Inflammation and Carotid Artery—Risk for Atherosclerosis Study (ICARAS)
http://www.100md.com
《循环学杂志》
the Departments of Angiology (M.S., W.M., S.S., J.A., R.N., E.T., B.K., C.M., M.P., E.M.), Medical and Chemical Laboratory Diagnostics (M.E., H.R., O.W.), Neurology (W.L.)
Cardiology (G.M.), University of Vienna, Medical School, Vienna, Austria.
Abstract
Background— Compelling evidence suggests that inflammation is fundamentally involved in the pathogenesis of atherosclerosis; however, temporal correlation between inflammation and morphological features of atherosclerosis progression has not been demonstrated unequivocally.
Methods and Results— We prospectively studied 1268 consecutive patients who were initially asymptomatic with respect to carotid artery disease. Patients underwent serial carotid ultrasound investigations at baseline and after a follow-up interval of a median of 7.5 months (range 6 to 9 months), with measurement of carotid flow velocities and categorization of carotid arteries as 0% to 29%, 30% to 49%, 50% to 69%, 70% to 89%, or 90% to 99% stenosed or occluded. High-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) were measured at baseline and follow-up. Progression of carotid atherosclerosis was found in 103 (8.1%) of 1268 patients. Hs-CRP and SAA, respectively, at baseline (P=0.004 and P=0.014) and follow-up (P<0.001 and P<0.001) and the change from baseline to follow-up (P<0.001 and P<0.001) were significantly associated with progressive atherosclerosis. Adjusted ORs (95% CI) for atherosclerosis progression with increasing quintiles of baseline hs-CRP were 1.65 (0.71 to 3.84), 1.87 (0.8 to 4.37), 3.32 (1.49 to 7.39), and 3.65 (1.65 to 8.08), and with increasing quintiles of baseline SAA, they were 0.86 (0.38 to 1.92), 0.99 (0.49 to 1.99), 1.72 (0.91 to 3.28), and 2.28 (1.24 to 4.20), respectively, compared with the lowest quintiles.
Conclusions— These findings supply evidence for a close temporal correlation between inflammation and morphological features of rapidly progressive carotid atherosclerosis, which suggests that elevation or increase of the inflammatory biomarkers hs-CRP and SAA identifies the presence of active atherosclerotic disease.
Key Words: inflammation ; atherosclerosis ; carotid arteries ; plaque ; amyloid
Introduction
Complications of atherosclerosis are the most common causes of morbidity and mortality in Western societies. Among these, stroke is the third-leading cause of death and the most common cause of permanent disability.1 Large-artery thromboembolism originating from atherosclerosis in the carotid arteries accounts for 30% of these events. Recently, a substantially increased risk for neurological events was noted in patients with rapid progression of atherosclerotic lesions in the carotid arteries, with 2-year stroke rates exceeding 10%.2 Prediction of the risk for progression of carotid atherosclerosis, however, remains a major unresolved issue.
Compelling evidence suggests that inflammation is fundamentally involved in the pathogenesis of atherosclerosis.3–6 In broad outline, atherosclerosis can be considered to be a form of chronic inflammation that results from an interaction between modified lipoproteins, monocyte-derived macrophages, T cells, and the normal cellular elements of the arterial wall.6 The inflammatory process leads to the development of complex lesions, or plaques, that progressively protrude into the arterial lumen, causing arterial stenoses and occlusions.3 The extent of inflammation as measured by specific biomarkers likely reflects the activity of the disease and thus may predict the individual’s risk for progression of atherosclerosis. In this context, numerous studies elucidated the association between inflammation and clinical surrogate markers of atherosclerosis progression, mainly the occurrence of cardiovascular adverse events such as myocardial infarction and stroke7–18; however, a direct temporal correlation between inflammation and morphological features of atherosclerosis progression has not been demonstrated unequivocally.
Among the panel of inflammatory biomarkers, high-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) represent powerful cardiovascular risk predictors.7–18 We hypothesized that inflammatory activity, measured by hs-CRP and SAA at baseline and follow-up, is associated with short-term progression of atherosclerotic lesions in the carotid bifurcation.
Methods
Study Design
We prospectively enrolled all consecutive patients who underwent duplex ultrasound investigations of the extracranial carotid arteries from March 2002 until March 2003 at our institution who were currently neurologically asymptomatic with respect to carotid obstructions. In the present study, we aimed to directly evaluate progression of atherosclerotic stenosis instead of measuring surrogate markers of carotid disease (such as intima-media thickness). Our ultrasound laboratory serves the departments of angiology and cardiology, and the main indications for performing carotid ultrasound were carotid bruits, known atherosclerotic disease in other vessel areas (coronary or peripheral artery disease), and patients scheduled for major cardiac surgery, thus meeting the requirements of a study sample of patients likely to exhibit carotid atherosclerosis and potentially progression of disease.
Inclusion and Exclusion Criteria
Patients who were initially asymptomatic with respect to carotid artery disease were eligible, with "asymptomatic" defined as the absence of transient ischemic attacks (TIA), amaurosis fugax, or stroke in patients’ recent history. Patients with a history of prior TIA or stroke were only eligible if the event occurred at least 12 months before inclusion and no residual or recurrent symptoms were identified by a neurologist. Exclusion criteria were patients’ refusal to participate in the study, symptomatic carotid artery disease that necessitated revascularization therapy, current infectious or inflammatory diseases, recent operations or endovascular interventions (within 14 days), patients after bilateral carotid occlusions, bilateral stent implantation, or bilateral carotid endarterectomy. The study complied with the Declaration of Helsinki and was approved by the local review board and institutional ethics committee. All patients had to provide written informed consent.
