Download Disclosures: None Conclusions

Association of osteopontin with cardiac fibrosis and diastolic dysfunction in heart failure
of hypertensive origin. A role for lysyl oxidase?
Begoña López PhD,1 Arantxa González PhD,1 Ramón Querejeta MD PhD,2 Javier Beaumont PhD,1 Susana Ravassa PhD,1 Javier Díez MD PhD1.
(1) Division of Cardiovascular Science, Center of Applied Medical Research, Pamplona, Spain. (2) Division of Cardiology, Donostia University Hospital, San Sebastián, Spain
Purpose
Myocardial fibrosis may play a role in the development and progression of chronic heart failure (HF) in hypertensive patients, contributing to left ventricular (LV) dysfunction by increasing LV chamber passive stiffness and subsequently impairing
diastolic function. Experimental and clinical studies have demonstrated that the effects of myocardial fibrosis on LV mechanics and function depend critically on the degree of collagen type I cross-linking and deposition. Of interest, abnormally high
levels of lysyl oxidase (LOX), the enzyme responsible for the formation of covalent bonds between adjacent collagen fibrils, have been reported in the myocardium of hypertensive patients with HF exhibiting increased collagen cross-linking and
deposition. On the other hand, the matricellular protein osteopontin (OPN) is involved in myocardial fibrosis through the stimulation of myofibroblasts and the inhibition of collagen degradation. However, there is no information about its potential role
in collagen cross-linking and deposition. Therefore, the aim of this study was to investigate whether OPN is associated with the enzyme LOX involved in collagen cross-linking and deposition in the failing human heart.
Methods
The study was performed in myocardial samples of 21 hypertensive patients with HF and 10 normotensive controls. All patients underwent a full clinical evaluation, an echocardiographic study and a diagnostic catheterism. The fraction of myocardial
area occupied by total collagen tissue (CVF) and by collagen type I fibers (CIVF) was assessed by histomorphology. The degree of collagen cross-linking, which determines the formation of insoluble stiff collagen, was assessed by colorimetric and
enzymatic procedures. The expression of OPN and LOX, was assessed by Western-blot. An in vitro study using adult human dermal fibroblasts (HDFa) stimulated with OPN or vehicle was performed to evaluate LOX mRNA (real time RT-PCR) and
protein (Western blot) levels as well as its activity (fluorimetric assay).
Results
Myocardial Osteopontin & Fibrosis
Myocardial Fibrosis
1.95 ± 0.07
7.71 ± 0.53
Collagen I volume fraction, %
2.03 ± 0.15
7.63 ± 0.52
<0.001
Insoluble collagen, μg/mg
0.95 ± 0.28
8.99 ± 0.22
<0.001
Soluble collagen, μg/mg
0.66 ± 0.17
2.76 ± 0.14
<0.001
1.43 ± 0.29
Collagen cross-linking
3.47 ± 0.23
Whereas a 100% of control subjects
exhibited absent or mild immunostaining
for OPN, a 100% of HF patients presented
moderate to severe immunostaining
(P<0.005).
<0.001
Heart failure patient
<0.001
Myocardial OPN was directly correlated
with CVF (Figure) and CIVF (r=0.600,
P<0.005). Additionally, it was directly
correlated with LOX (Figure), collagen
cross-linking (Figure), and insoluble
collagen (r=0.534, P<0.01) in all patients.
Values are expressed as mean ± SEM.
All the parameters related to collagen quantity and quality were
significantly increased in HF patients as compared with controls.
Myocardial expression of LOX was directly correlated with the
quantity of insoluble collagen (r=0.805, P<0.005) and collagen
cross-linking (r=0.759, P<0.005) in HF patients.
Control subject
9
17.5
P < 0.01
r = 0.566
6
P<0.05
r=0.460
8
Collagen cross-linking
Collagen volume fraction, %
Cardiac Osteopontin
P
Collagen Cross-linking
Lysyl Oxidase
Collagen Volume Fraction
15.0
Lysyl oxidase (ADU)
Parameters
Patients
Collagen volume fraction (%)
Controls
12.5
10.0
7.5
7
6
5
4
3
5
4
3
2
5.0
2
1
0
0
0
0 0.6
0.8
1.0
1.2
1.4
1.6
Osteopontin (ADU)
0
0.6
0.8
1.0
1.2
1.4
1.6
P < 0.05
r = 0.517
0
0.6
0.8
Osteopontin (ADU)
1.0
1.2
1.4
1.6
Osteopontin (ADU)
Conclusions
Left Ventricular Function
Osteopontin & Lysyl Oxidase
2.0
1.0
LV chamber stiffness
3.0
0.55
0.45
0.35
0.25
(mmHg/mL)
0.65
0.40
P < 0.05
r = 0.458
0.30
LV chamber stiffness
P < 0.05
(mmHg/mL)
P < 0.05
4.0
0.40
0.75
Pro-LOX protein (ADU)
LOX activity (Fold-change)
5.0
0.20
0.10
0
0
Vehicle
Osteopontin
Vehicle
Osteopontin
LOX activity was increased (P<0.05) in HDFa fibroblasts exposed to OPN
compared with unstimulated cells. Moreover, OPN induced an increase in
LOX mRNA levels (71.28 ± 1.68 vs 28.47 ± 1.67 AU; P<0.01) and in pro-LOX
protein levels in HDFa fibroblasts compared with unstimulated cells .
0.30
0.20
P < 0.005
r = 0.716
0.10
0
0
0
The main findings of this study are as follows:
(i) An excess of myocardial OPN is associated with increased
LOX, collagen cross-linking and collagen type I deposition in
hypertensive patients with HF.
(ii) OPN upregulates LOX activity and expression in human
fibroblasts.
(iii) Myocardial OPN and collagen cross-linking are associated
with increased LV stiffness and filling pressures in these
patients.
0.6
0.8
1.0
1.2
1.4
Osteopontin (ADU)
1.6
1.0
2.0
3.0
4.0
5.0
6.0
Cross-Linking
Myocardial OPN was directly correlated with LV chamber stiffness (KLV; Figure) and pulmonary
capillary wedge pressure (r=0.558, P<0.01) in all patients. Similarly, direct correlations were found of
both collagen cross-linking and LOX with KLV (Figure and r=0.459, P<0.05, respectively) and
pulmonary capillary wedge pressure (r=0.711, P<0.01 and r=0.598, P<0.01, respectively) in all
patients.
In conclusion, these results suggest that the stimulation of
the OPN-LOX axis might facilitate the deposition of stiff
collagen and the subsequent alteration of LV diastolic
mechanical properties and function in the hypertensive
failing heart.
Disclosures: None