Platelet-Activating Factor Acetylhydrolase Activity Indicates Angiographic Coronary Artery Disease Independently of Systemic Inflammation an
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循环学杂志 2005年第3期
the Department of Medicine, University of Freiburg, Freiburg, Germany (K.W., M.M.H., A.B.G., M.N.)
Cardiology Group Frankfurt-Sachsenhausen, Frankfurt, Germany (B.R.W.)
Clinical Institute of Medical and Chemical Laboratory Diagnostics, University of Graz, Graz, Austria (H.S., W.M.)
Division of Endocrinology and Diabetes, Department of Medicine, University of Ulm, Ulm, Germany (B.O.B.).
Abstract
Background— Platelet-activating factor acetylhydrolase (PAF-AH), also denoted as lipoprotein-associated phospholipase A2, is a lipoprotein-bound enzyme that is possibly involved in inflammation and atherosclerosis. This study investigates the relationship of PAF-AH activity to angiographic coronary artery disease (CAD), the use of cardiovascular drugs, and other established risk factors.
Methods and Results— PAF-AH activity, lipoproteins, sensitive C-reactive protein (sCRP), fibrinogen, serum amyloid A, and white blood cell count were determined in 2454 subjects with angiographically confirmed CAD and in 694 control subjects. PAF-AH activity was highly correlated with LDL cholesterol (r=0.517), apolipoprotein B (r=0.644), and non-HDL cholesterol (r=0.648) but not with sCRP or fibrinogen. PAF-AH activity was lower in women than in men and was affected by the intake of lipid-lowering drugs (–12%; P<0.001), aspirin (–6%; P<0.001), ;-blockers (–6%; P<0.001), and digitalis (+7%; P<0.001). Unlike sCRP, fibrinogen, and serum amyloid A, PAF-AH activity was not elevated in unstable angina, non–ST-elevation myocardial infarction, or ST-elevation myocardial infarction. When nonusers of lipid-lowering drugs were examined, PAF-AH activity was associated with the severity of CAD and the number of coronary vessels with significant stenoses. In individuals not taking lipid-lowering drugs and after adjustment for use of aspirin, ;-blocker, and digitalis, the odds ratio for CAD associated with increasing PAF-AH activity was 1.39 (95% CI 1.26 to 1.54, P<0.001), a finding that was robust against further adjustments.
Conclusions— PAF-AH activity is not an indicator of the systemic inflammation that accompanies acute coronary syndromes. PAF-AH activity is affected by a number of cardiovascular drugs; however, after such medication use was accounted for, PAF-AH activity was associated with angiographic CAD, complementary to sCRP and independently of established risk factors such as LDL cholesterol.
Key Words: lipoproteins ; C-reactive protein ; cholesterol
Introduction
Platelet-activating factor (PAF) is a potent lipid mediator that may be involved in inflammatory diseases1 and atherogenesis.2 In plasma, PAF is hydrolyzed and inactivated by PAF-acetylhydrolase (PAF-AH; EC 3.1.1.47), a Ca2+-independent phospholipase A2.3 Plasma PAF-AH is complexed to lipoproteins in vivo; hence, it is also denoted lipoprotein-associated phospholipase A2 (Lp-PLA2).4
Several reports link PAF-AH to atherogenesis and increased risk of coronary artery disease (CAD); however, the role of PAF-AH in inflammatory and atherosclerotic diseases remains to be established. PAF-AH may represent a potent antiinflammatory and antiatherogenic enzyme because it degrades PAF and proinflammatory oxidized phospholipids.5 Conversely, PAF-AH may also generate bioactive oxidized free fatty acids4 and lysophosphatidylcholine.6 In addition, phospholipase A2/PAF-AH activity may liberate arachidonic acid, a precursor of eicosanoids including prostaglandins and leukotrienes.7
Several studies show that PAF-AH may indicate CAD risk; however, in the Women’s Health Study, the predictive value of PAF-AH mass was not independent of lipoprotein risk factors, particularly LDL cholesterol (LDL-C).8 In line with this, Ballantyne et al9 reported that in the Atherosclerosis Risk in Communities (ARIC) study, PAF-AH mass was associated with CAD only in individuals presenting with LDL-C <130 mg/dL. In contrast, in a study by Caslake et al,10 PAF-AH mass appeared to be an independent predictor of angiographically proven CAD, even after adjustment for LDL-C and other risk factors, and in the West of Scotland Coronary Prevention Study,11 PAF-AH mass was a strong risk factor for incident CAD, independent of markers of inflammation and LDL-C. Similarly, in an investigation by Blankenberg et al,12 PAF-AH activity was positively associated with angiographic CAD after adjustment for LDL-C, HDL cholesterol (HDL-C), and triglycerides. In a recent study, risk prediction by PAF-AH mass was independent of total cholesterol/HDL-C ratio.13 However, it remains controversial whether PAF-AH will be just another inflammatory marker, like sensitive C-reactive protein (sCRP), or whether PAF-AH will be predictive of CAD risk independently of other risk factors. Therefore, this cross-sectional study set out to determine the relationship of PAF-AH activity with CAD in relationship to established inflammatory markers and other well-recognized risk factors for CAD in 3148 patients who underwent coronary angiography.
