The associations of fibroblast growth factor 23 and uncarboxylated matrix Gla protein with mortality in coronary artery disease: the Heart and Soul Study.
Context Other investigators recently identified novel regulators of mineral metabolism, which have been associated with cardiovascular disease and mortality in patients with end-stage renal disease. Contribution The authors studied 833 persons and found that those with higher levels of fibroblast growth factor 23 and lower levels of uncarboxylated matrix Gla protein had higher cardiovascular disease event rates and mortality after adjustment for traditional risk factors, C-reactive protein levels, and kidney function. Caution All study participants had coronary artery disease. Implication Better understanding of these relationships might lead to new therapies for cardiovascular disease. The Editors People with cardiovascular disease (CVD) are at high risk for recurrent CVD events and mortality (1), and many traditional CVD risk factors are not strongly associated with CVD events in this setting (2, 3). New insights into mechanisms for recurrent events might ultimately provide novel therapeutic targets or prevention strategies. Abnormalities in mineral metabolism are associated with CVD risk in the general population. For example, higher phosphorus levels promote arterial calcification in vitro (4, 5) and are associated with coronary artery calcification and CVD events in the general population, even among persons with normal kidney function (69). Similarly, lower serum vitamin D levels and higher serum intact parathyroid hormone levels are associated with CVD events in the general population (10, 11). Recently, several novel regulators of arterial calcification, including fibroblast growth factor 23 (FGF23), uncarboxylated matrix Gla protein (ucMGP), and fetuin-A, have been identified. Yet, the relationship of circulating levels of these proteins with CVD events and mortality is unknown. Fibroblast growth factor 23 is a bone-derived protein that promotes renal phosphorus wasting and inhibits conversion of 25-hydroxyvitamin D to the active 1,25-dihydroxyvitamin D form (1216). Studies of mice have suggested that elevated FGF23 levels may reflect a response to dietary phosphorus burden and serve as a marker of arterial calcification (17). Matrix Gla protein (MGP) is produced by chondrocytes and vascular smooth-muscle cells and prevents arterial calcification through local effects in the arterial wall (1821). Its precursor form is ucMGP, which has affinity for hydroxyapatite and may be depleted from blood in the setting of arterial calcification. Therefore, low serum levels may be a marker of vascular disease (22). Fetuin-A is a liver-secreted protein that complexes with calcium and phosphorus in circulation and prevents the precipitation of these minerals from serum (23, 24). In mice, knockouts of genes encoding FGF23, ucMGP, or fetuin-A result in arterial or soft-tissue calcification or both (2426). In humans, FGF23 and fetuin-A have been most extensively studied in cohorts with end-stage renal disease (ESRD), where higher FGF23 and lower fetuin-A levels are associated with mortality and CVD events (2730). Although the temporal association of ucMGP with mortality is uncertain, patients with ESRD or atherosclerosis have lower ucMGP levels than healthy controls (3134). Because these 3 proteins have complementary biological functions, we measured blood levels of all 3 and evaluated associations with mortality and CVD events in an ambulatory cohort with prevalent coronary artery disease (CAD) and a spectrum of kidney function from normal to moderate chronic kidney disease. On the basis of directions of association observed in cohorts with ESRD, we hypothesized that higher FGF23, lower ucMGP, and lower fetuin-A levels would each be associated with higher CVD events and mortality, independent of traditional CVD risk factors, kidney function, and one another. Methods Study Participants The Heart and Soul Study is an observational study designed to investigate the influence of psychosocial factors on the progression of CVD. Methods are described elsewhere (35). In brief, participants were recruited from outpatient clinics in the San Francisco Bay area if they met at least one of the following inclusion criteria: history of myocardial infarction (MI); angiographic evidence of greater than 50% stenosis in 1 or more coronary vessels; evidence of exercise-induced ischemia by treadmill or nuclear testing; and history of coronary revascularization or documented diagnosis of CAD by an internist or cardiologist. Participants were excluded if they could not walk 1 block, had MI within the past 6 months, or were likely to move out of the area within 3 years. The study protocol was approved by the institutional review boards of participating institutions. All participants provided written informed consent. From September 2000 to December 2002, a total of 1024 participants enrolled and had a baseline study appointment. Measurements Predictor Variables