# Circulating Tissue Inhibitor of Matrix  Metalloproteinase-4 (TIMP-4) in Systemic Sclerosis Patients with Elevated Pulmonary Arterial Pressure

Authors: Gialafos J. Elias; Moyssakis Ioannis; Psaltopoulou Theodora; Papadopoulos P. Dimitrios; Perea Despoina; Vlasis Kostantinos; Kostopoulos Charalampos; Votteas Vassilios; Sfikakis P. Petros
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Decreased levels of matrix metalloproteinases (MMPs) or excess levels of their tissue inhibitors (TIMPs) may contribute to dysregulation of extracellular matrix turnover in systemic sclerosis (SSc).  In a cross-sectional study of 106 SSc patients, we measured serum levels of TIMP-4 which is preferentially expressed in cardiovascular structures and searched for correlations with simultaneously performed echocardiography measurements of pulmonary artery systolic pressure (PASP), myocardial performance, and pulmonary function tests. TIMP-4, but not MMP-9, levels were significantly raised in patients with SSc than controls. However, in the subgroup of patients with PASP measurements lower to 40 mmHg  (n=69), TIMP-4 levels were comparable to controls irrespective of the presence of diffuse or limited skin involvement, or lung fibrosis. Individual PASP measurements suggestive of pulmonary hypertension were associated with increased TIMP-4 serum levels   (P=.03),  independently of age, extent of skin sclerosis, or lung fibrosis, suggesting a cardiopulmonary vasculature-specific role of TIMP-4 activation in SSc.


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## Body

## 1. Introduction

Systemic sclerosis
(SSc) is characterized  by excessive
accumulation of collagen and  other
components of extracellular  matrix in the skin and internal organs, being
perhaps the prototypic disorder of a generalized disruption of connective
tissue homeostasis [1]. Vasoconstriction
and structural changes of the blood vessels, including intimal proliferation
and obstruction, are expressed clinically as Raynaud’s phenomenon, digital
ulcers, renal disease, cardiac disease, and pulmonary hypertension (PH). Cardiopulmonary complications, including PH which
occurs in a significant proportion of patients either as an isolated
abnormality or secondary to pulmonary fibrosis, are currently the leading cause
of death in SSc [1, 2]. Although effective screening for PH has proven
difficult, many experts believe that early detection and intervention may alter
the natural history of the disease [3].Connective tissue turnover depends on the balance between the synthesis
and degradation of the extracellular matrix. Extracellular matrix degradation is regulated
mainly by matrix metalloproteinases (MMP-1 to MMP-28) and an important
mechanism for the regulation of their activity is via binding to a family of
homologous proteins, the tissue inhibitors of metalloproteinases (TIMP-1 to
TIMP-4). Several lines of evidence indicate that the balance between MMPs and
TIMPs levels governs connective tissue homeostasis, being a crucial determinant
in inflammation, fibrosis and angiogenesis [4, 5]. Fibroblasts derived from patients with SSc
produce increased amounts of TIMP-1, TIMP-2, and TIMP-3 [6, 7], whereas
expression of MMP-1, MMP-2, and MMP-3 genes is decreased in fibroblasts from
patients with early SSc compared to fibroblasts from healthy individuals or
patients with late-stage disease [6]. These and other results suggest that
excess levels of TIMPs, or decreased levels of MMPs may contribute to matrix
accumulation in SSc.TIMP-4 is the newest member in the mammalian TIMP family and differs
from the other 3 TIMPs by its expression pattern. TIMP-4 is abundantly expressed in human
cardiovascular structures, while all other tissues at the normal state,
including the lung parenchyma, are characterized by low or absent expression [8]. Animal studies have suggested an important
role of TIMP-4 in inflammatory diseases and cardiovascular pathologies
[4, 5]. Moreover, TIMP-4 myocardial
expression is remarkably increased in patients with aortic stenosis undergoing
surgery [9], and in dilated cardiomyopathy patients with deteriorating heart
failure [10]. On the other hand, MMP-9 is also found in cardiac
myocytes, cardiac fibroblasts, and endocardial cells [4]. Although among other TIMPs there is only
little specificity for inhibiting individual MMPs, key factors in every MMP
inhibition are the size, charge, and polarity of residue 2 in the particular structure of
TIMP-4 [11].Based on the above, we hypothesized that aberrant TIMP-4 and/or MMP-9
activation may play a role in cardiovascular complications of SSc. To test this hypothesis, we examined serum
levels of these molecules as
well as of B-type natriuretic peptide (BNP), an
established marker of SSc-related cardiovascular pathology [1, 12], and searched for correlations with
echocardiography measurements of pulmonary artery systolic pressure (PASP),
myocardial performance, and pulmonary function tests.


