Collagen Cross-Linking But Not Collagen Amount
Associates With Elevated Filling Pressures in Hypertensive
Patients With Stage C Heart Failure
Potential Role of Lysyl Oxidase
Begoña López, Ramón Querejeta, Arantxa González, Mariano Larman, Javier Díez
Abstract—We investigated whether the quality of myocardial collagen associates with elevated left-sided filling pressures
in 38 hypertensive patients with stage C chronic heart failure. Filling pressures were assessed invasively measuring
pulmonary capillary wedge pressure. Left ventricular chamber stiffness constant was calculated from the deceleration
time of the early mitral filling wave. The fraction of myocardial volume occupied by total collagen tissue and collagen
type I fibers was assessed histomorphologically. The degree of collagen cross-linking (CCL), which determines the
formation of insoluble stiff collagen, was assessed by colorimetric and enzymatic procedures. The expression of lysyl
oxidase (LOX), which regulates CCL, was assessed by Western blot. Compared with patients with normal pulmonary
capillary wedge pressure (ⱕ12 mm Hg; n⫽16), patients with elevated pulmonary capillary wedge pressure (⬎12
mm Hg; n⫽22) exhibited increases of left ventricular chamber stiffness constant, fraction of myocardial volume
occupied by total collagen tissue, fraction of myocardial volume occupied by collagen type I fibers, CCL, insoluble stiff
collagen, and LOX. Pulmonary capillary wedge pressure was correlated with left ventricular chamber stiffness constant
(r⫽0.639; P⬍0.001), insoluble stiff collagen (r⫽0.474; P⬍0.005), CCL (r⫽0.625; P⬍0.001), and LOX (r⫽0.410;
P⬍0.05) in all of the patients but not with fraction of myocardial volume occupied by total collagen tissue or fraction
of myocardial volume occupied by collagen type I fibers. In addition, CCL was correlated with insoluble stiff collagen
(r⫽0.612; P⬍0.005), LOX (r⫽0.538; P⬍0.01), left ventricular chamber stiffness constant (r⫽0.535; P⬍0.005), peak
filling rate (r⫽⫺0.343; P⬍0.05), ejection fraction (r⫽⫺0.430; P⬍0.01), and amino-terminal propeptide of brain
natriuretic peptide (r⫽0.421; P⬍0.05) in all of the patients. These associations were independent of confounding
factors. These findings indicate that, in hypertensive patients with stage C heart failure, it is only the quality of collagen
(ie, degree of cross-linking) that associates with elevated filling pressures. It is suggested that LOX-mediated excessive
CCL facilitates the increase in left ventricular stiffness with the resulting elevation of filling pressures in these patients.
(Hypertension. 2012;60:677-683.) ● Online Data Supplement
Key Words: collagen 䡲 filling pressures 䡲 heart failure
A
rterial hypertension has been long considered as one of
the most common etiologic conditions predisposing to
heart failure (HF).1 Furthermore, a recent systematic review
shows that HF development remains a major problem in
treated hypertensive patients.2 It has been shown that myocardial fibrosis, a common finding in postmortem studies and
endomyocardial biopsies of patients with hypertensive heart
disease (HHD),3 is associated with the increase of left
ventricular (LV) chamber stiffness4,5 and the development of
clinically overt HF in these patients.6,7 It has been proposed
that the increase in LV stiffness can contribute to compromised diastolic function and elevate left-sided filling pressures (FPs) in hypertensive patients.8
The definition of myocardial fibrosis is based on the
quantification of an excess of collagen fiber deposition, as
assessed by staining techniques. However, it must be considered that collagen-dependent LV chamber stiffness is influenced not only by the amount of collagen fibers but also by
their qualitative properties (ie, the degree of collagen crosslinking [CCL] among collagen fibrils that determines the
insolubility, stiffness, and resistance to degradation of the
resulting fibers; the relative proportions of collagen types I
and III fibers; and the diameter of the collagen fibers and their
spatial alignment).9 In fact, as demonstrated in different
experimental models of pressure overload, although LV
chamber stiffness is affected by changes in both collagen
Received March 29, 2012; first decision April 23, 2012; revision accepted July 1, 2012.
From the Division of Cardiovascular Sciences, Centre for Applied Medical Research (B.L., A.G., J.D.), and Department of Cardiology and Cardiac
Surgery, University of Navarra Clinic (JD), University of Navarra, Pamplona, Spain; Division of Cardiology (M.L., R.Q.), Donostia University Hospital,
San Sebastián, Spain.
The online-only Data Supplement is available with this article at ttp://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
112.196113/-/DC1.
