Osteoprotegerin and Cardiovascular Events in High-Risk Populations: Meta-Analysis of 19 Prospective Studies Involving 27450 Participants

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC 3.0 https://creativecommons.org/licenses/by-nc/3.0/ Osteoprotegerin and Cardiovascular Events in High-Risk Populations: Meta-Analysis of 19 Prospective Studies Involving 27450 Participants © 2018 The Authors Published version Tschiderer, Lena; Klingenschmid, Gerhard; Nagrani, Rajini; Willeit, Johann; Laukkanen, Jari; Schett, Georg; Kiechl, Stefan; Willeit, Peter Tschiderer, L., Klingenschmid, G., Nagrani, R., Willeit, J., Laukkanen, J., Schett, G., Kiechl, S., & Willeit, P. (2018). Osteoprotegerin and Cardiovascular Events in High-Risk Populations: MetaAnalysis of 19 Prospective Studies Involving 27450 Participants. Journal of the American Heart Association, 7(16), Article e009012. https://doi.org/10.1161/JAHA.118.009012 2018 Osteoprotegerin and Cardiovascular Events in High-Risk Populations: Meta-Analysis of 19 Prospective Studies Involving 27 450 Participants Lena Tschiderer, BSc, Dipl.-Ing.;* Gerhard Klingenschmid, MD;* Rajini Nagrani, PhD; Johann Willeit, MD; Jari A. Laukkanen, MD; Georg Schett, MD, PhD; Stefan Kiechl, MD; Peter Willeit, MD, MPhil, PhD Background-—Osteoprotegerin is a cytokine involved in bone metabolism as well as vascular calcification and atherogenesis. Although circulating osteoprotegerin levels are robustly associated with incident cardiovascular disease (CVD) in the general population, its relevance as a biomarker among populations at high CVD risk is less clear. Methods and Results-—Three independent reviewers systematically searched PubMed, EMBASE, and Web of Science to identify prospective studies that had recruited participants on the basis of having conditions related to high CVD risk. A total of 19 studies were eligible for inclusion, reporting on 27 450 patients with diabetes mellitus (2 studies), kidney disease (7 studies), preexisting heart disease (5 studies), or recent acute coronary syndromes (5 studies) at baseline. Over a mean follow-up of 4.2 years, 4066 CVD events were recorded. In a random-effects meta-analysis, the pooled risk ratio for CVD events comparing people in the top versus the bottom tertile of osteoprotegerin concentration was 1.30 (95% confidence interval, 1.12–1.50; P<0.001; I 2 =68.3%). There was evidence for presence of publication bias (Pvalue from Egger’s test=0.013). Correction for publication bias using the trim-and-fill method reduced the risk ratio to 1.21 (95% confidence interval, 1.03–1.42; P<0.001). The risk ratios did not vary significantly by population type, geographical region, statistical adjustment, sample or assay type, age, sex, or length of follow-up. Conclusions-—In populations at high CVD risk, elevated circulating osteoprotegerin levels are associated with a higher risk for future CVD events. The magnitude of association appears weaker than in the general population. (J Am Heart Assoc. 2018;7: e009012. DOI: 10.1161/JAHA.118.009012.) Key Words: cardiovascular disease •high-risk population •meta-analysis •osteoprotegerin •prospective cohorts Osteoprotegerin is a member of the tumor necrosis factor (TNF) receptor superfamily and is involved in bone homeostasis. 1 It inhibits osteoclastogenesis by binding to the receptor activator of nuclear factor-jB ligand (RANKL), which prevents RANKL from binding to the receptor activator of nuclear factor-jB (RANK). 2 Inhibition of the RANK/RANKL pathway results in less osteoclast differentiation as well as reduced activation and survival of mature osteoclasts. 2,3 TNFrelated apoptosis-inducing ligand, a protein that belongs to the TNF superfamily as well, also serves as an osteoprotegerin ligand. 4 Osteoprotegerin thereby contributes to maintaining the balance between bone resorption and bone formation. 2,5 In addition to its role in bone homeostasis, osteoprotegerin has been implicated in the development of cardiovascular diseases (CVDs). 