© 1997 American Heart Association, Inc.
Articles |
Soluble Vascular Cell Adhesion Molecule-1 as a Biohumoral Correlate of Atherosclerosis
From the CNR Institute of Clinical Physiology (R.D.C., G.B., G.L.), I Medical Clinic (G.D., R.P.), Institute of Radiology (R.P.), and II Medical Clinic (M.M., F.C.), the University of Pisa, Pisa, Italy.
Correspondence to Raffaele De Caterina, MD, PhD, Laboratory for Thrombosis and Vascular Research, CNR Institute of Clinical Physiology, Via Savi, 8-I-56126 Pisa, Italy. E-mail rdecater{at}po.ifc.pi.cnr.it.
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Abstract |
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Abstract Vascular cell adhesion molecule-1 (VCAM-1) is a protein expressed on the surface of activated endothelial cells and expressed in early atherosclerosis. Because part of the protein is shed in the circulation and can be detected in peripheral plasma [soluble (s)VCAM-1], we hypothesized that sVCAM-1 may be a circulating marker of the presence and severity of atherosclerosis in humans. We selected 11 patients with essential hypertension plus peripheral vascular disease (PVD) and matched them for age, gender, body mass index, and smoking habits with 11 patients with uncomplicated essential hypertension (UH) and 11 healthy controls. We evaluated plasma concentrations of sVCAM-1 along with those of the soluble form of two other endothelial leukocyte adhesion molecules [sE-selectin and s-intercellular adhesion molecule-1 (sICAM-1)] and other markers of endothelial dysfunction/damage [s-thrombomodulin, plasminogen activator inhibitor type I, and von Willebrand factor (vWF)]. We also measured insulin, glucose, fibrinogen, total and HDL cholesterol, and the urinary albumin excretion (UAE), which may also be related to atherosclerosis. Results of these assays were related to the echographic assessment of the maximum intima-media thickness (IMTmax) at the carotid bifurcation, as an index of atherosclerosis in the carotids. PVD patients had a clearly elevated IMTmax [2.7 (1.1-3.1) mm, median (range)] compared with both UH patients [1.2 (0.8-2.4) mm] and controls [1 (0.6-2) mm]. sVCAM-1 was clearly higher in PVD patients [990 (273-1808) ng/mL, median (range)] versus 340 (236-975) ng/mL in UH and 386 (204-835) ng/mL in controls, and it separated clinical categories better than sICAM-1, vWF, glucose, insulin, UAE, triglycerides, or total, LDL or HDL cholesterol. sVCAM-1 was also the best biohumoral correlate of IMTmax (R=.59; P<.001) in univariate analysis. Because many of the biohumoral variables assessed were mutually intercorrelated, they were entered in a multivariate analysis to assess their contribution in explaining IMTmax variability. sVCAM-1 remained the only independent predictor of IMTmax and totally abolished the contribution of other variables to IMTmax variability. Thus, sVCAM-1 is a good biohumoral correlate of overt atherosclerosis, independent of underlying hypertension, and may be an in vivo marker of endothelial activation. Its potential value as a surrogate for global risk assessment and its behavior in intervention studies remain to be determined.
Key Words: soluble VCAM-1 • soluble ICAM-1 • intima-media thickness • atherosclerosis
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Introduction |
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In vivo detection of endothelial dysfunction is an active area of present investigation, because it may offer pathogenetic insight, yield integrated information on the burden of risk factors and on the global functional state of vascular disease,1 2 and possibly detect subjects at risk.3 4 Circulating markers of endothelial damage, such as von Willebrand factor (vWF)5 6 or s-thrombomodulin,7 8 9 10 and of increased vascular permeability, such as urinary albumin excretion (UAE)11 12 have been shown to be altered in patients with vascular disease. Some of these alterations have been also detected in uncomplicated hypertension, including elevation of vWF,13 14 plasminogen activator inhibitor-type 1 (PAI-1),15 16 and microalbuminuria.17
The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) is an early manifestation of experimental cholesterol-induced atherosclerosis18 19 and occurs in human atherosclerotic plaques.20 21 Soluble forms of endothelial leukocyte adhesion molecules have been detected recently in the supernate of cytokine-stimulated cultured endothelial cells22 23 and the availability of commercial immunoassays for these molecules has allowed the assessment of serum or plasma concentrations, offering the possibility of in vivo detection of endothelial activation.24 25 26
We hypothesized that the soluble form of VCAM-1 (sVCAM-1) could be a marker of the presence and severity of atherosclerosis. We, therefore, undertook a comparative evaluation of soluble endothelial leukocyte adhesion molecules and of other potential markers of endothelial dysfunction and damage, or of metabolic derangements accompanying atherogenesis, in overt atherosclerosis with peripheral arterial disease. We undertook the same evaluation in patients with uncomplicated essential hypertension and in healthy control subjects to assess the potential of sVCAM-1, by itself and relative to other humoral indexes, to mark and predict the presence and severity of vascular disease.
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Methods |
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Subjects
Subjects enrolled in this study consisted of three groups.