Methods
Study Design and Participants
We studied 3148 white patients hospitalized for coronary angiography between June 1997 and January 2000.14 Informed written consent was obtained from each of the participants, and the study was approved by the ethics review committee at the Landes;rztekammer Rheinland-Pfalz.
Clinically relevant CAD was defined as the occurrence of a visible luminal narrowing (20% stenosis) in at least 1 of 15 coronary segments according to a classification of the American Heart Association.15 Angiograms were analyzed by visual analysis. To minimize the interobserver variability of visual analysis, all angiograms were assessed in a standardized fashion with enlarged projection. Furthermore, visual analysis was limited to 5 experienced angiographers. The final catheterization report was released only after approval by the senior cardiologist.14
Two thousand four hundred fifty-four patients were considered CAD positive, and 694 were CAD negative (controls). One thousand one hundred nineteen individuals had a history of 6 months or more of proven CAD. Severity of CAD was quantified with the Friesinger score16 and the number of major vessels with stenosis 50%. CAD patients were further stratified into individuals with stable CAD (n=1523) and patients with acute coronary syndrome (n=931; troponin T–negative unstable angina [n=627], non–ST-elevation myocardial infarction [NSTEMI; troponin T 0.1 μg/L; n=114], or ST-elevation myocardial infarction [STEMI; troponin T 0.1 μg/L; n=190]).
Laboratory Procedures
Blood sampling was done early in the morning before cardiac catheterization, after subjects had fasted. Blood samples were assayed for blood cell count and biochemistry as described previously.14 Measurement of PAF-AH activity was performed by the Azwell Auto PAF-AH kit (Azwell Inc)17 on a Hitachi 912 autoanalyzer: PAF-AH hydrolyzes the sn-2 position of the substrate [1-myristoyl-2-(4-nitrophenyl succinyl) phosphatidylcholine], producing 4-nitrophenyl succinate, which is immediately degraded to 4-nitrophenol and subsequently measured spectrophotometrically. LDL radius was estimated from free cholesterol, cholesterol esters, triglycerides, phospholipids, and apolipoprotein (apo) B18 after a combined ultracentrifugation-precipitation method (;-quantification).19 With this method, the LDL fraction includes particles with densities of 1.006 through 1.063 kg/L and thus comprises intermediate-density lipoproteins (IDL, 1.006 to 1.019 kg/L).20
Statistical Analysis
Correlation coefficients were calculated according to Spearman. Clinical and biological variables were compared by different methods depending on the nature of variables. Continuous variables were compared by univariate ANOVA between CAD patients and controls by adjustment for gender and drugs as indicated. Categorical variables were examined by logistic regression analysis, also with adjustment for gender.
The effect of medication on PAF-AH activity was assessed by univariate ANOVA. Marginal means were adjusted for age, gender, and status of CAD (CAD negative, CAD positive, unstable angina, NSTEMI, or STEMI) and for medication as indicated. Because lipid-lowering drugs (LLDs) had the highest impact on PAF-AH activity, we established quartile ranges of PAF-AH activity according to the values in the control subjects without CAD who were not using LLDs. We obtained risk ratios by comparing the prevalences of clinically relevant CAD in these quartiles by logistic regression analysis with adjustments as indicated. P<0.05 was considered statistically significant.
Results
Characteristics of Patients and Controls
PAF-AH activity, lipoproteins, sCRP, fibrinogen, white blood cell counts, and serum amyloid A (SAA) levels were available from 3148 individuals. Patients with stable CAD were significantly older than controls. Current or past smoking, type 2 diabetes mellitus, and hypertension were more prevalent in stable CAD patients than in control subjects. Stable CAD patients had higher systolic and diastolic blood pressure, higher fasting glucose, higher LDL-C, higher triglycerides, and lower HDL-C. Stable CAD patients were more often treated with ;-blockers, ACE inhibitors, calcium channel blockers, antiplatelet agents, and LLDs. Body mass index did not differ significantly between CAD patients and controls (Table 1).
PAF-AH, Lipoproteins, and Markers of Systemic Inflammation
PAF-AH activity was strongly correlated with total cholesterol, LDL-C, non-HDL-C, and apoB, with correlation coefficients that exceeded 0.5. Triglycerides and VLDL cholesterol were also positively correlated with PAF-AH activity. HDL-C, apoA-I, and LDL radius were weakly and inversely correlated with PAF-AH activity (Table 2). sCRP, fibrinogen, and lipoprotein(a) [Lp(a)] did not correlate with PAF-AH activity. Weak positive and negative associations were found between PAF-AH activity and white blood cell count and SAA, respectively. Analyses in subjects not using LLDs or in control subjects not using LLDs produced similar results (Table 2).
Activity of PAF-AH and Cardiovascular Medication
PAF-AH activity in male and female control subjects not using LLDs was 506±107 and 439±98 U/L (P<0.001 by ANOVA), respectively (data not shown), and males had higher PAF-AH activity than females in the entire study population (Figure, A). Both men and women taking LLDs (of whom 97.4% were taking statins, 2.4% fibrates, and 0.2% resins) had PAF-AH activity 12% lower than nonusers of LLDs (Table 3). Furthermore, the use of aspirin or other antiplatelet agents and ;-blockers decreased PAF-AH activity by 6%, respectively, whereas digitalis increased PAF-AH activity by 7% (Table 3). Use of AT1 receptor antagonists had a slightly decreasing effect, whereas ACE inhibitors (Table 3), calcium channel blockers, and antidiabetic drugs had no effect on PAF-AH activity (data not shown).