During the study period, 1512 patients underwent a duplex ultrasound investigation of the extracranial carotid arteries. Of these, 149 were not eligible because of symptomatic carotid disease (n=89), current infections or recent operations (n=51), or refusal to participate (n=9). We enrolled 1363 eligible patients in the study. All study participants were white. Of these, 95 patients (7%) had to be excluded because of missing duplex ultrasound follow-up data (28 deaths; 67 refused the repeated duplex ultrasound investigation), which left 1268 patients for the final analysis. Clinical baseline characteristics (age, gender, frequency of atherothrombotic risk factors, cardiovascular comorbidities, and baseline degree of stenosis) of the 95 patients with missing duplex ultrasound follow-up data were not significantly different from the study sample (data not shown).
Study End Point
The primary study end point was unilateral or bilateral progression of carotid atherosclerosis in the extracranial internal carotid arteries (ICAs) from baseline to a follow-up investigation after 6 to 9 months. We used the following categories to quantify the degree of ICA stenosis at baseline and follow-up: 0% to 29% (carotid plaques), 30% to 49% (advanced plaques), 50% to 69% (moderate stenosis), 70% to 89% (high-grade stenosis), 90% to 99% (subocclusive stenosis), and 100% (occlusion). Progression of atherosclerotic disease was defined as an increase of the degree of stenosis by at least one category. Progression of stenosis in either one or both ICAs was considered indicative of progressive disease. The secondary objective was the change in peak systolic velocity in the internal carotid artery (PSVICA) from baseline to follow-up, as a continuous surrogate marker of progressive carotid disease.
Clinical Data
After patient identification at the ultrasound laboratory, medical history and data from physical examination were recorded, including the following variables: age, gender, body mass index, arterial hypertension, blood pressure, diabetes mellitus, smoking habits, hyperlipidemia, family history of atherosclerosis, history of myocardial infarction, angina pectoris (according to the Canadian Cardiovascular Society [CCS] classification), peripheral artery disease (according to the Fontaine classification), history of prior cerebral accident, and current medication use. We screened for current infectious or inflammatory diseases by evaluating patients’ clinical history and current symptoms. Clinical suspicion for infectious or inflammatory diseases prompted further specific investigations according to clinical judgment. Completeness and accuracy of all data were ascertained by 2 independent observers.
Medication
Pharmacotherapy of patients with evidence of atherosclerosis followed a standard protocol: Patients received antithrombotic therapy with either acetylsalicylic acid 100 mg or clopidogrel 75 mg once daily. Patients with hyperlipidemia (LDL cholesterol >130 mg/dL) received inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins).
Color-Coded Duplex Sonography and Grading of ICA Stenosis
Duplex examinations at baseline and during follow-up were performed on an Acuson 128 XP10 with a 7.5-MHz linear-array probe by experienced technical assistants who were supervised by 2 of the authors. Two independent investigators determined progression of carotid atherosclerosis on the basis of the baseline and follow-up duplex investigations. All duplex operators were blinded with respect to patients’ clinical data and laboratory findings. Duplex grading of the carotid stenosis was done as described previously by measurement of the peak systolic and end-diastolic velocities in the ICAs and common carotid arteries.19–21 When these velocities are combined according to a recommended algorithm, the degree of ICA stenosis can be obtained in 6 categories, which correspond to NASCET (North American Symptomatic Carotid Endarterectomy Trial) angiographic degree.19–21 As a continuous surrogate marker of stenosis severity, we examined PSVICA, which is considered the most sensitive single flow parameter with respect to quantifying the degree of carotid stenosis.19–21 The accuracy of our classification of the degree of stenosis with respect to angiography was assessed previously in our duplex laboratory in an independent cohort that included 1006 carotid arteries.21 With angiography as the "gold standard," positive and negative predictive values ranged from 70% to 98%, and interobserver agreement was excellent with respect to the absolute degree of stenosis (=0.83, 95% CI 0.79 to 0.88) and with respect to progression of the disease (=0.85, 95% CI 0.80 to 0.89).
Laboratory Data
Antecubital venous blood samples were obtained at baseline and follow-up visits. Measurements included HDL, LDL, and total cholesterol; HbA1c; hs-CRP; SAA; and complete blood count. For determination of hs-CRP values, a high-sensitivity assay (N Latex CRP Mono, DADE Behring) with a detection level of 0.03 mg/dL and a coefficient of variation of 4.6% was used. SAA was measured by N Latex SAA (DADE Behring) with a detection level of 3.8 mg/L and a coefficient of variation of 6.4%. Treating physicians and ultrasonographers were blinded as to the hs-CRP and SAA values at baseline and follow-up.
Surveillance Protocol
Patients were scheduled for a follow-up visit 6 to 9 months after the initial presentation for clinical reexamination, neurological history with standard questionnaires, duplex examination, and blood sampling. Patients with clinical suspicion of neurological events during the follow-up period were further evaluated by a neurologist and underwent mandatory cranial computed tomography. If patients were symptomatic before the scheduled follow-up visit, which clinically prompted a duplex ultrasound investigation, the scheduled study follow-up ultrasound at 6 to 9 months was performed additionally and was considered for the study purposes to avoid any bias due to a shorter follow-up period.
