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Increased serum levels and tissue expression of matrix metalloproteinase-12 in patients with systemic sclerosis: correlation with severity of skin and pulmonary fibrosis and vascular damage
  1. Mirko Manetti1,
  2. Serena Guiducci2,
  3. Eloisa Romano2,
  4. Silvia Bellando-Randone2,
  5. Maria Letizia Conforti2,
  6. Lidia Ibba-Manneschi1,
  7. Marco Matucci-Cerinic2
  1. 1Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy
  2. 2Department of Biomedicine, Division of Rheumatology, AOUC and Excellence Centre for Research, Transfer and High Education DENOthe, University of Florence, Florence, Italy
  1. Correspondence to Dr Mirko Manetti, Department of Anatomy, Histology and Forensic Medicine, University of Florence, Largo Brambilla 3, 50134 Florence, Italy; mirkomanetti{at}yahoo.it

Abstract

Objective To determine serum concentrations and tissue expression of matrix metalloproteinase-12 (MMP-12) and their correlation with clinical features in patients with systemic sclerosis (SSc).

Methods Serum MMP-12 levels from 72 patients with SSc and 42 healthy volunteers were examined by ELISA. Immunohistochemical expression of MMP-12 was analysed in skin biopsies from 20 patients with SSc and 13 healthy subjects and lung biopsies from three patients with SSc-related interstitial lung disease (ILD) and five controls.

Results Circulating levels of MMP-12 were significantly increased in patients with SSc compared with healthy controls. Serum MMP-12 levels were significantly higher in both patients with limited cutaneous SSc and those with diffuse cutaneous SSc than in healthy controls, and correlated positively with the extent of skin involvement. MMP-12 levels were raised in SSc patients with ILD compared with patients without ILD, and correlated with severity of lung restriction. Increased serum levels of MMP-12 were also associated with the presence of digital ulcers and severity of nailfold capillary abnormalities. In contrast to almost undetectable MMP-12 expression in healthy skin, MMP-12 was strongly expressed in keratinocytes, dermal endothelial cells, fibroblasts/myofibroblasts and inflammatory cells in the skin of patients with SSc. Affected lung tissue from patients with SSc-related ILD showed strong MMP-12 expression in capillary vessels, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa, while faint expression was observed in normal lung tissue.

Conclusions MMP-12 levels are increased in patients with SSc and are associated with severity of skin and pulmonary fibrosis and peripheral vascular damage.

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Introduction

Systemic sclerosis (SSc, scleroderma) is a life-threatening connective tissue disease characterised by immune dysfunction, widespread microvascular damage, lack of angiogenesis and progressive fibrosis of the skin and internal organs.1,,3 Vasculopathy and tissue fibrosis significantly contribute to the morbidity of patients with SSc, and pulmonary fibrosis, which manifests clinically as interstitial lung disease (ILD), is a major cause of death.4 5

In SSc, fibrosis results from the overproduction and deposition of extracellular matrix (ECM) components by activated fibroblasts that differentiate into myofibroblasts and acquire the ability to contract.1 2 The development of fibrosis is accompanied by a continuous and dysbalanced tissue remodelling process that is regulated by a complex network of cell-matrix interactions and comprises synthetic and degradative mechanisms.6 ECM turnover is controlled by various proteolytic enzymes including matrix metalloproteinases (MMPs) and their tissue inhibitors which are produced by granulocytes, macrophages, vascular endothelial cells and fibroblasts.6 7 MMPs degrade a broad range of ECM components, are key regulators of cell migration and are involved in the control of growth factor, cytokine and chemokine activity.6 7 MMPs are known to play an important role in chronic inflammation, autoimmune diseases and aberrant fibrotic tissue remodelling.7,,9 However, the functions of MMPs in the pathogenesis of SSc have not been fully elucidated.

