GALECTIN-3 PREDICTS LONG TERM CARDIOVASCULAR DEATH IN HIGH-RISK CORONARY ARTERY DISEASE PATIENTS Table of Contents List of authors pag 2 Supplemental figure I pag 3 Supplemental figure II pag 4 Supplemental figure III pag 5 Supplemental figure IV pag 6 Supplemental figure V pag 7 Supplemental figure VI pag 8 Supplemental table I pag 9 Supplemental table II pag 10 Supplemental table III pag 11 Supplemental table IV pag 12 Supplemental table V pag 13 Supplemental table VI pag 14 Supplemental table VII pag 15 Supplemental table VIII pag 16 Supplemental table IX pag 17
Giuseppe Maiolino* 2, MD, PhD, Giacomo Rossitto* 2, MD, Luigi Pedon 1, MD, Maurizio Cesari 2, MD, PhD, Anna Chiara Frigo 3, MS, Matteo Azzolini 2, MD, Mario Plebani 4, MD, Gian Paolo Rossi 2, MD. 1 Divisione di Cardiologia Ospedale di Cittadella, Italy 2 Dept. of Medicine -Internal Medicine 4 University of Padua, Italy 3 Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Italy 4 Dept. of Medicine - Laboratory Medicine University of Padua, Italy SUPPLEMENTAL MATERIAL Figures 6, Tables 9 There is no conflict of interest and no financial disclosure concerning this manuscript to be disclosed. * These Authors have equally contributed to this manuscript. Correspondence to: Prof. Gian Paolo Rossi, MD. FACC, FAHA. DIMED- Clinica Medica 4 University Hospital via Giustiniani, 2 35126 Padova, Italy phone: 39-(0)49-821-7821 or 2279 Fax: 39-49-880-2252 E-mail: gianpaolo.rossi@unipd.it 2
Fig. I suppl: Occurrence of Acute Myocardial Infarction (AMI) in the whole cohort (top), CAD patients (mid) and CAD patients with preserved left ventricular ejection fraction (bottom). Kaplan-Meier curves for AMI in the high (dashed line), mid (dotted line) and low (solid line) Gal-3 tertiles. 3
Fig. II suppl: Occurrence of Stroke in the whole cohort (top), CAD patients (mid) and CAD patients with preserved left ventricular ejection fraction (bottom). Kaplan-Meier curves for Stroke in the high (dashed line), mid (dotted line) and low (solid line) Gal-3 tertiles. 4
Fig. III supplemental: Receiver-operating characteristic (ROC) curve for Galectin-3. The ROC analysis for Gal-3 showed a c-statistic of 0.689 (p < 0.0001) (solid line) with a 95% confidence interval of 0.654-0.722 (dotted lines). Y.I.: Youden Index. 5
Fig. IV suppl: Cardiovascular events, CAD patients. Cardiovascular death and events rate in patients with angiographically documented CAD by tertiles of Gal-3 (the absolute number of events is shown above each column). CV deaths X 2 = 26.3, p < 0.001; CV events X 2 = 6.1, p = 0.047; Fatal ischemic events X 2 = 7.8, p = 0.02. CV: cardiovascular. 6
Fig. V suppl: Cardiovascular deaths (top), CV events (mid), and fatal ischemic events (bottom) in the CAD patients. Kaplan-Meier curves show that patients in the high Gal-3 tertile (dashed line) had a significantly higher cardiovascular (CV) deaths, CV events, and fatal ischemic events than the patients in the mid (dotted line) and low (solid line) Gal-3 tertiles. 7