Essential Hypertension With Peripheral Vascular
Disease (PVD)
Inclusion criteria were the diagnosis of peripheral
arterial disease (ankle/arm pressure index <.96 on at
least one limb) associated with a history of intermittent claudication
and the concomitant diagnosis of essential hypertension. All such
patients had angiographic documentation of aorto-iliac-femoral
atherosclerosis. Essential hypertension was diagnosed
by conventional criteria,27 with routine clinical and
biohumoral exclusion of secondary forms. In all patients, the angiogram
excluded the presence of renal artery stenosis, and a renal
ultrasound examination showed kidneys of normal size, with no evidence
of cortical scarring or obstructive uropathy. Exclusion criteria were
as follows: age >70 years; fasting blood glucose >120 mg/dL;
abnormal oral glucose tolerance test, defined as a 2-hour value of
plasma glucose >200 mg/dL and any other value during the 2-hour
test 
200 mg/dL; serum creatinine >1.2
mg/dL; total cholesterol >300 mg/dL;
abnormal urinalysis and evidence of urinary tract infections; obesity
(body mass index >33 kg/m2); the clinical evidence
of congestive heart failure, ischemic heart disease (in all
cases by a
dipyridamole-echocardiographic stress
test), advanced chronic obstructive pulmonary disease, Fontaine
class IIb and higher (previous amputation, pain at rest,
ischemic trophic ulcers or gangrene, pain-free walking distance
<50 m), and the inability to obtain a reliable, accurate measurement
of the region of the carotid bifurcation by ultrasounds. A high
resolution carotid ultrasound examination (see below) also excluded the
presence of hemodynamically significant (>50%
internal diameter reduction) carotid stenoses. Patients were
chosen in chronologic order from a data base of recent (<2 years)
admissions to our clinic. Study population comprised 11 of a total
number of 160 patients who visited for peripheral
arterial disease. Age was (mean±SD) 58±8 years. Two
patients were female; two were active smokers; three were taking
lipid-lowering drugs (gemfibrozil or statins); and all were taking
aspirin or ticlopidine.
Uncomplicated Essential Hypertension (UH)
From the same database, we selected 11 gender- and age-matched
patients with uncomplicated essential hypertension, who underwent a
similar diagnostic work-up, with the exception of
angiography. PVD was excluded by the finding of a normal carotid,
aortic, and lower limb artery echo-Doppler examination and of an
ankle/arm index .96. Exclusion criteria were similar as for PVD
patients. Age was 56±9 years. Two patients were female and two were
smokers.
Two uncomplicated hypertensive patients had never taken
antihypertensive drugs, whereas the others from both groups, who were
taking calcium antagonists,
angiotensin-converting enzyme inhibitors, or
both, were withdrawn from any such medication, as well as from
lipid-lowering medications, for 2 weeks before the study.
Normotensive Controls
Eleven normal, sedentary, nonobese, gender- and age-matched
subjects served as controls. They were receiving no drugs and had
normal physical examinations, normal routine blood and urine tests,
normal blood pressure, and normal ECG, abdominal ultrasounds, and
ankle/arm pressure indexes. Age was 57±9 years. Two subjects were
smokers. Demographic characteristics of the patients recruited are
detailed in Table 1
and are in a parallel
study in the same patient population.28
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After recruitment, experimental evaluations were completed in a 2-week period. According to institutional guidelines, subjects were all informed of the investigational nature of the study and agreed to participate. The protocol was approved by the local Ethical Committee. All subjects were asked not to smoke the day of sampling.
Experimental Procedures
Vascular Echographic Data
For the measurement of carotid thickness, the sonographer (among
the authors of this study), blinded as to the clinical status of the
patient and of biohumoral results, demonstrated the interfaces required
for the measurement of arterial wall thickness as clearly
as possible, searching for the thickest interface at four to five sites
of the right and left common carotid artery (i.e., the
arterial segment extending from 8 to 16 mm below the
tip of the flow divider into the common carotid artery) and the carotid
bifurcation (bulb) using anterolateral, lateral, and posterolateral
fixed angles of examination. Measurements of the distance from the
leading edge of the first echogenic luminal bright line to the leading
edge of the second echogenic line were taken from frozen images, as
described by Pignoli et al.29 Patients who had arteries in
which references were not identifiable, tortuous, or calcified vessels
were excluded (see exclusion criteria, above). The ultrasound system
used was an AU 590 Asynchronous Scanner (Ansaldo, Genova, Italy), with
a linear 7.5-MHz probe and an axial resolution of 0.1 mm. Scanning
and measurements were obtained by the same instrument and probe, with
the observer unaware of the clinical status of the subject under
examination. All measurements were made with the image at the maximum
depth of the focus. Gains and image pre- and post-processing options
were set up by the operator for each individual patient and for each
artery under scrutiny to obtain the best possible image. During
scanning, distance measurements were recorded using software-driven
cursors with a digital display expressing distances in millimeters. The
operator spent on average about 20 minutes for complete scanning of
each subject plus 20 additional minutes for the measurements. Carotid
data measured at the far wall were expressed as (a) maximum
intima-media thickness (IMTmax), defined as the maximum of
the measurements, and (b) as the intima-media thickness (IMT), defined
as the average of the four to five determinations of the far wall
intima-media distance in the common carotid proximal to the
bifurcation. A wall thickening 1.6 mm was defined as a plaque.