Correspondence to Javier Díez, Área de Ciencias Cardiovasculares, CIMA, Avenida Pío XII 55, 31008 Pamplona, Spain. E-mail jadimar@unav.es
© 2012 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.112.196113
677
678
Hypertension
September 2012
quantity and quality, the effects of changes in collagen
quantity, particularly the abundance of collagen type I, are
modified by collagen quality, in particular the degree of
CCL.10–12
Therefore, a more complete understanding of the quantitative and qualitative variations of myocardial collagen matrix
and their impact on LV function is critical as we strive to
develop specific and effective therapies for HF. In this
conceptual framework, this study was designed to investigate
whether it is the quantity (as assessed by the amounts of total
or collagen type I fibers) or the quality (as assessed by the
degree of CCL and availability of insoluble collagen [insCol]) that associates with FPs in hypertensive patients with
HHD and chronic stage C HF. In addition, the expression of
lysyl oxidase (LOX), which regulates CCL,13 and the enzymes procollagen type I carboxy-terminal proteinase (PCP)
and furin, which participate in the activation of LOX,13 was
also assessed. Finally, a number of biomarkers related to
collagen were measured to investigate whether any association exists between their circulating levels and parameters
assessing the quantity and quality of myocardial collagen.
Methods
Subjects
All of the subjects gave written informed consent to participate in the
study, and the institutional review committee approved the study
protocol. The study conformed to the principles of the Helsinki
Declaration.
The population consisted of 38 hypertensive patients with a
previous clinical diagnosis of chronic stage C HF based on the
presence of ⱖ1 major and 2 minor Framingham criteria.14 The
included patients were in New York Heart Association functional
classes II to IV. Table S1 (available in the online-only Data
Supplement) shows the characteristics of these patients. (For further
details see the Expanded Methods section in the online-only Data
Supplement).
Three transvenous endomyocardial biopsies were taken from the
middle area of the interventricular septum from each patient during
the cardiac catheterization procedure. Septal endomyocardial biopsies were obtained from autopsies of 10 age- and sex-matched
subjects to assess control reference values of histomorphological
parameters. They were subjects without clinical history of cardiac
disease and with no LV hypertrophy.
Noninvasive Cardiac Studies
Two-dimensional echocardiographic-Doppler and pulsed-Doppler
imaging was performed in all of the patients. (For further details see
the Expanded Methods section in the online-only Data Supplement).
Nuclear Studies
To rule out microvascular ischemia, single photon emission computed tomography was performed in all of the patients. In addition,
peak filling rate (PFR) and time to PFR were calculated.15
Invasive Cardiac Studies
After significant coronary artery disease (ⱖ50% stenosis in a major
epicardial coronary artery) was discarded, pulmonary capillary
wedge pressure (PCWP) was measured. Values of PCWP ⬎12
mm Hg were considered as abnormally elevated.16
Biochemical Determinations
Plasma amino-terminal propeptide of brain natriuretic peptide and
aldosterone were measured using commercial kits. Circulating biomarkers related to collagen were measured in all of the patients using
commercial ELISA kits. Procollagen type I carboxy-terminal pro-
peptide and collagen type I carboxy-terminal telopeptide (CITP)
were measured as specific biomarkers of collagen type I synthesis
and degradation, respectively. Procollagen type III amino-terminal
propeptide was assessed as a marker of collagen type III turnover. In
addition, matrix metalloproteinases (MMPs) 1, 2, and 9 and tissue
inhibitors of MMPs (TIMPs) 1 and 2 were also measured as markers
of collagen degradation. Furthermore, MMP-1/TIMP-1, MMP-2/
TIMP-2, and MMP-9/TIMP-1 ratios were calculated as partial
indices of MMP activity. (For further details, see the Expanded
Methods section in the online-only Data Supplement).
Histomorphological Studies
The area of myocardium occupied by total collagen tissue or
collagen volume fraction (CVF) was determined by quantitative
morphometry in sections stained with collagen-specific picro-sirius
red, as reported previously.17 To distinguish between cross-linked
(insCol) and noncross-linked collagen (soluble collagen), colorimetric and enzymatic procedures were used, as described previously.18
The concentration of each form of collagen was related to the total
amount of protein. The degree of CCL was calculated as the ratio
between the insCol and noncross-linked collagen.
Immunohistochemical analysis of collagen type I was performed
on formalin-fixed and paraffin-embedded sections. Immunohistochemical staining was performed by the avidin peroxidase-labeled
dextran polymer method. Positive staining was visualized with DAB
Plus (Boehringer Mannheim Corp), and tissues were counterstained
with Harris hematoxylin (Sigma). The area of myocardium with
positive staining for collagen type I (CIVF) was analyzed by
quantitative morphometry, as described previously.6
Molecular Studies
The protein expression of LOX, PCP, and furin was analyzed in
myocardial samples from 23 patients. A 25-␮g sample of total
protein obtained from each biopsy was processed for Western blot,
as described previously.19 Data are expressed as arbitrary densitometric units relative to ␤-actin expression. (For further details see the
Expanded Methods section in the online-only Data Supplement).
