Takeki Suzuki, MD, MPH Lipoprotein-Associated Phospholipase A2 and Risk of Congestive Heart Failure in Older Adults: the Cardiovascular Health Study Suzuki Brief Title: Lp-PLA2 and Congestive Heart Failure

Background: Inflammation may be an etiologic factor in congestive heart failure (CHF). Lipoprotein associated phospholipase A2 (Lp-PLA2) is an inflammation marker associated with vascular risk. One previous study showed an association of Lp-PLA2 activity with CHF risk, but there were only 94 CHF cases and Lp-PLA2 antigen, which is available clinically in the US, was not measured. Methods and Results: We measured baseline Lp-PLA2 antigen and activity in 3991 men and women without baseline CHF or cardiovascular disease, participating in the Cardiovascular Health Study, a prospective observational study of adults 65 years old. Cox proportional hazards models adjusted for age, sex, clinic site, race, LDL and HDL cholesterol, body-mass index, systolic and diastolic blood pressure, hypertension, smoking status, pack-years and diabetes were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for incident CHF. Further models adjusted for coronary disease events during follow up and Creactive protein (CRP). 829 participants developed CHF over 12.1 years. Adjusted HRs for CHF with Lp-PLA2 in the fourth compared to first quartile, were 1.44 (CI 1.16-1.79) for Lp-PLA2 antigen and 1.06 (CI 0.84-1.32) for activity. Adjustment for incident coronary disease attenuated the HR for Lp-PLA2 antigen to 1.26 (CI 1.02-1.57), adjustment for CRP had minimal impact. Conclusions: Lp-PLA2 antigen was associated with risk of future CHF in older people, independent of CHF and coronary risk factors, and partly mediated by coronary disease events. Further clinical and basic research is needed to better understand the role of Lp-PLA2 in CHF. 2 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH Introduction Congestive heart failure (CHF) is a major public health problem in the United States. Approximately 5 million patients have CHF and 550,000 are newly diagnosed each year. Accumulating evidence supports that inflammation is an underlying pathophysiology of CHF. 3 Various inflammation markers such as C-reactive protein (CRP) and interleukin (IL)-6 are increased in patients with CHF. CRP and IL-6 have been shown to be associated with incident CHF. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is an inflammation marker used for cardiovascular risk assessment. It is synthesized by monocytes and macrophages, and, in the circulation, is bound to LDL. 12 Lp-PLA2 has proinflammatory properties through hydrolyzing oxidized phospholipids generating lysophosphatidylcholine and oxidized fatty acids. 14 Lp-PLA2 is strongly expressed in advanced coronary plaques suggesting a potential role in promoting plaque instability. However, Lp-PLA2 may also play an anti-inflammatory role through inhibition of PAF. LpPLA2 can be measured using an activity assay or a commercially available antigenic (mass) assay and the antigen and activity were measured in previous epidemiological studies. 19 In a previous study from the Cardiovascular Health Study (CHS), Lp-PLA2 activity and antigen were not correlated (r = 0.51), but this modest association points out the importance of considering both measures. Recently, a U.S. expert panel published a document on the clinical use of LpPLA2 in cardiovascular disease. 3 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH Several epidemiological studies reported that higher Lp-PLA2 is a risk marker for coronary heart disease (CHD) and ischemic stroke. 26 One study is available which reported an association of Lp-PLA2 activity with risk of CHF, but there were less than 100 cases and Lp-PLA2 antigen, which is available clinically in the U.S., was not measured. Thus, whether Lp-PLA2 antigen or activity are a risk factor for CHF is not clear. We examined the association of both Lp-PLA2 antigen and activity with risk of future CHF in the CHS. Methods Subjects The Cardiovascular Health Study (CHS) is a prospective population-based observational study of older adults 65 years old at baseline to evaluate risk factors for the development and progression of cardiovascular disease (CVD). The design, rationale and examination details have been described elsewhere. Briefly, participants were randomly selected from Medicare eligibility lists in four field centers: Forsyth County, North Carolina; Sacramento County, California, Allegheny County, Pennsylvania, and Washington County, Maryland. An initial primarily white cohort of 5201 was recruited between 1989 and 1990 and an additional 687 African-Americans (minority cohort) were recruited in 1992 and 1993. Persons were ineligible for participation if they were receiving active treatment for cancer, were wheelchair-bound or institutionalized, or were unable to participate in the examination. Comprehensive examinations and interviews were performed annually. The study was approved by institutional review boards at each site. Informed consent was obtained from all subjects. 