## 2. Patients and Methods

### 2.1. Study Population

One hundred and six consecutive patients (102 women) with SSc, aged
between 22 and 80 years (mean 54± 13 years) and with disease duration
ranging between 2 to 25 years (mean 11 ± 4 years) from date of the first
non-Raynaud’s phenomenon SSc manifestation, participated in this
cross-sectional study. Blood samples
were collected at the day of their regular follow-up which included lung
function tests and echocardiography. SSc patients with previous myocardial
infarction or stroke, valvular or congenital heart disease, hypertrophic
cardiomyopathy, previous history of arterial hypertension, chronic obstructive
pulmonary disease, or malignancies, as well those patients receiving bosentan
or intravenous iloprost during the last 4 weeks, were excluded. Seventeen patients (15%) were current
smokers. As shown in Table 1, medications
included calcium-channel blockers in 77%, angiotensin-converting enzyme inhibitors
in 55%, corticosteroids in 29%, cyclophosphamide in 22%, and mycophenolate
mofetil in 7% of patients.Table 1
Characteristics of patients with
systemic sclerosis (SSc).
All
patientsDiffuse
SScLimited
SScWomen/men (n)102/471/431/0Mean age± SD,54± 1352± 1356± 12years
(range)(22–80)(22–80)(25–77)Mean disease duration±
SD,11± 410± 312± 4years (range)(2–25)(2–20)(2–25)Medications,
% of patients(n)Calcium—channel blockers77% (82)76% (57)81% (25)Angiotensin—converting
enzyme inhibitors55% (58)63% (47)31%
(11)Corticosteroids29% (31)35% (26)16% (5)Cyclophosphamide22% (21)23% (17)13% (4)Mycophenolate
mofetil7% (7)8% (6)3% (1)Pulmonary function tests (Master-Screen Diffusion, Jaeger, Wuerzburg,
Germany) included spirometry, total lung
capacity (TLC), and carbon monoxide diffusing capacity (DLCO) measurements, as
described in [13, 14]. In all patients with both TLC and DLCO lower than 80% of predicted
(indicative of pulmonary fibrosis) high resolution computed
tomography of the lung [14] performed during the previous 6 months had confirmed
the presence of fibrosis.Complete echocardiographic examination (Hewlet-Packard Sonos 1000 ultrasound system, using a
2.5 MHz transducer) was performed as described in
detail elsewhere [15], and  established indices of myocardial performance for
right and left ventricles (Tei-index) were calculated, as described in [16]. PASP was considered elevated when exceeded the
level of 40 mm Hg [17]. There were 37
patients with elevated PASP; 18 of them had undergone right heart
catheterization during the previous year confirming the presence of PH secondary to SSc
[18] in all.Sera collected from 24 age-matched healthy subjects (54± 19 years,
23 women), who fulfilled the exclusion criteria employed for patients, served as
controls. All controls underwent a
complete examination comprising electrocardiogram, echocardiography and
exercise stress to exclude asymptomatic cardiac disease. Of the 24 control
subjects, 8 women were current smokers, a marginally higher frequency comparing
to 17 of 106 patients with SSc (P  =  .052). The study protocol was approved by Laikon Hospital and Alexandra Hospital
ethics committees and all
subjects gave informed consent.


### 2.2. Measurements of Circulating TIMP-4, MMP-9, and BNP Molecules

Circulating levels
of TIMP-4 and MMP-9were measured by quantitative sandwich
enzyme-linked immunosorbent assays (Quantikine human TIMP-4 and Quantikine
human MMP-9 total, respectively, R&D Systems Inc., Minneapolis, Minn, USA) according to the manufacturer’s instructions in patient’s and control sera
that had been kept at−70 C. BNP concentrations were measured immediately after
venipuncture in plasma samples from SSc patients using a sandwich immunoenzymatic assay
(Triage BNP test, Biosite, San Diego, Calif, USA), according to the manufacturer’s instructions.


### 2.3. Statistical Analysis

Comparisons for continuous variables between groups were
performed usingt-test or Mann-Whitney test,
in case of normal or skewed distribution, respectively. Age-adjusted partial correlation coefficients were
built to evaluate examined correlations. TIMP-4, MMP-9/TIMP-4 ratios, and BNP
were expressed as log10(TIMP-4), log10(MMP-9/TIMP-4), and log10(BNP),
respectively, when correlated or regressed because of their skewed distribution.
Bonferroni correction was used in cases of
multiple testing to avoid false positive associations. Multivariate regression analysis was used to assess the association of
TIMP-4 and/or MMP-9 levels with PASP, after controlling for possible
confounders, such as age, presence or not of lung fibrosis, type of skin
involvement (diffuse or limited), and BNP. The statistical package used was SPSS 13.0. Values
are expressed as mean ± SD and a P-value
< .05 was considered significant.