Statistical Analysis
To analyze the differences in histomorphological, biochemical, and
molecular parameters between controls and patients and between
patients with either increased or normal PCWP, a Student t test for
unpaired data was used once normality was demonstrated; otherwise,
the Mann-Whitney U test was performed. Categorical variables were
analyzed by the ␹2 test or Fisher exact test when necessary. The
correlation between continuously distributed variables was tested by
correlation coefficients and univariate regression analysis. The influence of confounding factors on correlations was excluded by
partial correlation analysis for quantitative parameters. Values are
expressed as mean⫾SEM and categorical variables as numbers and
percentages. A value of P⬍0.05 was considered statistically
significant.
Results
Clinical, Echocardiographic, and
Biochemical Characteristics
The clinical characteristics of the 2 groups of patients are
shown in Table S1 (available in the online-only Data Supplement). No significant differences in the assessed parameters were found between the 2 groups of patients.
Table 1 shows the echocardiographic parameters assessed
in the 2 groups of patients. The values of LV mass index
(LVMI), LV end-diastolic diameter (LVEDD), and LV enddiastolic volume (LVEDV) were higher in patients with
elevated PCWP than in patients with normal PCWP. Patients
with elevated PCWP exhibited lower values of LV ejection
fraction (LVEF), deceleration time, and PFR than patients
López et al
Collagen Cross-Linking and Heart Failure
Table 1.
Cardiac Parameters in Hypertensive Patients With
Heart Failure Classified According to PCWP
P Value
LVMI, g/m2
132.31⫾12.34
168.82⫾12.60
⬍0.05
0.36⫾0.02
0.34⫾0.02
NS
RWT
LVEDD, mm
51.98⫾2.23
58.92⫾1.96
⬍0.05
LVEDV, mL
134.65⫾13.77
178.16⫾13.51
⬍0.05
55.25⫾3.09
41.00⫾3.66
⬍0.01
LVEF, %
1.25⫾0.11
1.35⫾0.28
NS
IVRT, ms
103.13⫾2.74
106.73⫾4.73
NS
DT, ms
215.69⫾9.93
184.5⫾13.37
⬍0.05
0.117⫾0.01
0.188⫾0.02
⬍0.01
1.88⫾0.18
1.37⫾0.12
⬍0.05
0.039⫾0.003
0.037⫾0.001
NS
VE:VA ratio
KLV, mm Hg/mL
PFR, edv/s
Time to PFR, ms
PCWP indicates pulmonary capillary wedge pressure; LV, left ventricular;
LVMI, LV mass index; RWT, relative wall thickness; LVEDD, LV end-diastolic
diameter; LVEDV, LV end-diastolic volume; LVEF, LV ejection fraction; VE,
maximum early transmitral flow velocity in diastole; VA, maximum late
transmitral velocity flow in diastole; IVRT, isovolumic relaxation time; DT,
deceleration time; KLV, LV chamber stiffness constant; PFR, peak filling rate;
NS, nonsignificant. Values are expressed as mean⫾SEM.
with normal PCWP. The value of LV chamber stiffness
constant was increased in patients with elevated PCWP
compared with patients with normal PCWP. Although patients with elevated PCWP exhibited higher values of aminoterminal propeptide of brain natriuretic peptide than patients
with normal PCWP, plasma aldosterone levels were similar in
the 2 groups of patients (Table S1).
Histomorphological and Molecular
Myocardial Parameters
Compared with control subjects, the whole group of patients
exhibited higher values of CVF and CIVF (Table 2). Interestingly, a strong direct correlation (r⫽0.969; P⬍0.001) was
found between CVF and CIVF in all of the HF patients
(Figure S1). The amounts of insCol and noncross-linked
collagen were increased by 9-fold and 4-fold, respectively, in
the whole group of patients compared with controls (Table 2).
As a consequence, the degree of CCL was significantly higher in
the whole group of patients than in controls (Table 2).
Compared with patients with normal PCWP, patients
with elevated PCWP exhibited increased values of CVF
Table 2.
Histomorphological Parameters in Control Subjects
and Hypertensive Patients With HF
Parameters
Controls (n⫽10) HF Patients (n⫽38)
P Value
CVF, %
1.95⫾0.07
7.51⫾0.49
⬍0.001
CIVF, %
2.03⫾0.15
7.58⫾0.52
⬍0.001
Insoluble collagen, ␮g/mg
0.95⫾0.28
8.83⫾0.17
⬍0.001
Soluble collagen, ␮g/mg
0.66⫾0.17
2.82⫾0.09
⬍0.001
Collagen cross-linking
1.43⫾0.29
3.31⫾0.14
⬍0.001
HF indicates heart failure; CVF, total collagen volume fraction; CIVF, collagen
type I volume fraction. Values are expressed as mean⫾SEM.
P<0.01
Collagen cross-linking
PCWP
⬎12 mm Hg
(n⫽22)
4.4
3.8
3.2
2.6
0
Normal
Elevated
PCWP
Figure 1. Degree of collagen cross-linking in the myocardium of
heart failure patients with either normal or elevated pulmonary
capillary wedge pressure (PCWP).