Definitions
Diabetes mellitus was defined according to the 1997 criteria of the American Diabetes Association. Arterial hypertension was diagnosed in patients with resting blood pressure values above 140/90 mm Hg measured repetitively (at least twice) and was assumed to be present in patients taking antihypertensive drugs. Hyperlipidemia was defined as an elevation of LDL cholesterol above 130 mg/dL or total cholesterol above 200 mg/dL and was assumed to be present in all patients taking lipid-lowering medication. The diagnosis of peripheral artery disease was classified according to Fontaine.22 Coronary artery disease was recorded according to the CCS classification. History of myocardial infarction was defined according to the consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction.23 Stroke was defined as a neurological deficit that persisted longer than 24 hours evaluated by a neurologist according to the modified Rankin stroke scale.24 Mandatory cranial computed tomography or, if available, MRI was used for confirmation of the diagnosis.
Statistical Methods
Continuous data are presented as median and interquartile range (IQR; range from the 25th to the 75th percentile) or the total range. Discrete data are given as counts and percentages. We used 2 tests, Mann Whitney U tests, exact tests, and Spearman correlation coefficients for univariate analyses, as appropriate. Partial correlation was used to assess the correlation between changes in hs-CRP/SAA and changes in the degree of stenosis as measured by PSVICA. Multivariable logistic regression analysis was applied to assess the effect of inflammatory parameters on progression of the disease with adjustment for potential confounders. Hs-CRP and SAA were divided into quintiles before being entered into the models, to obtain clinically useful measures for the effect sizes. Separate models were calculated for hs-CRP and SAA at baseline and follow-up. Results of the logistic regression models are presented as the OR and 95% CI. Analyses that included the baseline degree of stenosis of both carotid arteries were adjusted for clustering by patient. Interaction was assessed with additive and multiplicative interaction terms, the linearity of the logit assumption was checked for continuous predictor variables, and an analysis of residuals was performed. Regression diagnostics and overall model fit were performed according to standard procedures.25 A 2-sided probability value <0.05 was considered statistically significant. Calculations were performed with Stata (release 8.0, Stata) and SPSS for Windows (version 10.0, SPSS Inc).
Results
Patient Characteristics
The inflammatory markers hs-CRP and SAA were significantly correlated at baseline (r=0.54, P<0.001). HbA1c (r=0.13, P<0.001 and r=0.10, P<0.001) and body mass index (r=0.17, P<0.001 and r=0.07, P=0.009) were also significantly correlated both with hs-CRP and SAA, respectively. Furthermore, current smokers (P<0.001 and P=0.81), patients with arterial hypertension (P=0.052 and P=0.030), and patients with a history of myocardial infarction (P=0.008 and P=0.030), stroke (P=0.042 and P=0.016), or peripheral artery disease (P=0.010 and P=0.053) had higher hs-CRP and SAA values, respectively, at baseline. The baseline extent of carotid artery stenosis (in categories) and the PSVICA were not significantly associated with hs-CRP or SAA (all P>0.2), respectively.
Progression of Carotid Atherosclerosis
During the median follow-up period of 7.5 months (range 6 to 9 months), progression of carotid lesions was found in 103 (8.1%) of 1268 patients. Of these, 44 patients (3.5%) showed progression of a left ICA stenosis, 52 patients (4.1%) showed progression of a right ICA stenosis, and 6 patients (0.5%) had progressive lesions in both carotid arteries. In the 1268 patients, the change in PSVICA from baseline to follow-up was a median of 0.01 m/s (IQR –0.01 to 0.08 m/s); in patients with a progressive lesion, the median increase in PSV was 0.56 m/s (IQR 0.35 to 1.08 m/s). Eight patients (0.6%) developed a de novo occlusion of a carotid artery; each of these patients had an ipsilateral subocclusive stenosis (90% to 99%) at baseline. None of the patients underwent a carotid revascularization procedure during the follow-up period. Fifteen patients (1.2%) experienced a stroke during follow-up (4/103 patients [3.9%] with progressive lesions versus 11/1165 [0.9%] patients with stable disease; P=0.028). The associations between baseline clinical characteristics and risk for progression of the disease are given in Table 2; age, current smoking, history of prior stroke, and the baseline degree of stenosis were risk factors for disease progression.
Inflammatory Biomarkers and Disease Progression
Levels of hs-CRP and SAA at baseline and follow-up and the change from baseline to follow-up were significantly associated with progression of carotid atherosclerosis as a categorical measure (Figure 1). Furthermore, the changes in hs-CRP and SAA from baseline to follow-up were significantly but weakly correlated to the changes in PSVICA during the same time interval as a continuous measure of carotid stenosis progression (Figure 1).
Partial correlation analyses (with adjustment for the same covariates listed above) confirmed a significant but weak correlation between the change in PSVICA from baseline to follow-up and the change in hs-CRP (r=0.061, P=0.021) and SAA (r=0.064, P=0.017), respectively.
We performed 2 sets of post hoc sensitivity analyses. First, the use of an even more conservative classification of carotid stenosis that categorized lesions as 0% to 49%, 50% to 69%, or 70% to 99% stenosed or as occluded revealed virtually identical associations between progression of disease and inflammatory biomarkers. Second, because we were aware of a potential difference between patients with and without prevalent cerebrovascular disease, we reanalyzed the data after excluding the 199 patients (16%) with a history of stroke. Univariate and multivariable analyses revealed significant associations between inflammatory parameters at baseline and follow-up with disease progression in 1069 patients without a history of stroke; the observed effect sizes were closely comparable to the results obtained in the entire patient sample of 1268 patients. Furthermore, testing for interaction between history of stroke, inflammatory parameters, and disease progression in the entire sample of 1268 patients by multiplicative interaction terms and log likelihood ratio tests revealed no significant effect modification.