Fasting (12-hour) serum and plasma samples were obtained at the baseline visit, frozen at 70 C, and later used for measurement of FGF23 (in 2007), ucMGP (in 2007), and fetuin-A (in 2004). Blood levels of each were measured twice in each participant, and results were averaged (the Appendix includes measurement details). Outcome Variables From the baseline examination to 1 December 2008, we conducted annual telephone interviews with participants (or their proxies), inquiring about hospitalizations, cardiac procedures, or death. Two independent, blinded adjudicators reviewed all events, medical records, electrocardiography results, death certificates, and coroner's reports. In the event of disagreement, the adjudicators conferred, reconsidered their classification, and requested consultation from a third blinded adjudicator, as necessary. We considered the composite of MI, stroke, transient ischemic attack, or heart failure as a CVD event. Myocardial infarction was defined by cardiac biomarkers, electrocardiography results, and cardiac symptoms or signs according to the American Heart Association criteria (36). We defined stroke as a new neurologic deficit not secondary to brain trauma, tumor, infection, or other cause. Transient ischemic attack was defined as a focal neurologic deficit (in the absence of head trauma) lasting more than 30 seconds and no longer than 24 hours, with rapid symptom evolution to the maximal level of deficit in less than 5 minutes and with subsequent complete resolution. We defined heart failure as hospitalization for a clinical syndrome involving at least 2 of the following changes from usual status: paroxysmal nocturnal dyspnea, orthopnea, elevated jugular venous pressure, pulmonary rales, third heart sound, and cardiomegaly or pulmonary edema on chest radiography. Mortality was determined by death certificates and coroner's reports. Other Measurements We determined age, sex, race or ethnicity, and smoking status by questionnaire. We instructed participants to bring their medication bottles to the baseline appointment, and study personnel recorded medications. Waist-to-hip ratio was calculated after measuring waist circumference midway between the lower rib margin and iliac crest and hip circumference at the level of the greater trochanters. We determined prevalent hypertension and diabetes by questionnaire. Systolic and diastolic blood pressures were measured after 5 minutes of rest in the supine position. We calculated estimated glomerular filtration rate (GFR) by incorporating creatinine concentrations, age, sex, and race in the abbreviated (4-variable) Modification of Diet in Renal Disease Study equation (37). High-sensitivity C-reactive protein levels were measured with the Roche assay (Indianapolis, Indiana) and the Beckman Extended Range assay (Galway, Ireland) (38). We measured total cholesterol, high-density lipoprotein cholesterol, calcium, and phosphorus levels by using standard clinical chemistry analyzers. Participants had transthoracic echocardiography at baseline, which we used to measure left ventricular ejection fraction. We did a symptom-limited, graded exercise treadmill test according to the standard Bruce protocol and defined peak exercise capacity as total number of metabolic equivalent tasks achieved. Statistical Analysis We excluded 5 participants (<1%) who were missing outcome data and 186 participants (18%) who did not have baseline blood specimens for measurement of fetuin-A, ucMGP, or FGF23, resulting in a final sample size of 833 participants. Compared with those of excluded participants, demographic characteristics and event rates were similar among participants who were retained in this analysis (Appendix Table 1). For descriptive statistics, we compared demographic and clinical variables among participants with and without CVD events or mortality during follow-up by using the t test or the KruskalWallis test for continuous variables and the chi-square test or Fisher exact test for categorical variables, as appropriate. We used graphical methods to determine the distribution of FGF23, ucMGP, and fetuin-A in the study sample. Linear regression was used to compare associations of FGF23, ucMGP, and fetuin-A with calcium, phosphorus, and other baseline cardiovascular risks. Appendix Table 1. Comparison of Variables in Persons Excluded Because of Missing Measurements We elected to evaluate tertiles of each protein as our primary predictor variables. The lowest tertile served as the reference category. Because the functional form was fairly linear for each, we also evaluated each protein on a continuous scale, to minimize the chance of a type II error. We used KaplanMeier curves to evaluate unadjusted time to events and Cox proportional hazards models to evaluate adjusted associations of each novel mineral regulator with CVD events and mortality. Three sequential models were developed: one adjusted for age, sex, and race; one adjusted for age, sex, race and other CVD risk factors (smoking, hypertension, diabetes, waist-to-hip ratio, 