(6.44⫾0.54% versus 8.48⫾0.75%; P⬍0.05), CIVF (6.43⫾0.65%
versus 8.61⫾0.87%; P⬍0.01), and insCol (8.34⫾0.23 versus
9.19⫾0.24 ␮g/mg; P⬍0.01) but decreased values of noncrosslinked collagen (2.99⫾0.13 versus 2.69⫾0.12 ␮g/mg; P⬍0.05).
The degree of CCL was higher in patients with elevated PCWP
than in patients with normal PCWP (3.63⫾0.23 versus
2.88⫾0.15, P⬍0.01; Figure 1).
The expression of LOX was enhanced in patients with
elevated PCWP compared with patients with normal PCWP
(5.61⫾0.42 versus 4.34⫾0.55 arbitrary densitometric units;
P⬍0.05; Figure 2). PCP expression was reduced in patients
with elevated PCWP compared with patients with normal
PCWP (21233⫾1463 versus 29234⫾1808 arbitrary densitometric units; P⬍0.05). The expression of furin did tend to be
higher (P⫽0.05) in patients with elevated PCWP than in
patients with normal PCWP (0.41⫾0.08 versus 0.24⫾0.11
arbitrary densitometric units).
Analysis of Associations
Correlations among collagen-related histomorphological and
molecular parameters and the main parameters assessing LV
6.6
P<0.05
5.8
LOX (A.D.U.)
Parameters
PCWP
ⱕ12 mm Hg
(n⫽16)
679
5.0
4.2
0
Normal
Elevated
PCWP
Figure 2. Protein expression of the enzyme lysyl oxidase (LOX)
in heart failure patients with either normal or elevated pulmonary
capillary wedge pressure (PCWP).
680
Hypertension
September 2012
Table 3.
Correlations of Collagen-Related Histomorphological and Molecular Parameters and Left Ventricular Function Parameters
in Hypertensive Patients With Heart Failure
CCL
P
Parameters
InsCol
r
CVF
CIVF
P
r
⬍0.005
P
r
LOX
P
r
P
r
PCWP, mm Hg
⬍0.001
0.474
NS
0.230
NS
0.235
⬍0.05
0.410
PFR, edv/s
⬍0.05
⫺0.343
NS
⫺0.266
NS
⫺0.261
NS
⫺0.293
NS
0.086
LVEF, %
⬍0.01
⫺0.430
NS
⫺0.274
NS
⫺0.083
NS
⫺0.084
⬍0.05
0.625
⫺0.418
CCL indicates collagen cross-linking; InsCol, insoluble collagen; CIVF, collagen type I volume fraction; CVF, total collagen volume fraction; LOX, lysyl oxidase; PCWP,
pulmonary capillary wedge pressure; PFR, peak filling rate; LVEF, left ventricular ejection fraction; NS, nonsignificant.
function are presented in Table 3. CCL was directly correlated with PCWP (Figure 3A) and inversely correlated with
PFR (Figure 3B) and LVEF (Figure 3C). In addition, insCol
and LOX were directly correlated with PCWP in all of the
patients. Finally, LOX was inversely associated with LVEF
in all of the patients. Of interest, PCWP was directly
correlated with LV chamber stiffness constant (r⫽0.639;
P⬍0.001) in all of the patients. All of these correlations
remained significant when we excluded the influence of a
number of potential confounding factors (ie, age, sex, LVMI,
LVEDD, and LVEDV) in partial correlation analysis. No
correlations were found between CVF and CIVF with LV
functional parameters in this study.
CCL was correlated with insCol (r⫽0.778; P⬍0.001) and
LOX (r⫽0.538; P⬍0.01) in all and 23 patients, respectively.
In addition, CCL was correlated with LV chamber stiffness
constant (r⫽0.535; P⬍0.005; Figure 4A) and amino-terminal
propeptide of brain natriuretic peptide (r⫽0.421; P⬍0.05;
Figure 4B) in all of the patients. These correlations remained
significant when we excluded the influence of a number of
potential confounding factors (ie, age, sex, LVMI, LVEDD,
and LVEDV) in partial correlation analysis.
Finally, a direct correlation was found between LOX and
furin (r⫽0.599; P⬍0.05) in all of the patients. This correlation remained significant when we excluded the influence of
a number of potential confounding factors (ie, age, sex,
LVMI, LVEDD, and LVEDV) in partial correlation analysis.