## 2.1. Study Population

One hundred and six consecutive patients (102 women) with SSc, aged
between 22 and 80 years (mean 54± 13 years) and with disease duration
ranging between 2 to 25 years (mean 11 ± 4 years) from date of the first
non-Raynaud’s phenomenon SSc manifestation, participated in this
cross-sectional study. Blood samples
were collected at the day of their regular follow-up which included lung
function tests and echocardiography. SSc patients with previous myocardial
infarction or stroke, valvular or congenital heart disease, hypertrophic
cardiomyopathy, previous history of arterial hypertension, chronic obstructive
pulmonary disease, or malignancies, as well those patients receiving bosentan
or intravenous iloprost during the last 4 weeks, were excluded. Seventeen patients (15%) were current
smokers. As shown in Table 1, medications
included calcium-channel blockers in 77%, angiotensin-converting enzyme inhibitors
in 55%, corticosteroids in 29%, cyclophosphamide in 22%, and mycophenolate
mofetil in 7% of patients.Table 1
Characteristics of patients with
systemic sclerosis (SSc).
All
patientsDiffuse
SScLimited
SScWomen/men (n)102/471/431/0Mean age± SD,54± 1352± 1356± 12years
(range)(22–80)(22–80)(25–77)Mean disease duration±
SD,11± 410± 312± 4years (range)(2–25)(2–20)(2–25)Medications,
% of patients(n)Calcium—channel blockers77% (82)76% (57)81% (25)Angiotensin—converting
enzyme inhibitors55% (58)63% (47)31%
(11)Corticosteroids29% (31)35% (26)16% (5)Cyclophosphamide22% (21)23% (17)13% (4)Mycophenolate
mofetil7% (7)8% (6)3% (1)Pulmonary function tests (Master-Screen Diffusion, Jaeger, Wuerzburg,
Germany) included spirometry, total lung
capacity (TLC), and carbon monoxide diffusing capacity (DLCO) measurements, as
described in [13, 14]. In all patients with both TLC and DLCO lower than 80% of predicted
(indicative of pulmonary fibrosis) high resolution computed
tomography of the lung [14] performed during the previous 6 months had confirmed
the presence of fibrosis.Complete echocardiographic examination (Hewlet-Packard Sonos 1000 ultrasound system, using a
2.5 MHz transducer) was performed as described in
detail elsewhere [15], and  established indices of myocardial performance for
right and left ventricles (Tei-index) were calculated, as described in [16]. PASP was considered elevated when exceeded the
level of 40 mm Hg [17]. There were 37
patients with elevated PASP; 18 of them had undergone right heart
catheterization during the previous year confirming the presence of PH secondary to SSc
[18] in all.Sera collected from 24 age-matched healthy subjects (54± 19 years,
23 women), who fulfilled the exclusion criteria employed for patients, served as
controls. All controls underwent a
complete examination comprising electrocardiogram, echocardiography and
exercise stress to exclude asymptomatic cardiac disease. Of the 24 control
subjects, 8 women were current smokers, a marginally higher frequency comparing
to 17 of 106 patients with SSc (P  =  .052). The study protocol was approved by Laikon Hospital and Alexandra Hospital
ethics committees and all
subjects gave informed consent.


## 2.2. Measurements of Circulating TIMP-4, MMP-9, and BNP Molecules

Circulating levels
of TIMP-4 and MMP-9were measured by quantitative sandwich
enzyme-linked immunosorbent assays (Quantikine human TIMP-4 and Quantikine
human MMP-9 total, respectively, R&D Systems Inc., Minneapolis, Minn, USA) according to the manufacturer’s instructions in patient’s and control sera
that had been kept at−70 C. BNP concentrations were measured immediately after
venipuncture in plasma samples from SSc patients using a sandwich immunoenzymatic assay
(Triage BNP test, Biosite, San Diego, Calif, USA), according to the manufacturer’s instructions.


## 2.3. Statistical Analysis

Comparisons for continuous variables between groups were
performed usingt-test or Mann-Whitney test,
in case of normal or skewed distribution, respectively. Age-adjusted partial correlation coefficients were
built to evaluate examined correlations. TIMP-4, MMP-9/TIMP-4 ratios, and BNP
were expressed as log10(TIMP-4), log10(MMP-9/TIMP-4), and log10(BNP),
respectively, when correlated or regressed because of their skewed distribution.
Bonferroni correction was used in cases of
multiple testing to avoid false positive associations. Multivariate regression analysis was used to assess the association of
TIMP-4 and/or MMP-9 levels with PASP, after controlling for possible
confounders, such as age, presence or not of lung fibrosis, type of skin
involvement (diffuse or limited), and BNP. The statistical package used was SPSS 13.0. Values
are expressed as mean ± SD and a P-value
< .05 was considered significant.