6 It is found in atherosclerotic plaques, 7,8 may regulate vascular calcification, 9,10 and may thereby influence cardiovascular risk. Furthermore, genetic studies have demonstrated associations of osteoprotegerin gene polymorphisms with CVD. 11–15 In a literature-based metaanalysis of 9 general population studies, we recently From the Department of Neurology, Medical University of Innsbruck, Austria (L.T., G.K., R.N., J.W., S.K., P.W.); Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland (J.A.L.); Central Finland Central Hospital, Jyv€ askyl€ a, Finland (J.A.L.); Faculty of Sport and Health Sciences, University of Jyv€ askyl€ a, Finland (J.A.L.); Department of Internal Medicine 3, University of Erlangen-Nuremberg, Erlangen, Germany (G.S.); and Department of Public Health and Primary Care, University of Cambridge, United Kingdom (P.W.). Accompanying Tables S1, S2 and Figures S1, S2 are available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.118.009012 *Dipl.-Ing. Tschiderer and Dr Klingenschmid share first authorship. Correspondence to: Peter Willeit, MD, MPhil, PhD, Department of Neurology, Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. E-mail: [email protected] Received February 23, 2018; accepted July 16, 2018. ª2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 1 SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 demonstrated robust positive associations between osteoprotegerin concentration and incident CVD. 16 However, it is unclear whether these associations equally apply to high-risk populations. Although several individual studies have investigated the predictive significance of osteoprotegerin in these settings, 17–35 the interpretation of their findings has been complicated by differing in scales of association, levels of adjustment, and outcome definitions. The principal aim of this report is to review comprehensively the available literature and to perform a meta-analysis of reported associations between osteoprotegerin and risk for future CVD in high-risk populations (ie, in studies that have recruited participants on the basis of having conditions related to high CVD risk). Secondary analyses will assess associations with coronary heart disease (CHD) events and stroke separately and will clarify whether the magnitude of association differs according to study-level characteristics. Methods Research Data Availability The database of published results from studies included in the meta-analysis is made available to other researchers for purposes of reproducing the results or replicating the procedure. 36 Literature Search, Study Selection, and Data Extraction We systematically sought PubMed, Web of Science, and EMBASE for prospective studies published between January 1970 and April 2017 that reported on associations of osteoprotegerin concentration with CVD outcomes (defined as nonfatal CHD [ie, myocardial infarction, unstable or stable angina, or coronary revascularization procedures], nonfatal stroke,orcardiovascular death).Wealsoscannedreferencelists of articles (including reviews) and corresponded with several study investigators. Table S1 provides a detailed description of search terms used in the literature search. Studies were eligible for inclusion if they (1) had a prospective design; (2) had recruited study participants on the basis of having preexisting conditions favoring risk of future CVD; and (3) had recorded incident CVD outcomes over a period of >1 month. Studies that did not report on the predefined outcome definition (including those reporting on the combination of CVD events and all-cause mortality) were excluded from the analysis. For each eligible study, 3 reviewers (L.T., G.K., P.W.) independently extracted the following pieces of information: type of baseline disease, study location, year of baseline, duration of follow-up, mean or median age at baseline, proportion of male participants, osteoprotegerin assay type (ELISAs versus immunofluorometric assays), osteoprotegerin assay manufacturers, and sample types (plasma versus serum). In addition, we extracted information on the statistical adjustment used, categorizing adjustments as “unadjusted”if no adjustment was employed; “+”for adjustment for age and sex; “++”for adjustment for age, sex, and non–blood-based risk factors, such as smoking, blood pressure, and diabetes mellitus; and “+++”for further adjustments for blood-based risk factors (eg, cholesterol and C-reactive protein). If a study reported different adjustment models, the most adjusted model was used to minimize the scope for confounding. If information about the same study was published twice or more often, we used the most recent publication. Study quality was evaluated using the Newcastle-Ottawa scale for cohort studies. 