MMP-12 (also known as macrophage metalloelastase) has broad substrate specificity for matrix macromolecules, recognising elastin, type IV collagen, fibronectin, vitronectin, laminins, entactin and α1-antitrypsin, and has been implicated in different pathological conditions including atherosclerosis, cancers, skin and kidney diseases.10,,14 Increasing evidence also indicates that MMP-12 is involved in types of pathological lung tissue remodelling such as chronic pulmonary inflammation and fibrosis.15,,17 Moreover, it has been shown that MMP-12 has an ability to convert plasminogen into angiostatin, a potent inhibitor of endothelial cell proliferation and angiogenesis.18 Indeed, overexpression of MMP-12 appears to correlate with reduced angiogenesis and vascular invasion in cancer, and suppresses tumour angiogenesis in animal models.18 19

We have recently shown that dermal fibroblasts and microvascular endothelial cells isolated from the skin of patients with SSc constitutively overexpress and release MMP-12 in vitro.20 21 Furthermore, we have shown that MMP-12 overproduction by SSc fibroblasts and endothelial cells accounts for endothelial urokinase-type plasminogen activator receptor (uPAR) cleavage leading to impaired endothelial cell proliferation, migration and angiogenesis.21 Moreover, a functional polymorphism in the MMP-12 gene promoter region has recently been implicated in the genetic predisposition to SSc susceptibility and clinical phenotype.22

In line with our previous reports, the aim of the present study was to investigate whether MMP-12 could serve as a biomarker reflecting the activity and severity of the fibrotic process and vascular modifications in SSc. The serum levels of MMP-12, their correlation with clinical features and measures of microvascular involvement in a well-characterised cohort of patients with SSc and the expression of MMP-12 in SSc skin and lung biopsies were investigated.

Methods

Patients, controls and serum samples

Serum samples were obtained from a total of 72 consecutive patients with SSc (64 women, 8 men; mean±SD age 61.9±12.5 years) recruited from the Division of Rheumatology, University of Florence. All patients fulfilled the criteria for SSc as suggested by LeRoy et al.23 Patients with symptoms overlapping with those of other autoimmune, rheumatic and/or connective tissue diseases were excluded from the study. Forty-two age- and sex-matched healthy individuals (38 women, 4 men; mean±SD age 60.8±13.2 years) were used as controls. Twenty-one age- and sex-matched subjects with primary Raynaud's phenomenon (19 women, 2 men; mean±SD age 58.5±11.4 years) were enrolled as an additional control group. All study subjects were of Italian European white ancestry, defined as all four grandparents being Italian white.

Fresh venous blood samples from patients and controls were drawn, allowed to clot for 30 min before centrifugation at 1500 g for 15 min and serum was collected and stored in aliquots at −80°C until used.

Clinical assessment

Patients were classified as having limited cutaneous SSc (lcSSc; n=47) or diffuse cutaneous SSc (dcSSc; n=25) according to LeRoy et al.23 Disease duration was calculated from the time of onset of the first clinical event (other than Raynaud's phenomenon) that was a clear manifestation of SSc. Patients were classified as being in the early (n=29) or late (n=43) stage of SSc according to disease duration (early lcSSc, disease duration <5 years; early dcSSc, disease duration <3 years).24

SSc patients were phenotypically assessed as previously recommended.25 Antinuclear antibodies were determined using indirect immunofluorescence and HEp-2 cells as antigen substrate. Anticentromere antibodies (ACA) were determined by their distinctive indirect immunofluorescence pattern on HEp-2 cells. Antitopoisomerase I (anti-Scl-70) antibodies were determined by immunoblot analysis. The extent of skin involvement was scored with the modified Rodnan skin thickness score (mRSS) by summing skin thickness measurements determined by palpation on a scale of 0–3 in 17 body areas.26 ILD was defined as the presence of typical features such as bibasilar interstitial fibrosis on a high-resolution CT (HRCT) scan of the chest, this procedure being carried out by an experienced radiologist in all patients with SSc. In addition, pulmonary function tests (forced vital capacity (FVC) and lung carbon monoxide transfer factor (TLCO)) were evaluated to examine the severity of pulmonary fibrosis. When FVC and TLCO were ≤70% of the predicted normal values, they were considered abnormal. Patients with SSc who were smokers or had other respiratory disorders that could have affected %FVC or %TLCO were excluded from the study. In the presence of TLCO reduction alone, patients were investigated for pulmonary arterial hypertension (PAH). Patients with a systolic pulmonary artery pressure >40 mm Hg at echocardiography underwent right heart catheterisation to confirm the presence of PAH. Confirmed PAH was defined as a mean pulmonary artery pressure >25 mm Hg at rest or >30 mm Hg during exercise with normal pulmonary capillary wedge pressure (<15 mm Hg).