The intraindividual correlation of carotid scanning values at the
common carotid and the bulb repeated at 1-month intervals was .84 and
.78, respectively.
Renal Parameters
Serum and urinary creatinine (this last with 24-hour
urine collection in duplicate samples) were determined by standard
colorimetric methods, as described.28
Urinary albumin excretion (UAE) was measured by determining
urinary albumin concentration by nephelometry (Istituto Behring
S.p.A., Scoppito, L'Aquila, Italy) as described.17 To
minimize the confounding influence of daily physical activity and to
facilitate the collection procedure, our outpatients collected urine
from 8:00 PM to 8:00 AM during three
consecutive nights as described.17
Lipid and Metabolic Parameters
Serum triglycerides and total serum
cholesterol were measured by enzymatic
colorimetric methods (GDP-PAP and Monotest
cholesterol CHOD-PAP respectively,
Boehringer-Mannheim, Mannheim, Germany). High-density
lipoprotein (HDL) cholesterol was measured in the
supernatant after precipitation of apolipoprotein B-containing
lipoproteins with MgCl2 by a similar enzymatic
colorimetric method (Carlo Erba, Milan, Italy). LDL
cholesterol was calculated with the Friedewald's formula.
Fasting plasma glucose was measured by the glucose oxidase method
(Boehringer Mannheim) and insulin by an immunoradiometric assay
(Medgenix Diagnostics, Fleurus, Belgium).
Indexes of Endothelial Dysfunction and of
Blood-Vessel Wall Interactions
Peripheral whole blood samples were obtained between
8:00 and 9:00 AM from patients and controls after an
overnight fast. Venous blood (9 mL) was collected with minimal venous
stasis through a 19-G needle in a syringe containing either 3.8%
sodium citrate (0.11 mol/L) to a final ratio (v:v) of one part
of sodium citrate to nine parts of blood (for fibrinogen, PAI-1, vWF,
and s-thrombomodulin measurements) or heparin (for soluble adhesion
molecules). Platelet-poor plasma was obtained by
centrifugation at 3000 revolutions per minute for 15
minutes, then aliquoted and immediately used for the functional assay
or stored at -80°C until assayed. Assays were performed in batch for
each assay within 45 days of sampling. Before each assay, frozen
samples were brought to room temperature slowly and gently mixed.
Soluble endothelial leukocyte adhesion molecules were measured by commercially available enzyme-linked immunosorbent assays (Bender, Vienna, Austria, for sE-selectin and soluble Intercellular Adhesion Molecule-1 (sICAM-1); R&D Systems Europe, Oxon, United Kingdom, for sVCAM-1) in heparinized plasma samples. Assays were performed in duplicate on duplicate samples, each assayed at least at two different dilutions. The development of a color reaction because of the conversion of the chromogenic substrate (tetramethyl-benzidine), directly proportional to the amount of the analyte assayed, was followed for 5 to 20 minutes with an ELISA plate reader (ETI-System, Sorin Biomedica, Saluggia, Italy) at 450 nm, up to an optimal reading of positive wells. The enzyme reaction was stopped by the addition of 4 N sulfuric acid. Results were calculated by interpolation of a standard curve consisting of at least five measurable points. Intra-assay and interassay precisions (coefficient of variation) for all of these assays are <5.9% and <10.2%, respectively, as reported by the manufacturers (Bender and R&D).
The antigenic levels of vWF or s-thrombomodulin were estimated by ELISA methods, using commercial kits (Diagnostica Stàgo, Asnières-sur-Seine, France).30 31 32 Fibrinogen (Clauss method) was evaluated by clotting rate on an automated device (Chromo Time System, Behringwerke, Scoppito, L'Aquila, Italy), as described previously.33
As an in vivo index of nitric oxide (NO) synthesis, plasma
concentrations of nitrites and nitrates
(NO2-/NO3-), the
stable NO metabolites, were evaluated by semiautomatic HPLC
analysis (Beckman Instruments Inc., Berkeley, Calif) according
to the method of Green, as modified by Noris et al.34
Briefly, plasma samples were treated with zincum sulfate (60
µMol/L final concentration) and centrifuged to
eliminate proteins. Supernates were eluted onto a Dowex AG 50 WX-8
column followed by a cadmium column, which catalyzes the reduction of
nitrate to nitrite (eluent: borate buffer, pH 8.5). The postcolumn
eluate reacted with Griess reagent (5% H3PO4,
1% sulfanilic acid, 0.1%
n-[1-naphthyl]-ethylenediamine, vol:vol:vol;
Sigma) to form a purple azo dye, and the color was detected by UV-VIS
detector at 
=504 nm. The absorbance peak area was measured, and
NO2-/NO3-
concentration in the sample was calculated by extrapolation from a
standard nitrate solution curve. Values were corrected for recovery
(ranging from 60 to 90%), as determined by the addition of known
amounts of standard nitrate to an additional aliquot of each plasma
sample.