Serum CITP was inversely associated with insCol
(r⫽⫺0.451; P⬍0.01), this association being independent of a
number of potential confounding factors (ie, age, sex, LVMI,
LVEDD, LVEDV, estimated glomerular filtration rate,
P<0.001
r=0.625
PCWP (mmHg)
30
25
20
15
Discussion
The 3 main findings of this study are as follows: (1) FPs are
associated with the quality (ie, the degree of cross-linking)
but not with the quantity of collagen present in the myocardium of patients with HHD and stage C HF; (2) the degree of
collagen cross-linking was associated with impairment of LV
diastolic and systolic function; and (3) the degree of crosslinking is associated with the expression of the enzyme LOX
in the myocardium of these patients. An increase in myocardial stiffness translates into a reduced compliance of the left
ventricle and the resulting increase in FPs.8 Other than
variations in intrinsic cardiomyocyte stiffness,20 changes in
the collagen matrix also influence myocardial stiffness. In
particular, Badenhorst et al12 demonstrated in experimental
models of pressure overload that myocardial and LV chamber
stiffness are affected by changes in both collagen quantity
and quality (ie, cross-linking), with the effects of changes in
C
4
80
70
P<0.05
r=-0.343
3
LVEF (%)
B
35
PFR (edv/s)
A
MMP-1, and TIMP-1) in partial correlation analysis. No
further associations were found between circulating biomarkers of collagen type I metabolism and insCol or CCL. In
addition, serum procollagen type I carboxy-terminal propeptide was directly correlated with both CVF (r⫽0.735;
P⬍0.001) and CIVF (r⫽0.689; P⬍0.001) in all of the
patients, with these associations being independent of a
number of potential confounding factors (ie, age, sex, LVMI,
LVEDD, and LVEDV) in partial correlation analysis. No
correlations were found between procollagen type III
amino-terminal propeptide and the quantity (ie, CVF) and
quality (ie, insCol and collagen cross-linking) of myocardial collagen.
2
P<0.01
r=-0.430
60
50
40
1
30
10
5
1
2
3
4
5
Collagen cross-linking
6
0
1
2
3
4
5
Collagen cross-linking
6
20
1
2
3
4
5
6
Collagen cross-linking
Figure 3. A, Direct correlation (y⫽4.65x⫹0.42) between the degree of collagen cross-linking (CCL) and pulmonary capillary wedge
pressure (PCWP) in all of the patients. B, Inverse correlation (y⫽⫺0.24x⫹2.23) between the degree of collagen cross-linking and peak
filling rate (PFR) in all of the patients. C, Inverse correlation (y⫽⫺8.07x⫹73.72) between the degree of collagen cross-linking and left
ventricular ejection fraction (LVEF) in all of the patients.
López et al
B
0.4
NT-proBNP (pg/mL)
K LV (mmHg/mL)
A
0.3
0.2
P<0.005
r=0.535
0.1
0.0
1
2
3
4
5
Collagen cross-linking
6
Collagen Cross-Linking and Heart Failure
681
3000
P<0.05
r=0.421
2500
2000
Figure 4. A, Direct correlation
(y⫽0.05x⫺0.01) between the degree of
collagen cross-linking and left ventricular chamber stiffness (KLV) in all of
the patients. B, Direct correlation
(y⫽329.48x⫹4.91) between the degree of
collagen cross-linking and amino-terminal
propeptide of brain natriuretic peptide
(NT-proBNP) in all of the patients.
1500
1000
500
0
1
2
3
4
5
6
Collagen cross-linking
collagen content being modified by collagen cross-linking. In
accordance with these data, we found that it is the degree of
CCL and the abundance of insCol but not the amount of total
or type I collagen fibers that associate with LV chamber
stiffness (ie, LV chamber stiffness constant) and FPs (ie,
PCWP) in patients with HHD and stage C HF. Thus, CCL
emerges as an important determinant of the function of the
left ventricle in these patients.
In this regard, an association has been reported between an
excess of CCL and diastolic dysfunction in patients with HF
and with preserved LVEF of different etiologies.21 On the
other hand, a reduction in CCL has been found to be
associated with improved systolic function in patients with
HF, with reduced LVEF submitted to support with LV assist
devices.22 Therefore, our observation that the increase of
CCL is associated with both diastolic (ie, reduced PFR) and
systolic (ie, reduced LVEF) dysfunction suggests that the
qualitative alterations of the myocardial collagen matrix have
a functional impact in patients with stage C HF of hypertensive origin. This is further reinforced by the finding that CCL
is also associated with the amino-terminal propeptide of brain
natriuretic peptide, an index of the severity of HF,23 in these
patients.
Cross-linking is a process whereby collagen fibrils are
covalently linked to one another resulting in insoluble fibers
with increased material stiffness and resistance to degradation
by MMPs. There are 2 major groups of collagen cross-links,
those derived from advanced glycation end product–mediated
glycation of lysine and hydroxylysine residues and those
initiated by the enzyme LOX that catalyzes the oxidation of
peptidyl lysine side chains of fibrillar collagens, resulting in
the formation of corresponding allysine aldehydes.24 Associations of enhanced myocardial LOX expression with increased CCL and LV stiffness have been reported in animals
with genetic25 or induced26,27 hypertension, as well as in
hypertensive patients without28 and with HF.18 Therefore, our
finding that LOX is associated with CCL in patients from this
study suggests that the altered regulation of this enzyme may
play a role in collagen-dependent disturbances of LV function
in stage C HF of hypertensive origin.
