Bio-molecular and Genetic Markers

for Vascular Disease

Yuhei Kawano, M.D., Ph.D.

Division of Hypertension and Nephrology

National Cardiovascular Center

Suita, Osaka, Japan

Risk factors for cardiovascular disease

• Risk factorsOlder age, Hypertension, Dyslipidemia, Diabetes,

Smoking, Family history of premature CV disease,

Obesity, Metabolic syndrome

• Subclinical organ damageLeft ventricular hypertrophy, Carotid thickning or plaque,

Decreased GFR, Microalbuminuria,

Increased PWV, Decreased ABI

• Established CV or renal diseaseCerebrovascular disease, Heart disease, Renal disease,

Peripheral artery disease

Bio-molecular and genetic factors are involved

Bio-molecular markers for CV disease

• Vasoactive substances

Renin, Angiotensin, Aldosterone, Catecholamines,

Endothelins, Natriuretic peptides, Adrenomedullin,

Prostaglandins, Nitric oxide, Kinins

• Inflammation and oxidative stress

CRP, Homocystein, Reactive oxygen species

• Appetite and glucose/lipid metabolism

Leptin, Ghrelin, Insulin, Adiponectin, PPAR-γ

• Tissue structure and remodelling

Matrix metalloprotease, TGF-β, TNF-α

Natriuretic peptides

Circulating hormone

GC-A

ANP

BNP

Vasodilation

Natriuresis

Reduction in BP and cardiac load

Local hormone

CNP

Inhibition of growth

GC-B

GC-A

GC-B

ANP

BNP

CNP

Local hormone

Inhibition of

hypertrophy

and fibrosis

Local hormone

Inhibition of remodeling（Cardioprotective effect）

0 10-10 10-9 10-8 10-770

80

90

100

Peptide concentration (mol/L)

Collagen synthesis

*

*

**

*

**

**ANP

BNP

CNP

0 10-10 10-9 10-8 10-770

80

90

100

Peptide concentration (mol/L)

DNA synthesis

*

*

**

*

*

ANP

BNP

CNP

3H

-pro

lin

e u

pta

ke (

%)

3H

-th

ym

idin

e u

pta

ke (

%)

*P

QuickTime?풋Graphics ?粒?풉플 퓸惹픽?합??푿?휖

0

20

40

60

80

100

0 10 20 30

Ev

en

t-F

ree

Ra

te (

%)

Time (months)

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

Se

ns

itiv

ity

1-Specificity

BNP 150 pg/mL

BNP >150 pg/mL

BNP

Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-GlyCys

Cys

Arg

Phe

GlyThrAsp-Thr-Phe-Gln-Tyr-Ile-Gln-His-Ala-Leu-Lys-Gln-Val-Thr

Lys

AspLys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr-NH2

• Adrenomedullin (AM), a 52-amino acid peptide, is a potent

vasodilator and natriuretic factor that was originally isolated from

human pheochromocytoma.

· Kitamura K, Kangawa K, et al: Biochem Biophys Res Commun, 1993

Adrenomedullin

Plasma Adrenomedullin and Atherosclerotic

Disease: Usefulness as a Predictor of

Future Cardiovascular Events

Takeshi Horio1, Hidenori Nishida1, Yoshihiko Suzuki2,

Kenji Kangawa3, Yuhei Kawano1

1Division of Hypertension and Nephrology, Department of Medicine, National

Cardiovascular Center, Suita, Japan2Department of Internal Medicine, Circulatory and Fluid Regulation,

Faculty of Medicine, University of Miyazaki, Miyazaki, Japan3National Cardiovascular Center Research Institute, Suita, Japan

July 14, 2007

C RA

L

UCSAVOID

R

A

CE

NTERNA

TI

ON

AL

The 39th Annual Scientific Meeting of the Japanese Atherosclerosis Society

Symposium 7: Atherosclerosis and Blood Biomarkers

Plasma AM levels in patients with and without IHD

and/or PAD

Values are mean ± SD.