## 3. Results

### 3.1. Circulating MMP-9 and TIMP-4 Levels and Extent of Skin or Pulmonary Fibrosis in SSc

Of the 106 patients, 75 had diffuse (truncal skin involvement) and 31
patients had limited SSc (skin sclerosis confined to hands, arms, feet, and
face), according to LeRoy’s classification [19]
(Table 1). Mean values of pulmonary
function tests were comparable between patients with diffuse and limited SSc
(Table 2); pulmonary  fibrosis was present in 46 patients
(34 with diffuse SSc).Table 2
Pulmonary function tests (mean± SD
of % predicted, number of patients with less
than 80% of predicted) in patients with systemic sclerosis (SSc).
All patientsDiffuse SScLimited SSc(n = 106)(n = 75)(n = 31)FEV186± 17, 3286± 18, 2688± 16, 6FVC87± 19, 3187± 20, 2590± 16, 6TLC80± 16, 4679± 16, 3481± 17, 12DLCO68± 21, 8167± 22, 5469± 19, 27FEV1: forced expiratory volume
at 1 second;
FVC: forced vital capacity;
TLC: total lung capacity;
DLCO: diffusing lung capacity
for carbon monoxide.MMP-9 levels were not different between the whole SSc patient group and
controls (530± 260 ng/mL versus 446 ± 201 ng/mL, resp.), but patients
with diffuse SSc had higher MMP-9 levels than controls
(Figure 1(a)) as well as than patients with limited
SSc (587 ± 266 ng/mL versus 393 ± 182 ng/mL, P=.0003).
No significant difference was noted
between patients with lung fibrosis and those without (548 ± 222 ng/mL versus 517 ± 287 ng/mL).(a) MMP-9, (b) TIMP-4, and (c) BNP
blood levels in SSc patients with diffuse (dSSc) and limited (lSSc) skin
involvement as well as in those patients with pulmonary fibrosis (PF+) compared
to healthy controls (Mann-Whitney test, NS denotes
nonsignificant).
(a)(b)(c)In contrast to MMP-9, TIMP-4 levels were raised in the whole SSc patient
group (2035± 1064 pg/mL, range 380–4961 pg/mL)
compared to controls (1484 ± 489 pg/mL, range 683–2661 pg/mL) as
well as in subgroups of patients with diffuse (2028 ± 1100 pg/mL, range
380–4961 ng/mL) or
limited SSc (2050 ± 987 ng/mL, range 694–4900 ng/mL). Also, TIMP-4 levels were significantly higher
in patients with pulmonary fibrosis (2157 ± 1068 ng/mL, range 846–4900 ng/mL) than
controls (Figure 1(b)).As shown in Table3, age-adjusted
partial correlations revealed no significant associations between individual
serum levels of MMP-9, TIMP-4, or their ratio with corresponding pulmonary function
tests. No significant differences, either for TIMP-4 or for MMP-9 mean levels,
were noted between patients receiving, or not, angiotensin-converting enzyme
inhibitors, or between patients receiving immunomodulatory drugs and the
remaining patients.Table 3
Age-adjusted partial correlations (r)  of  MMP-9, TIMP-4 and their ratio
with respiratory and cardiac indicators in 106 patients with systemic sclerosis.
Levels of significance are shown in parentheses.
MMP-9Log10(TIMP-4)Log10(MMP-9/TIMP-4)Pulmonary
function testsFEV1 (%predicted)−0.14
(NS)−0.09
(NS)FVC (%predicted)−0.14 (NS)−0.14 (NS)TLC (%predicted)−0.16(P=.070)−0.09 (NS)DLCO (%predicted)−0.19(P=.068)−0.70 (NS)Cardiac parametersPASP0.02 (NS)0.29(P=0.021)log10 (BNP)0.00 (NS)0.19 (NS)RV Tei Index0.02 (NS)0.15 (NS)LV  Tei Index−0.23(P=.046)0.17 (NS)LV  EF (%)0.07 (NS)−0.15
(NS)FEV1: forced expiratory volume
at 1 second;
FVC: forced vital capacity;
TLC: total lung capacity;
DLCO: diffusing lung capacity
for carbon monoxide;
PASP: pulmonary artery systolic
pressure;
BNP: B-type natriuretic peptide;
RV: right ventricular;
LV: left ventricular;
EF: ejection fraction;
NS denotes nonsignificant.


