Abstract
BACKGROUND:
Calcific aortic stenosis (AS) is an atherosclerosis-related process and the most common cause of valve disease requiring surgery.
OBJECTIVE:
To assess the association of inflammatory markers with AS in advanced atherosclerosis.
METHODS:
Consecutive patients with coronary artery disease (CAD) associated with AS were prospectively identified (mean transvalvular aortic gradient of 30 mmHg or greater). Subjects with aortic sclerosis (mean transvalvular aortic gradient of 10 mmHg or less) served as controls. All patients underwent clinical evaluation, echocardiography and coronary angiography.
RESULTS:
One hundred twenty-two patients with AS (85 men) and 101 with aortic sclerosis (76 men) of similar CAD severity were enrolled. The AS patients were older (mean [± SD] 71±7 years versus 66±7 years; P<0.001), had higher soluble vascular adhesion molecule-1 (s-VCAM-1) levels (1533±650 μg/L versus 1157±507 μg/L; P<0.001), but lower soluble intercellular adhesion molecule-1 (s-ICAM-1) (254±81 μg/L versus 293±84 μg/L; P<0.01) and soluble E-selectin (53±28 μg/L versus 62±29 μg/L; P<0.05) levels. The two groups did not differ with respect to C-reactive protein level (3±2.9 mg/L versus 3.4±2.6 mg/L; P not significant). Higher s-VCAM-1 (OR 1.09, 95% CI 1.04 to 1.14; P<0.001) and lower s-ICAM-1 (OR 0.82, 95% CI 0.72 to 0.94; P<0.001) levels were associated with AS after adjustment for age.
CONCLUSION:
Increased s-VCAM-1 levels were associated with calcific AS in patients with significant CAD.
Keywords: Adhesion molecules, Aortic stenosis, Calcification, VCAM-1
Calcific aortic stenosis (AS) is an atherosclerosis-related process, prevalent in 2% to 4% of the elderly population older than 65 years of age (1,2), and the most common cause of valve disease requiring surgery – currently, the only long-term effective treatment for AS.
C-reactive protein (CRP) and soluble adhesion molecule levels predict coronary risk in patients with or without coronary artery disease (CAD) (3,4), and are also associated with severe AS in patients without CAD (5,6). The aim of the present study was to assess the association of inflammatory markers with moderate to severe calcific AS in patients with CAD. Understanding the role of biomarkers in AS could provide a basis for novel medical therapeutic targets to slow AS progression.
METHODS
Patient population
Consecutive patients admitted to hospital due to a history of dyspnea, chest pain, fatigue or syncope, who fulfilled the inclusion criteria of angiographically significant CAD, plus either AS (mean transvalvular aortic gradient of 30 mmHg or higher) or nonobstructive aortic sclerosis (mean transvalvular aortic gradient of 10 mmHg or less) diagnosed by echocardiography, were prospectively enrolled. Excluded were patients with acute coronary syndrome within the three months before admission, rheumatic heart disease (defined by fusion of commissures between the valve cusps plus rheumatic mitral valve disease), prosthetic valves, congenital heart disease except for bicuspid aortic valve, moderate to severe aortic regurgitation (greater than 2/4), Marfan syndrome, infectious endocarditis, hypertrophic obstructive cardiomyopathy, known primary hyperparathyroidism, severe heart failure (New York Heart Association class IV), end-stage renal disease requiring dialysis, and those with other systemic disease or malignancy severely limiting prognosis. The definition of the study population and baseline data have been published (7). All patients gave their written, informed consent to participate in the study. The study protocol complied with the Declaration of Helsinki. It was approved by the local Scientific and Ethical Committee, and was registered with the National Institutes of Health and the United States Food and Drug Administration (www.ClinicalTrials.gov [registration number NCT00375336]).
Clinical evaluation
All patients underwent a detailed history of cardiovascular risk factors including medication. At physical examination, blood pressure, waist circumference and body mass index were assessed.
Laboratory parameters
Blood samples were drawn between 06:00 and 07:00, after a 12 h fast. The serum or plasma was separated within 30 min to 60 min of collection. Sample aliquots were frozen at 80°C and kept on site without freeze-thaw cycles.
After study completion, the following analytes were evaluated in one core laboratory: soluble (s) intracellular adhesion molecule (sICAM)-1, s-vascular adhesion molecule (sVCAM)-1 and serum s-E-selectin levels by ELISA (BioSource Europe SA, Belgium). Reproducibility of test results were in accordance with the values described by the manufacturer, with variation coefficients ranging from 3% to 6%. Plasma high-sensitivity CRP levels were assessed by the immunoturbidimetric method (Orion Diagnostica OY, Finland).
Echocardiography
Standard echocardiographic examination was performed in all patients (System FiVE, Vivid Five, GE Vingmed, Norway; iE33, Philips, USA). The data were stored on videotapes and reviewed by two cardiologists blinded to the other laboratory results. Discrepancies in evaluation were resolved by consensus among the observers. Aortic sclerosis was defined as higher echogenicity or thickening of valvular leaflets, a peak aortic flow velocity of 2.5 m/s or less, or a mean transaortic gradient of less than 10 mmHg.