Statistical Analysis
Log transformation was applied to UAE, insulin,
triglyceride, vWF, PAI-1, fibrinogen, IMTmax,
and adhesion molecule data, as they were distributed asymmetrically.
Descriptive statistics were arithmetic mean±SD in general and median
and range for skewed data. Comparisons among the three different groups
were performed by one-way analysis of variance and, in the
presence of significant F values, between-group differences assessed by
Scheffé's multiple contrasts. Linear regression
analysis, the calculation of Pearson's correlation
coefficients, and multiple linear regression analyses were
performed by standard methods with the aid of the Statview statistical
package (Abacus Concepts, Inc., Berkeley, Calif).
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Results |
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As a result of the randomization, the three patient categories were comparable for age, gender, prevalence of smokers, and body mass index (Table 1
). By definition, the ankle/arm pressure index was normal
(>.96) in control subjects and in patients with UH and was reduced
(.55±.13, mean±SD) in PVD patients (Table 1). Systolic,
diastolic, and mean arterial pressures were
higher (and there was a similar trend for TPR) in UH and PVD patients
compared with controls, with higher (P<.01) values for
systolic blood pressure in PVD patients (164±21 mm Hg)
compared with UH patients (150±10 mm Hg). Systolic blood
pressure in control subjects was 134±10 mm Hg.
Creatinine and creatinine clearance were
similar in control and UH subjects, but serum creatinine
was higher and creatinine clearance lower in PVD patients
(creatinine clearance, 119±19 mL/minute in controls;
109±28 mL/minute in UH; 88±13 mL/minute in PVD), although by the
prespecified inclusion criteria, no patients had overt renal
insufficiency (serum creatinine, >1.2 mg/dL) at
recruitment.
Maximum Intima-Media Thickness Is Elevated in PVD Patients Compared
With UH and With Controls
The distribution of values of IMTmax at the carotid
bifurcation in the three patient categories is shown in Fig 1. There was no significant increase in
this index of carotid atherosclerosis in UH patients,
whereas there was a clear-cut increase in PVD patients. This confirmed
the substantial lack of atherosclerotic complications in the UH group
and the subclinical involvement of the carotid bifurcation by
atherosclerosis in PVD patients. The mean intima-media
thickness was also highest in PVD patients (1.02±.26) compared with UH
patients (.83±.16 mm) and controls (.67±.17 mm), although
differences were not as striking.
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Metabolic Correlates of Insulin Resistance Are More
Elevated in PVD Than in UH Patients
Metabolic data in our patient population are reported
in Table 2. There was a trend for total
and LDL cholesterol to be highest in PVD patients and
lowest in controls, and the reverse appeared to occur for HDL
cholesterol. Triglycerides, fasting plasma
glucose, and insulin were significantly higher in PVD compared with
both normal controls and UH patients (Table 2). Of note, no patient in
the PVD group, despite statistically significant higher fasting plasma
glucose, was diabetic or had "impaired glucose tolerance," as
assessed by conventional criteria of fasting plasma glucose and the
oral glucose tolerance test. Furthermore, hemoglobin A1C
values were 5.2±.2% (normal values, 4% to 6%).
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Circulating Indexes of Endothelial Damage and
Dysfunction and Other Parameters
These are summarized in Table 3.
There were no differences in the three patient categories with regard
to PAI-1 and sTM. Fibrinogen was also not significantly different among
the three subject categories. Concentrations were (median and range)
314 (249-593) in controls, 322 (278-386) in UH patients, and 352
(256-479) in PVD patients. sE-selectin showed a trend toward higher
values in UH patients (Table 3). vWF and sICAM-1 were significantly
higher in PVD patients, but still with relatively large overlaps among
the three groups. sVCAM-1 on the other hand was remarkably and
selectively elevated in PVD patients (median (range) 990 (273-1808)
ng/mL in PVD patients, versus 386 (204-835) ng/mL in
controls), with no elevation attributable to the presence of pure
hypertension (340 (236-975) ng/mL) (Fig 2). UAE was also selectively elevated in
PVD patients (12±5.5 in controls, 11.8±4 in UH, and 47.9±47.2 in
PVD), as reported and analyzed separately
elsewhere.28
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sVCAM-1 Is the Strongest Biohumoral Correlate of
IMTmax
In univariate analysis, the correlation
coefficients of IMTmax with those metabolic
parameters and indexes of endothelial
damage/dysfunction that were elevated in PVD patients are shown in
Table 4. Neither vWF nor insulin, despite
being elevated in PVD patients, showed any significant correlation with
IMTmax. Significant correlations (R>.35;
P<.05) were found among UAE, glucose,
triglycerides, total and LDL
cholesterol, sICAM-1, and sVCAM-1 on the one hand and
IMTmax on the other (Table 4). Of these indexes, the
strongest correlation observed was, by far, that with sVCAM-1
(R=.59; P<.001; Fig 3), followed by that with
triglycerides, sICAM-1, and UAE (for all of these,
R>.42; P<.02). A weaker, but still significant
correlation, was found between sVCAM-1 and IMT (R=0.39;
P<.05), and sVCAM-1 was, again among variables
considered, the strongest biohumoral correlate of IMT (not shown). The
correlation between sVCAM-1 and IMTmax was even stronger
(R=.62; P<.001) by taking into account only UH
and PVD patients, thus demonstrating the existence of a relationship
between these two parameters, even within patients with
vascular disease.