IHD PAD IHD and/or PADQ u i c k T i m e ? 풋G r a p h i c s ? 粒? 풉 플 퓸 惹 픽 ? 합 ? ? 푿 ? 휖

(-) (+)0

5

10

15

20

Pla

sma

AM

lev

el (

fmol

/ml)

P

CV event-free Kaplan-Meier curves in the three

groups divided by tertiles of basal AM levelsQ u i c k T i m e ? 풋G r a p h i c s ? 粒? 풉 플 퓸 惹 픽 ? 합 ? ? 푿 ? 휖

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90

Cum

ulat

ive

even

t-fr

ee r

ate

(%)

Time (months)

Log-rank test, P=0.033

Lowest tertile(AM＜10.1 fmol/ml)

Middle tertile(AM: 10.1-13.1 fmol/ml)

Highest tertile(AM≧13.1 fmol/ml)

C-Reactive Protein in Essential Hypertension:

Association with Left Ventricular Mass Index and

Risk of Cardiovascular Disease

Department of Geriatric Medicine, Osaka University Graduate School of

Medicine1

Division of Hypertension and Nephrology, Department of Medicine,

National Cardiovascular Center2

Yoshio Iwashima1, Takeshi Horio2, Kei Kamide2, Hiromi Rakugi1,

Toshio Ogihara1, Yuhei Kawano2

(Hypertension 2007)

120

130

140

150

0.3

CRP Category (mg/dL)

LV

MI,

g/m

2

110

120

130

140

0.3

CRP Category (mg/dL)Male Female

Association between CRP categories and LVMI in Multivariate Analysis

Multiple regression analysis including age, BMI, diabetes, duration of hypertension, systolic and diastolic BP, heart

rate, T-chol, TG, HDL-chol, Ccr, and smoking status was performed.

Values are given as mean S.E.

F=3.73

p=0.025

F=3.36

p=0.036

Cu

mu

lati

ve

Ev

ent-

free

Su

rviv

al

Ra

te

Time, months

LVH/ CRP 0.1 mg/dL

Non-LVH/ CRP 0.1 mg/dL

LVH/ CRP

Cytokines from small and large adipose cells

Leptin Adiponectin

FFAAngiotensinogen

PAI-1

Abudominal

obesity

Visceral fat

accumulation

TNFα

Adiponectin

● Insulin sensitive, N=152 ● Insulin resistant, N=93

Adip

on

ectin

SBP(mmHg)

(mg/mL)

DBP(mmHg)

(mg/mL)

Ad

ipo

ne

ctin

0

5

10

15

20

25

80 100 120 1400

5

10

15

20

25

40 60 80 100

r=-0.35

p

0

5

10

15

20

25

0 0.5 1 1.5

Ankle-brachial index

Pla

sma

ad

ipo

nec

tin

(

g/m

L)

r=0.43

P

MMP-TIMP Imbalance and Left Ventricular

Diastolic Dysfunction in Patients with

Essential Hypertension

Horio T, Kamide K, Yoshihara F, Nakata H, Nakamura S,

Nakahama H, Kawano Y

Division of Hypertension and Nephrology,

National Cardiovascular Center, Suita, Japan

(AHA-HBPC 2007)

Association of MMP-2 and TIMP-1 with LV

diastolic function in all subjects

A/E DcT

MMP-2

TIMP-1

MMP-2/TIMP-1

R RP P

-0.372 0.011

0.301 0.044

-0.423 0.003

-0.402 0.006

0.357 0.015

-0.453 0.002

QuickTime?풋Graphics ?粒?풉플 퓸惹픽?합??푿?휖

0

0.5

1

1.5

2

0

0.5

1

1.5

2

100

150

200

250

300

350

100

150

200

250

300

350

Comparison of LV diastolic function between

two groups divided by MMP-2 / TIMP-1 ratio

A/E DcT

Low-M/T High-M/TMean± SD

DcT

(m

sec)

