Common iliac

External iliac

Superficial femoral

Carotid

Subclavian

Vertebral

Renal

21/21

15/16

18/18

5/8

1/4

2/4

9/10

6 (14%) 8 (19%) 27 (64%) 1 (3%)

8 6 6 8 3

1 3 0 1

4 (67%) 2 (33%)

5 (26%) 9 (47%) 4 (21%)

8.9 75 7 (17%)

7.8 78 6 (19%)

2 (6%) 2 (6%) 7 (19%) 4 (11%) 10 (28%) 11 (31%) 5.1 85 17 (47%)

4.4 68 0 (0%)

5.3 71 0 (0%)

(26%) (19%) (19%) (26%) (10%)

— 77 2 (15%)

(20%) (60%) (0%) (20%)

8.8 75 0 (0%)

CD40 ligand in peripheral arterial disease Table 3

623

Circulating levels of CD-40L, MCP-1 and hs-CRP in PAD patients and control subjects PAD, lengths V5 cm

CD-40L (pg/ml) MCP-1 (pg/ml) Hs-CRP (mg/dl)

PAD, lengths N5 cm

Control subjects

N = 25 (A)

N = 38 (B)

N = 30 (C)

ANOVA

A vs. B

B vs. C

3889 F 507 310 F 49 0.51 F 0.15

5430 F 459 375 F 49 0.64 F 0.16

3973 F 551 297 F 24 0.46 F 0.15

0.047 0.371 0.682

0.037 — —

0.037 — —

arteries studied, there was no difference in the numbers of involvement sites between patients with PAD lengths N5 and V 5 cm.

Circulating CD-40L, MCP-1 and hs-CRP levels The serum concentrations of CD-40L, MCP-1, and hs-CRP in PAD patients and control subjects are presented in Table 3. Patients with PAD lengths N 5 cm (N = 38) presented higher sCD40L than those with lesions V 5 cm (N = 25) (5430 F 459 vs. 3889 F 507 pg/ml, p = 0.037; also in Fig. 1, left upper panel) and control subjects (5430 F 459 vs. 3973 F 551 pg/ml, p = 0.037). However, there was no significant difference in circulating MCP-1

(375 F 49 vs. 310 F 49 pg/ml and 297 F 24 pg/ml, respectively, p = 0.371; also in Fig. 1, left upper panel) or hs-CRP (0.64 F 0.16 vs. 0.51 F 0.15 mg/ml and 0.46 F 0.15 mg/dl, respectively, p = 0.682; Fig. 1, right upper panel) across three groups. PAD patients with concurrent coronary lesions (N = 42) did not differ in CD40L (4927 F 463 vs. 4754 F 679 pg/ml and 3973 F 551 pg/ml, respectively, p = NS; Fig. 1, left lower panel), MCP-1 (360 F 52 vs. 323 F 29 pg/ml and 297 F 24 pg/ml, respectively, p = NS; Fig. 1, left lower panel) or hsCRP (0.70 F 0.16 vs. 0.31 F 0.10 mg/dl and 0.46 F 0.15 mg/dl, respectively, p = NS; Fig. 1, right lower panel) from those without (N = 17) and control subjects (N = 30).

1.0 p=0.037

p=0.037

P=NS

5000 4000 3000 2000 CD40L MCP-1

1000

serum concentration (mg/dl)

serum concentration (pg/ml)

7000 6000

0

0.8

0.6

0.4

0.2

Hs-CRP

0.0 PAD =<5cm N=25

PAD >5cm N=38

Contol N=30

PAD =<5cm N=25

PAD >5cm N=38

Contol N=30

1.0 P=NS

5000 4000 3000 2000 CD40L MCP-1

1000

serum concentration (mg/dl)

6000

serum concentration (pg/ml)

p-value

P=NS 0.8

0.6

0.4

0.2 Hs-CRP 0.0

0 CAD (-), N=15 CAD (+), N=42

Control, N=30

CAD (-), N=15 CAD (+), N=42

Control, N=30

Figure 1 Distribution of circulating CD40L, MCP-1 and hs-CRP in PAD patients with total lengths V 5 and N 5 cm (upper panels) and in PAD patients with and without concurrent CAD (lower panels) in comparison with peer normal angiographic control subjects.

624

Discussion In summary, we found that in comparison with angiographic control subjects, sCD40L was significantly elevated only in patients with more advanced angiographically documented PAD. However, the distribution of circulating CD40L in PAD was not affected by simultaneous coronary involvement or not. On the other hand, circulating MCP-1 was not related to the presence or disease severity of PAD. To our knowledge, this is the first study to document the relationship of sCD40L to angiographic PAD disease severity in patients with well-defined lower-limb angiographic studies in whom coronary angiograms were mostly obtained.