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To ascertain whether a reduction of renal clearance of circulating soluble adhesion molecules could account for their elevation, regression analysis was performed between parameters of renal function (serum creatinine and creatinine clearance) and soluble adhesion molecules. No significant correlation was found between serum creatinine, creatinine clearance, or its log transformation and soluble adhesion molecule concentrations (not shown) over a relatively wide range (72-145 mL/minute) of creatinine clearance values.
Intercorrelations Between Biohumoral Indexes and Multivariate
Analysis
The correlation coefficients at linear regression analysis
between the various biohumoral variables exhibiting significant
differences at ANOVA (including total and HDL cholesterol,
for which there was a trend toward significance) among the three
subject groups are also shown in Table 5.
There was a high degree of intercorrelation among many of the
variables considered. Highest degree of correlations
(P<.001) were between sVCAM-1 and UAE and between
triglycerides on the one hand and sICAM-1, UAE, and insulin
on the other. To sort out whether any of these variables
contributed independently to explaining IMTmax variability,
multiple regression analysis was performed, entering
IMTmax as the dependent (y) variable and all
the others as independent (x) variables. Results are
shown in Table 5. Only sVCAM-1 contributed significantly to
IMTmax variability.
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Discussion |
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The results contained in this article show that circulating levels of VCAM-1, an adhesion molecule involved in atherogenesis, are significantly higher in patients with peripheral vascular disease and hypertension than in patients with essential hypertension without peripheral vascular disease and in normal controls and distinguishes these three subject categories, matched for age, gender, smoking, and body mass index, better than a number of other in vivo indexes of endothelial dysfunction/damage or of metabolic correlates of atherosclerosis. Furthermore, our data show that sVCAM-1 is a strong correlate of atherosclerosis severity, as assessed by the echographic evaluation of the maximum intima-media thickness in the region of the carotid bifurcation.
Metabolic Indexes and Markers of
Endothelial Dysfunction in Atherosclerosis
We evaluated a number of possible biohumoral correlates of
atherosclerosis. Concentrations of glucose, insulin,
vWF, sICAM-1, sVCAM-1, and UAE were significantly higher in PVD
patients compared with either UH patients or control subjects. This
confirms previously reported associations, such as that of
vWF,13 UAE,11 and insulin35 36
with atherosclerosis. These elevations are thought to
reflect endothelial damage or dysfunction occurring in
overt atherosclerosis or the metabolic
derangement of the insulin resistance syndrome.37 The
elevation of triglycerides, fasting plasma glucose, and
insulin in PVD compared with both normal controls and UH patients
indeed strongly suggests that our PVD patients were more
insulin-resistant than the two other patient categories. These
occurred despite rigorous exclusion of diabetes mellitus at enrollment.
The occurrence of a higher prevalence of elevated
triglycerides in PVD compared with other subject categories
was also no surprise. In an unselected series of 220 patients of ours
with proven PVD, we have indeed observed that 31% had
triglyceride levels >170 mg/dL. The absence of any
increase in PAI-1 in our patients is noticeable in light of frequent
reports of elevated PAI-1 in subjects with high
triglycerides and/or insulin resistance38 and
of its prognostic value in infarct survivors.39
Fibrinogen, which has been shown in large series studies to be elevated
in atherosclerotic patients undergoing complications,40 41
was also not significantly different among the three subject
categories. Our relatively small sample size, and an additional
confounding effect of smoking, which appears to be a strong determinant
of fibrinogen,41 may account for such discrepancies. Also,
the nitrate measurement, as an index of NO
production,34 did not differ significantly among
subject groups. This agrees with previous findings with this in vivo
measurement of a marginal usefulness of such a parameter in
differentiating distinct clinical patient categories such as
hypertensives and normal controls.42 Overall, however, a
large variety of biohumoral indexes appears to differ significantly in
well selected and appropriately matched groups of atherosclerotic
versus control subjects. Of these, sVCAM-1 showed the most clear-cut
and selective elevations in PVD, with no influence because of
hypertension per se.
Correlation of Biohumoral Indexes With Carotid
Atherosclerosis
Previous studies have established that IMTmax at the
carotid bifurcation is a good indicator of the presence of systemic
atherosclerosis.43 The choice of obtaining
measurements of intima-media thickness in the region of the carotid
bifurcation as opposed to other sites accessible to ultrasonography has
a biologic rationale in the lower representation of medial
smooth muscle cells at this level.44 As a consequence,
evaluation of the same parameter on the common carotid
artery (from which IMT is obtained) is more likely to be confounded by
the possible presence of medial hypertrophy coexisting with
hypertension.45 Thus, it is likely that IMTmax
is the most reliable ultrasonographic index for the presence of carotid
atherosclerosis so far available, and there is evidence
that it can be an indirect estimate of the atherosclerotic burden
elsewhere.43 Despite recruitment of our patients was done
with the prespecified exclusion of patients with carotid
stenosis, IMTmax and, to a lesser extent, IMT were
both clearly elevated in the group of PVD patients compared with both
UH and normal controls, indicating the presence of subclinical carotid
atherosclerosis in our PVD patients. Many of the
investigated biohumoral parameters appeared to correlate
with IMTmax. The strongest correlate (r=.59;
P<.001) was observed for sVCAM-1. This is a novel,
interesting finding considering the completely different methodologic
approaches to measure these two variables and the fact that they
were independently obtained in a blinded fashion.