All patients reported the occurrence of Raynaud's phenomenon after exposure to low temperatures. At the time of blood drawing, the presence of digital ulcers (DU) on the fingertips and other finger areas was recorded. Nailfold videocapillaroscopy was performed in a blinded manner by an experienced rheumatologist for the analysis of microvascular abnormalities. Patients were allowed to adapt to room temperature (20–22°C) for at least 15 min before the examination started. The nailfolds of all 10 fingers were analysed for the following parameters: presence of enlarged and giant capillaries, pericapillary oedema, haemorrhages, loss of capillaries, ramified or bushy capillaries and disorganisation of the vascular distribution. According to nailfold videocapillaroscopic features, patients were classified as follows: ‘early’ pattern: few giant capillaries and capillary haemorrhages, relatively well preserved capillary distribution, no evident loss of capillaries; ‘active’ pattern: frequent giant capillaries and capillary haemorrhages, moderate loss of capillaries with some avascular areas, mild disorganisation of the capillary architecture, absent or some ramified capillaries; ‘late’ pattern: irregular enlargement of capillaries, few or absent giant capillaries, absence of haemorrhages, severe loss of capillaries with large avascular areas, severe disorganisation of the normal capillary distribution, frequent ramified/bushy capillaries.27

Patients with SSc were not taking corticosteroids, methotrexate, cyclophosphamide, ACE inhibitors, D-penicillamine, iloprost or other immunosuppressive treatment and disease-modifying drugs. Before blood sampling, they were washed out for 10 days from oral vasodilating drugs and for 2 months from intravenous alprostadil α-cyclodextrin.

Assay for serum MMP-12

Serum levels of MMP-12 (active and proactive forms) were measured by commercial quantitative colorimetric sandwich ELISA using an antigen-affinity purified antihuman MMP-12 capture antibody precoated 96-well plate (Antibodies-online GmbH, Atlanta, Georgia, USA) according to the manufacturer's protocol. Standards and serum samples were incubated for 2 h at room temperature. Serum MMP-12 was determined with biotinylated antigen-affinity purified antihuman MMP-12 detection antibody. The reaction was developed with streptavidin-horseradish peroxidase conjugate and tetramethylbenzidine, and then stopped by applying 2 M H2SO4. Optical density was measured by microtitre plate reader at 450 nm. Serum levels of MMP-12 were read off from a standard curve according to the manufacturer's instructions. The detection range of the assay was 0.156–10 ng/ml. Each sample was measured in duplicate.

Skin and lung biopsies

Full-thickness skin biopsies were obtained from the clinically involved skin of one-third of the distal forearm of 20 patients with SSc (17 women, 3 men). Nine patients had lcSSc and 11 patients had dcSSc.23 Prior to biopsy, patients had not received any disease-modifying antirheumatic drug treatment. Skin samples from the same forearm region of 13 age- and sex-matched healthy subjects who underwent surgery for traumatic lesions were used as controls. Lung biopsies were obtained from autopsy samples from three subjects who died from early severe dcSSc with rapidly progressive disease. Lung biopsies displayed the typical features of non-specific interstitial pneumonitis and SSc-related pulmonary fibrosis. As control lung tissues, biopsy samples were obtained from five individuals who underwent surgery for neoplastic pathologies. We carefully selected healthy specimens that appeared to have no inflammatory/neoplastic infiltration according to histopathological examination of H&E-stained tissue sections. All tissue specimens were fixed in 10% buffered formalin, dehydrated in graded alcohol series and embedded in paraffin.

