Coronary Vasomotor Control in Obesity and Morbid Obesity

JACC: CARDIOVASCULAR IMAGING VOL. 5, NO. 8, 212 212 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION ISSN 1936-878X/$36. PUBLISHED BY ELSEVIER INC. htt://dx.doi.org/1.116/j.jcmg.212.1.2 Coronary Vasomotor Control in Obesity and Morbid Obesity Contrasting Flow Resonses With Endocannabinoids, Letin, and Inflammation Alessandra Quercioli, MD,* Zoltan Pataky, MD, Fabrizio Montecucco, MD,* Sebastian Carballo, MD, Aurélien Thomas, PHD, Christian Staub, PHD, Vincenzo Di Marzo, PHD, Gabriella Vincenti, MD,* Giusee Ambrosio, MD, Osman Ratib, MD,# Alain Golay, MD, François Mach, MD,* Elisabetta Harsch, MD, Thomas H. Schindler, MD* Geneva, Switzerland; and Nales and Perugia, Italy OBJECTIVES This study sought to investigate abnormalities in coronary circulatory function in 2 different disease entities of obese (OB) and morbidly obese (MOB) individuals and to evaluate whether these would differ in severity with different rofiles of endocannabinoids, letin, and C-reactive rotein (CRP) lasma levels. BACKGROUND There is increasing evidence that altered lasma levels of endocannabinoids, letin, and CRP may affect coronary circulatory function in OB and MOB. METHODS Myocardial blood flow (MBF) resonses to cold ressor test from rest and during harmacologically induced hyeremia were measured with N-13 ammonia ositron emission tomograhy/comuted tomograhy. Study articiants (n 111) were divided into 4 grous based on their body mass index (BMI) (kg/m 2 ): 1) control grou (BMI: 2 to 24.9, n 3); 2) overweight grou (BMI: 25 to 29.9, n 31), 3) OB grou (BMI: 3 to 39.9, n 25); and 4) MOB grou (BMI 4, n 25). RESULTS The cold ressor test induced change in endothelium-related MBF resonse ( MBF) rogressively declined in overweight and OB grous when comared with the control grou [median:.19 (interquartile range [IQR].8,.27) and.11 (.3,.17) vs..27 (.23,.38) ml/g/min;.1, resectively], whereas it did not differ significantly between OB and MOB grous [median:.11 (IQR:.3,.17) and.9 (.1,.19) ml/g/min;.93]. Comared with control subjects, hyeremic MBF subjects comarably declined in the overweight, OB, and MOB grous [median: 2.4 (IQR 1.92, 2.63) vs. 1.94 (1.65, 2.3), 2.5 (1.67, 2.38), and 2.14 (1.78, 2.76) ml/g/min;.5, resectively]. In OB individuals, MBF was inversely correlated with increase in endocannabinoid anandamide (r.45,.44), but not with letin (r.2,.946) or with CRP (r.33,.168). Conversely, there was a significant and ositive correlation among MBF and elevated letin (r.43,.31) and CRP (r.55,.6), resectively, in MOB individuals that was not observed for endocannabinoid anandamide (r.7,.74). CONCLUSIONS Contrasting associations of altered coronary endothelial function with increases in endocannabinoid anandamide, letin, and CRP lasma levels identify and characterize OB and MOB as different disease entities affecting coronary circulatory function. (J Am Coll Cardiol Img 212;5: 85 15) 212 by the American College of Cardiology Foundation From the *Deartment of Secialties in Medicine, Division of Cardiology, University Hositals of Geneva, Geneva, Switzerland; Service of Theraeutic Education for Chronic Diseases, University Hositals of Geneva, Geneva,

86 JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 ABBREVIATIONS AND ACRONYMS 2-AG 2-arachidonoylglycerol AEA anandamide ANOVA analysis of variance BMI body mass index CAC coronary artery calcification CAD coronary artery disease CI confidence interval CON control subject(s) CPT cold ressor test CT comuted tomograhy CVR coronary vascular resistance EC endocannabinoid(s) HDL high-density liorotein hscrp high-sensitivity C-reactive rotein MBF myocardial blood flow MFR myocardial flow reserve MOB morbid obesity OB obesity OW overweight PET ositron emission tomograhy RPP rate-ressure roduct SBP systolic blood ressure As obesity has been recognized as a risk factor of cardiovascular morbidity and mortality, the worldwide global eidemic of obesity with an increasing revalence, not only in adults but also among children and adolescents, has raised major health concerns (1). Recent investigations have demonstrated a close association between obesity and an abnormal function of the coronary circulation (2,3), which is commonly regarded as a functional recursor of the coronary artery disease (CAD) rocess (4,5). Increasing attention has been directed to a novel concet that lasma roteins See age 816 originating from the adiose tissue, socalled adiocytokines, such as adionectin, letin, and/or endocannabinoids (EC) as well as metabolically triggered inflammation may alter vascular function (6 8). Whereas adionectin has been widely areciated to beneficially affect nitric oxide mediated, endothelium-deendent vasomotion (6), the role of letin altering coronary