P=0.014P=0.031

Low-M/T High-M/T

A/E

MMP-2

TIMP-1

MMP-2 / TIMP-1

LVMI

R P

-0.203 0.182

0.335 0.024

-0.360 0.015

RWT

R P

-0.168 0.273

0.437 0.002

-0.392 0.007

FS

R P

0.041 0.790

-0.205 0.178

0.152 0.321

Association of MMP-2 and TIMP-1 with LV

hypertrophy and systolic function in all subjects

Cardiovascular/Renal

Complications

Genetic Factor

Hypertension

Insulin ResistanceRAS ActivitySNS Activity

Salt Sensitivity

Genetic Influence on Hypertension

Salt Intake

Low Physical Activity

Mental Stress

Body Weight Gain

Drugs Life Style

Modification

Kamide K, et al.

Jpn Heat J 2004

Strategies of genetic analysis for hypertension in NCVC

1．Whole genome approach2．Candidate gene approach3．Sequence of critical genes

Mainly Association Study (Case-Control)

Gene selection

Study subjects

1．General population: about 4000 subjects2．Hypertensive subjects: about 1000 subjects

Gene Polymorphisms Relating to

Carotid Atherosclerosis

Role of MMP2 and RGS2 gene polymorphisms

Evaluation of carotid atherosclerosis Carotid IMT

Max IMT(mm): maximum IMT in the common

carotid artery

Takiuchi S et al. NCVC

A total of 866 hypertensive subjects

Study subjects

Association between 26223 A>C (exon 13(3’ UTR)/MMP-2) and Carotid

atherosclerosis

0

0.5

1

1.5

2

0

0.5

1

1.5

2

Ma

x I

MT

(m

m)

AA AC CC

*

*: p

genetic polymorphismsgenotype n M- IMT (mm) Plaque score

-638A>G AA 245 0.890.20 2.273.10

AG 416 0.920.23 2.873.77

GG 176 0.900.21 2.703.50

p= 0.273 p= 0.043

1026T>A TT 305 0.890.20 2.303.21

TA 407 0.920.23 2.903.80

AA 124 0.900.21 2.753.41

p =0.030 p =0.026

1891-1892del TC II 302 0.890.20 2.313.31

ID 413 0.920.23 2.883.70

DD 126 0.900.21 2.743.41

p= 0.042 p= 0.036M-IMT; maximal intima media thickness. p; comparison between common

homo and minor allele.

RGS2 SNPs and carotid atherosclerosis

Yang J, Kamide K, et al. ISH 2006

Gene Polymorphisms Relating to

Left Ventricular Hypertrophy

Role of IGF-1 and IGF-1 Receptor gene polymorphisms

IGF-1 level in EHT

Verdecchia P et al: Circulation, 1999

Trophic effect on Cardiomyocytes by IGF-1

Ang: angiotensin, ET: endothelin, PE:

phenylephrine

Pro

tein

synth

esi

s (%

incr

ease

)QuickTime?풋Graphics ?粒?풉플 퓸惹픽?합??푿?휖

Ang II ET-1 PE IGF-10

20

40

60

80

100

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LVH (-) LVH (+)0

100

200

300

400P

Comparison of LVMI, RWT in Each Genotype

QuickTime?풋Graphics ?粒?풉플 퓸惹픽?합??푿?휖

CC CT+TT75

100

125

150

175

AA AC+CC0.4

0.45

0.5

0.55

0.6

LV

MI(g

/m2)

RW

T

0.47± 0.09 0.46± 0.09

130± 39 119± 28

P=0.008

P=0.056

Promoter: C-328T Intron13: A275124C

Horio T et al. NCVC

P

Age

Sex (male)

BMI

SBP

DBP

HT Duration

C-328T: CC

Age

Sex (male)

BMI

SBP

DBP

HT Duration

A275124C: AA

χ2

LVH(LVMI≥125 g/m2)

Concentric Hypertrophy(RWT≥0.45)