Mean and peak transaortic gradients were calculated by the modified Bernoulli equation at the time of examination. Aortic regurgitation was evaluated according to the American Society of Echocardiography criteria (8). The left ventricular dimensions were measured from the two-dimensional, long-axis parasternal view using the leading edge to leading edge method. Ventricular ejection fraction was assessed by the biplane modified Simpson’s method.
Coronary angiography
Coronary angiography was performed with a femoral approach in all patients using 4 Fr or 5 Fr instruments. The coronary artery tree was divided into three compartments (left anterior descending artery, circumflex artery and right coronary artery) to diagnose three-vessel or left main CAD. The reference diameter of the artery within the assessed lesion had to be greater than 1.5 mm to label it as a ‘diseased artery’. In cases of left-dominant or balanced-dominant circulation, the assessment was modified according to actual coronary morphology. Significant CAD was defined as more than 50% diameter stenosis. Radiation exposure per patient per examination was 10 mSv. All examinations were evaluated by an interventional cardiologist blinded to the other study results. Quantitative analysis was used only in borderline cases.
Statistics
The categorical variables are described as n (%), continuous variables are shown as mean ± SD, or median (interquartile range) in case of non-Gaussian distribution. The categorical variables were compared by the χ2 test, the continuous variables by the t test or by the Mann Whitney U test, as appropriate.
First, the clinical, echocardiographic, angiographic and laboratory characteristics of the patients with AS and controls were compared, followed by an assessment of the association of s-ICAM-1, s-E-selectin and s-VCAM-1 levels with AS using multivariate logistic regression. Statististical data were evaluated using the Statgraphics Centurion software package, version XV.2 (Statpoint Inc, USA). P<0.05 was considered to be statistically significant.
RESULTS
A total of 223 patients with significant CAD, ranging in age from 50 to 87 years, were enrolled. Of these, 122 had AS and 101 with aortic sclerosis served as controls. The baseline data and comparisons are presented in Table 1. Dyslipidemia was treated by statins in only 60% of patients with AS and in 74% of patients with aortic sclerosis. Comparison of soluble adhesion molecule levels in patients with AS and aortic sclerosis is shown in Table 2. After adjustment for age and statin treatment, only s-VCAM-1 levels were positively associated with AS.
TABLE 1.
Comparison of baseline patient characteristics and selected laboratory characteristics
| Characteristic |
Aortic |
P | |
|---|---|---|---|
| Stenosis (n=122) | Sclerosis (n=101) | ||
| Age, years | 71±7 | 66±7 | 0.001 |
| Women | 37 (30) | 25 (25) | 0.30 |
| Hypertension | 97 (80) | 85 (85) | 0.36 |
| Diabetes mellitus | 43 (35) | 41 (41) | 0.34 |
| Metabolic syndrome | 15 (12) | 22 (22) | 0.020 |
| Current or past smokers | 70 (57) | 64 (63) | 0.18 |
| Dyslipidemia | 104 (85) | 93 (92) | 0.08 |
| Body mass index, kg/m2 | 29±4 | 29±3 | 0.94 |
| Waist circumference, cm | 101±17 | 103±9 | 0.19 |
| Systolic BP, mmHg | 142±17 | 136±17 | 0.007 |
| Diastolic BP, mmHg | 80±10 | 80±9 | 0.70 |
| Laboratory profile | |||
| Total cholesterol, mmol/L | 4.9±1.0 | 5.0±1.2 | 0.33 |
| HDL cholesterol, mmol/L | 1.21±0.35 | 1.24±0.39 | 0.49 |
| LDL cholesterol, mmol/L | 3.01±0.98 | 3.07±0.94 | 0.85 |
| Lipoprotein (a), g/L | 0.5±0.6 | 0.6±0.7 | 0.041 |
| Apolipoprotein AI, g/L | 1.3±0.3 | 1.2±0.3 | 0.06 |
| Apolipoprotein B, g/L | 0.9±0.2 | 0.9±0.2 | 0.78 |
| Creatinine, μmol/L | 93 (82–105) | 96 (85–107) | 0.19 |
| Urea, mmol/L | 7.4 (5.6–8.5) | 5.5 (4.6–6.6) | <0.001 |
| Uric acid, μmol/L | 365±99 | 316±93 | <0.001 |
| Homocysteine, μmol/L | 13.9±5 | 14.2±3.9 | 0.19 |
| Leukocytes, ×109/L | 7.5±1.9 | 7.1±1.7 | 0.08 |
| Sedimentation rate, mm/h | 20 (12–34) | 16 (11–25) | 0.14 |
| hs C-reactive protein (mg/L) | 1.94 (0.79–5.04) | 2.93 (1.32–5.34) | 0.25 |
| Echocardiography | |||
| AVAI cm2/m2 | 0.43±0.1 | – | |
| Maximal gradient, mmHg | 73±22 | – | |
| Mean gradient, mmHg | 47±15 | – | |
| AR, degree 0–1 | 115 (95) | 96 (95) | 1.00 |
| LV end diastolic diameter, mm | 49±7 | 50±8 | 0.21 |
| LV mass index, g/m2 | 150±38 | 116±33 | <0.001 |
| LV ejection fraction, % | 57±12 | 55±11 | 0.35 |
| E/Ealat | 11.2±5 | 8.5±3 | <0.001 |
| Bicuspid aortic valve | 9 (7) | 1 (1) | 0.020 |
| Coronary angiography | |||
| One vessel | 40 (33) | 28 (28) | 0.34 |
| ≥2 vessels | 61 (50) | 67 (66) | 0.013 |
| Left main coronary artery | 21 (17) | 6 (6) | 0.017 |
Continuous data are presented as mean ± SD. Categorical variables are presented as n (%). Variables with non-Gaussian distribution are presented as median (interquartile range). AR Aortic regurgitation; AVAI Aortic valve area index; BP Blood pressure; E Early diastolic transmitral flow velocity by pulsed wave Doppler imaging; Ealat Early lateral annulus velocity by tissue Doppler imaging; HDL High-density lipoprotein; hs High sensitivity; LDL Low-density lipoprotein; LV Left ventricular
TABLE 2.