circulatory function in humans remains controversial (2). As regards the role of EC in obesity, we have shown recently that increases in EC lasma levels such as of anandamide (AEA) and 2- arachidonoylglycerol (2-AG) are inversely associated with coronary circulatory dysfunction in obese individuals (3). This would suggest that elevated EC lasma levels exert adverse effects on the function of the coronary circulation (4). Systemic inflammation also seems to lay an imortant role in altering coronary circulatory function in cardiovascular risk individuals (9,1). Conversely, its role in affecting the function of the coronary circulation in individuals with an excess of body weight still remains a matter of ongoing debate (2,11). Notably, obesity and morbid obesity have been suggested to reresent 2 different disease entities with differences in adiocytokine rofile, liid and glucose metabolism, and systemic inflammation rather than a disease continuum (1,7). With this in mind, we aimed to investigate the resence of abnormalities in coronary circulatory function in 2 different disease entities of obese and morbidly obese individuals and to evaluate whether these would differ in severity with different rofiles of EC, letin, and high-sensitivity C-reactive rotein (hscrp) lasma levels. METHODS Study oulation and design. The study oulation consisted of 3 normal weight control subjects (CON) (body mass index [BMI]: 2 to 24.9 kg/m 2 ), 31 overweight (OW) (BMI: 25 to 29.9 kg/m 2 ), and 5 obese individuals (BMI: 3 kg/m 2 ) without arterial hyertension, smoking, and diabetes mellitus (Table 1) (3). The obese grou was subsequently subdivided according to their BMI into common obesity (OB) (BMI: 3. to 39.9 kg/m 2,n 25) and morbid obesity (MOB) (BMI: 4 kg/m 2,n 25). Whereas in the OB grou, no individuals had hyercholesterolemia, 4 individuals in the MOB grou had cholesterol lasma levels mildly above 24 mg/dl. Study alicants had been recruited by flyers and newsaer advertisements. A study rerequisite was the absence of any cardiac or vasoactive medication, a history of variant angina, a family history of remature CAD, or clinically manifested cardiovascular or any other systemic disease. The alicants subsequently underwent an initial screening visit that comrised a hysical examination, Switzerland; Deartment of Internal Medicine, Service of General Internal Medicine, University Hositals of Geneva, Geneva, Switzerland; Unit of Toxicology, Centre Universitaire Romand de Médecine Légale, University Hositals of Geneva, Geneva, Switzerland; Endocannabinoid Research Grou, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Nales, Italy; and the University of Perugia, School of Medicine, Division of Cardiology, Perugia, Italy; #Deartment of Medical Imaging and Information Science, Division of Nuclear Medicine, University Hosital of Geneva, Geneva, Switzerland. Suorted by research grant nos. 32B-122237 (Dr. Schindler) and 322B-134963 (Dr. Montecucco) from the Swiss National Science Foundation, with contributions of the Clinical Research Center, University Hosital, and Faculty of Medicine, Geneva and the Louis-Jeantet Foundation (Dr. Schindler); Swiss Heart Foundation (Dr. Schindler); the Sir Jules Thorn Trust Reg fund and Gustave and Simone Prévot fund (Dr. Montecucco); and fellowshi grants from the Novartis Foundation (Dr. Quercioli), and the Euroean Society of Cardiology and the Italian Society of Cardiology (Dr. Vincenti). Dr. Ambrosio has consulted for Menarini International, Merck, Schering-Plough, Angelini, and has served on the Seakers Bureau for Boehringer. All other authors have reorted that they have no relationshis relevant to the contents of this aer to disclose. Manuscrit received November 15, 211; revised manuscrit received January 24, 212, acceted January 26, 212.

JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 87 Table 1. Characteristics of Study Poulation (N 111) CON (n 3) OW (n 31) OB (n 25) MOB (n 25) BMI, kg/m 2 21.9 (2.7, 22.9) 26.7 (25.9, 28.2).1 31.7 (31., 34.9).1 45. (43.2, 47.5).1 Waist/hi ratio.85 (.81,.89).9 (.85,.93).19.94 (.91,.98).1.97 (.84, 1.6).1 Waist, cm 83. (8., 85.) 