### 3.2. Elevated Pulmonary Artery Pressure in SSc Is Associated with Increased TIMP-4 Levels

Thirty seven of 106 patients with SSc (21 with diffuse and 16 with
limited SSc) had PASP measurements equal or higher than 40 mm Hg in
echocardiography (range 40–85 mm Hg, mean ± SD 51 ± 12 mm Hg). Age,
disease duration, digital ulcers, arthritis, esophageal or intestinal
involvement, antibodies to Scl-70, and current treatment regimens were
comparable between patients with elevated PASP and the remaining patients. Of patients with elevated PASP, 22 had concomitant pulmonary fibrosis;
the remaining 15 patients, all with limited SSc, had DLCO reduction as an
isolated abnormality. As shown in Table4, echocardiography-derived
measurements of myocardial performance were significantly compromised in
patients with elevated PASP measurements secondary to either diffuse or limited
SSc, compared to the remaining patients. High BNP blood levels reflecting
abnormalities in the cardiopulmonary vasculature were found in many of the
studied patients with SSc (depicted
in Figure 1(c)). As expected, BNP levels were increased by
almost 5-fold in patients with elevated PASP than the remaining patients
(Table 4).Table 4
Echocardiography-derived measurements of myocardial performance and BNP
blood levels (mean± SD) in SSc patients with normal or elevated PASP.
RV Tei-indexLV  Tei-indexLV EF (%)BNP (pg/mL)PASP ≤40 mm Hg (all patients,n = 69)0.37±0.020.38±0.0161± 933± 23PASP >40 mm Hg (all patients,n = 37)0.41±0.03(P<.001)*0.41±0.02(P<.001)*60± 5163± 159
(P<.0001)*PASP >40 mm Hg
(diffuse SSc,n = 21)0.41±0.030.41±0.0260± 5167± 187PASP >40 mm Hg (limited SSc,n = 16)0.42±0.030.40±0.0261± 4164± 120SSc: systemic sclerosis;
PASP: pulmonary artery systolic
pressure;
BNP: B-type natriuretic peptide;
RV: right ventricular;
LV: left ventricular;
EF: ejection fraction.
*Comparing to patients with PASP ≤40.As shown in Figure2,
TIMP-4 serum levels were considerably higher in SSc patients with elevated PASP
measurements (2486 ± 1190 pg/mL, range 850–4961 pg/mL) than
the remaining patients (1792 ± 909 pg/mL, range 380–4862 pg/mL, P  = .003).
Notably, after excluding the 37 patients with PASP ≥40 mm Hg from the
whole SSc group, there were no significant differences in TIMP-4 levels between
the control group and patients with diffuse (1767 ± 929 pg/mL, n = 53) or
limited SSc (1875 ± 862 pg/mL, n = 16), or patients with lung fibrosis
(1916 ± 918 pg/mL, n = 24), or those without
(1726 ± 908 pg/mL, n = 45). TIMP-4 levels
differed significantly between SSc patients, when as criterion for abnormally
elevated PASP the level of 50 mm Hg (2880 ± 1174 pg/mL, n = 13, versus 1916 ± 998 pg/mL, P  = .002), or the level of
45 mm Hg
(2586  ±  1283 pg/mL, n = 23
versus 1882 ±
948 pg/mL, P  = .02) was considered.Figure 2
Patients with SSc and elevated pulmonary artery systolic pressure (PASP ≥40 mm Hg) have higher TIMP-4 levels and smaller MMP-9/TIMP-4 ratios
(bars show
SD) than the remaining patients (Mann-Whitney test).In contrast, MMP-9 serum levels were slightly lower in patients with
elevated PASP than the remaining patients (511± 265 ng/mL and 541 ± 258 ng/mL, resp.). MMP-9/TIMP-4 ratios were significantly smaller in patients with
elevated PASP (255 ± 192) than in those with PASP measurements lower than
40 mm Hg (402 ± 380)(Figure 2).As shown in Table3, age-adjusted partial correlation
coefficients between individual TIMP-4 serum levels and the corresponding
levels of PASP revealed a positive significant correlation. Since multiple
testing may result to false positive associations, the Bonferroni correction
was used, yielding the same results. On the other hand, echocardiographic
indicators of either global myocardial performance (Tei-indices), or of left
ventricle’s systolic function (ejection fraction) did not correlate
significantly with TIMP-4 circulating levels. In contrast, increased left
ventricle Tei-index, indicative of impaired performance, was associated
significantly with lower MMP-9 levels and MMP-9/TIMP-4 ratios
(Table 3).Finally, stepwise multivariate
linear regression analysis was performed to assess possible associations among
individual PASP measurements and the 3 corresponding clinical and laboratory
parameters under study for the 106 SSc patients. Using this model, we found
that increased PASP measurements were associated with
TIMP-4 (log transformed-continuous,β-coefficient = 0.180, P  = .031) and BNP
(log transformed-continuous, β-coefficient = 0.534, P < .001). In contrast, no significant
associations could be established with age (continuous), MMP-9 (continuous), SSc
type (diffuse, limited), or presence of lung fibrosis (no, yes) in our SSc patient cohort.