Estimated marginal means (±95% CI) of PAF-AH activity and inflammatory markers by absence of angiographic CAD (CAD–), stable CAD (CAD+), troponin T–negative unstable angina (UA), NSTEMI, and STEMI in men (squares) and women (circles). Data are adjusted for non-HDL cholesterol, LLDs, aspirin, ;-blockers, and digitalis. A, PAF-AH activity; B, C-reactive protein; C, serum amyloid A; and D, fibrinogen. Asterisks indicate statistical significance (P<0.05) of post hoc comparisons between respective patient group and individuals without CAD (CAD-). PAF-AH activity was significantly lower in females (P<0.001 by ANOVA).
Because LLDs had the most pronounced effect on PAF-AH activity, we investigated only those patients not receiving LLDs (n=1630). In these patients, PAF-AH activity was still significantly influenced by aspirin or other antiplatelet agents, ;-blockers, and digitalis after adjustment for other potentially confounding medication (Table 3).
PAF-AH in Stable CAD, Unstable Angina, NSTEMI, and STEMI
PAF-AH activity (adjusted for non-HDL-C, LLDs, aspirin, ;-blockers, and digitalis) was increased if CAD was present (Figure, A). However, in contrast to sCRP, SAA, and fibrinogen (Figure, B through D), PAF-AH activity showed no consistent association with the severity of myocardial ischemia (Figure, A).
PAF-AH in Angiographic CAD
We examined relative risks of angiographic CAD conferred by increasing quartiles of PAF-AH activity (Table 4). Because PAF-AH activity was not increased in unstable angina, NSTEMI, or STEMI, we included all CAD patients in this evaluation. The crude ORs of angiographic CAD with increasing quartiles of PAF-AH activity were not significantly different from 1.
Of the medications investigated, LLDs had the most pronounced impact on PAF-AH activity. Therefore, we restricted our further analysis to those individuals not taking LLDs. The characteristics of these patients did not differ from those of the entire study population (data not shown).
In the absence of LLDs, PAF-AH activity correlated with the severity of CAD (Friesinger score and the number of vessels with stenosis >50%; Table 2). In addition, the OR associated with increasing quartiles of PAF-AH activity was 1.31 (Table 4). Because aspirin or other antiplatelet agents, ;-blockers, and digitalis independently affected PAF-AH activity (Table 3), we adjusted for these drugs, which resulted in an OR with increasing quartiles of 1.39, the highest quartile having an OR of 2.67 compared with the lowest quartile (Table 4). This adjustment for drugs was done for all further analysis. If, in addition, LDL-C, HDL-C, log-normalized triglycerides, log-normalized Lp(a), and LDL radius were included, the OR was 1.26. If gender, age, body mass index, smoking, diabetes, hypertension, sCRP, fibrinogen, white blood cell count, SAA, and LDL-C were added, the OR across PAF-AH activity quartiles was 1.25 (Table 4). If we adjusted in the same way but without LDL-C, the OR estimates in patients with LDL-C 130 mg/dL or >130 mg/dL were 1.197 (95% CI 1.032 to 1.388, P=0.017) and 1.293 (95% CI 1.041 to 1.605, P=0.020), respectively (data not shown).
There were only 181 controls and 151 CAD patients using neither LLDs, aspirin or other antiplatelet agents, ;-blockers, or digitalis. In this small subgroup not taking any confounding drugs, the OR associated with increasing quartiles of PAF-AH activity was 1.65 (95% CI 1.23 to 2.11, P<0.001; data not shown) after adjustment for age, gender, body mass index, smoking, hypertension, and type 2 diabetes mellitus.
Discussion
The present study provides 3 major observations: First, PAF-AH activity is not an indicator of systemic inflammation accompanying myocardial ischemia. Second, not only LLDs but also other medications such as aspirin, ;-blockers, and digitalis may influence PAF-AH activity. Third, elevated PAF-AH activity indicates increased risk of CAD independently of other established risk factors, especially LDL-C.
PAF-AH activity was significantly lower in women, a finding that is in agreement with findings in 3106 Japanese healthy subjects21 and may, in general, be related to the lower cardiovascular risk found in females. In the present study, LLDs, aspirin, ;-blockers, and digitalis affected PAF-AH activity independently, whereas LLDs had the most pronounced effect. Blankenberg and colleagues12 previously reported a PAF-AH activity–decreasing effect of ACE inhibitors; however, this was not confirmed in the present study. Given the close relationship between PAF-AH activity and apoB-containing lipoproteins (Table 2), it is also not surprising that PAF-AH activity was lower in users than in nonusers of LLDs. In fact, in a placebo-controlled trial, it was recently shown that fluvastatin lowered PAF-AH activity in patients with type 2 diabetes mellitus, the decrease of PAF-AH activity being associated with the decrease of apoB in dLDL.22 However, the putative mechanisms by which aspirin, ;-blockers, and digitalis influence PAF-AH activity are not clear at present.