Immunohistochemistry

Immunohistochemistry was performed using an indirect immunoperoxidase method. Tissue sections (5 μm thick) were deparaffinised and boiled for 10 min in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval, and then treated with 3% H2O2 in methanol for 30 min at 4°C to block endogenous peroxidase activity. After blocking non-specific site binding with UltraV block (UltraVision Detection System; LabVision, Fremont, California, USA), the sections were incubated with a rabbit polyclonal antihuman MMP-12 antibody (2 μg/ml) which specifically recognises both the proactive and active forms of MMP-12 (catalogue number ab66157; Abcam, Cambridge, UK) in a humidified chamber overnight at 4°C. Tissue sections were incubated sequentially with biotinylated secondary antibodies and the avidin-biotin-peroxidase complex (UltraVision Detection System). Immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride substrate with nickel (Vector Laboratories, Burlingame, California, USA) as chromogen. Parallel sections were incubated with isotype- and concentration-matched normal IgG (Sigma, St Louis, Missouri, USA) to replace the primary antibodies as negative staining controls. Sections were examined under a light microscope (Eclipse E400; Nikon, Tokyo, Japan) and photographed by digital camera (Coolpix 2500; Nikon). Three fields (×20 magnification) from three random sections of each specimen were analysed by two blinded observers and scored for the intensity of the immunostaining (− negative, +/− weak, + moderate, ++ intense). When there was interobserver disagreement, the specimen was reviewed again by both observers and the disagreement resolved. Double immunofluorescence stainings with rabbit polyclonal anti-MMP-12 antibody and mouse monoclonal antibodies against CD31/pan-endothelial cell marker (1:20 dilution; Dako, Hamburg, Germany), α-smooth muscle actin (α-SMA) (1:50 dilution; Abcam) and CD45/leucocyte common antigen (1:100 dilution; Dako) were performed and examined by confocal microscopy as previously described.28

Statistical analysis

Statistical analysis was performed using the SPSS software for Windows Version 12.0 (SPSS, Chicago, Illinois, USA). Descriptive statistics were expressed as mean±SD or median and IQR for continuous variables and as number and percentage for categorical variables. The non-parametric Mann–Whitney U test for independent samples was used to analyse the serum MMP-12 differences between two groups. For all comparisons between two SSc subgroups, Bonferroni adjustment was used for multiple comparisons (total of 10 comparisons). p Values after this adjustment for multiple testing are termed ‘padj’. The Spearman rank correlation coefficient (r) was used to examine the relationship between two continuous variables. p Values are two-tailed, and p values <0.05 were considered statistically significant.

Results

Demographic, clinical and serological characteristics of the patients with SSc are shown in table 1.

Table 1

Demographic, clinical and serological characteristics of patients with SSc

Serum levels of MMP-12 are increased in patients with SSc

Circulating levels of MMP-12 were significantly increased in patients with SSc (median 2.88 ng/ml, IQR 2.25–3.74 ng/ml) compared with both healthy controls (median 1.08 ng/ml, IQR 0.38–2.12 ng/ml; p<0.0001; figure 1A) and subjects with primary Raynaud's phenomenon (median 1.55 ng/ml, IQR 0.81–2.38 ng/ml; p<0.0001). MMP-12 levels were similar in the healthy controls and the group with primary Raynaud's phenomenon (p=0.4). With regard to SSc subsets, serum MMP-12 levels in patients with lcSSc (median 2.85 ng/ml, IQR 2.18–3.56 ng/ml) and dcSSc (median 3.23 ng/ml, IQR 2.40–4.26 ng/ml) were both significantly higher than in healthy individuals (p<0.0001 for both; figure 1A) and subjects with primary Raynaud's phenomenon (p<0.0001 for both). Patients with dcSSc had higher MMP-12 levels than those with lcSSc, but their differences did not reach statistical significance (p=0.06; figure 1A).