Soluble CD40L in atherosclerosis and PAD PAD involving lower limbs, as defined by abnormal ankle/brachial index, affects 11—25% of the general population 55 years and older, men and women equally [18,19]. PAD is associated with increased cardiovascular mortality to the same extent as coronary or cerebrovascular diseases [20,21] and this is in proportion to PAD severity. Thus it is important to find out the factors involved in systemic atherogenesis for primary and secondary preventions. CD40 is a 50-kDa integral membrane protein of the tumor necrosis factor (TNF) receptor family, whereas CD40L is a trimeric 39-kDa member of the TNF family. Both are expressed by the major cellular players in atherosclerosis [4]. Henn et al. first found that these two also exist in platelets [22]. CD40L is cryptic in unstimulated platelets but is rapidly presented to the surface after platelet stimulation. Soluble CD40L is chiefly derived from circulating platelets on activation by agonists such as adenosine diphosphate, thrombin or collagen [12,13]. Expression of CD40L from platelets can be induced even by shearing stress [23]. Soluble CD40L, when ligated to it receptors CD40L, initiates a series of proinflammatory responses in cells at the atherosclerosis plaque. Studies have shown that ligation of CD40 on atheroma-associated endothelial cells, smooth muscle cells and macrophages mediates expression of cell adhesion molecules, chemokines (MCP-1, IL-8), cytokines (IL-1, IL-6, IL18, TNFa), matrix metalloproteinases, and tissue factors [4—11]. Interruption of CD40/CD40L interactions diminished formation and progression of mouse atheroma [24,25]. CD40L was also shown to inhibit endothelial cell migration, which could

W.-J. Lee et al. precipitate acute atherosclerotic event [11]. One recent study even reported that pre-procedural sCD40L level is predictive of enhanced inflammatory response and restenosis after coronary angioplasty [26]. CD40-L-induced pathways also participate in angiogenesis by stimulating expressions of growth factors [27—29]. High level of sCD40L released from thrombus can also bind to platelet glycoprotein IIbIIIa, stabilize thrombus and stimulate platelets in turn [5]. Therefore, platelets and CD40L are two major mediators bridging thrombosis, inflammation and atherosclerosis. Increased sCD40L was observed in patients with hypercholesterolemia, type 1 or 2 diabetes mellitus and acute coronary syndrome, conditions where platelets are activated [12—15,30,31]. The bresponse to injuryQ theory promotes that platelets and elements of the hemostatic/thrombotic systems are involved in the atherogenesis in a self perpetuating circle [32]. The dysfunctioned endothelium could recruit and activate platelets [17]. As platelets are subjects to be activated in PAD with associated physical and functional endothelial dysfunction, particularly those with advanced diseases, and the released CD40L is pro-atherogenetic, it is reasonable to speculate increased sCD40L in PAD. To date there are only rare reports on this issue [16,17]. In the study by Blann et al., sCD40L was found to be elevated in patients with PAD. However, the serum level was not correlated with clinical disease severity and angiographic disease extent or associated coronary diseases were not mentioned in that study. In the current study, we found that in comparison with peer angiographic control subjects, sCD40L was significantly elevated only in patients with more advanced disease extent of angiographically documented PAD. We do believe our study results imply clinical significance as sCD40L is also elevated in different pro-atherosclerotic conditions and risks [12,13,15,33] and our findings were established using angiographic control subjects in whom the demographic characteristics and hs-CRP were similar to PAD patients rather than using completely normal subjects with different baseline demographics. Therefore, elevated sCD4L in PAD patients might be a marker for more extensive disease involvements. Though CD40L release might be affected by medications, particularly antiplatelet agents [34,35], this possibility was ruled out in the current study as uses of aspirin, ticlopidine/ clopidogrel, alone or in combination, were similar across three groups and no glycoprotein 2a3a antagonists were ever used. Furthermore, there were reports that sCD40L level was seemingly not affected by aspirin use in the human body [14,26].

CD40 ligand in peripheral arterial disease We also found that the distribution of CD40L in PAD was not affected by simultaneous coronary involvement or not. The causal relationship of elevated sCD40L in severe PAD remains to be elucidated. In the study by Blann et al., the sCD40L failed to correlate with markers of platelet activation, implying other cellular sources of circulating CD40L [16]. Whether the elevated sCD40L in these patients might reflect the enhanced expressions of CD40L in the atherosclerotic plaques could not be ascertained. Our finding does argue for sCD40L as a surrogate marker for stratifying symptomatic advanced PAD. However, it needs further study to consolidate our conclusion. On the other hand, circulating MCP-1, a major inflammatory signal subsequent to CD40 stimulation, did not show significant change in PAD patients or subgroups. MCP-1 was ever reported to be elevated in PAD in one and the only study [36]. It is possible that the change in MCP-1 in the plaques might not be reflected in the serum concentrations or the changes in circulating MCP1 in PAD were masked by employing angiographic control subjects in the current study design as discussed above. Our finding might speak against a major role of circulating MCP-1 in stratifying disease extent in PAD patients.

CRP and PAD Elevated circulating hs-CRP, a marker of inflammation, was reported in patients with acute coronary syndrome as well as PAD [37], but not in others [38]. Unfortunately, we did not observe a significant change in hs-CRP level between PAD patients and control subjects in the current retrospective study, despite we had reported a positive finding in a prospective study [39]. This might also result from the fact that we used the peer angiographic control subjects who did have high incidence of risk factors and presented some sort of chest pain prior to elective coronary angiographic studies. Though these control subjects were free of disabling medical diseases, CAD and PAD after angiographic studies, detailed history-taking and physical exam, their mean serum hs-CRP of 0.46 mg/dl was higher than the normal range in this age group as reported by us and others [39]. In conclusion, in comparison with angiographic control subjects, sCD40L was significantly elevated only in patients with more advanced symptomatic PAD. Therefore, elevated sCD40L might serve as a marker for more advanced PAD disease involve-

625 ments. The distribution of CD40L in PAD was not affected by coronary involvement or not. Circulating MCP-1, on the other hand, did not play a significant disease-stratifying role in PAD.

Acknowledgments This study is sponsored by Taichung Veterans General Hospital grant # TCVGH-933107D and in part by Yen Tjing Ling Medical Foundation (grant# CI-93-11), Taipei, Taiwan.

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