9/26/2016 Macaque Model of Hormones and Atherosclerosis - Female - - PDF document

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9/26/2016 Macaque Model of Hormones and Atherosclerosis - Female - - PDF document

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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

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Hormones and Atherosclerosis - Insights From Studies of Nonhuman Primates

Susan E. Appt, DVM, Associate Professor of Pathology / Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, NC

Wake Forest Baptist Medical Center

Macaque Model of Female Reproductive Aging & Atherosclerosis

Wake Forest Baptist Medical Center

5 10 15 20 25 100 200 300 400 500 600

E2 Prog FSH AMH

  • 14 0 14

(pg/ml) (ng/ml) Ovulation Menses

Days

Macaques Experience Similar Menstrual Cycle Hormone Profiles to Women

Appt et al., 2009

Wake Forest Baptist Medical Center

Ovarian Follicle Populations Decline with Age in Macaques

(Nichols et al, 2004,Human Reproduction)

0.9 Years ~3 13 Years ~39 24 Years ~72

Women’s Equivalent Age

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Wake Forest Baptist Medical Center

Antimullerian Hormone is a Marker of Follicle Numbers in Macaques

500 1000 1500 2000 2500 3000 3500 4000 4500

5 10 15

Primordial Follicle #

r = 0.78 p<0.001

AMH (ng/ml)

0 5 10 15 2 4 6 8 10 12 14 5 10 15 20 25

AMH (ng/ml) Age (yrs) r =-0.32, p<0.001

Appt et al., 2009 Appt et. al. unpublished

Wake Forest Baptist Medical Center

  • Naturally and oophorectomized postmenopausal

macaques

  • Elevated FSH and LH
  • Lower estradiol, progesterone & AMH
  • Perimenopausal monkeys
  • Defined by irregular menstrual cycles
  • Significant elevations in peak FSH concentrations
  • Lower AMH

Postmenopausal Hormone Patterns are Similar between Macaques and Women

(Downs et al., 2006, Downs and Urbanski 2007)

Wake Forest Baptist Medical Center

Atherosclerosis

Wake Forest Baptist Medical Center

Macaques Have Similar Coronary Artery Anatomy to Women

A O R T A R C A L C X L A D

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Sources of Coronary Arte ry Blocks

Lum en Plaq ue Adventit ia Med ia Inte rnal Ela stic Lam ina Inti mal Area (Pla que Area )

Mea surem ent of Inti mal (Plaque ) Crosssec tional Area s

Clarkson et al.,2001

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Wake Forest Baptist Medical Center

Coronary Artery Atherosclerosis following Exposure to a Western Diet

Appt et. al. unpublished

No Plaque Complicated Plaque

Wake Forest Baptist Medical Center

Iliac Artery Biopsy Technique Allows Baseline

  • r Interim Measures of Atherosclerosis

r = ~ 0.90 r = ~ 0.85 r = ~ 0.85

Right Common Iliac Artery Left Common Iliac Artery

r > 0.80 r > 0.80

Right Common Iliac Artery Left Common Iliac Artery

r ~ 0.90

Wake Forest Baptist Medical Center

Surgical Menopause, Hormone Treatment and Atherosclerosis in Macaques

Wake Forest Baptist Medical Center

Surgical Menopause Increases Coronary Artery Atherosclerosis in Nonhuman Primates

Median Coronary Artery Plaque Size (mm2) p < 0.02

Adams et al., 1985

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Wake Forest Baptist Medical Center

Placebo CEE

Coronary Artery Plaque Size (mm2)

0.10 0.20 0.30 0.40 Baseline Placebo CEE CEE+MPA

Early Intervention Late Intervention*

p < .05 p = NS

*Comparable to approximately 6 patient-years after menopause. Karas and Clarkson, Menopausal Med, 2003

Timing of Initiation of HT Affects Coronary Atherosclerosis Outcomes in Oophorectomized Monkeys

Wake Forest Baptist Medical Center

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Plaque Size at Outcome (mm2)

High >0.60 Low <0.12 Middle 0.12-0.60 CEE Control

Baseline Tertile Baseline PQ size (mm2)

Anthony & Clarkson, JACC 2002

p=0. 71 p=0.0001 p=0.33

CEE Prevents Initiation / Progression of Small Atherosclerotic Plaques in Female Monkeys

Wake Forest Baptist Medical Center

E2 Reduced Arterial Pro-Inflammatory Gene Expression in Early, but Not Late Menopausal (Oophorectomized) Monkeys

1 2 3 4 5 6 7 8 Early Early E2

MCP-1

000 002 004 006 008 010 012 014 Early Early E2

TNFα

Relative mRNA Abundance (AU) P< 0.05 P< 0.05

Sophonsritsuk, Register T.C. et. al., 2012

05 .1 15 .2

0.02 0.04 0.06 0.08

Macrophage (CD68)

0.05 0.10 0.15 0.20 0.0002 0.0006 0.0014 0.0010

CTL CTL CTL E2 E2 E2 P< 0.05

Wake Forest Baptist Medical Center

Premenopausal Influences on Atherosclerosis Progression

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Wake Forest Baptist Medical Center

Premenopausal Oral Contraceptives Reduced Postmenopausal Atherosclerosis

(Subordinate Psychosocially Stressed Monkeys)

17

Premenopause Postmenopause

Iliac Artery Plaque Area (mm2) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

OC OC CEE Placebo Placeb ebo OVX

p < 0.05

Kaplan et al., 2002

Wake Forest Baptist Medical Center

Why Would Premenopausal Monkeys Benefit from Hormone Treatment?