## 3.1. Circulating MMP-9 and TIMP-4 Levels and Extent of Skin or Pulmonary Fibrosis in SSc

Of the 106 patients, 75 had diffuse (truncal skin involvement) and 31
patients had limited SSc (skin sclerosis confined to hands, arms, feet, and
face), according to LeRoy’s classification [19]
(Table 1). Mean values of pulmonary
function tests were comparable between patients with diffuse and limited SSc
(Table 2); pulmonary  fibrosis was present in 46 patients
(34 with diffuse SSc).Table 2
Pulmonary function tests (mean± SD
of % predicted, number of patients with less
than 80% of predicted) in patients with systemic sclerosis (SSc).
All patientsDiffuse SScLimited SSc(n = 106)(n = 75)(n = 31)FEV186± 17, 3286± 18, 2688± 16, 6FVC87± 19, 3187± 20, 2590± 16, 6TLC80± 16, 4679± 16, 3481± 17, 12DLCO68± 21, 8167± 22, 5469± 19, 27FEV1: forced expiratory volume
at 1 second;
FVC: forced vital capacity;
TLC: total lung capacity;
DLCO: diffusing lung capacity
for carbon monoxide.MMP-9 levels were not different between the whole SSc patient group and
controls (530± 260 ng/mL versus 446 ± 201 ng/mL, resp.), but patients
with diffuse SSc had higher MMP-9 levels than controls
(Figure 1(a)) as well as than patients with limited
SSc (587 ± 266 ng/mL versus 393 ± 182 ng/mL, P=.0003).
No significant difference was noted
between patients with lung fibrosis and those without (548 ± 222 ng/mL versus 517 ± 287 ng/mL).(a) MMP-9, (b) TIMP-4, and (c) BNP
blood levels in SSc patients with diffuse (dSSc) and limited (lSSc) skin
involvement as well as in those patients with pulmonary fibrosis (PF+) compared
to healthy controls (Mann-Whitney test, NS denotes
nonsignificant).
(a)(b)(c)In contrast to MMP-9, TIMP-4 levels were raised in the whole SSc patient
group (2035± 1064 pg/mL, range 380–4961 pg/mL)
compared to controls (1484 ± 489 pg/mL, range 683–2661 pg/mL) as
well as in subgroups of patients with diffuse (2028 ± 1100 pg/mL, range
380–4961 ng/mL) or
limited SSc (2050 ± 987 ng/mL, range 694–4900 ng/mL). Also, TIMP-4 levels were significantly higher
in patients with pulmonary fibrosis (2157 ± 1068 ng/mL, range 846–4900 ng/mL) than
controls (Figure 1(b)).As shown in Table3, age-adjusted
partial correlations revealed no significant associations between individual
serum levels of MMP-9, TIMP-4, or their ratio with corresponding pulmonary function
tests. No significant differences, either for TIMP-4 or for MMP-9 mean levels,
were noted between patients receiving, or not, angiotensin-converting enzyme
inhibitors, or between patients receiving immunomodulatory drugs and the
remaining patients.Table 3
Age-adjusted partial correlations (r)  of  MMP-9, TIMP-4 and their ratio
with respiratory and cardiac indicators in 106 patients with systemic sclerosis.
Levels of significance are shown in parentheses.
MMP-9Log10(TIMP-4)Log10(MMP-9/TIMP-4)Pulmonary
function testsFEV1 (%predicted)−0.14
(NS)−0.09
(NS)FVC (%predicted)−0.14 (NS)−0.14 (NS)TLC (%predicted)−0.16(P=.070)−0.09 (NS)DLCO (%predicted)−0.19(P=.068)−0.70 (NS)Cardiac parametersPASP0.02 (NS)0.29(P=0.021)log10 (BNP)0.00 (NS)0.19 (NS)RV Tei Index0.02 (NS)0.15 (NS)LV  Tei Index−0.23(P=.046)0.17 (NS)LV  EF (%)0.07 (NS)−0.15
(NS)FEV1: forced expiratory volume
at 1 second;
FVC: forced vital capacity;
TLC: total lung capacity;
DLCO: diffusing lung capacity
for carbon monoxide;
PASP: pulmonary artery systolic
pressure;
BNP: B-type natriuretic peptide;
RV: right ventricular;
LV: left ventricular;
EF: ejection fraction;
NS denotes nonsignificant.