Fig. VI suppl: Cardiovascular events, CAD patients with preserved LVEF. Cardiovascular death and events rate in patients with angiographically proven CAD and preserved LVEF (> 50%) by tertiles of Gal-3 (the absolute number of events is shown above each column). CV deaths X 2 = 4.2, p = 0.118; CV events X 2 = 1.5, p = 0.473; Fatal ischemic events X 2 = 5.0, p = 0.081. CV: cardiovascular. 8
Galectin- 3 Tertile Variable 1 (n=348, 34.4%) 2 (n=329, 32.5%) 3 (n=336, 33.2%) P Clinical condition/hx (%) Hx of stroke 4 (1.2) 5 (1.5) 9 (2.7) = 0.293 Hx of AMI 105 (30.2) 120 (36.6) 124 (36.9) = 0.112 Hx of Bypass 35 (10.1) 32 (9.7) 29 (8.7) = 0.811 Hx of PTCA 34 (9.8) 23 (7.1) 14 (4.2) = 0.016 Medications at baseline (%) Statins 110 (32.3) 95 (29.1) 105 (31.8) = 0.645 Oral antiplatelet agents 252 (73.9) 237 (72.7) 220 (66.7) = 0.088 Calcium channel blockers 136 (39.9) 128 (39.3) 113 (34.2) = 0.259 Beta- blockers 142 (41.6) 122 (37.4) 116 (35.2) = 0.213 ACE inhibitors 138 (40.5) 140 (42.9) 186 (56.4) < 0.001 Heparin 67 (19.6) 84 (25.8) 60 (18.2) = 0.041 Diuretics 69 (20.2) 97 (29.8) 161 (48.8) < 0.001 Digoxin 25 (7.3) 35 (10.7) 60 (18.2) < 0.001 Supplemental Table I. Past medical history and medications at baseline of the whole cohort classified by Galectin-3 tertiles. Results are expressed as absolute number (percentage); comparisons across Galectin-3 tertiles were made by Χ 2. AMI, acute myocardial infarction; Bypass, coronary artery bypass; Hx, history; PTCA, percutaneous transluminal coronary angioplasty; ACE, angiotensin converting enzyme. n = 1013. 9
Galectin- 3 Tertile: CAD Patients Variable 1 (n=267, 34.2%) 2 (n=255, 32.6%) 3 (n=260, 33.2%) P Age (yrs) 61 [54-67]* 65 [57-71] # 69 [63-74] <0.001 Gender M (%) 240 (89.9) 203 (79.6) 193 (74.2) <0.001 Risk factors Non- Smokers/Smokers/Ex (%) 85/51/130 (32/19/49) 96/36/123 (38/14/48) 97/35/128 (37/14/49) =0.306 Hypertension (%)* 142 (53.0) 156 (61.2) 171 (65.8) =0.018 Systolic BP (mmhg) 133 ± 18 135 ± 18 134 ± 18 0.214 Diastolic Diastolic BP (mmhg) BP (mmhg) 78 ± 10 77 ± 9 78 ± 10 0.237 Diabetes (%) 36 (13.5) 37 (14.7) 54 (20.9) =0.021 BMI (Kg/m 2 ) 26.7 ± 3.1 27.0 ± 4.0 26.8 ± 3.6 =0.156 Heart disease Left Ventricular EF (%) 65 [56-71] 64 [54-72] # 59 [47-68] <0.001 Atherosclerotic burden (Duke score) 32 [23-48] 37 [23-48] 37 [23-48] =0.022 Serum Creatinine (µμmol/l) 88 [71-97] 88 [71-97] # 96 [80-115] <0.001 egfr (ml/min) 87.5 ± 26.7* 79.9 ± 24.9 # 70.9 ± 30.5 <0.001 Serum K + (mmol/l) 4.2 ± 0.4 4.2 ± 0.4 4.3 ± 0.5 =0.012 Serum Na + (mmol/l) 140 ± 2 140 ± 2 139 ± 3 <0.001 Serum Glucose (mmol/l) 6.2 ± 2.1 6.2 ± 1.9 6.6 ± 2.3 =0.084 Total Cholesterol (mg/dl) 202 [181-237] 206 [177-234] 199 [173-226] =0.035 HDL- Cholesterol (mg/dl) 46 [40-51] 46 [40-52] # 43 [36-49] =0.003 LDL- Cholesterol (mg/dl) 131 [114-157] 131 [111-154] 131 [109-144] =0.014 Triglycerides (mg/dl) 126 [91-169] 122 [90-163] 126 [93-175] =0.503 Homocysteine (µμmol/l) 11.7 ± 7.2 12.3 ± 7.0 # 14.5 ± 8.5 <0.001 Supplemental Table II. Demographic and clinical characteristics of the CAD subjects classified by Galectin-3 tertiles (n=782). Results are expressed as mean ± SD, absolute numbers (percentage), or median and interquartile range; comparisons across Galectin-3 tertiles were made by ANOVA and Bonferroni tests, after log or square root transformation if needed, or Χ 2, as appropriate. BMI, body mass index; egfr, estimated glomerular filtration rate; K +, potassium; Na +, sodium; BP, Blood Pressure; HDL, high density lipoprotein; LDL, low density lipoprotein; EF, ejection fraction. Bonferroni test: * I vs II tertile, # II vs III tertile, I vs III tertile. P < 0.05 for significance. *Systolic and diastolic BP and BMI values did not differ across Gal-3 tertiles. 10