Discussion
This is the first large, longitudinal study that unequivocally confirms a temporal correlation between inflammation and morphological features of rapidly progressive atherosclerosis. We found that increasing quintiles of the inflammatory biomarkers hs-CRP and SAA at baseline and follow-up predicted short-term progression of atherosclerotic lesions in the carotid arteries. Consistently, the change of inflammatory parameters correlated with the change of the degree of carotid stenosis during a 6- to 9-month period.
Progressive atherosclerotic plaques are associated with a markedly increased risk for clinical complications in virtually any segment of the arterial circulation.2,26 These progressive or so-called vulnerable lesions are histologically heterogeneous and may be characterized by an inflammatory necrotic lipid core, fibrocalcified nodules, a thin fibrous cap of the atheroma, superficial erosion, thrombus apposition, or intraplaque hemorrhage.26,27 Acute-phase reactants like C-reactive protein (CRP) and SAA provide an indirect measure of the cytokine-dependent inflammatory process in the arterial wall, which is a particularly key feature of the inflammatory activity that surrounds the necrotic lipid core.5,26 However, CRP and SAA were described not merely as markers of atherosclerosis risk but also as directly promoting endothelial cell activation, adhesion molecule expression, and resultant endothelial dysfunction.28–31 Accumulating epidemiological data have evolved that confirm that elevation of these parameters heralds clinically relevant atherothrombotic events.7–18 Nevertheless, it remains unclear whether the extent of inflammation directly correlates with morphological features of atherosclerosis progression. The present observation provides considerable reassurance that progressive atherosclerotic lesions cause an inflammatory process that can be quantified by acute-phase reactants or vice versa, assuming a causal relationship, that inflammation triggers rapidly progressive atherosclerosis. It remains questionable whether elevated levels of inflammatory biomarkers uncover the presence of a single vulnerable lesion or identify a "vulnerable patient" exhibiting several coexisting high-risk lesions in different arterial segments. Actually, it appears more likely that elevation of hs-CRP and SAA indicates the systemic nature of progressive atherosclerotic disease,26,32–35 which suggests that patients with enhanced inflammation are generally at high risk for progression of atherosclerotic disease and may exhibit multiple vulnerable lesions. The concept of early identification of vulnerable patients who are susceptible to cardiovascular adverse events seems appealing, and measurement of inflammatory biomarkers may be a potent adjunctive tool for this purpose.
The present findings suggest that inflammatory biomarkers indicate a risk for progressive disease rather than being associated with the baseline category of carotid stenosis. From the panel of traditional cardiovascular risk factors, only smoking was found to predict outcome, whereas diabetes, hyperlipidemia, arterial hypertension, and body mass index were not associated with morphological disease progression. This may suggest that the established risk factors are more important for the initiation and generalization of atherosclerosis and thus are strongly associated with the presence and extent of atherosclerosis,36 whereas the inflammatory biomarkers specifically indicate rapidly progressive disease. Admittedly, the power of the study may have been inadequate to demonstrate such associations for the other traditional risk factors.
As an alternative to the degree of stenosis, intima-media thickness has been used in numerous studies to estimate progression of carotid disease. The implications of an increase in carotid intima-media thickness versus an increase in degree of stenosis may differ, particularly in a sample with prevalent atherosclerotic disease. Changes in the degree of stenosis appear to directly reflect progression of atherosclerosis, whereas intima-media thickness is considered only as a surrogate marker for carotid disease.
Acknowledging potential confounding factors, we performed 2 post hoc sensitivity analyses. Restricting the number of categories of the degree of carotid stenosis to 4 instead of 6 revealed almost identical findings. Similarly, excluding the 199 patients with prevalent strokes did not significantly affect the observed effect sizes. Taken together, the consistency of the findings with respect to different ultrasound methods of measurement, temporal coherence, biological gradients, and relatively large effect sizes in the entire patient sample and in patients without prevalent cerebrovascular disease argue against a chance finding. Furthermore, the present results are in line with numerous former cross-sectional studies.37–40
Despite major advances in treatment of stroke, early identification and prevention of incident events remain crucial, because more than two thirds of all strokes occur without prior symptoms. Recently, Bertges et al2 demonstrated that progression of carotid artery disease as indicated by duplex ultrasound investigations translates clinically into a substantially increased risk for ipsilateral neurological events. The present observations confirm these finding and suggest that in particular, patients with advanced carotid lesions and high levels of hs-CRP and SAA require close ultrasound surveillance and aggressive treatment of risk factors.
Study Limitations
We are aware of some methodological limitations of the present study. Potential selection bias inherent in a consecutive series of hospital-based duplex examinations cannot be ruled out, and the generalizability of these findings to younger individuals, other ethnicities and races, and unselected persons in the community remains to be determined.
Conclusions
These findings supply evidence of a close temporal correlation between inflammation and morphological features of rapidly progressive carotid atherosclerosis, which suggests that elevation or increase of the inflammatory biomarkers hs-CRP and SAA identifies the presence of active atherosclerotic disease.