PAF-AH activity did not correlate with any of the inflammatory markers examined (Table 2). Furthermore, unlike C-reactive protein, SAA, or fibrinogen, it did not respond to increasing clinical instability of CAD (Figure). It is therefore unlikely that PAF-AH behaves as an acute phase marker or that it plays a major role as a trigger of systemic inflammation associated with atherosclerosis. However, PAF-AH is diminished in septic conditions and may be associated with multiorgan failure.23 Expression of PAF-AH is inhibited by interferon- and lipopolysaccharides, whereas other cytokines, including interferon-, interleukin (IL)-1, IL-6, and tumor necrosis factor- had no effect.24 In moderate systemic inflammation, when the increase of C-reactive protein is mainly mediated by tumor necrosis factor-, IL-1 , and IL-6, inhibition of PAF-AH gene expression may thus not occur. Therefore, the finding that PAF-AH activity is not elevated in the acute coronary syndrome appears biologically plausible.
PAF-AH activity was strongly correlated with plasma lipoproteins. The strongest correlations were found with apoB and non-HDL-C (Table 2). Both apoB and non-HDL-C are associated with increased CAD risk.25 Total apoB concentration is a crude marker of the number of LDL particles and the number of triglyceride-rich particles, including VLDL and IDL.26 Non-HDL-C was suggested to be the best surrogate measure for apoB27 and was recently introduced as a secondary target of therapy.28 However, PAF-AH activity may be a good estimate for atherogenic lipoproteins as well: 80% of PAF-AH activity is associated with LDL and another 7% with VLDL.10 Thus, almost 90% of PAF-AH activity resides on VLDL and LDL, with a preference to atherogenic dense LDL (dLDL).22,29
The strong reduction in PAF-AH activity by LLDs may explain why there was no association between PAF-AH activity and angiographic CAD when the use of LLDs was disregarded; however, when we restricted the analysis to individuals not using LLDs, PAF-AH activity was associated with the severity of CAD, and there was an OR for CAD of 1.31 with each increasing quartile of PAF-AH activity. The OR increased to 1.39 if the use of aspirin or other antiplatelet agents, ;-blockers, and digitalis was taken into account (Table 4).
In the Women’s Health Study,8 the predictive value of PAF-AH mass for CAD was not independent of LDL-C. In line with this, Ballantyne et al9 reported that in the ARIC study, PAF-AH mass was associated with CAD only in individuals with LDL-C <130 mg/dL. In the present study, the OR of PAF-AH activity remained significant after adjustment for major lipoprotein risk factors, including LDL-C, HDL-C, triglycerides, Lp(a), and estimated LDL radius (Table 4). Both in subjects with LDL-C 130 and in those with LDL-C >130 mg/dL, PAF-AH activity was significantly associated with CAD. Thus, we could not confirm that the association between PAF-AH activity and CAD disappeared above an LDL-C threshold of 130 mg/dL. As we have shown previously, PAF-AH activity correlates well with apoB in dLDL but only weakly with the calculated LDL radius.22 Furthermore, the LDL radius in the present study was estimated from the density range 1.006 to 1.063 kg/L, thus comprising IDL as well. Although PAF-AH was shown to reside on Lp(a),30 it remains controversial how strongly PAF-AH activity is associated with Lp(a); recently, it was shown that the fluvastatin-induced decrease of PAF-AH activity in patients with type 2 diabetes was independent of Lp(a).22
One might speculate how PAF-AH mediates increased CAD risk independently of other risk factors. PAF-AH may liberate arachidonic acid, a precursor of proinflammatory eicosanoids including prostaglandins and leukotrienes,7 within the atherosclerotic plaque. PAF-AH resides on dLDL,22,29 and LDL particles are an important source for the essential compound arachidonic acid.31 Therefore, we have suggested that dLDL may contribute to atherogenesis by functioning as a vector that provides both the essential substrate and the crucial enzyme activity necessary for prostaglandin synthesis.22 This concept would acknowledge PAF-AH as having an active role in atherogenesis rather than being a mere risk marker. Therefore, PAF-AH may provide a new target for the treatment of cardiovascular disease, and consequently, specific inhibitors of PAF-AH are currently under investigation.32
We exclusively enrolled individuals with clinically indicated coronary angiography. The referral bias associated with this design may be viewed as a limitation of the present study; however, this approach may also be a strength of this investigation. The prevalence of clinically asymptomatic coronary atherosclerosis has been reported to be very high at or above 50 years of age.33 Hence, angiography-based recruitment of control subjects prevents individuals with significant yet clinically inapparent CAD being inadvertently allocated to the control group. Furthermore, control subjects in the present study may be representative of a population-based control group, because the major cardiovascular risk factors occur at similar frequencies in these controls compared with the general population; the prevalence of hypertension was close to that found in a random probability sample from Germany.34 Prima facie, diabetes mellitus appears 2 to 3 times more frequent than in the German population.35 This is most likely because we did not rely on self-reports. Rather, we measured fasting glucose and performed an oral glucose challenge in individuals not previously known to have type 2 diabetes mellitus. On the basis of clinical history or fasting glucose measurements, the National Health and Nutrition Examination Surveys 1999 to 2000 reports prevalences of type 2 diabetes of 9.2% and 19.3% in adults 40 to 59 years old or more than 60 years old, respectively.36 In the present study, 12.1% of the control subjects had diabetes mellitus according to this criterion, whereas another 5.6% were detected by elevated postchallenge glucose only.
