Role of lysyl oxidase-like-2 in cardiac fibrosis and diastolic dysfunction in

experimental and clinical HFPEF

K. Savvatis1, J. Yang2, M. Kasner1, S. Van Linthout1, P. Fan3, J. Diez4, L. Yao3, CP. Chang2, C. Tschoepe1

1 Charité, Dep. of Cardiology CVK, Berlin, Germany, 2Dep. of Cardiology, Indiana University, USA,

3Gilead Sciences, USA, 4Dep. of Cardiology, University of Navarra, Pamplona, Spain

collagen fibers

collagen cross-linkingLOXLOX-like enzymes 1-5

Collagen and cross-linking is increased in HFPEF

n=15

Collagen I

healthy HFPEF

Collagen volume fraction

health

y

HFPEF0

5

10

15

20

CV

F (%

)

*Collagen crosslinking

health

y

HFPEF0.0

0.5

1.0

1.5

2.0

Co

llag

en c

ross

linki

ng *

LOXL2

Question?

• Is LOXL2 expressed in clinical and experimental HFPEF?

• What is its relation to cardiac fibrosis and LV stiffness?

• Can its inhibition lead to improved remodelling and LV function?

Methods• 24 pts with HFPEF and 15 controls– LV-Biopsy (LOXL2, collagen, CVF, crosslinking)– Echocardiography– Invasive studies

• Experimental cardiac hypertrophy (TAC model)– anti-LOXL2 antibody 30 mg/kg ip 2x/week (Gilead)– Echo, conductance, molecular studies (at 10 weeks)

• Primary cardiac fibroblasts– Stimulation with TGF-β– LOXL2-knockdown with LOXL2-siRNA– Migration and differentiation studies

LOXL2 increases in HFPEF patients and correlates with fibrosis

LOXL2

health

y

HFPEF0

1

2

3

4

LO

XL

2 (A

rea

frac

tio

n %

) *

healthy

HFPEF

LOXL2 vs. collagen I

0 1 2 3 40.0

0.1

0.2

0.3

LOXL2 (AF %)

Co

llag

en

I (

AF

%)

r = 0.49

p = 0.033

LOXL2 vs. collagen crosslink ing

0 1 2 3 40

1

2

3

4

r = 0.67

p = 0.009

LOXL2 (Area fraction %)

Co

llag

en

cro

ss-lin

kin

g

LOXL2 vs. collagen I

0 1 2 3 40.0

0.1

0.2

0.3

LOXL2 (AF %)

Co

llag

en

I (

AF

%)

r = 0.49

p = 0.033

LOXL2 vs. collagen crosslink ing

0 1 2 3 40

1

2

3

4

r = 0.67

p = 0.009

LOXL2 (Area fraction %)

Co

llag

en

cro

ss-lin

kin

g

Crosslinking and LOXL2 are associated with impaired diastolic function

E/E' vs. collagen crosslinking

0 10 20 30 40 500

1

2

3

4

r = 0.64

p = 0.015

E/E'

Co

llag

en c

ross

linki

ng

E/E' vs. LOXL2

0 2 4 6 8 100

10

20

30

40

50

LOXL2 (Area fraction %)

E/E

'

r=0.55p=0.018

LVEDP vs. collagen crosslink ing

0 5 10 15 20 250

1

2

3

4

r = 0.67

p = 0.02

LVEDP (mmHg)

Co

llag

en c

ross

li nki

ng

0 1 2 3 40

10

20

30

LOXL2 vs. LVEDP

LOXL2 (area fraction %)

LV

ED

P (m

mH

g)

r=0.59p=0.019

LOXL2 correlates with collagen production in experimental cardiac hypertrophy

LOXL2 inhibition prevents cardiac fibrosis and improves LV function in stressed hearts

LOXL2 affects TGF-β signaling in cardiac fibroblasts

Primary cardiac fibroblasts transfected with LOXL2siRNA

Downregulation of downstream TGF-β mediators and fibroblast-differentiation markers (α-SMA, Col1A1, fibronectin1)

LOXL2 affects migratory capacity of cardiac fibroblasts

Primary cardiac fibroblasts transfected with LOXL2siRNA

Cardiac stress

TGFβ

Fibroblast Myofibroblast Collagen

Migration Crosslinking

Fibrosis

Stiffness

Diastolic and systolic dysfunction

LOXL2 LOXL2

Conclusion

• LOXL2 is expressed in the heart and is increased in patients with HFPEF and experimental cardiac hypertrophy

• LOXL2 correlates with the degree of fibrosis, collagen crosslinking and ventricular stiffness

• Inhibition of LOXL2 reduces cardiac fibrosis and inhibits fibroblast migration and collagen crosslinking

• LOXL2 inhibition can be a promising therapeutic target in HFPEF