Galectin- 3 Tertile - CAD patients Variable 1 (n=267, 34.2%) 2 (n=255, 32.6%) 3 (n=260, 33.2%) P Clinical condition/hx (%) Hx of stroke 3 (1.1) 5 (2.0) 5 (1.9) = 0.695 Hx of AMI 93 (34.7) 108 (42.5) 123 (47.3) = 0.012 Hx of Bypass 30 (11.2) 34 (13.3) 26 (10.0) = 0.496 Hx of PTCA 31 (11.7) 22 (8.7) 14 (5.4) = 0.040 Medications at baseline (%) Statins 97 (36.9) 86 (34.0) 94 (36.6) = 0.756 Oral antiplatelet agents 216 (82.1) 201 (79.4) 201 (78.2) = 0.521 Calcium channel blockers 114 (43.3) 108 (42.7) 95 (37.0) = 0.269 Beta- blockers 124 (47.1) 102 (40.3) 106 (41.2) = 0.233 ACE inhibitors 96 (36.5) 103 (40.7) 139 (54.1) < 0.001 Heparin 56 (21.3) 68 (26.9) 55 (21.4) = 0.231 Diuretics 39 (14.8) 62 (24.5) 107 (41.6) < 0.001 Digoxin 10 (3.8) 15 (5.9) 31 (12.1) = 0.001 Supplemental Table III. Past medical history and medications at baseline of the CAD patients classified by Galectin-3 tertiles. Results are expressed as absolute number (percentage); comparisons across Galectin-3 tertiles were made by Χ 2. AMI, acute myocardial infarction; Bypass, coronary artery bypass; Hx, history; PTCA, percutaneous transluminal coronary angioplasty; ACE, angiotensin converting enzyme. N = 782. 11
Galectin- 3 Tertile - CAD pts with LVEF > 50% Variable 1 (n=199, 33.4%) 2 (n=201, 33.8%) 3 (n=195, 32.8%) P Clinical condition/hx (%) Hx of stroke 0 (0.0) 5 (2.5) 4 (2.1) = 0.097 Hx of AMI 59 (29.6) 66 (32.8) 71 (36.4) = 0.361 Hx of Bypass 20 (10.1) 26 (12.9) 19 (9.7) = 0.535 Hx of PTCA 23 (11.7) 21 (10.4) 12 (6.2) = 0.151 Medications at baseline (%) Statins 70 (36.1) 72 (35.8) 64 (33.0) = 0.777 Oral antiplatelet agents 159 (82.0) 164 (81.6) 158 (81.4) = 0.991 Calcium channel blockers 96 (49.5) 97 (48.3) 86 (44.3) = 0.568 Beta- blockers 90 (46.4) 87 (43.3) 84 (43.3) = 0.776 ACE inhibitors 62 (32.0) 70 (34.8) 93 (47.9) = 0.003 Heparin 43 (22.2) 47 (23.4) 45 (23.2) = 0.953 Diuretics 22 (11.3) 38 (18.9) 58 (29.9) < 0.001 Digoxin 5 (2.6) 7 (3.5) 16 (8.2) = 0.019 Supplemental Table IV. Past medical history and medications at baseline of the CAD patients with preserved LVEF classified by Galectin-3 tertiles. Results are expressed as absolute number (percentage); comparisons across Galectin-3 tertiles were made by Χ 2. AMI, acute myocardial infarction; Bypass, coronary artery bypass; Hx, history; PTCA, percutaneous transluminal coronary angioplasty; ACE, angiotensin converting enzyme. 12
