Lysyl oxidase like 2 inhibition decreases cardiac fibrosis and improves diastolic dysfunction in experimental

1. 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

3. collagen fibers
collagen
cross-linking
LOX
LOX-like enzymes 1-5
Collagen and cross-linking is increased in HFPEF
n=15
Collagen I
healthy HFPEF
Collagen volume fraction
healthy
H
FPEF
0
5
10
15
20
CVF(%)
*
Collagen crosslinking
healthy
H
FPEF
0.0
0.5
1.0
1.5
2.0
Collagencrosslinking
*
LOXL2

4. 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?

5. 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

6. LOXL2 increases in HFPEF patients and correlates
with fibrosis
LOXL2
healthy
H
FPEF
0
1
2
3
4
LOXL2(Areafraction%)
*
healthy
HFPEF
LOXL2vs.collagenI
0 1 2 3 4
0.0
0.1
0.2
0.3
LOXL2(AF%)
CollagenI(AF%)
r=0.49
p=0.033
LOXL2vs.collagencrosslinking
0 1 2 3 4
0
1
2
3
4
r=0.67
p=0.009
LOXL2(Areafraction %)
Collagencross-linking
LOXL2vs.collagenI
0 1 2 3 4
0.0
0.1
0.2
0.3
LOXL2(AF%)
CollagenI(AF%)
r=0.49
p=0.033
LOXL2vs.collagencrosslinking
0 1 2 3 4
0
1
2
3
4
r=0.67
p=0.009
LOXL2(Areafraction %)
Collagencross-linking

7. Crosslinking and LOXL2 are associated with
impaired diastolic function
E/E' vs. collagen crosslinking
0 10 20 30 40 50
0
1
2
3
4
r = 0.64
p = 0.015
E/E'
Collagencrosslinking
E/E' vs. LOXL2
0 2 4 6 8 10
0
10
20
30
40
50
LOXL2 (Area fraction %)
E/E'
r=0.55
p=0.018
LVEDP vs. collagen crosslinking
0 5 10 15 20 25
0
1
2
3
4
r = 0.67
p = 0.02
LVEDP (mmHg)
Collagencrosslinking
0 1 2 3 4
0
10
20
30
LOXL2 vs. LVEDP
LOXL2 (area fraction %)
LVEDP(mmHg)
r=0.59
p=0.019

8. LOXL2 correlates with collagen production in
experimental cardiac hypertrophy

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

10. 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)

11. LOXL2 affects migratory capacity of cardiac
fibroblasts
Primary cardiac fibroblasts transfected with LOXL2siRNA

12. Cardiac stress
TGFβ
Fibroblast  Myofibroblast Collagen
Migration Crosslinking
Fibrosis
Stiffness
Diastolic and systolic dysfunction
LOXL2 LOXL2

13. 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