9/26/2016 Macaque Model of Hormones and Atherosclerosis - Female Reproductive Aging Insights From Studies of Nonhuman Primates & Atherosclerosis Susan E. Appt, DVM, Associate Professor of Pathology / Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, NC Wake Forest Baptist Medical Center Ovarian Follicle Populations Decline Macaques Experience Similar Menstrual with Age in Macaques Cycle Hormone Profiles to Women Ovulation (pg/ml) (ng/ml) 600 25 500 20 E2 400 15 Prog 300 FSH 10 200 AMH 0.9 Years 13 Years 24 Years 5 100 Menses Women’s ~3 ~39 ~72 0 0 Equivalent Age Days -14 0 14 (Nichols et al, 2004,Human Reproduction) Wake Forest Baptist Medical Center Appt et al., 2009 Wake Forest Baptist Medical Center 1
9/26/2016 Antimullerian Hormone is a Marker of Postmenopausal Hormone Patterns are Follicle Numbers in Macaques Similar between Macaques and Women • Naturally and oophorectomized postmenopausal 14 macaques Primordial Follicle # 4500 r = 0.78 12 r =-0.32, 4000 p<0.001 AMH (ng/ml) • Elevated FSH and LH p<0.001 3500 10 3000 8 • Lower estradiol, progesterone & AMH 2500 6 2000 1500 4 1000 2 • Perimenopausal monkeys 500 0 0 • Defined by irregular menstrual cycles 0 5 10 15 20 25 0 5 10 15 0 5 10 15 Age (yrs) • Significant elevations in peak FSH concentrations AMH (ng/ml) • Lower AMH Appt et al., 2009 Appt et. al. unpublished Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center (Downs et al., 2006, Downs and Urbanski 2007) Macaques Have Similar Coronary Artery Anatomy to Women Sources of Coronary Arte ry Blocks Atherosclerosis A O R T A R C A L C X 5 4 3 2 1 1 2 3 4 5 1 2 3 4 5 L A D Mea surem ent of Inti mal (Plaque ) Crosssec tional Area s Adventit ia Lum en Med ia Inte rnal Ela stic Lam ina Inti mal Area Plaq ue (Pla que Area ) Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Clarkson et al.,2001 2
9/26/2016 Coronary Artery Atherosclerosis Iliac Artery Biopsy Technique Allows Baseline following Exposure to a Western Diet or Interim Measures of Atherosclerosis No Plaque Complicated Plaque r = ~ 0.85 r > 0.80 r = ~ 0.85 r > 0.80 Left Right Right Left Common Appt et. al. unpublished Common Common Common Iliac Artery r ~ 0.90 r = ~ 0.90 Iliac Artery Iliac Artery Iliac Artery Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Surgical Menopause Increases Coronary Artery Atherosclerosis in Nonhuman Primates Surgical Menopause, Hormone Treatment and Atherosclerosis p < 0.02 in Macaques Median Coronary Artery Plaque Size (mm 2 ) Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Adams et al., 1985 3
9/26/2016 Timing of Initiation of HT Affects Coronary CEE Prevents Initiation / Progression of Small Atherosclerosis Outcomes in Oophorectomized Monkeys Atherosclerotic Plaques in Female Monkeys Early Intervention Late Intervention* 1 p=0. 71 p = NS Control Plaque Size at Outcome (mm2) 0.9 Coronary Artery Plaque Size 0.40 CEE p < .05 0.8 p=0.33 0.7 0.30 0.6 (mm 2 ) p=0.0001 0.5 0.20 0.4 0.3 0.2 0.10 0.1 0 0 Low Middle High Placebo CEE Baseline Placebo CEE CEE+MPA Baseline Tertile Baseline PQ size <0.12 0.12-0.60 >0.60 (mm 2 ) *Comparable to approximately 6 patient-years after menopause. Karas and Clarkson, Menopausal Med , 2003 Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Anthony & Clarkson, JACC 2002 E2 Reduced Arterial Pro-Inflammatory Gene Expression in Early, but Not Late Menopausal Premenopausal Influences on (Oophorectomized) Monkeys Atherosclerosis Progression TNF α MCP-1 Macrophage (CD68) 0.0014 014 8 0.20 .2 Abundance (AU) 0.08 Relative mRNA 012 7 0.0010 010 0.15 15 0.06 6 5 008 .1 0.04 0.10 4 P< 0.05 0.0006 006 P< 0.05 P< 0.05 3 004 0.02 0.05 05 2 0.0002 002 1 0 0 0 000 0 0 CTL E2 CTL E2 CTL E2 Early Early E2 Early Early E2 Wake Forest Baptist Medical Center Sophonsritsuk, Register T.C. et. al., 2012 Wake Forest Baptist Medical Center 4