Figure 1

Serum levels of matrix metalloproteinase-12 (MMP-12) are increased and correlate with the extent of skin fibrosis in patients with systemic sclerosis (SSc). (A) Serum MMP-12 levels in healthy controls, patients with SSc, limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc). (B) MMP-12 levels in SSc according to early and late disease stages. (C) Higher levels of MMP-12 in patients with SSc with modified Rodnan skin thickness score (mRSS) >10. Serum MMP-12 levels were determined by a specific ELISA. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and 90th percentiles. Lines inside the boxes represent the median, circles the outliers and asterisks the extreme values. (D) Correlation of serum MMP-12 levels with mRSS in patients with SSc. Data are shown as a scatterplot, each dot representing a subject. Correlation coefficient (r) and p value are indicated.

Serum levels of MMP-12 were significantly increased in both early-stage (median 2.48 ng/ml, IQR 2.17–2.82 ng/ml) and late-stage SSc (median 3.51 ng/ml, IQR 2.65–4.00 ng/ml) compared with healthy controls (p<0.0001 for both), as well as in late-stage compared with early-stage SSc (p<0.0001, padj<0.001; figure 1B).

No significant correlation of MMP-12 levels with age and sex was found. No significant differences in MMP-12 levels were detected between patients with and without ACA, nor in anti-Scl-70 antibody-positive patients compared with anti-Scl-70-negative patients.

Correlation of serum MMP-12 levels with severity of skin and pulmonary fibrosis

The possible association of MMP-12 levels with the extent of skin sclerosis assessed by mRSS was investigated. Patients with mRSS >10 had significantly increased circulating levels of MMP-12 (median 3.49 ng/ml, IQR 2.79–4.28 ng/ml) compared with patients with mRSS ≤10 (median 2.52 ng/ml, IQR 2.15–3.30 ng/ml; p<0.0001, padj<0.001; figure 1C). Moreover, MMP-12 levels correlated positively with mRSS (r=0.62, p=0.01; figure 1D).

Serum MMP-12 levels were raised in patients with SSc with ILD on HRCT scan (median 3.23 ng/ml, IQR 2.47–3.95 ng/ml) compared with patients without ILD (median 2.66 ng/ml, IQR 2.10–3.46 ng/ml; p=0.02; figure 2A). However, this difference did not remain statistically significant after Bonferroni adjustment for multiple testing (padj=0.2). In patients with SSc, serum MMP-12 levels correlated inversely with %FVC (r=−0.82, p=0.01; figure 2B).

Figure 2

(A) Increased levels of matrix metalloproteinase-12 (MMP-12) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD). Serum MMP-12 levels were determined by a specific ELISA. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and 90th percentiles. Lines inside the boxes represent the median, circles the outliers and asterisks the extreme values. (B) Correlation of serum MMP-12 levels with percentage forced vital capacity (%FVC) in patients with SSc. Data are shown as scatterplot, each dot representing a subject. Correlation coefficient (r) and p value are indicated.

Correlation of serum MMP-12 levels with severity of microvascular damage

We also evaluated the possible correlation of serum MMP-12 levels with measures of microvascular involvement. MMP-12 levels were significantly higher in patients with SSc with ‘late’ capillaroscopic pattern (median 3.80 ng/ml, IQR 3.41–4.53 ng/ml) than in those with ‘early’ (median 2.34 ng/ml, IQR 2.06–3.01 ng/ml) and those with ‘active’ (median 2.99 ng/ml, IQR 2.45–3.83 ng/ml) capillaroscopic patterns (p<0.0001 and p=0.009, respectively; figure 3A). Moreover, MMP-12 levels were increased in patients with ‘active’ capillaroscopic pattern compared with those with ‘early’ pattern (p=0.02; figure 3A). The difference in MMP-12 levels between the ‘early’ and ‘late’ capillaroscopic groups remained highly significant even after Bonferroni adjustment for multiple testing (padj<0.001). Circulating levels of MMP-12 were also significantly increased in SSc patients with DU (median 3.43 ng/ml, IQR 2.63–3.96 ng/ml) compared with SSc patients without DU (median 2.58 ng/ml, IQR 2.16–3.21 ng/ml; p=0.004, padj=0.04; figure 3B). No significant differences in MMP-12 levels were detected between SSc patients with PAH confirmed by right heart catheterisation (median 2.61 ng/ml, IQR 2.26–3.58 ng/ml) and those without PAH (median 2.94 ng/ml, IQR 2.23–3.78 ng/ml; p=0.5).

