Atherosclerosis in Egyptian patients with ankylosing spondylitis

Introduction Ankylosing spondylitis (AS) is a systemic infl ammatory disorder that causes arthritis of the spine, sacroiliac joints, and even the peripheral joints. It involves extraarticular structures including the eyes, lung, heart, vessels, kidneys, and nerves [1]. Studies have shown that patients with AS have several cardiovascular disease risk factors, which are driven in turn by systemic infl ammatory mediators [2]. Indeed, there is increased evidence that the underlying infl ammatory process in chronic infl ammatory disorders contributes toward various stages of atherothrombosis [3].


Introduction
Ankylosing spondylitis (AS) is a systemic infl ammatory disorder that causes arthritis of the spine, sacroiliac joints, and even the peripheral joints. It involves extraarticular structures including the eyes, lung, heart, vessels, kidneys, and nerves [1]. Studies have shown that patients with AS have several cardiovascular disease risk factors, which are driven in turn by systemic infl ammatory mediators [2]. Indeed, there is increased evidence that the underlying infl ammatory process in chronic infl ammatory disorders contributes toward various stages of atherothrombosis [3].
Among traditional Framingham risk factors, AS has been associated with a high prevalence of metabolic syndrome including dyslipidemia and high ratio of low-density lipoprotein cholesterol ( LDL-C) to high-density lipoprotein cholesterol ( HDL-C) [4]. Moreover, infl ammatory mechanisms underlying AS may be the key factors that lead to atherosclerosis and vascular disease. Sustained systemic infl ammation in AS is accompanied by elevated serum levels of C-reactive protein (CRP) [5], which is an acute-phase protein with documented proatherogenic eff ects [6].
von Willebrand factor (vWF) plays an important role in platelet adhesion to subendothelial structure; it is considered an indirect measure of endothelial dysfunction [7]. vWF levels are also associated with infl ammation. vWF levels are elevated in a number of infl ammatory disorders such as rheumatoid arthritis and vasculitis. Several cytokines or other mediators of infl ammation induce endothelial vWF secretion [8].
Arterial duplex of the common carotid artery is usually used to evaluate the extent of subclinical atherosclerosis by measuring the intima-media thickness (IMT) [9]. Stress echocardiography is a noninvasive technique to evaluate the patients with suspected coronary Atherosclerosis in Egyptian patients with ankylosing spondylitis Dahlia

Objective
The objective of this study was to assess the presence of atherosclerosis in Egyptian patients with AS and its relation to disease activity.

Patients and methods
Thirty patients with AS of at least 18 years of age and 30 age-matched and sex-matched controls were included. Assessment of medical history, clinical examinations, and assessment of AS disease activity using BASDAI, BASMI, and BASFI as well as dobutamine echocardiography were performed only for patients. Complete blood count, ESR, C-reactive protein, lipid profi le, serum von Willebrand factor ( vWF) Ag level by ELISA, ECG, and carotid duplex were performed for all participants.

Results
In patients, 11 had active disease and 19 were in remission. A hypertensive response ( HTNR) appeared in eight patients; six of them had active disease. There was a signifi cant increase in the level of vWF in actively diseased patients than inactive patients and controls. Carotid intima-media thickness (IMT) was signifi cantly increased in AS patients than controls. Levels of low-density lipoprotein were signifi cantly higher in AS patients than the controls and in AS patients receiving biologics than those not receiving biologics. In the inactive group, vWF and IMT were signifi cantly increased in patients receiving biologics. vWF correlated positively with BASDI, BASMI, BASFI scores, ESR, and carotid IMT and negatively with high-density lipoprotein.
artery disease by visualization of its functional consequences [10]. It is the most frequently used technique to assess systolic wall motion. Both physical exercise and pharmacological stress can be used. Resting wall motion abnormalities mainly represent infarcted myocardium, whereas those induced by stress refl ect ischemia [11].
Th e aim of the present study was to assess the presence of atherosclerosis in Egyptian patients with AS and its relation to disease activity.

Clinical evaluation
Th e present cross sectional case-control study was carried out on 60 participants: 30 AS patients diagnosed according to the modifi ed New York criteria 1984 [12] and 30 healthy age-matched and sex-matched volunteers selected randomly as a control group.
Th e patients were recruited randomly from the Rheumatology Outpatient Clinic and the Rheumatology In-patient Department of Ain shams university hospital. All participants provided written informed consent to participate, which was approved by the Medical Ethics Committee. Patients with diabetes mellitus, obesity, hypertension, heart failure, manifestations of ischemia (angina, TIA, or claudication pain), and smokers were excluded from the study.
Patients were subjected to the following procedures: full assessment of medical history and a thorough clinical examination including general, systemic [Especially (esp.) blood pressure (BP) measurement and BMI calculation], and musculoskeletal examinations. Assessment of AS disease activity was performed using Bath ankylosing spondylitis disease activity index (BASDAI) [13], BASMI [14], and BASFI [15] scores.