9/26/2016 Premenopausal Oral Contraceptives Reduced Postmenopausal Atherosclerosis (Subordinate Psychosocially Stressed Monkeys) Why Would Premenopausal OVX Iliac Artery Plaque Area (mm2) 1 Monkeys Benefit from Hormone 0.9 Treatment? 0.8 Placeb ebo p < 0.05 0.7 0.6 Placebo 0.5 0.4 CEE 0.3 0.2 OC OC 0.1 0 Premenopause Postmenopause Wake Forest Baptist Medical Center Kaplan et al ., 2002 17 Wake Forest Baptist Medical Center Ovarian Follicle Varies Significantly between Plasma AMH Prior to Consumption of a Western Diet Predicted Iliac Artery Atherosclerosis Extent 2 years Later Monkeys of the same Age Iliac Artery Plaque Area (mm 2 ) 14 1 12 p = 0.02 AMH (ng/ml) 0.9 10 0.8 8 0.7 6 0.6 4 0.5 2 0 0.4 0 5 10 15 20 25 0.3 Age (yrs) 0.2 0.1 0 Could this be an Indication of variation in ovarian aging Lowest Middle High and subsequent risk for atherosclerosis, irrespective of AMH (ng/ml) biologic age ? Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Appt et. al., Menopause 2012 5
9/26/2016 Premenopausal Monkeys with Low AMH Prior to Monkeys with Low AMH Prior to OVX and 2 years Western Diet Develop Larger Iliac Artery Plaques Exposure to Western Diet Develop Larger Coronary Artery Plaques p < 0.05 0.4 0.7 p = 0.04 Iliac IA (mm2)/IELL SQRT 0.35 0.6 p = 0.04 0.3 n = 19 0.5 0.25 Coronary 0.4 0.2 Artery AMH LO Mean IA 0.15 AMH HI 0.3 (SQRT mm 2 ) 0.1 0.2 0.05 0.1 0 2.5 years 4.5 years 0 Years Consuming Western Diet AMHLO AMHHI Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Premenopausal Monkeys with Low AMH Prior to 4 Premenopausal Monkeys with Low AMH Prior to Western years of Western Diet Have Greater Arterial Stiffness Diet Develop Larger Coronary Artery Plaques 4 years later and Diastolic Blood Pressure 8 0.70 p = 0.06 7 Plaque Area (SQRT mm 2 ) r = - 0.39 0.60 6 p = 0.03 0.50 5 AMH (ng/ml) 4 0.40 SQRT 3 0.30 2 0.20 1 0 0.10 4 9 14 19 PWV (m/s) 0.00 Low AMH High AMH Predictors of PWV SBP R 2 = 0.37, DBP R 2 = 0.43, p’s < 0.004) Whole body fat (R 2 = 0.44, p=0.002) Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center 6
9/26/2016 AMH Protein and AMHR2 mRNA are Present AMH is Correlated with Markers of Inflammation in NHP Arteries • In a separate study of premenopausal monkeys • Immunohistochemistry consuming a western diet (n=49) • Staining is seen consistently in the smooth muscle cell layer • Significant inverse relationship between baseline • Arterial smooth muscle cells are integral in arterial responses to serum AMH and plasma monocyte chemoattractant stressors such as changes in blood pressure, inflammation and protein-1 (MCP-1) after 30 months of diet (r = -0.34, oxidative stress. p=0.02) • Using human TGFß / BMP signaling pathway PCR • AMH and iliac artery gene expression of inflammatory array, AMHR2 mRNA expression was identified in NHP markers were correlated (p’s <= 0.05) arteries and ovary • IL-6 r = -0.34 • IL-2 r = -0.37 • CD 68 r = -0.36 Melissa Wellons 1 , Philip Owens 1 , David Caudell, Susan E. Appt 1 Vanderbilt University, Wake Forest School of Medicine Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Antimullerian Hormone Protein Expression in Summary Female Macaque Arteries AMH VVG H & E NPC • In Postmenopausal monkeys, estrogen treatment A B C D Carotid m p m p m m prevents atherosclerosis progression when lesions are small and uncomplicated E F G H • Premenopausal monkeys with low ovarian reserve m p m p m p A. Aorta m p (AMH) prior to challenge with Western diet appear to be at greater risk for atherosclerosis, arterial stiffness, increased diastolic blood pressure and arterial I J K L m p m p m p Iliac m p inflammation • AMH protein and AMHR2 mRNA are present in M N O P Coronary Macaque arteries p p m p m p m m Melissa Wellons 1 , Philip Owens 1 , David Caudell, Susan E. Appt Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center 1 Vanderbilt University, Wake Forest School of Medicine 7
9/26/2016 Thomas B. Clarkson Jr, DVM, Conclusions Professor of Comparative Medicine • Together these data suggest a potential window of opportunity during pre- and perimenopause to decrease postmenopausal atherosclerosis progression • Raise the question of whether AMH is an marker of vascular aging and susceptibility to CHD Wake Forest Baptist Medical Center 29 Wake Forest Baptist Medical Center Ovarian Follicle Number Prior to Western Diet is Inversely Related to Iliac Artery Atherosclerosis 2 years Later Additional Slides 8000 Primordial Follicle # r = - 0.41 7000 p = 0.10 6000 5000 4000 3000 2000 1000 0 0 0.5 1 1.5 2 Plaque Area mm 2 Wake Forest Baptist Medical Center Wake Forest Baptist Medical Center Appt et al. unpublished 8
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