Figure 3

Association of matrix metalloproteinase-12 (MMP-12) levels with severity of peripheral vascular involvement in patients with systemic sclerosis (SSc). (A) MMP-12 levels in SSc according to ‘early’, ‘active’ and ‘late’ nailfold capillaroscopy patterns. (B) Increased levels of MMP-12 in patients with SSc with digital ulcers (DU). Serum MMP-12 levels were determined by a specific ELISA. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and 90th percentiles. Lines inside the boxes represent the median, circles the outliers and asterisks the extreme values.

Increased expression of MMP-12 in the skin and lung of patients with SSc

Whether the expression of MMP-12 was increased in the fibrotic skin and lung of patients with SSc was assessed by immunohistochemical analysis. Consistent with previous reports,29 MMP-12 expression was almost undetectable in healthy control skin (figure 4A,B). In the affected skin of patients with SSc, a strong expression of MMP-12 could be detected in epidermal keratinocytes and in the papillary and reticular dermis where it was found in fibroblasts, microvascular endothelial cells and perivascular infiltrating mononuclear cells (figure 4C–F). Moreover, in SSc skin, epithelial cells of sweat glands were strongly immunoreactive for MMP-12 (figure 4E). The results of immunostaining intensity analysis on skin sections are summarised in table S1 in the online supplement. No significant differences in cutaneous expression of MMP-12 were observed between patients with lcSSc and those with dcSSc (see table S1 in online supplement). In order to further characterise the nature of MMP-12-expressing cells in skin from patients with SSc, double immunofluorescence analyses were performed using antibodies against cell type-specific markers (figure 4G–L). In agreement with the findings obtained by immunoperoxidase methodology, in SSc skin a strong MMP-12 expression was observed in endothelial cells of capillary vessels that were identified as either MMP-12/CD31-double positive cells or MMP-12-positive cells lining the vessel lumen and surrounded by α-SMA-positive pericytes (figure 4H–J,L). Moreover, CD45-positive inflammatory/immune cells and α-SMA-positive myofibroblasts were also immunopositive for MMP-12 in SSc skin (figure 4I,K).

Figure 4

Increased expression of matrix metalloproteinase-12 (MMP-12) in the skin of patients with systemic sclerosis (SSc). (A–F) Immunoperoxidase staining. In healthy control skin (A, B), MMP-12 expression is almost undetectable in the epidermis (e), papillary and reticular dermis. Note the absence of immunostaining for MMP-12 in microvascular endothelial cells (arrowheads). In the affected skin of patients with SSc (C), MMP-12 expression is found in epidermal keratinocytes (e), dermal microvascular endothelial cells (arrowheads) and fibroblasts. (D) Higher magnification view showing immunopositive capillary vessels (arrowheads) and fibroblasts (arrows) in SSc papillary dermis. (E) In the deep reticular dermis of SSc skin, strong MMP-12 expression is observed in epithelial cells of sweat glands and periglandular capillary vessels (arrowheads). (F) Note the intense immunostaining for MMP-12 in microvascular endothelial cells (arrowheads) and perivascular infiltrating mononuclear cells (asterisk) in SSc reticular dermis. Original magnification ×20 (A, B, C, E), ×40 (B inset, D, F). (G–L) Immunofluorescence and confocal laser scanning microscopy (CLSM) analysis. (G–J) Representative microphotographs of double immunostaining of MMP-12 (green) and α-smooth muscle actin (α-SMA, red) in skin from (G) controls and (H–J) patients with SSc. In all skin sections, pericytes surrounding the endothelial cells of capillary vessels show α-SMA positivity. (G) Faint expression of MMP-12 in capillary vessels (arrowheads) and fibroblasts (white arrows) of healthy skin. (H–J) In SSc skin, strong MMP-12 expression is evident in endothelial cells of capillary vessels (arrowheads, H, I, J), fibroblasts (white arrows, H, I), α-SMA-positive myofibroblasts (yellow arrows, I) and perivascular inflammatory infiltrates (asterisks, J). (K) Double immunofluorescence analysis of SSc skin with anti-MMP-12 (green) and anti-CD45 (red) antibodies. In SSc dermis, MMP-12 is strongly expressed in CD45-positive leucocytes (arrows) observed around capillary vessels (arrowheads). (L) Double immunofluorescent staining of MMP-12 (green) and CD31/pan-endothelial marker (red) in SSc skin. Arrowheads indicate double immunopositive capillary endothelial cells, asterisks indicate MMP-12-positive perivascular inflammatory cells. Original magnification ×63 (G–L).