37 The meta-analysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 38 Statistical Analyses We conducted analyses according to a predefined statistical analysis plan. The primary outcome was CVD events (as defined above); secondary outcomes were CHD events and stroke events. Because studies reported effect estimates on different scales (eg, per standard deviation or across quartiles), we converted risk ratios and 95% confidence intervals (CIs) to reflect a comparison of the top versus bottom tertiles of baseline osteoprotegerin distribution using methods described elsewhere. 39 One study 29 did not provide sufficient information on the osteoprotegerin distribution—a prerequisite for converting its risk ratio—and we therefore estimated its distribution on the basis of comparable study populations. 26,28,30 We pooled study-specific risk ratios using random-effects meta-analysis (sensitivity analyses used fixedClinical Perspective What Is New? •In the present report, we systematically reviewed and combined the published evidence on the relevance of circulating osteoprotegerin concentration to cardiovascular events in high-risk populations. •Our meta-analysis demonstrated a significant positive association between osteoprotegerin concentration and cardiovascular disease risk. •The magnitude of association was similar in various clinically significant subgroups, including those defined by age and sex. What Are the Clinical Implications? •This work highlights the potential of osteoprotegerin as a biomarker for cardiovascular disease risk. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 2 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 effect meta-analysis). The I 2 statistic was used to assess heterogeneity across studies. 40 Subgroup analyses were conducted using meta-regression across prespecified studylevel characteristics. 40 We evaluated whether publication bias was present by visually inspecting a funnel plot and applying Egger’s asymmetry test. 41 We estimated a risk ratio corrected for publication bias using the trim-and-fill method, which imputes artificial studies to achieve symmetry of the funnel plot. 42 In addition, to evaluate the influence of single studies on the overall result, we performed a leave-one-out crossvalidation, which reestimates the pooled risk ratio while omitting each study in turn. All statistical tests were 2-sided; P<0.05 was deemed as statistically significant. Data were analyzed using the statistical software Stata, version 14.1 (StataCorp). Because our analysis relied entirely on data available in the published literature, approval by the institutional review board of the project was not required. Results General Characteristics of Included Studies Of 2602 records retrieved from PubMed, Web of Science, and EMBASE, we excluded 1001 duplicates and 1318 records after review of titles and abstracts (Figure 1). When reviewing the full text of the remaining 283 articles, we excluded a Figure 1. Study flow diagram. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 3 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 further 264 additional articles, leaving 19 prospective studies 17–35 eligible for inclusion in the meta-analysis. Patients were recruited on the basis of having diabetes mellitus in 2 studies, kidney disease in 7 studies, preexisting heart disease in 5 studies, and recent acute coronary syndromes in 5 studies. Details on the definitions of baseline conditions are provided in Table S2. Twelve studies were based in Europe, 4 were based in Asia, and 3 were located in multiple continents (Table 1). Of the 19 prospective studies, 7 were nested in a trial. The weighted mean age was 60.9 years; 68.3% of patients were men. The average quality of the studies assessed by the Newcastle-Ottawa scale for cohort studies was 7.6. For measuring osteoprotegerin concentrations, 15 studies used ELISAs