One conclusion arising from our analysis is the high degree of intercorrelation among various parameters examined. sVCAM-1, for instance, appeared to be highly correlated with UAE, triglycerides, and vWF and, to a lesser extent, sICAM-1 and insulin, which were all, in univariate analysis, correlates of IMTmax as well. The high degree of intercorrelation explains the loss of predictive power of many such parameters on IMTmax in multiple regression analysis. Because of this, only sVCAM-1 was an independent predictor of the variability of IMTmax at multivariate analysis. The interconnection of sVCAM-1 with a number of weaker correlates of IMTmax raises the possibility that sVCAM-1 may be a useful in vivo biohumoral marker or surrogate of the severity of atherosclerosis, integrating bits of information otherwise present in a number of other less strongly related biohumoral variables. At the time of the preparation of this study, another report showed increased expression of sVCAM-1 in dyslipidemic subjects, especially in hypertriglyceridemia.46 Such report is consistent with the associations found by us of sVCAM-1 with lipid parameters. However, the predictive value for sVCAM-1, stronger than either triglycerides or LDL cholesterol, with regard to IMTmax in our series, suggests that atherosclerosis, rather than its lipid risk factors, are more directly related to elevation of sVCAM-1.
Biologic Significance of sVCAM-1 in Relation to the Present
Findings
sVCAM-1 is the shed soluble portion of VCAM-1, a member of the
immunoglobulin family thought to be relevant in supporting the stable
attachment of the monocyte to arterial
endothelium through its binding to the monocyte
integrin VLA-4. Antibodies to VCAM-1 inhibit monocyte adhesion to
endothelial cells in vitro,18 and VCAM-1
expression marks the early development of
cholesterol-induced atherosclerosis in the
rabbit,18 19 preceding the appearance of foam cells in the
arterial intima.19 These data suggest a
pathogenetic role for VCAM-1 in early atherogenesis. In addition,
VCAM-1 has been shown to be present on the surface of advanced
atherosclerotic plaques, where it might contribute to lesion
progression.20 21 VCAM-1 is not entirely specific for
endothelial cells, being also found in follicular
dendritic cells47 and smooth muscle cells.19
However, vascular endothelium, for its extension, its
blood-tissue interface location, and its ability to release sVCAM-1, is
a likely source of the circulating molecule.22 23
Upon endothelial "activation" in vitro by inflammatory cytokines and bacterial lipopolysaccharide, the concerted surface expression of E-selectin, ICAM-1, and VCAM-1 takes place, together with the release of other soluble products such as interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor.48 49 Pathophysiologically relevant stimuli able to elicit the expression of adhesion molecules in vivo are largely unknown, although a selective increase in VCAM-1 (as opposed to E-selectin) has been reported for lysophosphatidylcholine, a component of oxidized LDL,50 and the advanced glycation end-products of diabetes.51 On the other hand, antioxidants,52 some n-3 fatty acids,53 or nitric oxide54 appear also to be at least relatively selective in reducing VCAM-1 expression. Epidemiologic and experimental evidence linking oxidized LDL55 and advanced glycation end-products56 to atherosclerosis on the one hand and antioxidants,55 n-3 fatty acids,57 or L-arginine58 to protection from atherosclerosis on the other, would, therefore, support the speculation that such interventions may indeed be effective, at least in part, through a relatively selective regulation of VCAM-1 surface expression,52 of which sVCAM-1 would be the in vivo detectable counterpart. These data are a theoretic basis to expect VCAM-1, rather than other adhesion molecules, as a relatively specific index of atherosclerosis compared with other conditions of endothelial activation.
Preliminary results showing differential elevations of different adhesion molecules in disease states different from atherosclerosis would support such a conclusion. Thus, E-selectin has been found to be elevated in patients with septic shock59 and hypertension26 (this last condition only showing a trend to higher values also in our data) and sICAM-1 in some malignancies60 61 62 and immune-inflammatory diseases.63 64
Limitations of the Present Study
Despite the successful match for age, gender, and body mass index
and the prespecified exclusion of diabetic patients, our PVD patients
showed significantly higher plasma glucose, insulin, and
insulin-glucose ratio than hypertensive patients and control subjects.