A faint expression of MMP-12 was observed in normal lung tissues (figure 5A,B). Affected lung tissues from patients with SSc-related ILD showed strong MMP-12 expression in microvascular endothelial cells, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa, which are typical of SSc alveolitis (figure 5C–F).

Figure 5

Increased expression of matrix metalloproteinase-12 (MMP-12) in lung tissues from patients with systemic sclerosis (SSc). (A, B) Faint expression of MMP-12 is observed in normal lung tissue. (C–F) In the fibrotic lung of patients with SSc with interstitial lung disease, MMP-12 is abundantly expressed in capillary vessels, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa. (D, F) Higher magnification views of SSc lung sections showing MMP-12-immunopositive alveolar macrophages (arrows) and fibroblasts (arrowheads). Original magnification ×20 (A–C, E), ×40 (D, F).

Discussion

In this cross-sectional study we show for the first time that serum levels and tissue expression of MMP-12 are increased in patients with SSc. Remarkably, serum MMP-12 levels were raised in patients with dcSSc and in those with lcSSc, and were associated with a greater extent of skin involvement, disease duration, higher prevalence of pulmonary fibrosis and worse pulmonary function. Moreover, the increase in serum MMP-12 levels in patients with SSc was associated with the severity of nailfold capillary abnormalities and the presence of DU. In contrast to almost undetectable MMP-12 expression in healthy skin and lung, MMP-12 was found to be strongly expressed in different cell types of SSc skin and lung tissues including fibroblasts, microvascular endothelial cells and inflammatory cells. Taken together, our results suggest that MMP-12 may play an important role in the development and progression of skin sclerosis, pulmonary fibrosis and peripheral vascular damage in patients with SSc.