Laboratory evaluation
Patients' venous blood samples (8 ml each) were collected by venipuncture; 5 ml was placed in EDTA for a complete blood count and for the determination of the erythrocyte sedimentation rate. A 3 ml aliquot was placed in a clean tube and serum was separated and kept until used.
Complete blood count: [Beckman Coulter counter T660 Automated Hematology Analyzer (Beckman instrument incorporation, California, USA)]. First hour erythrocyte sedimentation rate was estimated using the Westergren method. CRP was determined by ELISA. Blood urea nitrogen, serum creatinine, serum alanine, and aspartate aminotransferase ( AST and ALT) were measured using the calorimetric method. Lipid profi le [total cholesterol, triglycerides (TG), LDL, HDL] was measured using the enzymatic method. Serum vWF Ag level was assessed by ELISA [16].

Radiological assessment
Plain X ray chest posteroanterior view and common carotid arterial duplex were obtained [17]. A duplex ultrasound system was used to assess the common carotid arteries by a single observer. Longitudinal high-resolution B-mode ultrasound scans were used over both right and left common carotid arteries and were R-synchronized and recorded. Th e offl ine measurements were performed 1 cm proximal to the carotid bulb in the far wall. Sites of carotid plaques were avoided. Th e IMT was defi ned as the distance between the fi rst and the second echogenic lines from the lumen, taking the average of 10 measurements on both sides. Values of IMT were expressed in mm [17].
Cardiovascular assessment included ECG and dobutamine stress echocardiography [18], which was performed only for patients.
Dobutamine stress echocardiography is a valuable diagnostic tool in patients with suspected or known coronary artery disease for the detection of myocardial ischemia and myocardial viability [10]. Two-dimensional echocardiography evaluates global and regional left ventricular function. Left ventricular regional wall motion is analyzed using the 16-segment model recommended by the American Society of Echocardiography [18]. Dobutamine is a sympathomimetic agent with predominant β-receptor stimulation. Its eff ect on the β1 receptor is more pronounced than that on the β2 receptor. Th e eff ects on β1 receptors are minor. Β-receptor stimulation exerts positive inotropic, chronotropic eff ects on the heart [18]. Oxygen and metabolic supplies to the myocardium depend on coronary blood fl ow. Reduced coronary blood fl ow provides myocardial ischemia as a result of oxygen and metabolite deprivation. Under resting conditions, basal coronary artery fl ow is maintained at normal levels until coronary artery stenosis becomes severe; under conditions of stress, coronary blood fl ow normally increases, mediated by the increased demand for oxygen and metabolites. Th e ability of coronary arteries to increase coronary blood fl ow is reduced in signifi cantly stenosed vessels [19].
Th e vital parameters of the patient (heart rate, BP, ECG, oxygen saturation) are monitored throughout the procedure. Th e patient is positioned optimally (usually left lateral decubitus) for proper image acquisition. At baseline, the resting images are acquired (parasternal long axis and short axis, apical two-chamber and four-chamber views), which are digitized and stored. Dobutamine infusion is then initiated at 5 μg/kg/min and increased every 3 min to 10, 20, 30, 40, and 50 μg/kg/min. At the peak dobutamine dose, 0-25 mg atropine is added as a bolus at intervals of 1 min up to a maximum dose of 1 mg until an end point is reached.
Th e end points are as follows: (1) Target heart rate (85% of age -predicted maximal heart rate). (2) Development of new regional wall motion abnormality.

Findings of dobutamine stress echocardiography
In order to interpret stress echocardiography, all 16 left ventricular segments are evaluated using a scoring system. Normokinesia is graded with a score of 1, hypokinesia with 2, akinesia with 3, and dyskinesia with 4. A new wall motion abnormality with an increase in score of more than 1 in more than one segment is considered to be a marker for ischemia [18].
In healthy normotensive adults, increased BP response to exercise may be associated with a higher risk of developing hypertension at rest and increased incidence of hypertensive left ventricular hypertrophy [20].
Th e data collected were coded, tabulated, and analyzed statistically on an IBM computer using Statistical Package for Social Science version 17.0 (SPSS Inc. Released 2008. SPSS Statistics for Windows, Version 17.0. Chicago: SPSS Inc). Quantitative variables were described as mean, SD, and range. Qualitative variables were described as number and percentage. Th e unpaired t-test was used to compare two groups for quantitative variables. Th e χ 2 -test was used to compare qualitative variables between groups. Th e Spearman correlation test was used to rank diff erent variables against each other positively or inversely. P-value 0.05 or less was considered statistically signifi cant, P-value more than 0.05 was considered statistically insignifi cant, and P-value more than 0.001 or less was considered highly statistically signifi cant.