This suggests a gradient of insulin resistance possibly related to the
presence of overt atherosclerosis, physical
deconditioning, or both. At variance from PAI-1, another biohumoral
correlate of insulin resistance, plasma triglyceride
concentration, was higher in the two patient groups, and the increase
in triglycerides in PVD patients was quantitatively
substantial. Total and LDL cholesterol showed a trend to be
higher, and HDL cholesterol lower, in PVD and UH patients
than in controls. Because the three experimental groups were
unbalanced to their regard, it is possible that higher values of
sVCAM-1 in PVD patients are the result of these factors rather than to
the presence of atherosclerosis per se, as recently
suggested.46 Indeed, as an example, elevated
triglycerides were significantly correlated with sVCAM-1
(P<.01; see Table 4). All such parameters may
theoretically contribute causally to the development of
atherosclerosis and are indeed "risk factors" in
epidemiologic studies. Therefore, even if it is their presence, rather
than overt atherosclerosis, to cause increased sVCAM-1,
this would not negate the interest in sVCAM-1 concentrations as an
integrated index of the atherosclerotic risk factor burden. In an
attempt to ascertain their link to sVCAM-1 concentrations, they were
all included in our multivariate analysis of
the variability of IMTmax. In such analysis,
sVCAM-1 remained the only independent predictor of IMTmax
variability. In other words, taking into account sVCAM-1 as a correlate
of IMTmax in multivariate analysis
eliminated the independent contribution of other indexes, such as serum
triglycerides.
Another limitation of our study is the selection of our patient populations, for which patients with overt PVD were taken and matched with selected UH patients and controls. Such selection probably accounts for the little overlap of sVCAM-1 values between PVD patients and UH patients and controls. It is likely that more over- lapping values would be found if atherosclerotic patients are chosen by less stringent criteria. The present study, therefore, does not indicate a clinical value for sVCAM-1 in discriminating patients on the basis of a single plasma measurement, which is unlikely to be the case for any parameter investigated to this purpose. Rather, our findings suggest a role for sVCAM-1 as a more direct indicator of the atherosclerotic burden than other soluble markers and a role of VCAM-1 as a transducer of atherosclerotic risk factors.
Last, our study does not conclusively prove the increased expression of VCAM-1 on endothelial cell surface as the cause for increased sVCAM-1 levels in plasma. PVD patients, despite the a priori exclusion of renal insufficiency, had higher serum creatinine and lower creatinine clearance than the two other subject categories. Because a reduction of renal clearance of circulating soluble adhesion molecules could theoretically account for their elevation, regression analysis was performed between these parameters of renal function and soluble adhesion molecules. No significant correlation was found between serum creatinine or creatinine clearance and soluble adhesion molecules, thus suggesting little or no influence of changes in renal function on sVCAM-1 or sICAM-1 within the variation found in our subjects. On the other hand, sE-selectin was not higher at all in PVD patients, indicating some selectivity in the behavior of plasma levels of these molecules according to the clinical selection of patients. These considerations and the higher proteinuria in PVD patients, which would increase the urinary loss of these proteins, suggest production rather than the renal excretion rate as a more important determinant of plasma levels. Careful analyses of the clearance pathways of soluble adhesion molecules from the circulation are presently lacking in the literature and certainly now are warranted, in light of the increasing interest in soluble adhesion molecules as markers of disease.
Conclusion
Our search for biohumoral correlates of
atherosclerosis indicates sVCAM-1, the circulating form
of an endothelial leukocyte adhesion molecule
supporting the adhesion of monocytes to the
endothelium, as a good such correlate, predicting
intima-media thickness in the carotids better than previously reported
soluble markers. Further research on the evaluation of sVCAM-1 as a
marker for disease extension or severity on its role in predicting the
presence or complications of atherosclerosis, as well
as the impact of therapeutic or preventive interventions on this
potential surrogate marker for atherosclerosis, appears
now warranted.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was partially supported by grants from the Italian Ministero della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche. The authors express gratitude to Walter Bernini for valuable technical help, Dr Alessandra Bertolotto for assay of lipid parameters, Dr Ferdinando De Negri for assays of coagulation and fibrinolytic parameters, Dr Marina Noris and Dr Giuseppe Remuzzi at Mario Negri Institute for Pharmacologic Research, Bergamo, for in vivo assay of nitric oxide, and Dr Vitantonio Di Bello for cardiac echocardiographic assessments. Thanks also to Bender, Vienna, Austria-Prodotti Gianni, Milano, Italy and to R&D Systems Europe, Oxon, United Kingdom-SPACE, Milano, Italy, for generous gifts of immunoassay kits for sE-selectin/sICAM-1 and sVCAM-1, respectively.
Received January 7, 1997; accepted March 21, 1997.