and 4 studies used immunofluorometric assays. Ten studies had measured osteoprotegerin concentration in plasma, and 9 had measured osteoprotegerin concentration in serum. In total, the studies involved 27 450 participants and reported on 4066 CVD outcomes recorded over a weighted mean follow-up duration of 4.2 years (Table 2). One study reported unadjusted effect estimates; another 3 studies reported effect estimates adjusted for age, sex, and non–blood-based markers; 14 studies reported multivariable adjusted effect estimates (including blood-based markers); and 1 study reported unadjusted risk ratios for CVD events and multivariable adjusted risk ratios for stroke. Table 1. Design Features of Contributing Studies Study Acronym or First Author Location Year of Baseline, Range Study Quality, NOS Mean Age, y Male Sex, % Osteoprotegerin Assay Type (Manufacturer) Sample Type Populations with diabetes mellitus at baseline Anand 17 United Kingdom NR 7 52.7 60.6 ELISA (Biomedica) Plasma FINNDIANE 18 Finland 1997–2004 8 36.9 49.8 IFMA (R&D Systems) Serum Populations with kidney disease at baseline ALERT 19 * Multicenter 1997 7 49.6 65.8 ELISA (Biomedica) Serum CRISIS 20 United Kingdom 2002–2010 6 63.8 61.8 ELISA (BioVendor) Plasma Kuzniewski 21 Poland 2004 8 60.0 56.5 ELISA (BioVendor) Plasma Nakashima 22 Japan 2003 7 62.1 56.3 ELISA (Immundiagnostik) Plasma Nishiura 23 Japan 2000–2006 7 58.9 65.7 ELISA (Immundiagnostik) Serum Speer 24 Hungary 2004–2007 7 63.4 61.2 ELISA (Immundiagnostik) Serum Yilmaz 25 Turkey 2009–2013 7 48.9 51.9 ELISA (RayBiotech) Serum Populations with preexisting heart disease at baseline CLARICOR 26 * Denmark 1999–2000 9 65.4 69.4 IFMA (R&D Systems) Serum CORONA 27 * The Netherlands 2003–2005 8 72.0 76.7 ELISA (R&D Systems) Plasma Jono 28 Japan 1999–2000 8 63.1 82.7 ELISA (Cosmo Bio) Serum PEACE 29 * Multicenter 1996–2000 6 63.7 81.0 ELISA (R&D Systems) Plasma Pedersen (1) 30 Norway 2000–2001 9 62.0 † 71.9 ELISA (R&D Systems) Serum Populations with recent acute coronary syndromes at baseline MERLIN-TIMI36 31 * Italy 2004–2006 8 64.0 64.9 IFMA (R&D Systems) Plasma OPTIMAAL 32 Multicenter 1998–1999 9 67.8 70.3 ELISA (R&D Systems) Plasma PLATO 33 * Multicenter 2006–2008 6 62.0 71.6 ELISA (NR) Plasma PRACSIS 34 Sweden 1996–2001 9 65.0 70.7 ELISA (R&D Systems) Serum Pedersen (2) 35 Denmark 2006–2008 9 63.5 41.3 IFMA (R&D Systems) Plasma Total 1996–2013 7.6 60.9 68.3 Summary statistics are ranges, weighted means, or sums, as appropriate. ALERT indicates Assessment of Lescol in Renal Transplantation Study; CLARICOR, Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease; CORONA, Controlled Rosuvastatin Multinational Trial; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; FINNDIANE, Finnish Diabetic Nephropathy Study; IFMA, immunofluorometric assay; MERLIN-TIMI36, Metabolic Efficiency With Ranolazine for Less Ischemia in Non–ST-Elevation Acute Coronary Syndromes Trial; NOS, Newcastle-Ottawa scale; NR, not reported; OPTIMAAL, Optimal Trial in Myocardial Infarction With Angiotensin II Antagonist Losartan; PEACE, Prevention of Events With Angiotensin Converting Enzyme Inhibition Trial; PLATO, Platelet Inhibition and Patient Outcomes Trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. *Nested in clinical trial. † Median. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 4 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 Overall Association of Osteoprotegerin With Cardiovascular Events Figure 2 shows the forest plot of the association of baseline osteoprotegerin concentration with incident CVD events. The pooled relative risk for CVD events was 1.30 (95% CI, 1.12– 1.50; P<0.001) for a comparison of individuals in the top versus the bottom tertile of baseline osteoprotegerin concentration. There was a high degree of between-study heterogeneity (I 2 =68.3%; P<0.001). In comparison, a fixedeffect meta-analysis yielded a pooled risk ratio of 1.15 (95% CI, 1.10–1.21; P<0.001). There was evidence for publication bias, as indicated by the funnel plot (Figure 3) and a significant Egger’s asymmetry test (P=0.013). Using the trim-and-fill method, 5 additional artificial studies were included into the meta-analysis to generate a symmetric funnel