LOX is secreted from fibrogenic cells as a 50-kDa proenzyme that appears to have little or no enzymatic activity and
is processed in the extracellular environment to produce the
32-kDa catalytically active enzyme and a 18-kDa propeptide.29 Previous evidence suggests that the processing of the
pro-LOX precursor into the LOX active enzyme is mainly
accomplished by the metalloproteinase PCP30 and to a lesser
degree by the serine protease furin.31 Here we report that
LOX is associated with furin but not with PCP in the
myocardium of patients with HHD and HF, thus suggesting
that the regulation of this convertase is altered in HF
hypertensive patients with elevated PCWP. Interestingly, it
has been reported that microRNA-24 regulates protein and
mRNA levels of furin in cardiac fibroblasts.32 Furthermore,
microRNA-24 downregulation has been found to be associated with fibrosis in hypertrophic hearts.33 Further studies are
required to elucidate the potential contribution of furin in
collagen alterations of HF patients.
In addition, regarding the potential usefulness of circulating biomarkers of collagen metabolism to evaluate the quality
of myocardial collagen, CITP levels were found to be
inversely correlated with insCol, this correlation being independent of MMP-1 and TIMP-1. Taking into account that
collagen cross-linking determines collagen insolubility24 and
that insCol seems to be resistant to degradation by MMPs,34
it can be assumed that the more insCol is formed the less
collagen fibers will be degraded, thus contributing to fibrosis.
On the other hand, the above association would suggest that
serum CITP may constitute a biomarker of the process of
collagen cross-linking in the myocardium. Nevertheless, we
are aware that further studies are necessary to definitively test
this hypothesis.
Several limitations need to be acknowledged. First, this
was a study involving a relatively small number of patients,
but because of the nature of the goals under investigation, it
was adequately powered. Second, it must be recognized that
therapy may have confounded the findings and their interpretation. Nevertheless, none of the patients were treated with
the loop diuretic torasemide that has been shown to reduce
myocardial LOX and CCL in stage C HF patients.18 Third,
we performed biopsies of the right side of the interventricular
septum to assess the characteristics of collagen tissue. However, as we have shown previously,19 in terms of deposition
of collagen fibers, the septum is representative of the free
wall.
Perspectives
Data here presented suggest that a shift in treatment strategies
for HF of hypertensive origin directed more specifically at
affecting the process of collagen cross-linking rather than
reducing the content of collagen may be warranted in the
attenuation of the adverse functional impact of myocardial
682
Hypertension
September 2012
collagen matrix alterations. In this conceptual framework,
although some recent clinical data suggest that pharmacological interference with advanced glycation end product–mediated cross-linking is not associated with beneficial clinical
effect in HF patients,35 other findings suggest that inhibition
of LOX-mediated cross-linking results in functional and
clinical benefits in these patients.18 Therefore, a deeper
knowledge of the underlying mechanisms involved in LOX
dysregulation in the failing human heart are required to
generate both diagnostic and therapeutic strategies targeting
the enzyme to identify and interfere with, respectively, its
detrimental actions in HF patients.
12.
13.
14.
15.
Acknowledgments
We acknowledge Sonia Martínez and María González for their
technical assistance in biochemical determinations.
Sources of Funding
This work was funded through the agreement between the Foundation for Applied Medical Research and Unión Temporal de Empresas
Proyecto Centro de Investigación Médica Aplicada, the Red
Temática de Investigación Cooperativa en Enfermedades Cardiovasculares from the Instituto de Salud Carlos III, Ministry of Economy
and Competitiveness, Spain (grant RD06/0014/0008), and the European Union (MEDIA project grant HEALTH-2010-261409 and
EU-MASCARA project grant HEALTH-2011-278249). A.G. is a
recipient of a Ramón y Cajal contract from the Ministry of Economy
and Competitiveness, Spain (RYC-2010-05797).
16.
17.
Disclosures
18.
References
19.
None.
1. Levy D, Larson MG, Vasan RS, Kannel WB, Ho KK. The progression
from hypertension to congestive heart failure. JAMA. 1996;275:
1557–1562.
2. Tocci G, Sciarretta S, Volpe M. Development of heart failure in recent
hypertension trials. J Hypertens. 2008;26:1477–1486.
3. Weber KT. Fibrosis and hypertensive heart disease. Curr Opin Cardiol.
2000;15:264–272.
4. Díez J, Querejeta R, López B, González A, Larman M, Martínez Ubago
JL. Losartan-dependent regression of myocardial fibrosis is associated
with reduction of left ventricular chamber stiffness in hypertensive
patients. Circulation. 2002;105:2512–2517.
5. Brilla CG, Rupp H, Maisch B. Effects of ACE inhibition versus non-ACE
inhibitor antihypertensive treatment on myocardial fibrosis in patients
with arterial hypertension: retrospective analysis of 120 patients with left
ventricular endomyocardial biopsies. Herz. 2003;28:744–753.