Acknowledgments
The authors are indebted to Angelika Haumer, Edyta Madroszkiewicz, Irene Liegler, Irene Mlekusch, Karin Pikesch, Sonja Fasching, and Sylvia Kaiser for excellent technical assistance.
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Cardiology (G.M.), University of Vienna, Medical School, Vienna, Austria.
Abstract
Background— Compelling evidence suggests that inflammation is fundamentally involved in the pathogenesis of atherosclerosis; however, temporal correlation between inflammation and morphological features of atherosclerosis progression has not been demonstrated unequivocally.
Methods and Results— We prospectively studied 1268 consecutive patients who were initially asymptomatic with respect to carotid artery disease. Patients underwent serial carotid ultrasound investigations at baseline and after a follow-up interval of a median of 7.5 months (range 6 to 9 months), with measurement of carotid flow velocities and categorization of carotid arteries as 0% to 29%, 30% to 49%, 50% to 69%, 70% to 89%, or 90% to 99% stenosed or occluded. High-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) were measured at baseline and follow-up. Progression of carotid atherosclerosis was found in 103 (8.1%) of 1268 patients. Hs-CRP and SAA, respectively, at baseline (P=0.004 and P=0.014) and follow-up (P<0.001 and P<0.001) and the change from baseline to follow-up (P<0.001 and P<0.001) were significantly associated with progressive atherosclerosis. Adjusted ORs (95% CI) for atherosclerosis progression with increasing quintiles of baseline hs-CRP were 1.65 (0.71 to 3.84), 1.87 (0.8 to 4.37), 3.32 (1.49 to 7.39), and 3.65 (1.65 to 8.08), and with increasing quintiles of baseline SAA, they were 0.86 (0.38 to 1.92), 0.99 (0.49 to 1.99), 1.72 (0.91 to 3.28), and 2.28 (1.24 to 4.20), respectively, compared with the lowest quintiles.
Conclusions— These findings supply evidence for a close temporal correlation between inflammation and morphological features of rapidly progressive carotid atherosclerosis, which suggests that elevation or increase of the inflammatory biomarkers hs-CRP and SAA identifies the presence of active atherosclerotic disease.
Key Words: inflammation ; atherosclerosis ; carotid arteries ; plaque ; amyloid
Introduction
Complications of atherosclerosis are the most common causes of morbidity and mortality in Western societies. Among these, stroke is the third-leading cause of death and the most common cause of permanent disability.1 Large-artery thromboembolism originating from atherosclerosis in the carotid arteries accounts for 30% of these events. Recently, a substantially increased risk for neurological events was noted in patients with rapid progression of atherosclerotic lesions in the carotid arteries, with 2-year stroke rates exceeding 10%.2 Prediction of the risk for progression of carotid atherosclerosis, however, remains a major unresolved issue.
Compelling evidence suggests that inflammation is fundamentally involved in the pathogenesis of atherosclerosis.3–6 In broad outline, atherosclerosis can be considered to be a form of chronic inflammation that results from an interaction between modified lipoproteins, monocyte-derived macrophages, T cells, and the normal cellular elements of the arterial wall.6 The inflammatory process leads to the development of complex lesions, or plaques, that progressively protrude into the arterial lumen, causing arterial stenoses and occlusions.3 The extent of inflammation as measured by specific biomarkers likely reflects the activity of the disease and thus may predict the individual’s risk for progression of atherosclerosis. In this context, numerous studies elucidated the association between inflammation and clinical surrogate markers of atherosclerosis progression, mainly the occurrence of cardiovascular adverse events such as myocardial infarction and stroke7–18; however, a direct temporal correlation between inflammation and morphological features of atherosclerosis progression has not been demonstrated unequivocally.
Among the panel of inflammatory biomarkers, high-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA) represent powerful cardiovascular risk predictors.7–18 We hypothesized that inflammatory activity, measured by hs-CRP and SAA at baseline and follow-up, is associated with short-term progression of atherosclerotic lesions in the carotid bifurcation.
Methods
Study Design
We prospectively enrolled all consecutive patients who underwent duplex ultrasound investigations of the extracranial carotid arteries from March 2002 until March 2003 at our institution who were currently neurologically asymptomatic with respect to carotid obstructions. In the present study, we aimed to directly evaluate progression of atherosclerotic stenosis instead of measuring surrogate markers of carotid disease (such as intima-media thickness). Our ultrasound laboratory serves the departments of angiology and cardiology, and the main indications for performing carotid ultrasound were carotid bruits, known atherosclerotic disease in other vessel areas (coronary or peripheral artery disease), and patients scheduled for major cardiac surgery, thus meeting the requirements of a study sample of patients likely to exhibit carotid atherosclerosis and potentially progression of disease.
Inclusion and Exclusion Criteria
Patients who were initially asymptomatic with respect to carotid artery disease were eligible, with "asymptomatic" defined as the absence of transient ischemic attacks (TIA), amaurosis fugax, or stroke in patients’ recent history. Patients with a history of prior TIA or stroke were only eligible if the event occurred at least 12 months before inclusion and no residual or recurrent symptoms were identified by a neurologist. Exclusion criteria were patients’ refusal to participate in the study, symptomatic carotid artery disease that necessitated revascularization therapy, current infectious or inflammatory diseases, recent operations or endovascular interventions (within 14 days), patients after bilateral carotid occlusions, bilateral stent implantation, or bilateral carotid endarterectomy. The study complied with the Declaration of Helsinki and was approved by the local review board and institutional ethics committee. All patients had to provide written informed consent.