98. (92.62, 13.).1 111. (16., 117.5).1 124. (116., 14.2).1 Total fat amount, g 12,872 (1,59, 15,961) 2,341. (17,323, 23,628).1 29,31 (27,328, 35,281).1 6,871 (5,9, 66,44).1 Percentage of body fat 19.7 (14.8, 26.1) 23.6 (21.5, 28.3).8 29.7 (28.5, 31.4).1 47.8 (4.9, 5.7).1 Android/gynoid fat mass.89 (.8, 1.18) 1.43 (1.12, 1.89).1 1.59 (1.42, 1.83).1 1.69 (1.19, 2.19).1 Central fat distribution.4 (.38,.45).48 (.43,.54).1.52 (.5,.56).1.56 (.46,.61).1 Age, yrs 4 (3, 46) 44 (33, 51).159 44 (36, 57).158 42 (35, 54).233 Female/male 9/12 11/19 8/17 1/15 AEA, ng/ml.56 (.49,.71).52 (.42,.67).267.65 (.5,.73).179.7 (.58,.77).2 2-AG, ng/ml 1.5 (.6, 5.47) 1.4 (.72, 2.9).65 1.92 (1.21, 2.66).755 2.67 (1.37, 5.23).576 Adionectin, g/ml 3.78 (2.3, 5.4) 3.25 (1.67, 5.27).43 2.76 (.5, 4.5).25 2.11 (.4, 3.6).41 Letin 5.47 (2.3, 11.2) 12.66 (8.4, 25.84).1 16.3 (9.4, 22.9).1 11.5 (59.8, 147.5).1 Liid status Cholesterol levels, mg/dl 191 (168, 23) 27 (17, 23).723 211 (172, 226).533 187 (158, 213).96 LDL level, mg/dl 122 (15, 145) 129 (111, 152).41 122 (99, 152).899 119 (13, 137).969 HDL level, mg/dl 58 (41, 66) 46 (39, 55).1 44 (37, 49).8 42 (39, 46).17 Triglyceride level, mg/dl 54 (48, 11) 93 (67, 12).12 123 (95, 191).1 122 (83, 167).1 Glucose level, mg/dl 93 (82, 11) 97 (88, 13).15 97 (92, 18).2 13 (96, 118).11 Insulin, mui/l 3.2 (1.9, 5.2) 6.1 (3.3, 1.9).4 8.1 (5.2, 1.6).1 28.6 (17.3, 38.1).1 HOMA.7 (.44, 1.3) 1.4 (.7, 2.8).8 1.9 (1.2, 3.9).1 8.3 (4.5, 13.2).1 hscrp levels, mg/l.9 (.9, 3.) 1. (.9, 2.).847 2.9 (1.4, 5.2).8 8. (4.9, 13.).1 HbA lc, % 5.2 (4.9, 5.5) 5.2 (5., 5.4).961 5.4 (5., 5.7).338 5.45 (5.4, 5.7).8 s are median (Q1, Q3). Central fat distribution is calculated as trunk fat mass/total body fat mass 1. P values versus CON (Mann-Whitney U test for indeendent samles). 2-AG 2-arachidonoylglycerol; AEA anandamide; BMI body mass index; CON control subjects; HbA lc glycosylated hemoglobin; HDL high-density liorotein; HOMA Homeostasis Model Assessment; hscrp high-sensitivity C-reactive rotein; LDL low-density liorotein; MOB morbid obesity; OB obesity; OW overweight; Q quartile. electrocardiogram, blood ressure measurements, and routine blood chemistry in a fasting state. Physical examination revealed normal findings in all alicants and they had normal resting electrocardiograms. Each study articiant then underwent dual x-ray absortiometry (Hologic QDR45A, Hologic, Bedford, Massachusetts) to measure body comosition, total fat amount burden, and fat distribution as described reviously (12). Subsequently, N-13 ammonia ositron emission tomograhy (PET)/comuted tomograhy (CT) measurements of myocardial blood flow (MBF) at rest and during vasomotor stress were erformed in a fasting state to assess coronary circulatory function. Twenty-one control (CON), 26 OW, 17 OB, and 13 MOB subjects were art of a revious investigation assessing the effect of elevated EC lasma levels in obesity on coronary circulatory function (3). Routine blood chemistry of EC such as AEA and 2-AG, letin and adionectin lasma levels were determined (3). The study was aroved by the University Hositals of Geneva Institutional Review Board (No. 7-183), and each articiant signed the aroved informed consent form. Assessment of coronary artery calcification and myocardial erfusion with PET/CT. Prior to PET flow studies, we used 64-slice multidetector comuted tomograhy of the Biograh HiRez TruePoint PET/CT scanner (Siemens, Erlangen, Germany) to determine coronary artery calcium score (13). Following, myocardial erfusion was determined with N-13 ammonia PET/CT (3). In all study articiants, visual evaluation and olar ma analysis of the N-13 ammonia distribution at rest and during vasomotor stress revealed homogeneous tracer utake. In addition, MBF was determined in ml/g/min from serial transaxial N-13 ammonia PET/CT image acquisition in conjunction with a 2-comartment tracer kinetic model (3). N-13 ammonia PET/CT determined leftventricular MBFs at rest, then during the cold ressor test (CPT), and during harmacologically induced hyeremia with standard infusion of diyridamole (14 mg/kg/min) (3,4). Heart rate, blood ressure, and a 12-lead electrocardiogram were recorded continuously during each MBF measurement. From the average of heart rate and systolic blood ressure during the first 2 min of

88 JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 each image acquisition, the rate-ressure roduct (RPP) was derived as an index of cardiac work. To account for ossible interindividual variations in coronary driving ressure, an index of global coronary vascular resistance (CVR) was determined as the ratio of mean arterial blood ressure (mm Hg) to MBF (ml/g/min). In addition, MBF was normalized to the RPP, and thus myocardial work (averaged during the first 2 min of image acquisition; MBF divided by RPP multilied by 1 3 ) was determined. Statistical analysis. Because continuous variables are not always normally distributed, they are resented as median and interquartile range (25th to 75th ercentile: quartile 1, quartile 3). For comarison of differences, we used the Mann-Whitney U test for indeendent samles (SAS Institute, Cary, North Carolina). A comarison of CPT-induced change in MBF and diyridamole MBFs among the different grous was erformed by 1-way analysis of variance (ANOVA) followed by Scheffe multile comarison test. Pearson correlation coefficients (r), assuming a linear regression and the standard error of the estimate (SEE), were calculated to investigate the associations between CPT- and diyridamoleinduced changes in MBFs and laboratory arameters. Multivariate analysis was erformed with stewise forward regression of variables with significance on univariate analysis. All test rocedures were 2-tailed, and.5 was considered statistically significant. RESULTS Patient characteristics and metabolic rofile. The clinical characteristics, anthroometrical measurements, and dual x-ray absortiometry determined total fat amount burden and fat distribution of the study grous are given in Table 1. For the entire study oulation, coronary artery calcification (CAC) was found in 14% (15 of 111). In the CON grou, the revalence of CAC was 17% (5 of 3) with a coronary artery calcium score of 6.8 6.7. Conversely, in the OW, OB, and MOB grous only 12% (4 of 31), 16% (4 of 25), and 8% (2 of 25) had CAC with coronary artery calcium score of 96.5 94.4, 11.9 9.2, and 57.7 27.4, resectively. Regarding the CAC distribution, the highest revalence was in the left anterior descending artery (n 14), followed by the right coronary (n 7) and left circumflex (n 5) arteries, resectively. Correlations among fat arameters, EC, and adiocytokines. In the entire study oulation, Pearson regression analysis denoted a significant correlation between BMI and total fat amount as estimated by dual x-ray absortiometry (r.95;.1). Significant and ositive correlations were noted among BMI, total fat, ercentage of body fat, or trunk fat amount and letin, AEA, and hscrp, resectively, whereas they correlated inversely with adionectin (Table 2). Further, central fat distribution, as the trunk fat and total fat ratio, was significantly associated with letin and AEA and Table 2. Entire Study Poulation Letin, ng/ml Adionectin, g/ml AEA, ng/ml 2-AG, ng/ml hscrp, mg/l Waist, cm r.54 r.2 r.16 r.16 r.31.1.133.24.22.13 Waist/hi ratio r.17 r.24 r.24 r.24 r.8.168.43.43.4.479 BMI, kg/m 2 r.69 r.29 r.35 r.14 r.54.1.3.1.167.1 Total fat amount, g r.73 r.23 r.33 r.13 r.59.1.19.1.27.1 Percentage of body fat r.75 r.15 r.21 r.9 r.62.1.135.34.39.1 Trunk fat amount, g r.74 r.27 r.41 r.19 r.49.1.8.1.68.1 Central fat distribution r.27 r.23 r.32 r.14 r.6.1.25.1.175.596 Android/gynoid fat mass r.12 r.21 r.37 r.17 r.7.258.44.1.18.537 Central fat distribution trunk fat mass/total fat mass 1. Android/gynoid fat mass trunk fat amount/leg fat amount. Abbreviations as in Table 1.

JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 89 inversely with adionectin. As regards the android/ gynoid fat mass distribution, it significantly correlated with AEA and inversely with adionectin. The waist circumference was significantly associated with letin and hscrp, whereas the waist/hi ratio correlated inversely with adionectin and ositively with AEA and 2-AG, resectively. As regards the OB grou, only central fat distribution and android/gynoid fat mass ratio were inversely correlated with letin (r.58;.11 and r.53;.24), whereas android/gynoid fat mass ratio also correlated ositively with AEA (r.51;.3). In the MOB grou again, there were significant associations among total fat amount and AEA (r.52;.11) and 2-AG (r.45;.31), whereas ercentage of body fat correlated with letin (r.43;.35) and hscrp (r.62;.2), resectively. Also, trunk fat amount correlated with adionectin (r.43;.47), AEA (r.65;.1), and 2-AG (r.66;.1), resectively. Finally, there were also significant associations among android/gynoid fat mass distributions and letin (r.49;.2), and 2-AG (r.48;.23). Hemodynamic arameters. At baseline, heart rate did not differ significantly among the CON, OW, and OB grous, but it was significantly higher in the MOB grou (Table 3). Systolic blood ressure (SBP) among grous was comarable. Due to the higher heart rate in the MOB grou, however, the resting RPP was significantly higher in the MOB subjects than in CON and OW subjects, whereas it did not differ significantly between the OB and MOB grous. Symathetic stimulation with CPT induced a significant increase in heart rate and SBP among grous, so that the RPP was significantly higher during CPT than at baseline. The increase in the RPP ( RPP) as a result of the CPT-induced Table 3. MBF and Hemodynamic Findings During PET/CT Exam Grous CON OW OB MOB MBF, ml/min/g MBF at rest.69 (.65,.75).7 (.62,.76).761.68 (.57,.79).914.83 (.71,.98).3 NMBF at rest.92 (.85, 1.13).96 (.83, 1.9).332.88 (.78, 1.3).25.98 (.9, 1.17).382 MBF during CPT 1. (.89, 1.18).86 (.78, 1.4).26.75 (.71,.9).1.93 (.81, 1.8).141 MBF to CPT from rest.27 (.23,.38).19 (.8,.27).5.11 (.3,.17).1.9 (.1,.19).1 NMBF during CPT 1.5 (.92, 1.15).93 (.84, 1.6).53.78 (.71,.87).1.77 (.71,.98).1 MBF during hyeremia 2.4 (1.92, 2.63) 1.94 (1.65, 2.3).6 2.5 (1.67, 2.38).14 2.14 (1.78, 2.42).53 MFR 3.4 (2.97, 3.82) 2.88 (2.31, 3.27).7 3.15 (2.36, 3.28).5 2.53 (2.13, 2.76).1 MBF hyeremia/nmbf rest 2.33 (1.94, 2.7) 1.93 (1.57, 2.52).55 2.18 (1.26, 2.76).12 2.4 (1.76, 2.4).1 CVR, mm Hg/ml/min/g At rest 117 (12, 134) 13 (116, 138).45 119 (19, 149).357 14 (94, 128).99 During CPT 96 (76, 11) 117 (99, 134).2 132 (117, 151).1 119 (99, 138).1 Change to CPT from rest 23 ( 33, 15) 9( 31, 4).49 6 ( 9, 13).1 7 ( 4, 18).1 Pharmacologic vasodilation 33 (27, 39) 43 (35, 5).1 43 (37, 5).1 43 (37, 55).2 Hemodynamics at rest Heart rate, beats/min 62 (54, 7) 63 (55, 68).928 66 (57, 73).48 67 (66, 74).6 SBP, mm Hg 114 (13, 123) 12 (113, 128).51 122 (16, 133).233 12 (11, 132).21 RPP 6,788 (6,159, 7,756) 7,134 (6,527, 8,24).44 7,488. (6,738, 9,21).14 8,284 (7,293, 8,964).3 CPT Heart rate, beats/min 72 (64, 83) 69 (6, 78).26 72 (62, 77).456 76 (7, 84).224 SBP, mm Hg 134 (122, 141) 136 (127, 148).324 148 (126, 156).88 148 (141, 158).3 RPP 9,98 (8,511, 1,913) 9,525 (8,34, 1,764).98 9,45 (8,628, 11,53).597 11,376 (1,293, 12,528).3 RPP, CPT-rest 2,448 (1,811, 3,27) 1,92 (1,329, 2,951).148 1,786 (1,336, 3,279).597 3,96 (1,84, 4,75).285 Pharmacologic vasodilation Heart rate, beats/min 84 (73, 92) 84 (8, 91).669 87 (81, 95).461 95 (83, 12).65 SBP, mm Hg 111 (13, 118) 117 (111, 122).31 12 (111, 129).1 12 (118, 131).2 RPP 8,91 (8,129, 1,375) 9,84 (8,692, 1,998).125 1,512 (9,366, 11,538).13 11,88 (1,438, 12,365).1 RPP, harmacologic-rest 1,887 (1,13, 2,668) 2,147 (1,549, 3,738).349 2,392 (1,648, 4,51).297 3,324 (2,35, 4,314).19 s are median (Q1, Q3). values versus CON (Mann-Whitney U test for indeendent samles). CPT cold ressor test; CT comuted tomograhy; CVR coronary vascular resistance; MBF myocardial blood flow; MFR myocardial flow reserve; NMBF normalized myocardial blood flow; PET ositron emission tomograhy; RPP rate-ressure roduct; SBP systolic blood ressure; other abbreviations as in Table 1.

81 JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 symathetic stimulation was not significantly different among the CON, OW, OB, and MOB subjects (Table 3), signifying comarable increases in myocardial workload among the study grous. During harmacologic vasodilation with diyridamole to induce hyeremic flows, the heart rate significantly increased among study grous, whereas SBP mildly decreased in the CON, OW, and OB grous and remained unchanged in the MOB grou. The change in RPP ( RPP) during diyridamole stimulation was comarable among the study grous (Table 3). Myocardial blood flow. At baseline, MBF was comarable among the CON, OW, and OB grous, but it was significantly higher in the MOB grou (Table 3). When adjusted for RPP, the normalized MBF at rest did not differ significantly among grous. The endothelium-related change of MBF during CPT from rest ( MBF to CPT) and the normalized MBF during CPT were significantly less in the OW, OB, and MOB grous than in the CON grou, whereas they were comarable between the OB and MOB grous, resectively (Table 3). The grou comarison between the MBF to CPT and normalized MBF during CPT in CON subjects was significant as comared with OW, OB, and MOB subjects, resectively (.1 by ANOVA). In addition, changes of CVR widely mirrored those of MBF for each study grou (Table 3). Thus, differences in hemodynamic resonses can be widely ruled out as a ossible cause for the observed alteration in MBF during CPT. Diyridamole-stimulated hyeremic MBFs were significantly lower in OW, OB, and MOB subjects than in CON subjects (Table 3, Fig. 1). The MFR, or total coronary vasodilator caacity, declined from the CON grou to the OW and OB grous, but was comarable between the OW and OB grous. In addition, the MFR was significantly less in the MOB grou than in the CON, OW, and OB grous, resectively. The grou comarison of diyridamole MBFs and MFR in the CON grou was significantly different from those in the OB and MOB grous (.15 and.2 by ANOVA). The CVR during diyridamole stimulation, accounting for ossible interindividual variations in coronary driving ressure, was significantly higher in the OW, OB, and MOB grous than in the CON grou and thus confirmed the reduced vasodilatory caacity in individuals with increasing body weight (Table 3). Also here, the grou comarison of CVR during diyridamole stimulation in the CON grou was significantly different from those in the OW, OB, and MOB grous (.2 by ANOVA). Figure 1. N-13 Ammonia PET/CT Perfusion Images Examle of a normal stress and rest myocardial erfusion study on short-, horizontal-, and vertical-axis images of N-13 ammonia ositron emission tomograhy (PET)/comuted tomograhy (CT) in a 55-year-old morbidly obese woman (body mass index: 43 kg/m 2 ). Conversely, the myocardial flow reserve (MFR myocardial blood flow [MBF] stress/mbf rest) of 1.91 is mildly reduced, signifying an imairment of the coronary vasodilatory caacity.

JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 811 Determinants of MBF to CPT and hyeremic MBF. In the entire study oulation, on univariate analysis age, total fat amount, sex, AEA, 2-AG, SBP, and triglyceride lasma levels were inversely, whereas HDL cholesterol was ositively correlated with endothelium-related changes in MBF from rest to CPT ( MBF) (Table 4). By multivariate analysis, only total fat amount and sex remained indeendently associated with MBF (beta:.38 [95% confidence interval (CI):.1 to.1];.1, and beta:.338 [95% CI:.212 to.46],.3). Further, hyeremic MBFs correlated inversely with age, sex, AEA, and 2-AG, but ositively with HDL cholesterol lasma levels, resectively. Conversely, only age, sex, and 2-AG lasma levels roved to be indeendently associated with hyeremic MBFs, resectively (beta:.234 [95% CI:.18 to.2],.16; beta:.22 [95% CI:.48 to.7];.43, and beta:.194 [95% CI: 1.61 to.9];.54). As regards the OB grou, the univariate analysis demonstrated sex, AEA, and 2-AG lasma levels as well as SBP to be significantly and inversely associated with MBF (Table 5, Fig. 2A). By multivariate analysis, only 2-AG lasma levels were indeendently associated with MBF (beta:.484 [95% CI:.21 to.1];.3). Further, univariate analysis showed hyeremic MBFs to be significantly associated only with 2-AG lasma levels. Conversely, the univariate analysis in MOB (Table 5, Fig. 2B) demonstrated letin and hscrp lasma levels to be significantly and ositively associated with MBF. By multivariate analysis, only increases in hscrp lasma levels were associated with MBF in an indeendent fashion (beta:.522 [95% CI:.2 to.13];.14). Regarding the hyeremic MBFs, they correlated with total fat amount, sex, SBP, HDL cholesterol, and hscrp lasma levels, resectively, whereas on multivariate analysis only sex was indeendently associated with MBFs during diyridamole stimulation (beta:.459 [95% CI:.691 to.61];.22). DISCUSSION The results of the current study rovide several new findings. At first, there was a rogressive decrease in endothelium-deendent MBF resonses to CPT from normal weight CON subjects to OW and OB subjects, whereas it did not differ significantly between the OB and MOB grous. In addition, hyeremic MBFs were comarably altered among grous with increases in body weight. Thus, desite marked increases in body weight from OB to MOB subjects, there was no further rogressive worsening of coronary circulatory function. Secondly, increases in EC lasma levels of AEA and 2-AG were inversely associated with an imairment of coronary endothelial function in OB subjects, which is sug- Table 4. Entire Study Poulation MBF to CPT Univariate Analysis Hyeremic MBF Coefficient Beta (95% CI) Coefficient Beta (95% CI) Age, yrs.22 (.6 to.1).37.286 (.2 to.4).3 Total fat amount, g.37 (.1 to.1).1.74 (.1 to.1).457 Male.252 (.164 to.24).9.299 (.496,.116).2 AEA, ng/ml.295 (.483 to.14).3.234 ( 1.165 to.16).19 2-AG, ng/ml.22 (.18 to.1).28.21 (.48 to.2).37 Adionectin, g/ml.171 (.2 to.24).87.177 (.4 to.67).78 Letin, ng/ml.152 (.1 to.1).128.38 (.2 to.2).74 SBP, mm Hg.23 (.6 to.1).36.147 (.14 to.2).133 Total cholesterol, mg/dl.8 (.1 to.1).424.26 (.2 to.3).797 LDL cholesterol, mg/dl.17 (.2 to.1).297.34 (.3 to.2).74 HDL cholesterol, mg/dl.239 (.1 to.5).15.278 (.3 to.15).4 Triglycerides, mg/dl.295 (.1 to.1).3.95 (.2 to.1).346 Glucose, mg/dl.98 (.2 to.1).322.95 (.5 to.2).34 HOMA.2 (.2 to.1).74.98 (.43 to.17).388 hscrp, mg/l.114 (.9 to.2).255.12 (.15 to.17).96 values determined by analysis of variance. CI confidence interval; other abbreviations as in Tables 1 and 3.

812 JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 Table 5. Obesity and Morbid Obesity Grous OB MOB MBF to CPT Hyeremic MBF MBF to CPT Hyeremic MBF Coefficient Beta (95% CI) Coefficient Beta (95% CI) Coefficient Beta (95% CI) Coefficient Beta (95% CI) Age, yrs.27 (.8 to.2).257.31 (.34 to.7).19.27 (.8 to.2).194.8 (.19 to.24).775 Total fat amount, g.12 (.1 to.1).625.27 (.1 to.1).28.7 (.1 to.1).748.67 (.1 to.1).28 Male.46 (.498 to.9).43.32 ( 1.723 to.353).182.3 (.194 to.29).14 1.22 ( 1.379 to.537).2 AEA, ng/ml.45 (.66 to.1).44.18 ( 2.53 to.961).455.7 (.386 to.278).74.17 ( 1.238 to.462).293 2-AG, ng/ml.63 (.23 to.5).4.46 (.8 to.1).57.26 (.56 to.5).224.19 (.13 to.44).223 Adionectin, g/ml.5 (.26 to.26).985.2 (.98 to.15).944.33 (.26 to.6).112.19 (.125 to.37).22 Letin, ng/ml.2 (.7 to.6).946.1 (.31 to.21).698.43 (.1 to.2).31.13 (.5 to.3).483 SBP, mm Hg.51 (.9 to.1).21.11 (.25 to.16).654.14 (.7 to.4).515.81 (.17 to.41).2 Total cholesterol, mg/dl.18 (.2 to.1).462.1 (.6 to.6).992.1 (.1 to.1).978 4.15 (.53 to.135).353 LDL cholesterol, mg/dl.24 (.2 to.1).344.3 (.6 to.6).915. (.2 to.2).991.39 (.12 to.3).158 HDL cholesterol, mg/dl.5 (.5 to.7).838.21 (.14 to.33).41.12 (.4 to.7).584.44 (.26 to.3).22 Triglycerides, mg/dl.8 (.1 to.1).74.6 (.4 to.3).85.23 (.2 to.1).298.22 (.1 to.5).171 Glucose, mg/dl.2 (.2 to.2).95.1 (.9 to.6).629.29 (.1 to.3).183.19 (.3 to.3).347 HOMA.11 (.37 to.24).657.4 (.117 to.133).888.12 (.9 to.14).612.13 (.35 to.16).396 hscrp, mg/l.33 (.22 to.4).168.34 (.87 to.16).163.55 (.2 to.13).7.34 (.3 to.24).2 values determined by analysis of variance. Abbreviations as in Tables 1, 3, and 4. gestive of adverse effects of ECs on the coronary endothelium, but this association was not more observed in MOB subjects. Thirdly, elevations in letin and hscrp lasma levels were correlated with endothelium-related MBF resonses to CPT in the MOB grou, although ositively, whereas there was no such association in the OB grou. Increases in