Results
Th irty AS patients were included in the study (they were all men). Th eir mean ± SD age was 34.4 ± 9.3 years. Th irty age-matched and sex-matched healthy volunteers (all were also men) were included as the control group. Th eir mean ± SD age was 27.7 ± 7.1 years. Th ere was no signifi cant diff erence between the groups in sex, age, or the ir BMI.
We then divided the patients according to the BASDAI score into patients with active disease (BASDAI³4), and patients in remission (BASDAI<4). Patients with disease activity were found to have higher ESR (P = 0.006), CRP (P = 0.017) and vWF (P = 0.001) levels than those in remission ( Table 2).
AS patients receiving antitumor necrosis factor (TNF) therapy were found to have signifi cantly higher levels of LDL (P = 0.021) than AS patients not receiving anti-TNF therapy group, but no signifi cant diff erence atherosclerosis [21]. Atherosclerosis has been shown to be increased in chronic infl ammatory diseases including AS [22].
Markers of infl ammation (e.g. CRP), rather than traditional coronary heart disease risk parameters, may better predict the risk for vascular events in autoimmune diseases [2].
In the present study, we found signifi cantly higher levels of ESR and CRP in AS patients compared with healthy controls and in active AS patients than those in remission. In addition, ESR correlated signifi cantly with all the activity scores used in our study (BASMI, BASFI, and BASDAI scores). Similarly, it has been reported that ESR and CRP levels are elevated in ∼75% of AS patients and correlated with disease activity [23].
Impaired endothelial function, the fi rst step in atherosclerosis, may be refl ected by changes in various endothelial biomarkers of hemostasis [22]. Plasma vWf was postulated previously to be a useful marker of endothelial injury in atherosclerosis because it is specifi c for endothelial cells, is stable, may be relevant to the disease process, and is simple to assay [24].
In our study, we found that vWF levels were signifi cantly higher in the AS patient group compared with the controls; in addition, the levels were signifi cantly higher in the active cases compared with those in remission. Our results are in agreement with those of Taylan et al. [22], who found that vWF was higher in the sera of AS patients compared with controls and also with those of a previous Russian study that found that signs of endothelial injury (increased level of circulating endothelial cells and vWF activity) and endothelial dysfunction were found in patients with AS [25].  was found between both groups in other lipid parameters (total cholesterol, TG, HDL), ESR, CRP, vWF, or carotid IMT (Table 4), although statistically signifi cantly higher levels of vWF (P = 0.045) and greater carotid IMT (P = 0.009) were found in patients who were in remission because they were receiving anti-TNF therapy than those in remission but had not received anti-TNF treatment.