## 3.2. Elevated Pulmonary Artery Pressure in SSc Is Associated with Increased TIMP-4 Levels

Thirty seven of 106 patients with SSc (21 with diffuse and 16 with
limited SSc) had PASP measurements equal or higher than 40 mm Hg in
echocardiography (range 40–85 mm Hg, mean ± SD 51 ± 12 mm Hg). Age,
disease duration, digital ulcers, arthritis, esophageal or intestinal
involvement, antibodies to Scl-70, and current treatment regimens were
comparable between patients with elevated PASP and the remaining patients. Of patients with elevated PASP, 22 had concomitant pulmonary fibrosis;
the remaining 15 patients, all with limited SSc, had DLCO reduction as an
isolated abnormality. As shown in Table4, echocardiography-derived
measurements of myocardial performance were significantly compromised in
patients with elevated PASP measurements secondary to either diffuse or limited
SSc, compared to the remaining patients. High BNP blood levels reflecting
abnormalities in the cardiopulmonary vasculature were found in many of the
studied patients with SSc (depicted
in Figure 1(c)). As expected, BNP levels were increased by
almost 5-fold in patients with elevated PASP than the remaining patients
(Table 4).Table 4
Echocardiography-derived measurements of myocardial performance and BNP
blood levels (mean± SD) in SSc patients with normal or elevated PASP.
RV Tei-indexLV  Tei-indexLV EF (%)BNP (pg/mL)PASP ≤40 mm Hg (all patients,n = 69)0.37±0.020.38±0.0161± 933± 23PASP >40 mm Hg (all patients,n = 37)0.41±0.03(P<.001)*0.41±0.02(P<.001)*60± 5163± 159
(P<.0001)*PASP >40 mm Hg
(diffuse SSc,n = 21)0.41±0.030.41±0.0260± 5167± 187PASP >40 mm Hg (limited SSc,n = 16)0.42±0.030.40±0.0261± 4164± 120SSc: systemic sclerosis;
PASP: pulmonary artery systolic
pressure;
BNP: B-type natriuretic peptide;
RV: right ventricular;
LV: left ventricular;
EF: ejection fraction.
*Comparing to patients with PASP ≤40.As shown in Figure2,
TIMP-4 serum levels were considerably higher in SSc patients with elevated PASP
measurements (2486 ± 1190 pg/mL, range 850–4961 pg/mL) than
the remaining patients (1792 ± 909 pg/mL, range 380–4862 pg/mL, P  = .003).
Notably, after excluding the 37 patients with PASP ≥40 mm Hg from the
whole SSc group, there were no significant differences in TIMP-4 levels between
the control group and patients with diffuse (1767 ± 929 pg/mL, n = 53) or
limited SSc (1875 ± 862 pg/mL, n = 16), or patients with lung fibrosis
(1916 ± 918 pg/mL, n = 24), or those without
(1726 ± 908 pg/mL, n = 45). TIMP-4 levels
differed significantly between SSc patients, when as criterion for abnormally
elevated PASP the level of 50 mm Hg (2880 ± 1174 pg/mL, n = 13, versus 1916 ± 998 pg/mL, P  = .002), or the level of
45 mm Hg
(2586  ±  1283 pg/mL, n = 23
versus 1882 ±
948 pg/mL, P  = .02) was considered.Figure 2
Patients with SSc and elevated pulmonary artery systolic pressure (PASP ≥40 mm Hg) have higher TIMP-4 levels and smaller MMP-9/TIMP-4 ratios
(bars show
SD) than the remaining patients (Mann-Whitney test).In contrast, MMP-9 serum levels were slightly lower in patients with
elevated PASP than the remaining patients (511± 265 ng/mL and 541 ± 258 ng/mL, resp.). MMP-9/TIMP-4 ratios were significantly smaller in patients with
elevated PASP (255 ± 192) than in those with PASP measurements lower than
40 mm Hg (402 ± 380)(Figure 2).As shown in Table3, age-adjusted partial correlation
coefficients between individual TIMP-4 serum levels and the corresponding
levels of PASP revealed a positive significant correlation. Since multiple
testing may result to false positive associations, the Bonferroni correction
was used, yielding the same results. On the other hand, echocardiographic
indicators of either global myocardial performance (Tei-indices), or of left
ventricle’s systolic function (ejection fraction) did not correlate
significantly with TIMP-4 circulating levels. In contrast, increased left
ventricle Tei-index, indicative of impaired performance, was associated
significantly with lower MMP-9 levels and MMP-9/TIMP-4 ratios
(Table 3).Finally, stepwise multivariate
linear regression analysis was performed to assess possible associations among
individual PASP measurements and the 3 corresponding clinical and laboratory
parameters under study for the 106 SSc patients. Using this model, we found
that increased PASP measurements were associated with
TIMP-4 (log transformed-continuous,β-coefficient = 0.180, P  = .031) and BNP
(log transformed-continuous, β-coefficient = 0.534, P < .001). In contrast, no significant
associations could be established with age (continuous), MMP-9 (continuous), SSc
type (diffuse, limited), or presence of lung fibrosis (no, yes) in our SSc patient cohort.