letin and hscrp lasma levels therefore were associated with relatively higher, though diminished, endothelium-mediated MBF increases to CPT in the MOB grou, stressing some beneficial effects on coronary endothelial function. These contrasting associations of altered coronary endothelial function with increases in AEA, letin, and hscrp lasma levels, however, confirm the hyothesis that OB and MOB reflect 2 different disease entities, rather than a simle continuation of increases in body weight, affecting coronary circulatory function. Metabolic rofile. As exected, total cholesterol and low-density liorotein cholesterol were similar among grous with increasing body weight (1). Conversely, HDL cholesterol rogressively decreased and triglyceride increased from CON subjects to OW and OB subjects, but no further alterations were noted between the OB and MOB grous. When regarding lasma markers of the insulin-resistance syndrome and chronic inflammation, they also continuously increased but across the whole sectrum of increasing body weight. Notably, aart from glucose lasma levels, there was a striking and nonlinear increase in insulin resistance, lasma levels of insulin, and hscrp from the OB to MOB grous. Consequently, a marked increase of body weight from the OB to MOB grous did not further alter the liid rofile, HDL cholesterol, or triglycerides, whereas lasma glucose levels were only mildly affected. This observation can be exlained by differences in adiose tissue distribution and metabolic activity between the OB and MOB grous. Visceral adiose tissue accumulation exerts more deleterious metabolic effects than subcutaneous fat accumulation does, mainly due to the higher fatty acid suly from the abdominal area, which contributes to metabolic abnormalities (1). Contrary to intra-abdominal fat deots, subcutaneous adiose tissue accumulation may be rather rotective owing to a lower liolytic resonse to catecholamines, a higher antiliolytic sensitivity to insulin, and an enhanced liorotein liase activity (14). Also, distinct visceral adiose tissue, such as round ligament and mesenteric adiose tissues, which ose a higher sensibility to effects of insulin in stimulating liorotein liase activity and liogenesis and in inhibiting liolysis, is likely to have contributed to reduced metabolic effects at the exense of an increased fat accumulation (1,14,15). This also rovides a rationale why lasma glucose levels were only slightly higher in MOB subjects than in OB subjects

JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 813 A AEA (ng/ml) y = -.66x +.68 1.1 r=-.45, SEE=.11 =.44 1..9.8.7.6.5.4.3 -.3 -.1.1.3.5 Letin (ng/ml) -.3 12 1 8 6 4 2 y = -38.45x + 24.65, r=-.17, SEE=.13 =.946 -.1.1.3.5 CRP (mg/l) -.2 2 18 16 14 12 1 8 6 4 2 y = -12.153x + 5.614 r=-.33, SEE=.12 =.168.2.4 B AEA (ng/ml) 1.3 1.2 1.1 1.9.8.7.6.5.4 y = -.96x +.73 r=.7, SEE=.18 =.74.3 -.2.2.4 Letin (ng/ml) -.2 25 2 15 1 5 y = 183.8x + 85.88 r=.43, SEE=51. =.31.2.4 CRP (mg/l) -.2 5 45 4 35 3 25 2 15 1 5 y = 4.5x + 7.63 r=.55, SEE=.11 =.6.2.4 MBF to CPT Figure 2. Relationshis Among AEA, Letin, Inflammation, and Coronary Endothelial Function Correlations among (A) anandamide (AEA), letin, and high-sensitivity C-reactive rotein (CRP) lasma levels and change of endotheliumrelated myocardial blood flow ( MBF) during cold ressor testing (CPT) in the obese grou, resectively, and (B) corresondingly in the morbid obesity grou. SEE standard error of the estimate. desite a rogressive increase in insulin lasma levels and insulin resistance. When looking secifically at EC and adionectin, lasma levels of EC mildly but rogressively increased, whereas adionectin declined with increases in body weight (1,3). Interestingly, the EC, AEA, and 2-AG were ositively, whereas adionectin lasma levels were inversely, associated with the total fat amount but also with surrogate markers for visceral adiose tissue burden such as waist/hi ratio, central fat, and android/gynoid fat distribution. Thus, aart from the total fat amount increase, the visceral adiose tissue aears to reflect a redominant source of alterations in EC and adionectin lasma levels in individuals with increasing body weight as reorted reviously (1,3). The rogressive decrease of adionectin lasma levels in obese individuals with increasing body weight has been related to a state of metabolic stress associated with increases in catecholamines, glucocorticoids, and insulin, which of all exert inhibitory effects on the exression and release of adionectin from the adiose tissue (1). As regards letin lasma levels, they were significantly higher in OB subjects than in CON and OW subjects (2). Notably, there was a 7-fold higher increase in letin lasma levels in MOB subjects versus OB subjects. Plasma levels of letin have been shown to correlate more closely with total and subcutaneous tissue than with visceral adiose tissue (1). For this reason, circulating letin lasma levels are generally higher in women, who commonly develo more subcutaneous fat than men do (1). In the current study, the striking increase in letin lasma levels was correlated with the total ercentage of body fat and an android attern of fat distribution in MOB subjects, suggesting the increase in adiose tissue as a whole and also visceral fat mass distribution as redominant sources of the marked increase in letin lasma levels. This may be artly surrising (1) but most likely is related to the lower revalence of women versus men in the MOB grou. Coronary circulatory function and interrelations. When regarding the MFR or total coronary vasodilator caacity, it significantly declined from the CON to the OW grou with a mild increase from the OW to the OB grou, whereas it was then lowest in the MOB grou. The relatively low MFR in the MOB grou was rimarily related to the augmentation in resting MBF induced by higher resting heart rates, SBP, and resulting RPP, which are indicative of the myocardial workload. This finding also accords with revious investigations in the assessment of coronary circulatory function in