Association of soluble adhesion molecule serum levels with calcific aortic stenosis
| Soluble adhesion molecule |
Aortic |
OR | 95% CI | P* | |
|---|---|---|---|---|---|
| Stenosis (n=122) | Sclerosis (n=101) | ||||
| Soluble E-selectin, μg/L | 49 (33–68) | 58 (38–73) | 0.99 | 0.98–1.01 | 0.28 |
| Soluble intercellular adhesion molecule-1, μg/L | 243 (202–284) | 276 (238–337) 0.82 | 0.82 | 0.72–0.94 | <0.001 |
| Soluble vascular adhesion molecule-1, μg/L | 1348 (1102–1833) | 1066 (820–1419) | 1.09 | 1.04–1.14 | <0.001 |
Variables with non-Gaussian distribution are presented as median (interquartile range).
P-values are adjusted for age and statin treatment
DISCUSSION
Increased s-VCAM-1 levels were independently associated with calcific AS in a large sample of prospectively identified patients with significant CAD and sclerosis or stenosis of the aortic valve. Our results suggest s-VCAM-1 levels could be a potential marker of calcific AS. Speculatively, it may reflect the process of neoangiogenesis in the severely calcified valve; however, further studies are required to confirm whether it predicts progression of valve calcification. To our knowledge, the present study is the first to assess soluble adhesion molecule levels in patients with calcific AS in the context of advanced atherosclerosis.
Studies regarding valvular expression showed VCAM-1 and ICAM-1 to be predominantly expressed in the endothelial cells of neovessels in the majority of excised, severely stenotic, calcific tricuspid aortic valves (9). While the normal valve is an avascular tissue supplied with nutrients by diffusion (10), neovascularization is necessary for the initiation and progression of calcification. Neovascular density was been found to be most prominent in moderately stenotic, as opposed to mildly or severely stenotic valves (11), but neoangiogenesis always accompanied areas of ossification in severe AS (12).
Elevated levels of soluble adhesion molecules have been reported in a variety of inflammatory disorders. In patients with AS, one previous study (13) reported higher levels of s-E-selectin, s-VCAM-1 and s-ICAM-1 compared with controls with an intact valve. Ghaisas et al (6) found higher s-E-selectin but not adhesion molecule levels in 16 patients with severe AS versus healthy controls, with levels strongly correlated with valvular expression. However, patients with significant CAD were excluded from those studies. In our study, s-VCAM-1, but not s-E-selectin or s-ICAM-1 levels were higher in patients with calcific AS than in those with mildly diseased, sclerotic valves. Higher CRP levels were found in patients with severe AS (14) or mitral calcification (15); however, others (16) did not demonstrate an association between CRP and valve calcification or its progression, which is in accordance with our study. Thus, in patients with advanced atherosclerosis, the s-VCAM-1 level could signal incident AS, speculatively reflecting neoangiogenesis activity in the diseased aortic valve.
We are aware that the cross-sectional nature of the present study cannot assess cause-effect relationships. Furthermore, it is difficult to associate rapidly changing parameters with chronic processes such as calcification. However, in the design, attention was given to this aspect by excluding patients with acute coronary syndrome diagnosed within the past three months.
CONCLUSION
In patients with significant CAD, higher s-VCAM-1 levels were associated with the moderate to severe calcific AS. Speculatively, s-VCAM-1 could be a marker of increased neovascularization activity, a proposed important pathogenetic factor, even in the late stage of calcific AS in these patients, which could help define a potential new treatment target. Further follow-up is required to confirm whether s-VCAM-1 levels predict the rate of AS progression.
Acknowledgments
This study was supported by grant NR/8306-5 from the Internal Grant Agency, Ministry of Health, Czech Republic (KL, GS, RC, JR) and by the Cardiovascular Research Project of the Charles University of Prague, Czech Republic (Nr. MSM0021620817) entitled ‘The invasive approach to myocardial salvage and regeneration’ (GS, KL, RR), and registered with the National Institutes of Health and the Unites States Food and Drug Administration No. NCT00375336 (www.ClinicalTrials.gov).
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