## 4. Discussion

In the present
study, we found that TIMP-4 serum levels are increased in patients with either
diffuse or limited SSc as well as in patients with pulmonary fibrosis. Because a relatively large number of patients
were available,
appropriate comparisons between patient subgroups were possible. No significant
differences in TIMP-4 levels were noted between diffuse or limited skin
involvement, or between patients with lung fibrosis and those without,
suggesting a not convincing association of increased TIMP-4 serum levels with
the extent of fibrosis characterizing SSc.
However, further analysis showed that increased TIMP-4 circulating
levels were higher in patients with elevated PASP measurements in
echocardiography, irrespective of skin involvement extent or lung fibrosis.
PASP was considered elevated when reaching or exceeding the level of 40 mm Hg
in echocardiography, as also reported in other studies using noninvasive
assessments of pulmonary pressure
[15, 17, 20].
Clearly, echocardiography is not valid for the definite diagnosis of PH,
but performing right cardiac catheterization in every patient was not possible.
However, similarly significant associations between elevated PASP and TIMP-4
levels were also obtained when higher thresholds suggestive of PH, that is,
45 mm Hg [21, 22] or
50 mm Hg [18], were applied in our patient cohort.Moreover,
individual TIMP-4 levels correlated positively with the corresponding PASP
measurements in our 106 patients with SSc. Treatment with angiotensin-converting enzyme inhibitors, known to influence TIMP-4
expression [23, 24], appeared not to affect this result. Age adjustment was applied in statistical analyses
because MMP-9 may decrease [25],
whereas TIMP-4 [25],
BNP [26], PASP [17], and
echocardiographic indices of myocardial performance [27] may increase with
age. Finally, despite the limitation that serum measurements were
performed only once, multivariate
linear regression analysis revealed significant associations of PASP elevations and increases of TIMP-4 serum levels, but not with the presence of diffuse or limited SSc, or the presence
of lung fibrosis in this cohort. In addition to TIMP-4, PASP was associated with increased BNP blood levels,
as expected. Previous studies have shown
that BNP levels are directly related to the severity of PH in SSc [12], and may
be considered an independent predictor of PH in these patients [28].To the best of our
knowledge, no previous studies have examined TIMP-4 in patients with systemic
connective tissue diseases or in patients with PH. Regarding TIMP-1 and TIMP-2
serum levels, both have been found elevated in SSc [29–34], and probably
increased TIMP-2 levels correlate with cardiopulmonary complications
[32, 33]. In a larger study examining both TIMP-1 and
TIMP-2, only TIMP-1 levels were significantly elevated in diffuse and limited
SSc compared to patients with primary Raynaud’s phenomenon or controls, and no
association with organ disease was found [34].On the other hand,
MMP-9 levels were significantly raised only in our subgroup of patients with
diffuse SSc, in accordance with previous findings [35]. Overexpression of TGF-beta in scleroderma skin
[36, 37]
may contribute to local MMP-9 induction and proteolytic activation [38],
thus resulting in increased circulating levels in patient with extended skin
sclerosis. Such increases explain perhaps the trend toward significance of the
inverse correlation of MMP-9 levels with DLCO and TLC measurements, since the
majority of our patients with pulmonary fibrosis had diffuse SSc.Patients with
elevated PASP appeared to have lower MMP-9 mean levels, as also reported in SSc
patients with PH [21]. Since bosentan [21] and iloprost
[39] may attenuate MMP-9
expression, patients receiving such treatments were excluded
from our study. Those patients
with elevated PASP, most likely due to increased TIMP-4, had significantly smaller MMP-9/TIMP-4 ratios than
the remaining patients, suggesting that different remodeling mechanisms of
extracellular matrix may operate. Since MMP-9/TIMP-4 ratio reflects better the
proteolytic activity, a decreased “net MMP activity” may favor decreased
degradation of extracellular matrix components [40] within the cardiopulmonary
vasculature in these patients. As shown
in an experimental model of PH associated with marked inflammatory component
[41], therapeutic inhibition of MMP activity by TIMP-1 gene transfer aggravated
PH, indicating that MMPs play a protective role against pulmonary artery
remodeling.Moreover, lower
individual MMP-9 levels and smaller MMP-9/TIMP-4 ratios were associated with
impaired left ventricle myocardial performance, further implying a role of
TIMP-4/MMP-9 interactions in cardiopulmonary vasculature abnormalities in SSc. Interestingly, cardiac remodeling in
erythropoietin-transgenic mice, characterized by a stiffer left ventricle with
diastolic dysfunction, is associated with decreased MMP-9 and increased TIMP-4
expression, followed by a shift in collagen mRNA expression from type III to
type I [42]. It should be noted,
however, that TIMPs and MMPs play also a complex role in regulating
angiogenesis. For example, while TIMP-4 can induce apoptosis in cardiac
fibroblasts [43], it may also act as an inhibitor of capillary endothelial cell
migration, but not of proliferation or of angiogenesis in vivo [44]. On the other hand, mice
hyperexpressing the profibrotic cytokine TGF-beta develop myocardial fibrosis and
have a 2.5 increase of TIMP-4 myocardial expression compared to nontransgenic control
mice [45].


## 5. Conclusion

The results
presented herein may suggest that activation of TIMP-4, perhaps by leading to
enhanced interactions with MMPs, plays a role in the increased stiffening
within the cardiopulmonary vasculature in SSc. Whether this abnormality is a potential therapeutic target deserves further
investigation. As reported recently, TIMP-4
gene was identified as one of 8 candidate genes for SSc in a pilot study using DNA pooling and genetic
association analysis methods [46]. Prospective studies to examine whether serum TIMP-4 measurements may be used to identify high-risk SSc
patients for cardiopulmonary complications, perhaps in combination with other
biomarkers [3, 47], are warranted.



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*Source: 164134-2009-01-25.xml*