Acknowledgments
The technical assistance of Isolde Friedrich, Gabi Herr, Silvia Beheim, Ursula Discher, and Brigitte Haas is gratefully acknowledged. We thank the LURIC study team involved in patient recruitment and sample and data handling and the laboratory staff at the Ludwigshafen General Hospital and the Universities of Freiburg and Ulm, Germany.
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Abstract
Background— Platelet-activating factor acetylhydrolase (PAF-AH), also denoted as lipoprotein-associated phospholipase A2, is a lipoprotein-bound enzyme that is possibly involved in inflammation and atherosclerosis. This study investigates the relationship of PAF-AH activity to angiographic coronary artery disease (CAD), the use of cardiovascular drugs, and other established risk factors.
Methods and Results— PAF-AH activity, lipoproteins, sensitive C-reactive protein (sCRP), fibrinogen, serum amyloid A, and white blood cell count were determined in 2454 subjects with angiographically confirmed CAD and in 694 control subjects. PAF-AH activity was highly correlated with LDL cholesterol (r=0.517), apolipoprotein B (r=0.644), and non-HDL cholesterol (r=0.648) but not with sCRP or fibrinogen. PAF-AH activity was lower in women than in men and was affected by the intake of lipid-lowering drugs (–12%; P<0.001), aspirin (–6%; P<0.001), ;-blockers (–6%; P<0.001), and digitalis (+7%; P<0.001). Unlike sCRP, fibrinogen, and serum amyloid A, PAF-AH activity was not elevated in unstable angina, non–ST-elevation myocardial infarction, or ST-elevation myocardial infarction. When nonusers of lipid-lowering drugs were examined, PAF-AH activity was associated with the severity of CAD and the number of coronary vessels with significant stenoses. In individuals not taking lipid-lowering drugs and after adjustment for use of aspirin, ;-blocker, and digitalis, the odds ratio for CAD associated with increasing PAF-AH activity was 1.39 (95% CI 1.26 to 1.54, P<0.001), a finding that was robust against further adjustments.
Conclusions— PAF-AH activity is not an indicator of the systemic inflammation that accompanies acute coronary syndromes. PAF-AH activity is affected by a number of cardiovascular drugs; however, after such medication use was accounted for, PAF-AH activity was associated with angiographic CAD, complementary to sCRP and independently of established risk factors such as LDL cholesterol.
Key Words: lipoproteins ; C-reactive protein ; cholesterol
Introduction
Platelet-activating factor (PAF) is a potent lipid mediator that may be involved in inflammatory diseases1 and atherogenesis.2 In plasma, PAF is hydrolyzed and inactivated by PAF-acetylhydrolase (PAF-AH; EC 3.1.1.47), a Ca2+-independent phospholipase A2.3 Plasma PAF-AH is complexed to lipoproteins in vivo; hence, it is also denoted lipoprotein-associated phospholipase A2 (Lp-PLA2).4
Several reports link PAF-AH to atherogenesis and increased risk of coronary artery disease (CAD); however, the role of PAF-AH in inflammatory and atherosclerotic diseases remains to be established. PAF-AH may represent a potent antiinflammatory and antiatherogenic enzyme because it degrades PAF and proinflammatory oxidized phospholipids.5 Conversely, PAF-AH may also generate bioactive oxidized free fatty acids4 and lysophosphatidylcholine.6 In addition, phospholipase A2/PAF-AH activity may liberate arachidonic acid, a precursor of eicosanoids including prostaglandins and leukotrienes.7
Several studies show that PAF-AH may indicate CAD risk; however, in the Women’s Health Study, the predictive value of PAF-AH mass was not independent of lipoprotein risk factors, particularly LDL cholesterol (LDL-C).8 In line with this, Ballantyne et al9 reported that in the Atherosclerosis Risk in Communities (ARIC) study, PAF-AH mass was associated with CAD only in individuals presenting with LDL-C <130 mg/dL. In contrast, in a study by Caslake et al,10 PAF-AH mass appeared to be an independent predictor of angiographically proven CAD, even after adjustment for LDL-C and other risk factors, and in the West of Scotland Coronary Prevention Study,11 PAF-AH mass was a strong risk factor for incident CAD, independent of markers of inflammation and LDL-C. Similarly, in an investigation by Blankenberg et al,12 PAF-AH activity was positively associated with angiographic CAD after adjustment for LDL-C, HDL cholesterol (HDL-C), and triglycerides. In a recent study, risk prediction by PAF-AH mass was independent of total cholesterol/HDL-C ratio.13 However, it remains controversial whether PAF-AH will be just another inflammatory marker, like sensitive C-reactive protein (sCRP), or whether PAF-AH will be predictive of CAD risk independently of other risk factors. Therefore, this cross-sectional study set out to determine the relationship of PAF-AH activity with CAD in relationship to established inflammatory markers and other well-recognized risk factors for CAD in 3148 patients who underwent coronary angiography.
Methods
Study Design and Participants
We studied 3148 white patients hospitalized for coronary angiography between June 1997 and January 2000.14 Informed written consent was obtained from each of the participants, and the study was approved by the ethics review committee at the Landes;rztekammer Rheinland-Pfalz.