plot (Figure S1). This correction for publication bias yielded a relative risk of 1.21 (95% CI, 1.03–1.42; P=0.020). Reestimated pooled risk ratios by omitting each study in turn remained significant for all omissions (Figure 4). A subset of studies had published risk ratios separately on the secondary outcomes CHD and stroke. When comparing the top versus the bottom tertile of baseline Table 2. Follow-Up Data in the Contributing Studies Study Acronym or First Author Maximum Follow-Up, y No. of Participants No. of Events Adjustment of Reported Risk RatioCVD CHD Stroke Populations with diabetes mellitus at baseline Anand 17 1.5* 510 16   Unadjusted FINNDIANE 18 10.5* 1903 190 152 71 ++ Populations with kidney disease at baseline ALERT 19† 6.7* 1889  285  +++ CRISIS 20 3.8* 463 108   +++ Kuzniewski 21 7.0 69 31   +++ Nakashima 22 6.0 151 40   ++ Nishiura 23 3.5* 99 27   +++ Speer 24 2.6 98 23   +++ Yilmaz 25 3.0 ‡ 291 87   +++ Populations with preexisting heart disease at baseline CLARICOR 26† 2.6* 4063 623 303 146 +++ CORONA 27† 3.0 1464 318 255  +++ Jono 28 5.1* 225 101   +++ PEACE 29† 7.0 3767 1290  NR Unadjusted/+++ § Pedersen (1) 30 6.1 ‡ 1025 60 103  +++ Populations with recent acute coronary syndromes at baseline MERLIN-TIMI36 31† 0.9 ‡ 4463 544 336  +++ OPTIMAAL 32 2.3* 234 26   +++ PLATO 33† 2.4 5123 432   ++ PRACSIS 34 10.1 897 150 107 43 +++ Pedersen (2) 35 2.3 ‡ 716  51  +++ Total 4.2 27 450 4066 1592 260 Summary statistics are weighted means or sums, as appropriate. ++ indicates adjusted for age, sex, and non–blood-based risk factors; +++, further adjusted for blood-based risk factors; ALERT, Assessment of Lescol in Renal Transplantation Study; CHD, coronary heart disease; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; CVD, cardiovascular disease; FINNDIANE, Finnish Diabetic Nephropathy Study; MERLIN-TIMI36, Metabolic Efficiency With Ranolazine for Less Ischemia in Non–ST-Elevation Acute Coronary Syndromes Trial; NR, study did not report the number of stroke events (despite reporting hazard ratios for stroke); OPTIMAAL, Optimal Trial in Myocardial Infarction With Angiotensin II Antagonist Losartan; PLATO, Platelet Inhibition and Patient Outcomes Trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. *Mean. † Nested in clinical trial. ‡ Median. § Study reported unadjusted risk ratios for the outcome cardiovascular events and multiple adjustment for the outcome stroke. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 5 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 osteoprotegerin concentration, the risk ratio was 1.24 (95% CI, 0.94–1.64; 8 studies; 1592 events; P=0.128) for CHD and 1.21 (95% CI, 0.97–1.50; 4 studies; 260 events; P=0.090) for stroke (Figure 5). The I 2 value for betweenstudy heterogeneity was high for CHD (71.7%; P=0.001) and low for stroke (0%; P=0.661). Corresponding risk ratios using fixed-effect meta-analysis were 1.14 (95% CI, 0.99– 1.32; P=0.063) for CHD and 1.21 (95% CI, 0.97–1.50; P=0.090) for stroke. Findings According to Study-Level Characteristics Figure 6 presents risk ratios pooled according to study-level characteristics. There were no significant differences in the strength of association according to population type, geographical region, statistical adjustment, sample type, and assay type (all P>0.05). Furthermore, there was no evidence that the strength of association differed according to mean age, sex distribution, or length of follow-up of the study population (Pvalues from meta-regression: 0.354, 0.170, and 0.564, respectively). Discussion In this literature-based meta-analysis, we analyzed 19 highrisk population studies involving a total of 27 450 participants recruited between 1996 and 2013. Our analysis identified positive associations between osteoprotegerin concentrations and cardiovascular risk. Individuals with a high osteoprotegerin concentration (ie, in the top tertile of baseline osteoprotegerin distribution) had a relative risk of 1.30 (95% CI, 1.12-1.50) for CVD events when compared with individuals with osteoprotegerin levels in the bottom tertile. This relative risk remained stable under multivariable adjustment and across various study-level characteristics. The between-study