The MMPs are a family of proteases that exert various biological effects in cell migration, in ECM and vascular remodelling, as well as inflammatory and immune processes by degrading collagen and other extracellular macromolecules, cytokines, growth factors and their receptors.7 30 MMP-12 has recently emerged as a critical player in chronic pulmonary pathologies characterised by intense tissue remodelling such as asthma, chronic obstructive pulmonary disease and pulmonary fibrosis.31 Alveolar macrophages are the principal source of MMP-12 in the lung, and MMP-12 protein levels and enzymatic activity are increased in patients with chronic pulmonary pathologies and are inversely correlated with measures of lung function.31 32 Indeed, MMP-12 silencing has been proposed as a potential therapeutic approach to treat pathological lung tissue remodelling.31 Interestingly, we observed increased MMP-12 expression in alveolar macrophages and pulmonary fibroblasts from patients with SSc. Circulating levels of MMP-12 were increased in SSc patients with ILD compared with those without ILD, although this difference did not remain statistically significant after correction for multiple testing. However, most interesting was the finding that MMP-12 levels correlated inversely with %FVC which is used as a surrogate measure for severity of lung restriction. Indeed, although a variety of pulmonary function test measures have been used to study ILD in SSc, only %FVC has been validated as an outcome measure in randomised controlled trials.33 Moreover, MMP-12 was found to be strongly expressed in dermal fibroblasts/myofibroblasts in affected SSc skin, but not in any of healthy control skin biopsies, and serum MMP-12 levels correlated positively with mRSS in patients with SSc. MMP-12 has been shown to act as a downstream mediator in fibrotic processes driven by transforming growth factor-β and interleukin-13, both of which are implicated in the pathogenesis of SSc-related fibrosis.1 16 34 Mice carrying a targeted deletion of the MMP-12 gene were resistant to the induction of pulmonary fibrosis and exhibited decreased expression of different profibrotic genes.17 31 In addition, it has been shown that MMP-12-dependent migration of extrapulmonary myofibroblast precursors may contribute to post-transplant fibrosis in the lung.35 There is also evidence to suggest that MMP-12 may promote ECM accumulation and tissue fibrosis by reducing the expression of specific collagen-degrading MMPs such as MMP-2 and MMP-13.34 In a recent study, increased serum levels of MMP-7 (matrilysin) have also been correlated with pulmonary involvement in patients with SSc.36

Besides ECM remodelling, it appears that MMP-12 also plays a crucial role in vascular remodelling and regulation of angiogenesis.18,,21 MMP-12 exerts potent antiangiogenic effects through the conversion of plasminogen to angiostatin which inhibits endothelial cell migration and proliferation.18 Furthermore, MMP-12 may suppress angiogenesis through the proteolysis and subsequent inactivation of uPAR, a cell surface receptor that regulates multiple steps in the angiogenic process.20 21 37 Indeed, we have previously shown that overproduction of MMP-12 accounts for uPAR cleavage leading to impaired angiogenesis in microvascular endothelial cells isolated from the skin of patients with SSc.21 Moreover, MMP-12 silencing could in part restore the ability of SSc endothelial cells to produce capillary structures in vitro.38 In this context, the association of serum MMP-12 levels with the severity of SSc-related peripheral vascular damage appears to be of major importance. In fact, MMP-12 levels were significantly higher in patients with SSc with DU than in patients without DU. In addition, serum MMP-12 levels progressively increased from the ‘early’ to the ‘late’ capillaroscopic patterns, suggesting that MMP-12 may actively participate in the derangement of the peripheral microcirculation in patients with SSc. It has also been reported that MMP-12 promotes vascular smooth muscle cell proliferation and contributes to intimal thickening in proliferative vasculopathies such as restenosis and atherosclerosis.39 We did not find any association between MMP-12 levels and SSc-related PAH, although our cohort included a relatively small number of patients with PAH. Further analyses using larger cohorts of SSc patients with PAH are therefore required to definitely rule out the possible involvement of MMP-12 in SSc-related PAH.

In summary, we demonstrate that high serum levels of MMP-12 are associated with the extent of skin involvement and severity of pulmonary fibrosis and vascular damage in SSc, suggesting that increased levels of MMP-12 may be a potential marker for patients with increased risk for sever, life-threatening disease. However, further studies examining the longitudinal changes of serum MMP-12 levels in patients with SSc will be required and are warranted. Translational studies on preclinical animal models will further investigate the role of MMP-12 in the pathophysiology of SSc. Together with previous reports,20,,22 38 the results of our study suggest that modulation of MMP-12 expression and activity might offer new targeted therapeutic strategies to control the progression of fibrosis and peripheral vasculopathy in SSc.

References

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Supplementary materials

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Footnotes

  • MM and SG contributed equally to this work.

  • LI-M and MM-C are joint senior authors.

  • Funding This study was supported by grants from the University of Florence (Progetti di Ricerca di Ateneo, ex 60% to LI-M and MM-C).

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval The study was approved by the local Institutional Review Board. The study complied with the principles of the Declaration of Helsinki and all subjects gave written informed consent.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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