6. Querejeta R, López B, González A, Sánchez E, Larman M, Martínez
Ubago JL, Díez J. Increased collagen type I synthesis in patients with
heart failure of hypertensive origin: relation to myocardial fibrosis. Circulation. 2004;110:1263–1268.
7. Goikoetxea MJ, Beaumont J, González A, López B, Querejeta R, Larman
M, Díez J. Altered cardiac expression of peroxisome proliferatoractivated receptor-isoforms in patients with hypertensive heart disease.
Cardiovasc Res. 2006;69:899–907.
8. Weber KT, Sun Y, Tyagi SC, Cleutjens JP. Collagen network of the
myocardium: function, structural remodeling and regulatory mechanisms.
J Mol Cell Cardiol. 1994;26:279–292.
9. Brower GL, Gardner JD, Forman MF, Murray DB, Vloshenyuk T, Levick
SP, Janicki JS. The relationship between myocardial extracellular matrix
remodeling and ventricular function. Eur J Cardiothor Surg. 2006;30:
604–610.
10. Mukherjee D, Sen S. Collagen phenotypes during development and
regression of myocardial hypertrophy in spontaneously hypertensive rats.
Circ Res. 1990;67:1474–1480.
11. Norton GR, Tsotetsi J, Trifunovic B, Hartford C, Candy GP, Woodiwiss
AJ. Myocardial stiffness is attributed to alterations in cross-linked
20.
21.
22.
23.
24.
25.
26.
27.
28.
collagen rather than total collagen or phenotypes in spontaneously hypertensive rats. Circulation. 1997;96:1991–1998.
Badenhorst D, Maseko M, Tsotetsi OJ, Naidoo A, Brooksbank R, Norton
GR, Woodiwiss AJ. Cross-linking influences the impact of quantitative
changes in myocardial collagen on cardiac stiffness and remodelling in
hypertension in rats. Cardiovasc Res. 2003;57:632–641.
López B, González A, Hermida N, Valencia F, de Teresa E, Díez J. Role
of lysyl oxidase in myocardial fibrosis: from basic science to clinical
aspects. Am J Physiol Heart Circ Physiol. 2010;299:H1–H9.
Ho KL, Pinsky JL, Kannel WB, Levy D. The epidemiology of heart
failure: the Framingham Study. J Am Coll Cardiol. 1993;22(suppl
A)6A–13A.
Klocke FJ, Baird MG, Lorell BH, Bateman TM, Messer JV, Berman DS,
O’Gara PT, Carabello BA, Russell RO Jr, Cerqueira MD, St John Sutton
MG, DeMaria AN, Udelson JE, Kennedy JW, Verani MS, Williams KA,
Antman EM, Smith SC Jr, Alpert JS, Gregoratos G, Anderson JL,
Hiratzka LF, Faxon DP, Hunt SA, Fuster V, Jacobs AK, Gibbons RJ,
Russell RO; American College of Cardiology; American Heart Association; American Society for Nuclear Cardiology. ACC/AHA/ASNC
guidelines for the clinical use of cardiac radionuclide imaging: executive
summary–a report of the American College of Cardiology/American
Heart Association Task Force on Practice Guidelines (ACC/AHA/ASNC
Committee to Revise the 1995 Guidelines for the Clinical Use of Cardiac
Radionuclide Imaging). J Am Coll Cardiol. 2003;42:1318–1333.
Davidson CJ, Bonow RO. Cardiac catheterization. In: Libby P, Bonow
RO, Mann DL, Zipes DP, eds. Braunwald’s Heart Disease. A Textbook of
Cardiovascular Medicine. Philadelphia, PA: Saunders Elsevier; 2008:
439–464.
Querejeta R, Varo N, López B, Larman M, Artiñano E, Etayo JC,
Martínez Ubago JL, Gutierrez-Stampa M, Emparanza JI, Gil MJ, Monreal
I, Mindán JP, Díez J. Serum carboxy-terminal propeptide of procollagen
type I is a marker of myocardial fibrosis in hypertensive heart disease.
Circulation. 2000;101:1729–1735.
López B, Querejeta R, González A, Beaumont J, Larman M, Díez J.
Impact of treatment on myocardial lysyl oxidase expression and collagen
cross-linking in patients with heart failure. Hypertension. 2009;53:
236–242.
López B, González A, Querejeta R, Larman M, Díez J. Alterations in the
pattern of collagen deposition may contribute to the deterioration of
systolic function in hypertensive patients with heart failure. J Am Coll
Cardiol. 2006;48:89–96.
Makarenko I, Opitz CA, Leake MC, Neagoe C, Kulke M, Gwathmey JK,
del Monte F, Hajjar RJ, Linke WA. Passive stiffness changes caused by
upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts. Circ Res. 2004;95:708–716.
Kasner M, Westermann D, Lopez B, Gaub R, Escher F, Kühl U, Schultheiss HP, Tschöpe C. Diastolic tissue Doppler indexes correlate with the
degree of collagen expression and cross-linking in heart failure and
normal ejection fraction. J Am Coll Cardiol. 2011;57:977–985.