heterogeneity was high (I 2 =68.3%). Although studies varied in terms of population type, geographical region, level of adjustment, sample type, assay type, proportion of men, mean age, and length of follow-up, none of these characteristics significantly influenced the strength of association of osteoprotegerin with future CVD risk. However, our analysis identified significant publication bias resulting from predominantly strong positive results in small studies. After correcting for publication bias, the relative risk was reduced to 1.21, Figure 2. Combined relative risk for cardiovascular events in the top vs the bottom tertile of osteoprotegerin (OPG) concentration. Sizes of data markers indicate the weight of each study in the analysis. The I 2 value was 68.3% (P<0.001). CI indicates confidence interval; CLARICOR, Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease; CORONA, Controlled Rosuvastatin Multinational Trial; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; FINNDIANE, Finnish Diabetic Nephropathy Study; MERLIN-TIMI36, Metabolic Efficiency With Ranolazine for Less Ischemia in Non–ST-Elevation Acute Coronary Syndromes Trial; OPTIMAAL, Optimal Trial in Myocardial Infarction With Angiotensin II Antagonist Losartan; PEACE, Prevention of Events With Angiotensin Converting Enzyme Inhibition Trial; PLATO, Platelet Inhibition and Patient Outcomes Trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 6 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 but remained significant, with a 95% CI ranging from 1.03 to 1.42. In addition, we confirmed with the leave-one-out crossvalidation method that our overall result was not driven by a single study, highlighting the robustness of our finding. We have previously demonstrated in a literature-based meta-analysis that osteoprotegerin is associated with incident CVD in people recruited from the general community. 16 A combination of findings from 9 general population studies yielded a pooled relative risk for CVD of 1.83 (95% CI, 1.462.30) for a comparison of extreme osteoprotegerin tertiles. In comparison, the present meta-analysis of studies involving individuals at high CVD risk yielded an association directionally concordant but significantly weaker (Figure S2). Three distinct features of high-risk populations may contribute to this weaker association. First, most high-risk individuals received (multi-)drug treatment. It has been demonstrated that in vivo treatment with antidiabetic medication, 43–46 statins, 47,48 heparins, 49,50 or glucocorticoids 51 and in vitro treatment with irbesartan 52 or different immunosuppressive therapies 53 affect circulating osteoprotegerin levels. Second, circulating osteoprotegerin levels differ in people with preexisting diseases. For instance, increased osteoprotegerin levels can be found in patients with preexisting CVD, such as severe peripheral artery disease, 54 heart failure, 55 and ST-segment–elevation acute myocardial infarction. 56 Moreover, serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease. 57 Patients with type 1 or type 2 diabetes mellitus exhibit elevated osteoprotegerin levels. 43,44 High osteoprotegerin values have also been linked to poor glycemic control 58,59 and severity of diabetic nephropathy. 60,61 In patients with chronic renal failure, levels of osteoprotegerin are higher compared with healthy controls, 62 are inversely correlated with glomerular filtration rate, 63 and correlate with time on maintenance hemodialysis in patients with end-stage renal disease. 64 Third, associations of osteoprotegerin may be Figure 3. Funnel plot of reported associations between osteoprotegerin (OPG) concentration and risk of cardiovascular events. The dotted lines show pseudo 95% confidence intervals around the overall pooled estimate. The Pvalue from Egger’s asymmetry test of associations was 0.013. CLARICOR indicates Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease; CORONA, Controlled Rosuvastatin Multinational Trial; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; FINNDIANE, Finnish Diabetic Nephropathy Study; MERLIN-TIMI36, Metabolic Efficiency With Ranolazine for Less Ischemia in Non–ST-Elevation Acute Coronary Syndromes Trial; OPTIMAAL, Optimal Trial in Myocardial Infarction With Angiotensin II Antagonist Losartan; PEACE, Prevention of Events With Angiotensin Converting Enzyme Inhibition Trial; PLATO, Platelet Inhibition and Patient Outcomes Trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 7 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 attenuated because of the dominance of other factors more relevant to CVD risk in high-risk patients, including highly prevalent traditional CVD risk factors as well as factors related to quality of clinical care, treatment response, or medication adherence. 