During the study period, 1512 patients underwent a duplex ultrasound investigation of the extracranial carotid arteries. Of these, 149 were not eligible because of symptomatic carotid disease (n=89), current infections or recent operations (n=51), or refusal to participate (n=9). We enrolled 1363 eligible patients in the study. All study participants were white. Of these, 95 patients (7%) had to be excluded because of missing duplex ultrasound follow-up data (28 deaths; 67 refused the repeated duplex ultrasound investigation), which left 1268 patients for the final analysis. Clinical baseline characteristics (age, gender, frequency of atherothrombotic risk factors, cardiovascular comorbidities, and baseline degree of stenosis) of the 95 patients with missing duplex ultrasound follow-up data were not significantly different from the study sample (data not shown).
Study End Point
The primary study end point was unilateral or bilateral progression of carotid atherosclerosis in the extracranial internal carotid arteries (ICAs) from baseline to a follow-up investigation after 6 to 9 months. We used the following categories to quantify the degree of ICA stenosis at baseline and follow-up: 0% to 29% (carotid plaques), 30% to 49% (advanced plaques), 50% to 69% (moderate stenosis), 70% to 89% (high-grade stenosis), 90% to 99% (subocclusive stenosis), and 100% (occlusion). Progression of atherosclerotic disease was defined as an increase of the degree of stenosis by at least one category. Progression of stenosis in either one or both ICAs was considered indicative of progressive disease. The secondary objective was the change in peak systolic velocity in the internal carotid artery (PSVICA) from baseline to follow-up, as a continuous surrogate marker of progressive carotid disease.
Clinical Data
After patient identification at the ultrasound laboratory, medical history and data from physical examination were recorded, including the following variables: age, gender, body mass index, arterial hypertension, blood pressure, diabetes mellitus, smoking habits, hyperlipidemia, family history of atherosclerosis, history of myocardial infarction, angina pectoris (according to the Canadian Cardiovascular Society [CCS] classification), peripheral artery disease (according to the Fontaine classification), history of prior cerebral accident, and current medication use. We screened for current infectious or inflammatory diseases by evaluating patients’ clinical history and current symptoms. Clinical suspicion for infectious or inflammatory diseases prompted further specific investigations according to clinical judgment. Completeness and accuracy of all data were ascertained by 2 independent observers.
Medication
Pharmacotherapy of patients with evidence of atherosclerosis followed a standard protocol: Patients received antithrombotic therapy with either acetylsalicylic acid 100 mg or clopidogrel 75 mg once daily. Patients with hyperlipidemia (LDL cholesterol >130 mg/dL) received inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins).
Color-Coded Duplex Sonography and Grading of ICA Stenosis
Duplex examinations at baseline and during follow-up were performed on an Acuson 128 XP10 with a 7.5-MHz linear-array probe by experienced technical assistants who were supervised by 2 of the authors. Two independent investigators determined progression of carotid atherosclerosis on the basis of the baseline and follow-up duplex investigations. All duplex operators were blinded with respect to patients’ clinical data and laboratory findings. Duplex grading of the carotid stenosis was done as described previously by measurement of the peak systolic and end-diastolic velocities in the ICAs and common carotid arteries.19–21 When these velocities are combined according to a recommended algorithm, the degree of ICA stenosis can be obtained in 6 categories, which correspond to NASCET (North American Symptomatic Carotid Endarterectomy Trial) angiographic degree.19–21 As a continuous surrogate marker of stenosis severity, we examined PSVICA, which is considered the most sensitive single flow parameter with respect to quantifying the degree of carotid stenosis.19–21 The accuracy of our classification of the degree of stenosis with respect to angiography was assessed previously in our duplex laboratory in an independent cohort that included 1006 carotid arteries.21 With angiography as the "gold standard," positive and negative predictive values ranged from 70% to 98%, and interobserver agreement was excellent with respect to the absolute degree of stenosis (=0.83, 95% CI 0.79 to 0.88) and with respect to progression of the disease (=0.85, 95% CI 0.80 to 0.89).
Laboratory Data
Antecubital venous blood samples were obtained at baseline and follow-up visits. Measurements included HDL, LDL, and total cholesterol; HbA1c; hs-CRP; SAA; and complete blood count. For determination of hs-CRP values, a high-sensitivity assay (N Latex CRP Mono, DADE Behring) with a detection level of 0.03 mg/dL and a coefficient of variation of 4.6% was used. SAA was measured by N Latex SAA (DADE Behring) with a detection level of 3.8 mg/L and a coefficient of variation of 6.4%. Treating physicians and ultrasonographers were blinded as to the hs-CRP and SAA values at baseline and follow-up.
Surveillance Protocol
Patients were scheduled for a follow-up visit 6 to 9 months after the initial presentation for clinical reexamination, neurological history with standard questionnaires, duplex examination, and blood sampling. Patients with clinical suspicion of neurological events during the follow-up period were further evaluated by a neurologist and underwent mandatory cranial computed tomography. If patients were symptomatic before the scheduled follow-up visit, which clinically prompted a duplex ultrasound investigation, the scheduled study follow-up ultrasound at 6 to 9 months was performed additionally and was considered for the study purposes to avoid any bias due to a shorter follow-up period.