Clinically relevant CAD was defined as the occurrence of a visible luminal narrowing (20% stenosis) in at least 1 of 15 coronary segments according to a classification of the American Heart Association.15 Angiograms were analyzed by visual analysis. To minimize the interobserver variability of visual analysis, all angiograms were assessed in a standardized fashion with enlarged projection. Furthermore, visual analysis was limited to 5 experienced angiographers. The final catheterization report was released only after approval by the senior cardiologist.14
Two thousand four hundred fifty-four patients were considered CAD positive, and 694 were CAD negative (controls). One thousand one hundred nineteen individuals had a history of 6 months or more of proven CAD. Severity of CAD was quantified with the Friesinger score16 and the number of major vessels with stenosis 50%. CAD patients were further stratified into individuals with stable CAD (n=1523) and patients with acute coronary syndrome (n=931; troponin T–negative unstable angina [n=627], non–ST-elevation myocardial infarction [NSTEMI; troponin T 0.1 μg/L; n=114], or ST-elevation myocardial infarction [STEMI; troponin T 0.1 μg/L; n=190]).
Laboratory Procedures
Blood sampling was done early in the morning before cardiac catheterization, after subjects had fasted. Blood samples were assayed for blood cell count and biochemistry as described previously.14 Measurement of PAF-AH activity was performed by the Azwell Auto PAF-AH kit (Azwell Inc)17 on a Hitachi 912 autoanalyzer: PAF-AH hydrolyzes the sn-2 position of the substrate [1-myristoyl-2-(4-nitrophenyl succinyl) phosphatidylcholine], producing 4-nitrophenyl succinate, which is immediately degraded to 4-nitrophenol and subsequently measured spectrophotometrically. LDL radius was estimated from free cholesterol, cholesterol esters, triglycerides, phospholipids, and apolipoprotein (apo) B18 after a combined ultracentrifugation-precipitation method (;-quantification).19 With this method, the LDL fraction includes particles with densities of 1.006 through 1.063 kg/L and thus comprises intermediate-density lipoproteins (IDL, 1.006 to 1.019 kg/L).20
Statistical Analysis
Correlation coefficients were calculated according to Spearman. Clinical and biological variables were compared by different methods depending on the nature of variables. Continuous variables were compared by univariate ANOVA between CAD patients and controls by adjustment for gender and drugs as indicated. Categorical variables were examined by logistic regression analysis, also with adjustment for gender.
The effect of medication on PAF-AH activity was assessed by univariate ANOVA. Marginal means were adjusted for age, gender, and status of CAD (CAD negative, CAD positive, unstable angina, NSTEMI, or STEMI) and for medication as indicated. Because lipid-lowering drugs (LLDs) had the highest impact on PAF-AH activity, we established quartile ranges of PAF-AH activity according to the values in the control subjects without CAD who were not using LLDs. We obtained risk ratios by comparing the prevalences of clinically relevant CAD in these quartiles by logistic regression analysis with adjustments as indicated. P<0.05 was considered statistically significant.
Results
Characteristics of Patients and Controls
PAF-AH activity, lipoproteins, sCRP, fibrinogen, white blood cell counts, and serum amyloid A (SAA) levels were available from 3148 individuals. Patients with stable CAD were significantly older than controls. Current or past smoking, type 2 diabetes mellitus, and hypertension were more prevalent in stable CAD patients than in control subjects. Stable CAD patients had higher systolic and diastolic blood pressure, higher fasting glucose, higher LDL-C, higher triglycerides, and lower HDL-C. Stable CAD patients were more often treated with ;-blockers, ACE inhibitors, calcium channel blockers, antiplatelet agents, and LLDs. Body mass index did not differ significantly between CAD patients and controls (Table 1).
PAF-AH, Lipoproteins, and Markers of Systemic Inflammation
PAF-AH activity was strongly correlated with total cholesterol, LDL-C, non-HDL-C, and apoB, with correlation coefficients that exceeded 0.5. Triglycerides and VLDL cholesterol were also positively correlated with PAF-AH activity. HDL-C, apoA-I, and LDL radius were weakly and inversely correlated with PAF-AH activity (Table 2). sCRP, fibrinogen, and lipoprotein(a) [Lp(a)] did not correlate with PAF-AH activity. Weak positive and negative associations were found between PAF-AH activity and white blood cell count and SAA, respectively. Analyses in subjects not using LLDs or in control subjects not using LLDs produced similar results (Table 2).
Activity of PAF-AH and Cardiovascular Medication
PAF-AH activity in male and female control subjects not using LLDs was 506±107 and 439±98 U/L (P<0.001 by ANOVA), respectively (data not shown), and males had higher PAF-AH activity than females in the entire study population (Figure, A). Both men and women taking LLDs (of whom 97.4% were taking statins, 2.4% fibrates, and 0.2% resins) had PAF-AH activity 12% lower than nonusers of LLDs (Table 3). Furthermore, the use of aspirin or other antiplatelet agents and ;-blockers decreased PAF-AH activity by 6%, respectively, whereas digitalis increased PAF-AH activity by 7% (Table 3). Use of AT1 receptor antagonists had a slightly decreasing effect, whereas ACE inhibitors (Table 3), calcium channel blockers, and antidiabetic drugs had no effect on PAF-AH activity (data not shown).