65 Altogether, differences in medical treatment, patient histories, disease severity, multimorbidities, and clinical course of disease among high-risk patients may obscure associations of osteoprotegerin levels with risk for future CVD and might result in reverse causation bias. The pathophysiological role of osteoprotegerin in CVD development is multifaceted and not completely understood. It is considered to reflect the overall activity of the osteoprotegerin/RANK/RANKL signaling pathway and regulate calcification in both the bone and the vasculature. 9,10 Osteoprotegerin is expressed in a variety of human tissues 1 ; in the vessel wall, it is mainly secreted by endothelial 66 and vascular smooth muscle cells. 67 Beneficial effects of osteoprotegerin on the cardiovascular system were reported by several earlier studies. For instance, osteoprotegerin deficiency in mice led to early-onset osteoporosis and arterial calcification. 68 Furthermore, osteoprotegerin inactivation in apolipoprotein E–deficient knockout mice increased plaque calcification. 69 In in vitro studies, osteoprotegerin was found to inhibit calcification in vascular smooth muscle cells 70 and act as a survival factor in endothelial cells. 71 In contrast, several lines of evidence from experimental studies in animals and cell cultures suggested harmful effects of osteoprotegerin in agreement with the positive association with CVD risk in our meta-analysis. 6 Osteoprotegerin not only contributes to systemic inflammation, 72 but also to vasculature-specific inflammation by increasing macrophage infiltration 73 and leukocyte adhesion to endothelial cells. 74,75 Moreover, it promotes vascular medial fibrosis 76 and may exert indirect proatherosclerotic effects by blocking TNF-related apoptosisinducing ligand. 77 Atherosclerotic plaques that highly express osteoprotegerin exhibit more calcification, 8 but studies yielded conflicting results about its relevance to plaque stability and conversion to a symptomatic plaque. 52,73,78,79 These inconsistent reports emphasize the wide-ranging aspects of osteoprotegerin in the complexity of regulatory processes in atherogenesis and call for more experimental studies to improve our understanding of this pathway in human disease. Although our meta-analysis shows positive associations of baseline osteoprotegerin concentration and CVD risk, its incorporation in clinical routine entails some analytical issues. Figure 4. Reestimated pooled risk ratios for cardiovascular outcomes omitting one study in each turn. CI indicates confidence interval; CLARICOR, Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease; CORONA, Controlled Rosuvastatin Multinational Trial; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; FINNDIANE, Finnish Diabetic Nephropathy Study; MERLIN-TIMI36, Metabolic Efficiency With Ranolazine for Less Ischemia in Non–ST-Elevation Acute Coronary Syndromes Trial; OPG, osteoprotegerin; OPTIMAAL, Optimal Trial in Myocardial Infarction With Angiotensin II Antagonist Losartan; PEACE, Prevention of Events With Angiotensin Converting Enzyme Inhibition Trial; PLATO, Platelet Inhibition and Patient Outcomes Trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. DOI: 10.1161/JAHA.118.009012 Journal of the American Heart Association 8 Osteoprotegerin and CVD Events in High-Risk Populations Tschiderer et al SYSTEMATIC REVIEW AND META-ANALYSIS Downloaded from http://ahajournals.org by on September 10, 2018 Table S2. Detailed baseline conditions of contributing studies Study acronym or first author Baseline diseases Populations with diabetes at baseline Anand1 Type 2 diabetes