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L. B. Goldstein, R. Adams, K. Becker, C. D. Furberg, P. B. Gorelick, G. Hademenos, M. Hill, G. Howard, V. J. Howard, B. Jacobs, et al. Primary Prevention of Ischemic Stroke : A Statement for Healthcare Professionals From the Stroke Council of the American Heart Association Circulation, January 2, 2001; 103(1): 163 - 182. [Full Text] [PDF]
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 N. Kaul, S. Devaraj, and I. Jialal {{alpha}}-Tocopherol and Atherosclerosis Experimental Biology and Medicine, January 1, 2001; 226(1): 5 - 12. [Abstract] [Full Text]
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L. B. Goldstein, R. Adams, K. Becker, C. D. Furberg, P. B. Gorelick, G. Hademenos, M. Hill, G. Howard, V. J. Howard, B. Jacobs, et al. Primary Prevention of Ischemic Stroke : A Statement for Healthcare Professionals From the Stroke Council of the American Heart Association Stroke, January 1, 2001; 32(1): 280 - 299. [Full Text] [PDF]
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P. Stenvinkel, B. Lindholm, M. Heimburger, and O. Heimburger Elevated serum levels of soluble adhesion molecules predict death in pre-dialysis patients: association with malnutrition, inflammation, and cardiovascular disease Nephrol. Dial. Transplant., October 1, 2000; 15(10): 1624 - 1630. [Abstract] [Full Text] [PDF]
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S. H. Jacobson, P. Thylen, and J. Lundahl Three monocyte-related determinants of atherosclerosis in haemodialysis Nephrol. Dial. Transplant., September 1, 2000; 15(9): 1414 - 1419. [Abstract] [Full Text] [PDF]
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J. A. de Lemos, C. H. Hennekens, and P. M. Ridker Plasma concentration of soluble vascular cell adhesion molecule-1 and subsequent cardiovascular risk J. Am. Coll. Cardiol., August 1, 2000; 36(2): 423 - 426. [Abstract] [Full Text] [PDF]
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 A. D. Blann and G. Y. H. Lip Editorial: Cell Adhesion Molecules in Cardiovascular Disease and Its Risk Factors--What Can Soluble Levels Tell Us? J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1745 - 1747. [Full Text]
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T. Murase, N. Kume, H. Kataoka, M. Minami, T. Sawamura, T. Masaki, and T. Kita Identification of Soluble Forms of Lectin-Like Oxidized LDL Receptor-1 Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 715 - 720. [Abstract] [Full Text] [PDF]
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M. Roque, J. T. Fallon, J. J. Badimon, W. X. Zhang, M. B. Taubman, and E. D. Reis Mouse Model of Femoral Artery Denudation Injury Associated With the Rapid Accumulation of Adhesion Molecules on the Luminal Surface and Recruitment of Neutrophils Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 335 - 342. [Abstract] [Full Text] [PDF]
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 C. Ferri, G. Desideri, M. Valenti, C. Bellini, M. Pasin, A. Santucci, and Giancarlo De Mattia Early Upregulation of Endothelial Adhesion Molecules in Obese Hypertensive Men Hypertension, October 1, 1999; 34(4): 568 - 573. [Abstract] [Full Text] [PDF]
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L. E. P. Rohde, C. H. Hennekens, and P. M. Ridker Cross-Sectional Study of Soluble Intercellular Adhesion Molecule-1 and Cardiovascular Risk Factors in Apparently Healthy Men Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1595 - 1599. [Abstract] [Full Text] [PDF]
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O. Johansen, I. Seljeflot, A. T. Hostmark, and H. Arnesen The Effect of Supplementation With Omega-3 Fatty Acids on Soluble Markers of Endothelial Function in Patients With Coronary Heart Disease Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1681 - 1686. [Abstract] [Full Text] [PDF]
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 W. Dichtl, L. Nilsson, I. Goncalves, M. P. S. Ares, C. Banfi, F. Calara, A. Hamsten, P. Eriksson, and J. Nilsson Very Low-Density Lipoprotein Activates Nuclear Factor-{kappa}B in Endothelial Cells Circ. Res., May 14, 1999; 84(9): 1085 - 1094. [Abstract] [Full Text] [PDF]
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 A. D BLANN and G. Y H LIP Cell adhesion molecules in cardiovascular disease: what can soluble levels tell us? Heart, January 1, 1999; 81(1): 101 - 102. [Full Text]
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L. E. Rohde, R. T. Lee, J. Rivero, M. Jamacochian, L. H. Arroyo, W. Briggs, N. Rifai, P. Libby, M. A. Creager, and P. M. Ridker Circulating Cell Adhesion Molecules Are Correlated With Ultrasound-Based Assessment of Carotid Atherosclerosis Arterioscler Thromb Vasc Biol, November 1, 1998; 18(11): 1765 - 1770. [Abstract] [Full Text] [PDF]
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 M-C. Boffa and M. Karmochkine Review : Thrombomodulin: an overview and potential implications in vascular disorders Lupus, January 1, 1998; 7(2_suppl): S120 - S125. [Abstract] [PDF]
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P. Ziccardi, F. Nappo, G. Giugliano, K. Esposito, R. Marfella, M. Cioffi, F. D'Andrea, A. M. Molinari, and D. Giugliano Reduction of Inflammatory Cytokine Concentrations and Improvement of Endothelial Functions in Obese Women After Weight Loss Over One Year Circulation, February 19, 2002; 105(7): 804 - 809. [Abstract] [Full Text] [PDF]
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A. Jager, V. W.M. van Hinsbergh, P. J. Kostense, J. J. Emeis, G. Nijpels, J. M. Dekker, R. J. Heine, L. M. Bouter, and C. D.A. Stehouwer C-Reactive Protein and Soluble Vascular Cell Adhesion Molecule-1 Are Associated With Elevated Urinary Albumin Excretion but Do Not Explain Its Link With Cardiovascular Risk Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 593 - 598. [Abstract] [Full Text] [PDF]
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