814 JACC: CARDIOVASCULAR IMAGING, VOL. 5, NO. 8, 212 obesity (3,16). An increase in myocardial workload in obese individuals, associated with higher resting MBF, is commonly related to an activation of the symathetic nervous system and renin-angiotensinaldosterone system (16). Addressing secifically the OB grou, increases in EC such as AEA and 2-AG lasma levels were inversely correlated with alterations in coronary circulatory function. These findings confirm and extend a recent reort (3), emhasizing that, aart from effects related to obesity such as low HDL cholesterol, insulin resistance, and inflammation, increases in EC lasma levels lay a ivotal role in mediating coronary circulatory dysfunction in OB subjects. As mentioned before, we did not observe a further worsening of coronary circulatory function from OB to MOB subjects. This somehow astounding observation may be suorted by recent results in the assessment of brachial artery function in severe obesity (11). Flowmediated and, thus, endothelium-deendent vasodilation was aradoxically higher in severely obese individuals than in obese and normal weight individuals. This aradoxical reservation in flowmediated vasodilation in individuals with severe obesity was suggested to be related to an enhanced inflammatory environment associated with a greater mobilization of endothelial rogenitor cells and reduced activation of the immune system (11). In addition, another investigation in 29 morbidly obese individuals with various traditional cardiovascular risk factors, including the metabolic syndrome (7), demonstrated that carotid-femoral ulse-wave velocity, as a reflection of arterial stiffness, was only mildly elevated. Interestingly, elevations in hscrp were inversely related to ulse-wave velocity, when the oosite might have been exected (9,1). Contrary to common oinion, therefore, metabolically triggered chronic microinflammation in severe obesity may be associated with reserved flowmediated vasodilation and relatively low arterial stiffness than was assumed reviously. In this direction, the results of the current study rovide first evidence that increases in letin and hscrp lasma levels correlated ositively with altered and endothelium-related MBF resonses to CPT in MOB subjects. Arterial increases in letin concentrations and a metabolically triggered systemic inflammation, therefore, confer some beneficial effects on coronary endothelial function against adverse effects of body fat on the endothelium in MOB subjects. On multivariate analysis, increases in hscrp lasma levels remained indeendently associated with the CPT-induced change in MBFs, signifying in articular inflammatory factors to mediate direct rotective effects on the coronary endothelium in MOB subjects. Visceral adiose tissue is characterized by an infiltration of macrohages, which have been demonstrated as a major source of inflammatory cytokines such as tumor necrosis factor-alha, interleukin-6, and interleukin-1 in obesity (1). For examle, increases in anti-inflammatory interleukin-1 have been shown to rotect vascular endothelial function by reducing increases in sueroxide formation within the arterial wall in an exerimental model (17). Also clinical studies lend further evidence of a otential vascular rotective role of interleukin-1 (18,19). If interleukin-1 serum levels were increased in CAD atients with elevated CRP lasma levels, no imairment of acetylcholine-stimulated forearm blood flow resonse was noted (18). This reorted reservation of endothelium function in the resence of inflammatory-triggered increases in interleukin-1 lasma levels in CAD atients (18) might also rovide a mechanistic link between a better clinical outcome after acute coronary syndromes and reduced increased risk associated with elevated CRP lasma levels (19). Because we did not measure interleukin-1 lasma levels and other arameters otentially involved in the adiose tissue triggered inflammatory rocess, further investigations are certainly needed to identify the exact mechanism of the inflammatory resonse mediating rotective effects on the coronary endothelium in MOB. CONCLUSIONS Contrasting associations of altered coronary endothelial function with increases in AEA, letin, and hscrp identify and characterize OB and MOB as different disease entities, rather than as a simle continuation of increases in body weight, affecting coronary circulatory function. Acknowledgments The authors thank Christina Laemmli, Stehan Dewarrat, and Claude Ponsolle for assisting in the PET studies, the cyclotron staff for N-13 ammonia roduction, and Katia Galan for erforming the laboratory measurements. Rerint requests and corresondence: Dr. Thomas Hellmut Schindler, Deartment of Secialties of Medicine, Division of Cardiology, 6th Floor, Nuclear Cardiology and Cardiac PET/CT, University Hositals of Geneva, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland. E-mail: thomas.schindler@hcuge.ch.

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