Estimated marginal means (±95% CI) of PAF-AH activity and inflammatory markers by absence of angiographic CAD (CAD–), stable CAD (CAD+), troponin T–negative unstable angina (UA), NSTEMI, and STEMI in men (squares) and women (circles). Data are adjusted for non-HDL cholesterol, LLDs, aspirin, ;-blockers, and digitalis. A, PAF-AH activity; B, C-reactive protein; C, serum amyloid A; and D, fibrinogen. Asterisks indicate statistical significance (P<0.05) of post hoc comparisons between respective patient group and individuals without CAD (CAD-). PAF-AH activity was significantly lower in females (P<0.001 by ANOVA).
Because LLDs had the most pronounced effect on PAF-AH activity, we investigated only those patients not receiving LLDs (n=1630). In these patients, PAF-AH activity was still significantly influenced by aspirin or other antiplatelet agents, ;-blockers, and digitalis after adjustment for other potentially confounding medication (Table 3).
PAF-AH in Stable CAD, Unstable Angina, NSTEMI, and STEMI
PAF-AH activity (adjusted for non-HDL-C, LLDs, aspirin, ;-blockers, and digitalis) was increased if CAD was present (Figure, A). However, in contrast to sCRP, SAA, and fibrinogen (Figure, B through D), PAF-AH activity showed no consistent association with the severity of myocardial ischemia (Figure, A).
PAF-AH in Angiographic CAD
We examined relative risks of angiographic CAD conferred by increasing quartiles of PAF-AH activity (Table 4). Because PAF-AH activity was not increased in unstable angina, NSTEMI, or STEMI, we included all CAD patients in this evaluation. The crude ORs of angiographic CAD with increasing quartiles of PAF-AH activity were not significantly different from 1.
Of the medications investigated, LLDs had the most pronounced impact on PAF-AH activity. Therefore, we restricted our further analysis to those individuals not taking LLDs. The characteristics of these patients did not differ from those of the entire study population (data not shown).
In the absence of LLDs, PAF-AH activity correlated with the severity of CAD (Friesinger score and the number of vessels with stenosis >50%; Table 2). In addition, the OR associated with increasing quartiles of PAF-AH activity was 1.31 (Table 4). Because aspirin or other antiplatelet agents, ;-blockers, and digitalis independently affected PAF-AH activity (Table 3), we adjusted for these drugs, which resulted in an OR with increasing quartiles of 1.39, the highest quartile having an OR of 2.67 compared with the lowest quartile (Table 4). This adjustment for drugs was done for all further analysis. If, in addition, LDL-C, HDL-C, log-normalized triglycerides, log-normalized Lp(a), and LDL radius were included, the OR was 1.26. If gender, age, body mass index, smoking, diabetes, hypertension, sCRP, fibrinogen, white blood cell count, SAA, and LDL-C were added, the OR across PAF-AH activity quartiles was 1.25 (Table 4). If we adjusted in the same way but without LDL-C, the OR estimates in patients with LDL-C 130 mg/dL or >130 mg/dL were 1.197 (95% CI 1.032 to 1.388, P=0.017) and 1.293 (95% CI 1.041 to 1.605, P=0.020), respectively (data not shown).
There were only 181 controls and 151 CAD patients using neither LLDs, aspirin or other antiplatelet agents, ;-blockers, or digitalis. In this small subgroup not taking any confounding drugs, the OR associated with increasing quartiles of PAF-AH activity was 1.65 (95% CI 1.23 to 2.11, P<0.001; data not shown) after adjustment for age, gender, body mass index, smoking, hypertension, and type 2 diabetes mellitus.
Discussion
The present study provides 3 major observations: First, PAF-AH activity is not an indicator of systemic inflammation accompanying myocardial ischemia. Second, not only LLDs but also other medications such as aspirin, ;-blockers, and digitalis may influence PAF-AH activity. Third, elevated PAF-AH activity indicates increased risk of CAD independently of other established risk factors, especially LDL-C.
PAF-AH activity was significantly lower in women, a finding that is in agreement with findings in 3106 Japanese healthy subjects21 and may, in general, be related to the lower cardiovascular risk found in females. In the present study, LLDs, aspirin, ;-blockers, and digitalis affected PAF-AH activity independently, whereas LLDs had the most pronounced effect. Blankenberg and colleagues12 previously reported a PAF-AH activity–decreasing effect of ACE inhibitors; however, this was not confirmed in the present study. Given the close relationship between PAF-AH activity and apoB-containing lipoproteins (Table 2), it is also not surprising that PAF-AH activity was lower in users than in nonusers of LLDs. In fact, in a placebo-controlled trial, it was recently shown that fluvastatin lowered PAF-AH activity in patients with type 2 diabetes mellitus, the decrease of PAF-AH activity being associated with the decrease of apoB in dLDL.22 However, the putative mechanisms by which aspirin, ;-blockers, and digitalis influence PAF-AH activity are not clear at present.
PAF-AH activity did not correlate with any of the inflammatory markers examined (Table 2). Furthermore, unlike C-reactive protein, SAA, or fibrinogen, it did not respond to increasing clinical instability of CAD (Figure). It is therefore unlikely that PAF-AH behaves as an acute phase marker or that it plays a major role as a trigger of systemic inflammation associated with atherosclerosis. However, PAF-AH is diminished in septic conditions and may be associated with multiorgan failure.23 Expression of PAF-AH is inhibited by interferon- and lipopolysaccharides, whereas other cytokines, including interferon-, interleukin (IL)-1, IL-6, and tumor necrosis factor- had no effect.24 In moderate systemic inflammation, when the increase of C-reactive protein is mainly mediated by tumor necrosis factor-, IL-1 , and IL-6, inhibition of PAF-AH gene expression may thus not occur. Therefore, the finding that PAF-AH activity is not elevated in the acute coronary syndrome appears biologically plausible.