4 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH Self-reported health behaviors, medical history, anthropometric measures, current medication use, seated blood-pressure readings, electrocardiography recordings, and fasting blood chemistry measures were obtained at baseline for both cohorts. Common carotid intima-media thickness (IMT) was measured at baseline in a standard manner as previously described. In previous CHS reports, IMT was an independent predictor of CHF and Lp-PLA2 was significantly higher in participants with higher IMT. Echocardiograms were obtained at baseline for the original cohort and again for members of both cohorts in 1994 to 1995. All participants in the original and minority cohorts were included in the primary analysis, except for 80 participants with baseline history of CHF, 115 with valvular heart disease by echocardiography (92 with aortic stenosis and 23 with severe mitral regurgitation), and 1190 with baseline CVD. Baseline CVD was defined as having one of the following at baseline: history of myocardial infarction, angina, stroke, transient ischemic attack, claudication, coronary artery bypass surgery, leg artery bypass, carotid endarterectomy, coronary angioplasty, or lower extremity angioplasty. Baseline CVD and CHF were adjudicated by a combination of self report of physician diagnosis as well as review of medical records. Secondary analysis was performed among participants with baseline CVD. Laboratory Methods Phlebotomy was performed on the morning of enrollment after 8-12 hours fast. Fibrinogen, total and HDL cholesterol, triglyceride, glucose and creatinine were measured at the central laboratory as previously reported. LDL cholesterol was calculated for those with triglycerides <400 mg/dL. CRP was measured by an in-house validated high-sensitivity enzyme-linked immunosorbent assay (ELISA). Interleukin-6 (IL-6) was measured by high-sensitivity ELISA (R&D Systems, Minneapolis, MN, USA). The interassay coefficients of variation were 6% for 5 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH CRP and 7% for IL-6. 35 Elevated CRP was defined as >3.0 mg/L corresponding to the “high risk category” in the American Heart Association/Centers for Disease Control (AHA/CDC) consensus statement. Elevated IL-6 and fibrinogen were defined as values in the top tertile of the distribution ( 2.04 pg/mL and >338 mg/dL, respectively). Plasma Lp-PLA2 antigen (or “mass”) was determined at the Laboratory for Clinical Biochemistry Research (University of Vermont, Burlington, VT) using a commercially available enzyme-linked immunosorbant assay (ELISA) kit (second generation PLAC Test; diaDexus Inc., South San Francisco, CA, USA). Plasma Lp-PLA2 activity was measured at GlaxoSmithKline (Research Triangle Park, NC) by high throughput radiometric assay using a tritium-labeled form of platelet activating factor [3H PAF] as substrate in a 96-well microplate, as previously described. The interassay coefficients of variation were 6.3 % for Lp-PLA2 antigen and 7.5 % for Lp-PLA2 activity. Adjudication of Incident Congestive Heart Failure Events Our outcome was incident CHF, which was assessed and validated as previously reported. 38 Subjects were interviewed every 6 months and follow-up examinations were conducted annually at each study center until May 31, 1998, after which telephone follow up continued. Self-report of a physician diagnosis of CHF was confirmed by review of medical records, with validation requiring a constellation of symptoms (shortness of breath, fatigue, orthopnea, paroxysmal nocturnal dyspnea), physical signs (edema, pulmonary rales, gallop rhythm, displaced left ventricular apical impulse), chest X-ray results (cardiomegaly and pulmonary edema), and treatment of CHF using diuretic agents, digitalis, or vasodilators (nitroglycerin, hydralazine, or 6 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH angiotensin-converting enzyme inhibitors). The CHS Events Committee adjudicated the index event of congestive heart failure by reviewing all pertinent data on hospitalization or outpatient visits, including