Discussion
Growing evidence suggests the role of infl ammation in the pathogenesis of CVD, especially in Correlation between caro tid intima-media thickness (IMT) and von willebrand factor (vWF) leve ls.
Moreover, AS patients were reported to have a high risk for thrombosis because of elevated levels of prothrombogenic factors (fi brinogen, vWF, platelets) and subnormal fi brinolytic blood activity [26].
vWF levels were signifi cantly correlated with ESR, CRP, and all the activity scores used in our study (BASMI, BASFI, and BASDAI scores). Bernardo et al. [8] suggested that infl ammatory cytokines may stimulate the vWF release and inhibit its cleavage, resulting in the accumulation of hyper reactive vWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium.
Plasma vWF levels have been proposed as a risk factor for cardiovascular events [27]. In addition, a correlation was found between vWF with IMT of the common carotid artery in the RA patients [28]. Similarly, a positive correlation was found in our study between levels of vWF with carotid IMT, and patients with HTNR of dobutamine echo were found to have signifi cantly higher levels of vWF than those with normal echo fi ndings. Th ese results suggest a potential linkage between infl ammation, endothelial damage, and the development of atherosclerosis.
In addition to the direct eff ect of infl ammation on endothelial cells, infl ammation can increase CVD through deterioration of the lipid profi le, which is supported by the fi ndings of the study carried out by Burger and Dayer [29], in which a decrease in HDL-C and apolipoprotein A1 levels and increase in TGs and apolipoprotein B levels were observed during an acute-phase response. Also, an association was found between the increase in lipids such as LDL-C and proinfl ammatory cytokines such as C RP, IL-6, and TNFα [30].
In the present study, LDL levels were found to be signifi cantly higher in patients with AS compared with the controls. Also, HDL levels were found to be lower in the patient group, although the diff erence was not statistically signifi cant. Th ese results are in agreement with those of Malesci et al. [4], who found lower levels of HDL and higher levels of LDL in AS patients compared with the controls. Also, lower HDL-C in AS patients was reported by the studies carried out by Divecha et al. [2] and van Halm et al. [21].
Th ere is considerable debate on the eff ect of anti-TNF therapy on the plasma lipids in diff erent infl ammatory disorders. Our data showed signifi cantly higher LDL-C levels in AS patients receiving anti-TNF than those who are not receiving anti-TNF.
Popa et al. [31] found that the plasma concentration of total cholesterol, LDL-C, and the atherogenic index was increased after 1 year of therapy with infl iximab in RA patients and concluded that long-term therapy with infl iximab may lead to a more proatherogenic pattern of plasma lipids.
In contrast, Vis et al. [32] investigated the short-term eff ect of infl iximab on the lipid profi le in RA patients and found that it was associated with a signifi cant increase in both total cholesterol and HDL-C levels, but without a signifi cant eff ect on the atherogenic index. Also, Tam et al. [33] found that after 14 weeks of infl iximab treatment in patients with RA, total cholesterol, HDL-C, LDL-C, TGs, and apolipoprotein B levels all increased signifi cantly from baseline, although the atherogenic index remained unchanged.
Furthermore, no signifi cant changes in plasma lipids were found either after 14 weeks [34] or after 48 weeks of infl iximab therapy [35] in RA patients, and also after 14 weeks of infl iximab therapy in AS patients [36].
Graces et al. [37] reported that it seems to be a class eff ect as they found that in patients with RA or AS treated with anti-TNF blockers, infl iximab treatment increased the total cholesterol and LDL-C levels, but had no eff ect on HDL-C and TGs production, whereas etanercept increased HDL-C signifi cantly but had no eff ect on total cholesterol or LDL-C levels.
In our study, we found that patients with AS showed greater carotid IMT than their matched healthy controls (P = 0.037). Our results are in agreement with those of Mathieu et al. [5], Gonzalez-Juanatey et al. [38], and Bodnár et al. [17] as they reported signifi cantly increased carotid IMT in AS patients compared with healthy controls. However, these results are not in agreement with those of Choe et al. [39] and Sari et al. [40], who found no diff erence in carotid IMT between AS patients and healthy controls, which could be attributed to the younger age group in the former study and the diff erent sex distribution of the study carried out by sari as about half the participants were women and in addition, no diff erence in lipid profi le was found between their patients and controls.
Th ere are confl icting results on the eff ect of anti-TNF therapy on carotid IMT in patients with diff erent infl ammatory disorders. Del Porto et al. [41] observed signifi cant carotid IMT reduction in RA patients after 12 months of receiving anti-TNF blockers and attributed this to their role in reducing infl ammation. However, Gonzalez-Juanatey et al. [38] reported worsening of carotid IMT during 2-3 years of TNFα blocking therapy.
Our study showed no diff erence in carotid IMT between AS patients receiving anti-TNF and those not receiving anti-TNF. However, we found signifi cantly increased carotid IMT in AS patients who were in remission following anti-TNF therapy in comparison with those in remission but not on anti-TNF therapy. Our study may be underpowered by the small number of patients using anti-TNF and that we do not have baseline data for the patients before the use of anti-TNF.
In our study, dobutamine echo showed a HTNR in eight (27%) of the 30 AS patients included in our study and was found to be signifi cantly higher in patients with active disease than those in remission. Similarly, in a study by Yildirir et al. [42], echocardiographic examination of AS patients showed signifi cantly increased incidence of diastolic dysfunction among AS patients, particularly an abnormal relaxation pattern.

Conclusion
We found a higher prevalence of atherosclerosis in AS patients, which was related to disease activity and not disease duration. vWF is a useful marker of atherosclerosis in AS patients and it was also correlated positively with disease activity scores and IMT. Patients with AS receiving anti-TNF blockers may be at a higher risk of developing atheroscleros is.