Follow- up cases available vs lost Overall model statistics Adjusted R 2 = 0.011 F = 1.246 p = 0.181 Variables in the model β P Variables in the model β P Age - 0.040.453 Left Ventricular EF (%) 0.077.106 Gender 0.034.479 Atherosclerotic burden (Duke score) 0.039.378 Smoke (0N1Y) - 0.039.395 Hx of AMI 0.063.180 BMI 0.026.574 Hx of Bypass - 0.065.123 egfr - 0.011.843 Hx of PTCA - 0.041.317 Serum K + (mmol/l) - 0.074.089 Hx of Diabetes 0.021.712 Serum Na + (mmol/l) 0.073.094 Statins 0.041.339 Serum Glucose (mmol/l) 0.029.619 Oral antiplatelet agents - 0.022.606 HDL- Cholesterol (mg/dl) LDL- Cholesterol (mg/dl) 0.055.198 Beta- blockers 0.005.905-0.040.346 ACE inhibitors 0.003.948 Triglycerides (mg/dl) 0.055.200 Heparin 0.017.714 Systolic Blood Pressure (mmhg) Diastolic Blood Pressure (mmhg) - 0.031.584 Diuretics - 0.018.697-0.049.390 Digoxin 0.005.910 Gal- 3-0.025.591 Supplemental Table V. Multiple regression analysis showing no difference between cases lost and those available at follow-up 13
CV Events HR 95%CI Wald P Model 1 Age ( increase) 1.22 (1.03-1.45) 5.04 = 0.025 LVEF ( increase ) 0.80 (0.70-0.91) 11.07 = 0.001 CAD Duke Index Score 1.12 (1.04-1.21) 9.86 = 0.002 s- K + 1.46 (1.01-2.11) 4.14 = 0.042 Tot cholesterol (Ln increase) 0.39 (0.18-0.83) 5.92 = 0.015 Galectin- 3 (Ln increase) 1.15 (0.77-1.72) 0.447 = 0.504 Model 2 Age ( increase) 1.26 (1.06-1.49) 6.99 = 0.008 LVEF ( increase ) 0.76 (0.67-0.86) 18.83 < 0.001 CAD Duke Index Score 1.09 (1.02-1.17) 6.70 = 0.010 s- K + 1.43 (1.00-2.05) 3.84 = 0.050 Tot cholesterol (Ln increase) 0.37 (0.17-0.77) 6.95 = 0.008 Galectin- 3 (Ln increase) 1.16 (0.78-1.73) 0.507 = 0.477 Supplemental Table VI. Predictors of CV events at Cox regression analysis. Model 1, adjusted for age; gender; left ventricular ejection fraction; coronary atherosclerotic burden (Duke score); total, HDL and LDL (high and low density lipoprotein, respectively) cholesterol; BMI (body mass index); hypertension; diabetes; serum sodium; serum potassium; egfr (estimated glomerular filtration rate); homocysteine; history of myocardial infarction, revascularization by PTCA (Percutaneous transluminal coronary angioplasty), peripheral vascular disease; use of ACE-inhibitors, beta-blockers, diuretics, digoxin, heparin. Model 2, adjusted for aforementioned variables excluding drug therapy. CI, confidence interval; HR, hazard ratio; LVEF, left ventricular ejection fraction; s-na +, serum sodium; ACE, angiotensin converting enzyme. P for significance < 0.05; n = 1013. 14
Fatal Ischemic Events HR 95%CI Wald P Model 1 Age ( increase) 1.73 (1.28-2.35) 12.43 < 0.001 s- Na + 0.87 (0.77-0.98) 5.00 = 0.025 Digoxin 3.55 (1.58-7.94) 9.38 = 0.002 ACE inhibitors 2.21 (1.05-4.63) 4.38 = 0.036 Heparin 0.27 (0.08-0.92) 4.40 = 0.036 Galectin- 3 (Ln increase) 2.28 (1.09-4.74) 4.81 = 0.028 Model 2 Age ( increase) 1.70 (1.29-2.25) 14.09 < 0.001 LVEF ( increase ) 0.70 (0.54-0.91) 6.88 = 0.009 Galectin- 3 (Ln increase) 2.03 (1.01-4.06) 3.94 = 0.047 Supplemental Table VII. Predictors of fatal ischemic events at Cox regression analysis. Model 1, adjusted for age; gender; left ventricular ejection fraction; coronary atherosclerotic burden (Duke score); HDL and LDL (high and low density lipoprotein cholesterol, respectively); BMI (body mass index); hypertension; diabetes; serum sodium; serum potassium; egfr (estimated glomerular filtration rate); homocysteine; history of myocardial infarction, revascularization by PTCA (Percutaneous transluminal coronary angioplasty), peripheral vascular disease; use of ACE-inhibitors, beta-blockers, diuretics, digoxin, heparin. Model 2, adjusted for aforementioned variables excluding drug therapy. CI, confidence interval; HR, hazard ratio; LVEF, left ventricular ejection fraction; s-na +, serum sodium; ACE, angiotensin converting enzyme. P for significance < 0.05; n = 1013. 15