history, physical examination, report of chest X-ray, and medication use. This analysis includes validated events through June 30, 2003. Statistical Analysis Baseline characteristics were compared between those who developed CHF and those who didn’t by using chi square tests for discrete values and t tests for continuous data. Lp-PLA2 antigen or activity was divided into quartiles (Quartile1-4, 1 being the lowest, 4 being the highest values) based on sex (men and women) and race (African American and non-African American). Kaplan-Meier curves with the endpoint of CHF were constructed based on Lp-PLA2 antigen or activity quartiles. A log-rank test was performed to examine differences among the four groups. The associations of these categories of Lp-PLA2 antigen or activity level with incident CHF were assessed using Cox proportional hazards models. Hazard ratios (HRs) and 95% confidence intervals (CIs) for incident CHF were calculated for each Lp-PLA2 quartile compared to the 1 quartile. Models were first adjusted for age, sex, clinic site and race. Additional adjustments included LDL and HDL cholesterol, body-mass index, systolic and diastolic blood pressure, hypertension status, smoking status and pack-years, and diabetes status. Incident validated CHD was added to the model as a time-dependent covariate to assess mediation. Incident CHD was defined as incident MI, angina, angioplasty, coronary artery bypass surgery, or CHD death. Additional variables were added individually to evaluate potential biological pathways of LpPLA2 and incident CHF: baseline serum creatinine, statin and aspirin use, CRP, IL-6, left 7 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH ventricular (LV) mass by electrocardiography, and common carotid IMT. In secondary analysis, we replicated the above models among participants with baseline CVD. We did not adjust for incident CHD in secondary analysis since these participants had already had baseline CHD. Stratified analyses were subsequently performed on the basis of sex and race (African American and non-African American). In addition, since it has been reported that Lp-PLA2 was more strongly associated with vascular events in those with low LDL cholesterol and in those subjects, those with both elevated Lp-PLA2 and CRP were at the greatest risk for CHD, we evaluated incident CHF stratified by the levels of LDL (above or below median), HDL (above or below median), and CRP (above or below 3 mg/L). To evaluate the combined predictive value of Lp-PLA2 and other inflammation markers for incident CHF, participants were cross-classified by Lp-PLA2 and inflammation markers (CRP >3 mg/L, and IL-6 and fibrinogen in tertiles) and interactions between Lp-PLA2 and these inflammation markers were assessed by calculating the relative excess risk due to interaction (RERI) , as well as the RERI%, defined as the proportion of disease related to Lp-PLA2 and the inflammation marker, either singly or in combination, attributable to their interaction. The Delta method was used to calculate P-values and 95% confidence intervals used to assess significance of the RERI. Statistical analyses were performed at the Cardiovascular Health Study Coordinating Center using Stata, Release 10 (Stata Co, College Station, Texas). 8 by gest on A ril 2, 2017 http://circheure.ahajournals.org/ D ow nladed from Takeki Suzuki, MD, MPH Results Baseline characteristics of the 3991 participants are shown in Table 1. There were 829 incident CHF cases over 12.1 years of follow-up (incidence rate of 19.1 per 1000 person-years). Those who developed CHF were older, more likely to be male, and to have diabetes, hypertension and greater LV mass. Smoking was relatively uncommon and was similar between the two groups. Baseline Lp-PLA2 antigen and activity, along with other inflammation markers, were higher in those who developed CHF. There were 1190 participants with baseline CVD evaluated in secondary analyses. Patterns of association of baseline risk factors with CHF were similar to those without baseline CVD (data not shown). Associations between Lp-PLA2 Antigen/Activity and Incident CHF Kaplan-Meier curves for time to CHF by Lp-PLA2 antigen or activity quartiles are shown in Figure 1. Those with the highest Lp-PLA2 antigen were more likely to develop CHF during follow-up, with the 10-year cumulative incidence rate ranging from 11.7 per 1000 person-years in the first quartile to 19.5 per 1000 person-years in the 4 quartile (P=0.0001) for difference among groups. There were no significant differences in CHF incidence among the quartiles of