Definitions
Diabetes mellitus was defined according to the 1997 criteria of the American Diabetes Association. Arterial hypertension was diagnosed in patients with resting blood pressure values above 140/90 mm Hg measured repetitively (at least twice) and was assumed to be present in patients taking antihypertensive drugs. Hyperlipidemia was defined as an elevation of LDL cholesterol above 130 mg/dL or total cholesterol above 200 mg/dL and was assumed to be present in all patients taking lipid-lowering medication. The diagnosis of peripheral artery disease was classified according to Fontaine.22 Coronary artery disease was recorded according to the CCS classification. History of myocardial infarction was defined according to the consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction.23 Stroke was defined as a neurological deficit that persisted longer than 24 hours evaluated by a neurologist according to the modified Rankin stroke scale.24 Mandatory cranial computed tomography or, if available, MRI was used for confirmation of the diagnosis.
Statistical Methods
Continuous data are presented as median and interquartile range (IQR; range from the 25th to the 75th percentile) or the total range. Discrete data are given as counts and percentages. We used 2 tests, Mann Whitney U tests, exact tests, and Spearman correlation coefficients for univariate analyses, as appropriate. Partial correlation was used to assess the correlation between changes in hs-CRP/SAA and changes in the degree of stenosis as measured by PSVICA. Multivariable logistic regression analysis was applied to assess the effect of inflammatory parameters on progression of the disease with adjustment for potential confounders. Hs-CRP and SAA were divided into quintiles before being entered into the models, to obtain clinically useful measures for the effect sizes. Separate models were calculated for hs-CRP and SAA at baseline and follow-up. Results of the logistic regression models are presented as the OR and 95% CI. Analyses that included the baseline degree of stenosis of both carotid arteries were adjusted for clustering by patient. Interaction was assessed with additive and multiplicative interaction terms, the linearity of the logit assumption was checked for continuous predictor variables, and an analysis of residuals was performed. Regression diagnostics and overall model fit were performed according to standard procedures.25 A 2-sided probability value <0.05 was considered statistically significant. Calculations were performed with Stata (release 8.0, Stata) and SPSS for Windows (version 10.0, SPSS Inc).
Results
Patient Characteristics
The inflammatory markers hs-CRP and SAA were significantly correlated at baseline (r=0.54, P<0.001). HbA1c (r=0.13, P<0.001 and r=0.10, P<0.001) and body mass index (r=0.17, P<0.001 and r=0.07, P=0.009) were also significantly correlated both with hs-CRP and SAA, respectively. Furthermore, current smokers (P<0.001 and P=0.81), patients with arterial hypertension (P=0.052 and P=0.030), and patients with a history of myocardial infarction (P=0.008 and P=0.030), stroke (P=0.042 and P=0.016), or peripheral artery disease (P=0.010 and P=0.053) had higher hs-CRP and SAA values, respectively, at baseline. The baseline extent of carotid artery stenosis (in categories) and the PSVICA were not significantly associated with hs-CRP or SAA (all P>0.2), respectively.
Progression of Carotid Atherosclerosis
During the median follow-up period of 7.5 months (range 6 to 9 months), progression of carotid lesions was found in 103 (8.1%) of 1268 patients. Of these, 44 patients (3.5%) showed progression of a left ICA stenosis, 52 patients (4.1%) showed progression of a right ICA stenosis, and 6 patients (0.5%) had progressive lesions in both carotid arteries. In the 1268 patients, the change in PSVICA from baseline to follow-up was a median of 0.01 m/s (IQR –0.01 to 0.08 m/s); in patients with a progressive lesion, the median increase in PSV was 0.56 m/s (IQR 0.35 to 1.08 m/s). Eight patients (0.6%) developed a de novo occlusion of a carotid artery; each of these patients had an ipsilateral subocclusive stenosis (90% to 99%) at baseline. None of the patients underwent a carotid revascularization procedure during the follow-up period. Fifteen patients (1.2%) experienced a stroke during follow-up (4/103 patients [3.9%] with progressive lesions versus 11/1165 [0.9%] patients with stable disease; P=0.028). The associations between baseline clinical characteristics and risk for progression of the disease are given in Table 2; age, current smoking, history of prior stroke, and the baseline degree of stenosis were risk factors for disease progression.
Inflammatory Biomarkers and Disease Progression
Levels of hs-CRP and SAA at baseline and follow-up and the change from baseline to follow-up were significantly associated with progression of carotid atherosclerosis as a categorical measure (Figure 1). Furthermore, the changes in hs-CRP and SAA from baseline to follow-up were significantly but weakly correlated to the changes in PSVICA during the same time interval as a continuous measure of carotid stenosis progression (Figure 1).
Partial correlation analyses (with adjustment for the same covariates listed above) confirmed a significant but weak correlation between the change in PSVICA from baseline to follow-up and the change in hs-CRP (r=0.061, P=0.021) and SAA (r=0.064, P=0.017), respectively.
We performed 2 sets of post hoc sensitivity analyses. First, the use of an even more conservative classification of carotid stenosis that categorized lesions as 0% to 49%, 50% to 69%, or 70% to 99% stenosed or as occluded revealed virtually identical associations between progression of disease and inflammatory biomarkers. Second, because we were aware of a potential difference between patients with and without prevalent cerebrovascular disease, we reanalyzed the data after excluding the 199 patients (16%) with a history of stroke. Univariate and multivariable analyses revealed significant associations between inflammatory parameters at baseline and follow-up with disease progression in 1069 patients without a history of stroke; the observed effect sizes were closely comparable to the results obtained in the entire patient sample of 1268 patients. Furthermore, testing for interaction between history of stroke, inflammatory parameters, and disease progression in the entire sample of 1268 patients by multiplicative interaction terms and log likelihood ratio tests revealed no significant effect modification.