2.529

20.737

6.491

6.849

6.653

2.287

4.723

1.472

5.640

16.815

2.700

2.962

4.524

4.417

0.112

Gene Polymorphisms Relating to

Chronic Kidney Disease

Role of gene polymorphisms of RAAS, SNS, oxidative stress, vasoactive peptides

Data were analyzed by adjusting confounding factors, Age, Sex, BMI, BP,

DM, lipid profiles

Related Gene Polymorphisms to Hypertensive Renal Dysfunction (Crn >1.4 mg/dl)

- Logistic regression analysis -

C2 P

(I/D) 5.26 0.022

4.82 0.028

3.51 0.061

2.37 0.124

5.89 0.015

2.94 0.087

3.63 0.057

4.55 0.033

3.81 0.051

3.42 0.064

8.30 0.004

ACE G12568C

MLR C850G

MTHFR C677T

SOD3 C-2450A

SOD3 C-1780T

TSC C1784T

ECE1 C-388A

ECE1 T65251C

UTS2 G5865A

HGF A70202T

NPR1 G2979C

Data were analyzed by adjusting confounding factors, Age, Sex, BMI, BP,

DM, lipid profiles

Related Gene Polymorphisms to CKD (CCr

Related Gene Polymorphisms to Hypertensive Renal Damage

- Working Hypothesis -

ACE G12568C

MLR C850G

SOD3 C-1780T

ECE1 T65251C

NPR1 G2979C

Angiotensin II

RAAS

Aldosterone

Oxidative Stress

ROS

Vasoactive Peptide

Endothelin-1

ANP

Nephrosclerosis

MMP2 A26223C

Carotid Atherosclerosis

Renal Damage

ACE G12568C

MLR C850G

SOD3 C-1780T

ECE1 T65251C

NPR1 G2979C

Cardiac Hypertrophy

IGF-1 R C-328T

IGF-1 R A275124C

Gene polymorphisms related to

hypertensive CV complications

HGF T43839A

IGF-1 R G263743A

MMP2 A26223C

TSC T8776C

GNB3 C825T

Clusterin 6316del T

RGS2 A-638CRGS2 T1026A

Case Age IGF1R IGF1R MMP2 SNPs CVDC-328T A275124C A26223C CKD

ACE I/D 3/31 56 CC AA CC DD Stroke2 67 CC AA CC DD AMI3 77 CC AA CC DD AMI

MLR C850G 1/24 76 CC AA CC CC Stroke5 56 CC AA CC CC None

SOD3 C-1708T 1/26 86 CC AA CC CT Effort AP7 58 CC AA CC CT None

ECE1 T65251C 2/28 56 CC AA CC GG Stroke9 60 CC AA CC GG Stroke

NPR1 G2979C 1/310 71 CC AA CC GC DAA11 69 CC AA CC GC None12 60 CC AA CC GC None

Is it possible to predict CV complications

using combination of SNPs ?

Total 8/12

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0

10

20

30

40

50

0

Association of the combination of MT1-MMP (G-724A), ETA (T54202C),

and AT1 (A1166C) gene polymorphisms with LV diastolic dysfunction

OR: 6.600 (P=0.0001)

Number of SNPs 0 (n=49) 1 (n=316) 2 (n=301) 3 (n=70)

・MT1-MMP: G-724A (GA+AA)

・ETA receptor: T54202C (TC+CC)

・AT1 receptor: A1166C (AA)

(SNPs)

Prevalence of

LV diastolic

dysfunction (%)

Conclusion

• Evaluation of bio-molecular and genetic

markers may be useful to identify high risk

patients and to manage them with

appropriate strategies for effective

cardiovascular protection.

Our Hypertension Research Team

Kawano YHorio TKamide KTakiuchi SIwashima YMiwa YYoshihara FNakamura SYasuda HNishida H

Division of Hypertension and Nephrology

Division of Preventive Cardiology

Okamura TOkayama AKokubo Y

Research Institute

Kangawa KTokudome TMiyata TYang JOkuda TKokame KHanada HMorisaki T

General Director

Tomoike H

Thank You!