Li YY, Feng Y, McTiernan CF, Pei W, Moravec CS, Wang P, Rosenblum
W, Kormos RL, Feldman AM. Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after
support with left ventricular assist devices. Circulation. 2001;104:
1147–1152.
Kim HN, Januzzi JL Jr. Natriuretic peptide testing in heart failure.
Circulation. 2011;123:2015–2019.
Reiser K, McCormick RJ, Bucker RB. Enzymatic and nonenzymatic
cross-linking of collagen and elastin. FASEB J. 1992;6:2439–2449.
Hermida N, López B, González A, Dotor J, Lasarte JJ, Sarobe P, BorrásCuesta F, Díez J. A synthetic peptide from transforming growth factor-␤1
type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats. Cardiovasc Res. 2009;81:601–609.
Yu Q, Horak K, Larson DF. Role of T lymphocytes in hypertensioninduced cardiac extracellular matrix remodeling. Hypertension. 2006;48:
98–104.
Zibadi S, Vazquez R, Moore D, Larson DF, Watson RR. Myocardial lysyl
oxidase regulation of cardiac remodeling in a murine model of dietinduced metabolic syndrome. Am J Physiol Heart Circ Physiol. 2009;
297:H976–H982.
Brilla CG, Funck RC, Rupp H. Lisinopril-mediated regression of myocardial fibrosis in patients with hypertensive heart disease. Circulation.
2000;102:1388–1393.
López et al
29. Trackman PC, Bedell-Hogan D, Tang J, Kagan HM. Post-translational
glycosylation and proteolytic processing of a lysyl oxidase precursor.
J Biol Chem. 1992;267:8666–8671.
30. Uzel MI, Shih SD, Gross H, Kessler E, Gerstenfeld LC, Trackman PC.
Molecular events that contribute to lysyl oxidase enzyme activity and
insoluble collagen accumulation in osteosarcoma cell clones. J Bone
Miner Res. 2000;15:1189–1197.
31. Borell A, Eichenberger D, Farjanel J, Kessler E, Glyzal C, Hulmes DJS,
Sommert P, Font B. Lysyl-oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.
J Biol Chem. 2001;276:48944–48949.
32. Wang J, Huang W, Xu R, Nie Y, Cao X, Meng J, Xu X, Hu S, Zheng Z.
MicroRNA-24 regulates cardiac fibrosis after myocardial infarction.
J Cell Mol Med. January 19, 2012. DOI: 10.1111/j.1582-4934.2012.01523.x.
Collagen Cross-Linking and Heart Failure
683
http://onlinelibrary.wiley.com/doi/10.1111/j.1582-4934.2012.01523.x/pdf.
Accessed July 17, 2012.
33. Wang J, Xu R, Lin F, Zhang S, Zhang G, Hu S, Zheng Z. MicroRNA:
novel regulators involved in the remodeling and reverse remodeling of the
heart. Cardiology. 2009;113:81–88.
34. Aronson D. Cross-linking of glycated collagen in the pathogenesis of
arterial and myocardial stiffening of aging and diabetes. J Hypertens.
2003;21:3–12.
35. Hartog JW, Willemsen S, van Veldhuisen DJ, Posma JL, van Wijk LM,
Hummel YM, Hillege HL, Voors AA; BENEFICIAL investigators.
Effects of alagebrium, an advanced glycation endproduct breaker, on
exercise tolerance and cardiac function in patients with chronic heart
failure. Eur J Heart Fail. 2011;13:899–908.
Novelty and Significance
What Is New?
●
FPs are associated with the quality (ie, the degree of CCL) but not with
the quantity of collagen present in the myocardium of patients with HHD
and stage C HF.
●
The association of LOX with CCL in patients from this study suggest that
disregulation of this enzyme may play a role in disturbances of LV
function in stage C HF of hypertensive origin, thus pointing to LOX as a
potential target for novel therapeutic strategies in HF.
What Is Relevant?
Summary
Data here presented point to an excess of CCL as responsible for
alterations of LV compliance and function in patients with HF of
hypertensive origin. Because of the potential involvement of LOX in
this collagen alteration, the possibility to develop either genetic or
biochemical and imaging markers of myocardial LOX expression
and/or activity could help to explore its contribution to the
diagnostic and prognostic handling of patients with HHD and likely
other cardiac diseases also evolving with HF. In this setting,
circulating CITP emerges as a potential biomarker of the amount of
myocardial insoluble collagen. This would represent the first
biomarker of the type of myocardial collagen that, because of its
physicochemical properties, more negatively impacts on LV function. Finally, a deeper knowledge of LOX structure/function, as well
as of the underlying mechanisms involved in myocardial LOX
dysregulation, may help to generate effective therapeutic strategies targeting the enzyme to prevent the qualitative alterations of
the myocardial collagen matrix in HF patients.