Discussion
This is the first large, longitudinal study that unequivocally confirms a temporal correlation between inflammation and morphological features of rapidly progressive atherosclerosis. We found that increasing quintiles of the inflammatory biomarkers hs-CRP and SAA at baseline and follow-up predicted short-term progression of atherosclerotic lesions in the carotid arteries. Consistently, the change of inflammatory parameters correlated with the change of the degree of carotid stenosis during a 6- to 9-month period.
Progressive atherosclerotic plaques are associated with a markedly increased risk for clinical complications in virtually any segment of the arterial circulation.2,26 These progressive or so-called vulnerable lesions are histologically heterogeneous and may be characterized by an inflammatory necrotic lipid core, fibrocalcified nodules, a thin fibrous cap of the atheroma, superficial erosion, thrombus apposition, or intraplaque hemorrhage.26,27 Acute-phase reactants like C-reactive protein (CRP) and SAA provide an indirect measure of the cytokine-dependent inflammatory process in the arterial wall, which is a particularly key feature of the inflammatory activity that surrounds the necrotic lipid core.5,26 However, CRP and SAA were described not merely as markers of atherosclerosis risk but also as directly promoting endothelial cell activation, adhesion molecule expression, and resultant endothelial dysfunction.28–31 Accumulating epidemiological data have evolved that confirm that elevation of these parameters heralds clinically relevant atherothrombotic events.7–18 Nevertheless, it remains unclear whether the extent of inflammation directly correlates with morphological features of atherosclerosis progression. The present observation provides considerable reassurance that progressive atherosclerotic lesions cause an inflammatory process that can be quantified by acute-phase reactants or vice versa, assuming a causal relationship, that inflammation triggers rapidly progressive atherosclerosis. It remains questionable whether elevated levels of inflammatory biomarkers uncover the presence of a single vulnerable lesion or identify a "vulnerable patient" exhibiting several coexisting high-risk lesions in different arterial segments. Actually, it appears more likely that elevation of hs-CRP and SAA indicates the systemic nature of progressive atherosclerotic disease,26,32–35 which suggests that patients with enhanced inflammation are generally at high risk for progression of atherosclerotic disease and may exhibit multiple vulnerable lesions. The concept of early identification of vulnerable patients who are susceptible to cardiovascular adverse events seems appealing, and measurement of inflammatory biomarkers may be a potent adjunctive tool for this purpose.
The present findings suggest that inflammatory biomarkers indicate a risk for progressive disease rather than being associated with the baseline category of carotid stenosis. From the panel of traditional cardiovascular risk factors, only smoking was found to predict outcome, whereas diabetes, hyperlipidemia, arterial hypertension, and body mass index were not associated with morphological disease progression. This may suggest that the established risk factors are more important for the initiation and generalization of atherosclerosis and thus are strongly associated with the presence and extent of atherosclerosis,36 whereas the inflammatory biomarkers specifically indicate rapidly progressive disease. Admittedly, the power of the study may have been inadequate to demonstrate such associations for the other traditional risk factors.
As an alternative to the degree of stenosis, intima-media thickness has been used in numerous studies to estimate progression of carotid disease. The implications of an increase in carotid intima-media thickness versus an increase in degree of stenosis may differ, particularly in a sample with prevalent atherosclerotic disease. Changes in the degree of stenosis appear to directly reflect progression of atherosclerosis, whereas intima-media thickness is considered only as a surrogate marker for carotid disease.
Acknowledging potential confounding factors, we performed 2 post hoc sensitivity analyses. Restricting the number of categories of the degree of carotid stenosis to 4 instead of 6 revealed almost identical findings. Similarly, excluding the 199 patients with prevalent strokes did not significantly affect the observed effect sizes. Taken together, the consistency of the findings with respect to different ultrasound methods of measurement, temporal coherence, biological gradients, and relatively large effect sizes in the entire patient sample and in patients without prevalent cerebrovascular disease argue against a chance finding. Furthermore, the present results are in line with numerous former cross-sectional studies.37–40
Despite major advances in treatment of stroke, early identification and prevention of incident events remain crucial, because more than two thirds of all strokes occur without prior symptoms. Recently, Bertges et al2 demonstrated that progression of carotid artery disease as indicated by duplex ultrasound investigations translates clinically into a substantially increased risk for ipsilateral neurological events. The present observations confirm these finding and suggest that in particular, patients with advanced carotid lesions and high levels of hs-CRP and SAA require close ultrasound surveillance and aggressive treatment of risk factors.
Study Limitations
We are aware of some methodological limitations of the present study. Potential selection bias inherent in a consecutive series of hospital-based duplex examinations cannot be ruled out, and the generalizability of these findings to younger individuals, other ethnicities and races, and unselected persons in the community remains to be determined.
Conclusions
These findings supply evidence of a close temporal correlation between inflammation and morphological features of rapidly progressive carotid atherosclerosis, which suggests that elevation or increase of the inflammatory biomarkers hs-CRP and SAA identifies the presence of active atherosclerotic disease.
Acknowledgments
The authors are indebted to Angelika Haumer, Edyta Madroszkiewicz, Irene Liegler, Irene Mlekusch, Karin Pikesch, Sonja Fasching, and Sylvia Kaiser for excellent technical assistance.
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