Wake Forest Baptist Medical Center

Ovarian Follicle Varies Significantly between Monkeys of the same Age

2 4 6 8 10 12 14 5 10 15 20 25

AMH (ng/ml) Age (yrs)

Could this be an Indication of variation in ovarian aging and subsequent risk for atherosclerosis, irrespective of biologic age ?

Wake Forest Baptist Medical Center

Plasma AMH Prior to Consumption of a Western Diet Predicted Iliac Artery Atherosclerosis Extent 2 years Later

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Lowest Middle High

AMH (ng/ml) Iliac Artery Plaque Area (mm2)

p = 0.02

Appt et. al., Menopause 2012

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Wake Forest Baptist Medical Center

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 2.5 years 4.5 years

Iliac IA (mm2)/IELL SQRT

Years Consuming Western Diet AMH LO AMH HI

Premenopausal Monkeys with Low AMH Prior to Western Diet Develop Larger Iliac Artery Plaques

p = 0.04 p < 0.05

Wake Forest Baptist Medical Center

0.1 0.2 0.3 0.4 0.5 0.6 0.7 AMHLO AMHHI

Monkeys with Low AMH Prior to OVX and 2 years Exposure to Western Diet Develop Larger Coronary Artery Plaques

Coronary Artery Mean IA (SQRT mm2)

p = 0.04 n = 19

Wake Forest Baptist Medical Center

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 p = 0.06

Plaque Area (SQRT mm2)

High AMH Low AMH

Premenopausal Monkeys with Low AMH Prior to Western Diet Develop Larger Coronary Artery Plaques 4 years later

Wake Forest Baptist Medical Center

Premenopausal Monkeys with Low AMH Prior to 4 years of Western Diet Have Greater Arterial Stiffness and Diastolic Blood Pressure

1 2 3 4 5 6 7 8 4 9 14 19

r = - 0.39 p = 0.03 AMH (ng/ml) SQRT PWV (m/s)

Predictors of PWV SBP R2 = 0.37, DBP R2 = 0.43, p’s <0.004) Whole body fat (R2 = 0.44, p=0.002)

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Wake Forest Baptist Medical Center

AMH is Correlated with Markers of Inflammation

  • In a separate study of premenopausal monkeys

consuming a western diet (n=49)

  • Significant inverse relationship between baseline

serum AMH and plasma monocyte chemoattractant protein-1 (MCP-1) after 30 months of diet (r = -0.34, p=0.02)

  • AMH and iliac artery gene expression of inflammatory

markers were correlated (p’s <= 0.05)

  • IL-6 r = -0.34
  • IL-2 r = -0.37
  • CD 68 r = -0.36

Wake Forest Baptist Medical Center

AMH Protein and AMHR2 mRNA are Present in NHP Arteries

  • Immunohistochemistry
  • Staining is seen consistently in the smooth muscle cell layer
  • Arterial smooth muscle cells are integral in arterial responses to

stressors such as changes in blood pressure, inflammation and

  • xidative stress.
  • Using human TGFß / BMP signaling pathway PCR

array, AMHR2 mRNA expression was identified in NHP arteries and ovary

Melissa Wellons1, Philip Owens1, David Caudell, Susan E. Appt

1Vanderbilt University, Wake Forest School of Medicine

Wake Forest Baptist Medical Center

Antimullerian Hormone Protein Expression in Female Macaque Arteries

Melissa Wellons1, Philip Owens1, David Caudell, Susan E. Appt

1Vanderbilt University, Wake Forest School of Medicine

AMH VVG H & E B C F G J K N O Iliac Carotid

  • A. Aorta

Coronary

M A E I NPC D H L P p p p p m m m m p m p m p m p m m m p m p m p m p m p m p m Wake Forest Baptist Medical Center

Summary

  • In Postmenopausal monkeys, estrogen treatment

prevents atherosclerosis progression when lesions are small and uncomplicated

  • Premenopausal monkeys with low ovarian reserve

(AMH) prior to challenge with Western diet appear to be at greater risk for atherosclerosis, arterial stiffness, increased diastolic blood pressure and arterial inflammation

  • AMH protein and AMHR2 mRNA are present in

Macaque arteries

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Wake Forest Baptist Medical Center

Conclusions

  • Together these data suggest a potential window
  • f 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

29 Wake Forest Baptist Medical Center

Thomas B. Clarkson Jr, DVM, Professor of Comparative Medicine

Wake Forest Baptist Medical Center

Additional Slides

Wake Forest Baptist Medical Center

Ovarian Follicle Number Prior to Western Diet is Inversely Related to Iliac Artery Atherosclerosis 2 years Later

1000 2000 3000 4000 5000 6000 7000 8000 0.5 1 1.5 2

Plaque Area mm2

r = - 0.41 p = 0.10

Primordial Follicle #

Appt et al. unpublished

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Wake Forest Baptist Medical Center

2 4 6 8 10 12 14 16 18 20 0.0 1.0 2.0

PWV is Associated with Atherosclerosis

PWV (m/s) Plaque Area (SQRT mm2) r = 0.44 p = 0.01 n = 33

Appt et. al. unpublished

Wake Forest Baptist Medical Center

0.05 0.1 0.15 0.2 0.25 Iliac Artery Abdominal Aorta Ovary Relative mRNA Expression n = 33 n = 1 n = 1

AMHR2 Relative mRNA Expression in Tissue Collected from Premenopausal Macaques

Appt et. al. unpublished

Wake Forest Baptist Medical Center

AMHR2 Relative mRNA Expression in Tissue Collected from Pre- and Postmenopausal Macaques

0.005 0.01 0.015 0.02 0.025 Premenopausal Ovx p = 0.13

Appt et. al. unpublished