PAF-AH activity was strongly correlated with plasma lipoproteins. The strongest correlations were found with apoB and non-HDL-C (Table 2). Both apoB and non-HDL-C are associated with increased CAD risk.25 Total apoB concentration is a crude marker of the number of LDL particles and the number of triglyceride-rich particles, including VLDL and IDL.26 Non-HDL-C was suggested to be the best surrogate measure for apoB27 and was recently introduced as a secondary target of therapy.28 However, PAF-AH activity may be a good estimate for atherogenic lipoproteins as well: 80% of PAF-AH activity is associated with LDL and another 7% with VLDL.10 Thus, almost 90% of PAF-AH activity resides on VLDL and LDL, with a preference to atherogenic dense LDL (dLDL).22,29
The strong reduction in PAF-AH activity by LLDs may explain why there was no association between PAF-AH activity and angiographic CAD when the use of LLDs was disregarded; however, when we restricted the analysis to individuals not using LLDs, PAF-AH activity was associated with the severity of CAD, and there was an OR for CAD of 1.31 with each increasing quartile of PAF-AH activity. The OR increased to 1.39 if the use of aspirin or other antiplatelet agents, ;-blockers, and digitalis was taken into account (Table 4).
In the Women’s Health Study,8 the predictive value of PAF-AH mass for CAD was not independent of LDL-C. In line with this, Ballantyne et al9 reported that in the ARIC study, PAF-AH mass was associated with CAD only in individuals with LDL-C <130 mg/dL. In the present study, the OR of PAF-AH activity remained significant after adjustment for major lipoprotein risk factors, including LDL-C, HDL-C, triglycerides, Lp(a), and estimated LDL radius (Table 4). Both in subjects with LDL-C 130 and in those with LDL-C >130 mg/dL, PAF-AH activity was significantly associated with CAD. Thus, we could not confirm that the association between PAF-AH activity and CAD disappeared above an LDL-C threshold of 130 mg/dL. As we have shown previously, PAF-AH activity correlates well with apoB in dLDL but only weakly with the calculated LDL radius.22 Furthermore, the LDL radius in the present study was estimated from the density range 1.006 to 1.063 kg/L, thus comprising IDL as well. Although PAF-AH was shown to reside on Lp(a),30 it remains controversial how strongly PAF-AH activity is associated with Lp(a); recently, it was shown that the fluvastatin-induced decrease of PAF-AH activity in patients with type 2 diabetes was independent of Lp(a).22
One might speculate how PAF-AH mediates increased CAD risk independently of other risk factors. PAF-AH may liberate arachidonic acid, a precursor of proinflammatory eicosanoids including prostaglandins and leukotrienes,7 within the atherosclerotic plaque. PAF-AH resides on dLDL,22,29 and LDL particles are an important source for the essential compound arachidonic acid.31 Therefore, we have suggested that dLDL may contribute to atherogenesis by functioning as a vector that provides both the essential substrate and the crucial enzyme activity necessary for prostaglandin synthesis.22 This concept would acknowledge PAF-AH as having an active role in atherogenesis rather than being a mere risk marker. Therefore, PAF-AH may provide a new target for the treatment of cardiovascular disease, and consequently, specific inhibitors of PAF-AH are currently under investigation.32
We exclusively enrolled individuals with clinically indicated coronary angiography. The referral bias associated with this design may be viewed as a limitation of the present study; however, this approach may also be a strength of this investigation. The prevalence of clinically asymptomatic coronary atherosclerosis has been reported to be very high at or above 50 years of age.33 Hence, angiography-based recruitment of control subjects prevents individuals with significant yet clinically inapparent CAD being inadvertently allocated to the control group. Furthermore, control subjects in the present study may be representative of a population-based control group, because the major cardiovascular risk factors occur at similar frequencies in these controls compared with the general population; the prevalence of hypertension was close to that found in a random probability sample from Germany.34 Prima facie, diabetes mellitus appears 2 to 3 times more frequent than in the German population.35 This is most likely because we did not rely on self-reports. Rather, we measured fasting glucose and performed an oral glucose challenge in individuals not previously known to have type 2 diabetes mellitus. On the basis of clinical history or fasting glucose measurements, the National Health and Nutrition Examination Surveys 1999 to 2000 reports prevalences of type 2 diabetes of 9.2% and 19.3% in adults 40 to 59 years old or more than 60 years old, respectively.36 In the present study, 12.1% of the control subjects had diabetes mellitus according to this criterion, whereas another 5.6% were detected by elevated postchallenge glucose only.
Acknowledgments
The technical assistance of Isolde Friedrich, Gabi Herr, Silvia Beheim, Ursula Discher, and Brigitte Haas is gratefully acknowledged. We thank the LURIC study team involved in patient recruitment and sample and data handling and the laboratory staff at the Ludwigshafen General Hospital and the Universities of Freiburg and Ulm, Germany.
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