FINNDIANE2 Type 1 diabetes Populations with kidney disease at baseline ALERT3 Renal transplant recipients (did not report on cause of kidney disease) CRISIS4 All cause chronic kidney disease Kuzniewski5 Chronic glomerulonephritis, pyelonephritis, polycystic kidney disease, diabetic nephropathy, unknown cause Nakashima6 Long term hemodialysis patients (did not report on cause of kidney disease) Nishiura7 Consecutive hemodialysis patients (diabetic nephropathy, chronic glomerulonephritis, nephrosclerosis, other cause) Speer8 Chronic hemodialysis patients (vascular-tubulointerstitial [including hypertension], diabetes mellitus, glomerulonephritis, other cause) Yilmaz9 Chronic kidney disease (did not report on cause of kidney disease) Populations with pre-existing heart disease at baseline CLARICOR10 Stable coronary artery disease CORONA11 Chronic heart failure of ischemic cause (NYHA class II to IV, LV ejection fraction ≤40% [≤35% if NYHA II]) Jono12 Stable coronary artery disease PEACE13 Stable coronary artery disease Downloaded from http://ahajournals.org by on September 10, 2018 Pedersen (1)14 Stable angina pectoris Populations with recent acute coronary syndromes at baseline MERLIN-TIMI3615 Non-ST elevation acute coronary syndromes OPTIMAAL16 Acute myocardial infarction complicated with heart failure during the acute phase PLATO17 Acute coronary syndromes PRACSIS18 Acute coronary syndromes (unstable angina, non-ST-segment elevation myocardial infarction, or ST-segment elevation myocardial infarction) Pedersen (2)19 ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention ALERT, Assessment of Lescol in Renal Transplantation Study; CLARICOR, Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease; CORONA, Controlled Rosuvastatin Multinational Trial; CRISIS, Chronic Renal Insufficiency Standards Implementation Study; FINNDIANE, Finnish Diabetic Nephropathy Study; MERLIN-TIMI36, Metabolic Efficiency with Ranolazine for Less Ischemia in Non–STelevation acute coronary syndromes trial; NYHA, New York Heart Association; OPTIMAAL, Optimal Trial in Myocardial Infarction with Angiotensin II Antagonist Losartan; PEACE, Prevention of Events with Angiotensin Converting Enzyme Inhibition Trial; PLATO, PLATelet inhibition and patient Outcomes trial; PRACSIS, Prognosis and Risk in Acute Coronary Syndrome in Sweden. Downloaded from http://ahajournals.org by on September 10, 2018 Figure S1. Funnel plot including artificial studies generated with the ‘trim and fill’ method. Full study names are listed in the footnote of Table S2. The dotted lines show pseudo 95% confidence intervals around the overall pooled estimate. The P value from Egger’s asymmetry test of associations was 0.013. Blue dots indicate artificial studies included by the ‘trim and fill’ method. OPG, osteoprotegerin. Downloaded from http://ahajournals.org by on September 10, 2018 Figure S2. Comparison of combined relative risk for cardiovascular events in the top vs. the bottom tertile of osteoprotegerin concentration of high-risk populations and general population results20. Sizes of data markers indicate the weight of each study in the analysis. Study-specific relative risks were pooled using random-effects meta-analysis. Full names of studies in high-risk populations are listed in the footnote of Table S2. The overall I2 value was 87.0% (P<0.001). BRUN, Bruneck Study; CAIFOS, Calcium Intake Fracture Outcome study; CCHS, Copenhagen City Heart Study; CI, confidence interval; FRAM-OC, Framingham StudyOffspring Cohort; OPG, osteoprotegerin; Tromso, The Tromso Study. Downloaded from http://ahajournals.org by on September 10, 2018 Supplemental References: 1. Anand DV, Lahiri A, Lim E, Hopkins D, Corder R. The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects. J Am Coll Cardiol. 2006;47:1850–1857. 2. Gordin D, Soro-Paavonen A, Thomas MC, Harjutsalo V, Saraheimo M, Bjerre M, Forsblom C, Flyvbjerg A, Groop P-H. Osteoprotegerin is an independent predictor of vascular events in Finnish adults with type 1 diabetes. Diabetes Care. 2013;36:1827– 1833. 3. Svensson M, Dahle DO, Mjoen G, Weihrauch G, Scharnagl H, Dobnig H, Marz W, Jardine A, Fellstrom B, Holdaas H. 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