Cardiovascular Mortality - CAD pts HR 95%CI Wald P = Model 1 Age ( increase ) 1.44 (1.12-1.87) 7.79 = 0.005 LVEF ( increase ) 0.68 (0.54-0.86) 10.23 = 0.001 CAD Duke Index Score 1.25 (1.04-1.49) 5.71 = 0.017 History of PTCA 2.43 (1.05-5.65) 4.28 = 0.039 Digoxin therapy 2.49 (1.21-5.16) 6.06 = 0.014 Galectin- 3 (Ln increase) 1.87 (1.04-3.33) 4.42 = 0.036 Model 2 Age ( increase) 1.55 (1.22-1.96) 13.01 < 0.001 LVEF ( increase ) 0.59 (0.47-0.74) 21.26 < 0.001 Diabetes 1.72 (1.01-2.92) 3.98 = 0.046 Galectin- 3 (1 Ln increase) 1.82 (1.04-3.20) 4.33 = 0.037 Supplemental Table VIII. Predictors of cardiovascular mortality at Cox regression analysis in CAD patients. Model 1, adjusted for age; gender; left ventricular ejection fraction; coronary atherosclerotic burden (Duke score); HDL and LDL (high and low density lipoprotein cholesterol, respectively); BMI (body mass index); hypertension; diabetes; serum sodium; serum potassium; egfr (estimated glomerular filtration rate); homocysteine; history of myocardial infarction, revascularization by PTCA, peripheral vascular disease; use of ACE-inhibitors, beta-blockers, diuretics, digoxin, heparin. Model 2, adjusted for aforementioned variables excluding drug therapy. CI, confidence interval; HR, hazard ratio; CAD, coronary artery cardiovascular disease; LVEF, left ventricular ejection fraction, PTCA, percutaneous transluminal coronary angioplasty. P for significance < 0.05; n = 782. 16
Fatal Ischemic Events - CAD pts with LVEF > 50% HR 95%CI Wald P Model 1 History of PTCA 7.94 (2.38-26.32) 11.33 = 0.001 Digoxin 13.18 (2.72-62.50) 10.28 = 0.001 ACE inhibitors 3.88 (1.28-11.77) 5.70 = 0.017 Galectin- 3 (Ln increase) 5.44 (1.86-15.93) 9.53 = 0.002 Model 2 History of PTCA 5.99 (2.11-16.95) 11.36 = 0.001 Galectin- 3 (Ln increase) 5.65 (1.96-16.28) 10.29 = 0.001 Supplemental Table IX. Predictors of fatal ischemic events at Cox regression analysis in CAD patients with preserved ejection fraction. Model 1 adjusted for age; gender; left ventricular ejection fraction; coronary atherosclerotic burden (Duke score); HDL and LDL (high and low density lipoprotein cholesterol, respectively); BMI (body mass index); hypertension; diabetes; serum sodium; serum potassium; egfr(estimated glomerular filtration rate); homocysteine; history of myocardial infarction, revascularization by PTCA (Percutaneous transluminal coronary angioplasty), peripheral vascular disease; use of ACE-inhibitor/ angiotensin-ii type-1 receptor blockers, beta-blockers, diuretics, digoxin, heparin. Model 2, adjusted for aforementioned variables excluding drug therapy. CI, confidence interval; HR, hazard ratio. PTCA, percutaneous transluminal coronary angioplasty. P for significance < 0.05; n = 595. 17