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Inactivation of urokinase-type plasminogen activator receptor (uPAR) gene induces dermal and pulmonary fibrosis and peripheral microvasculopathy in mice: a new model of experimental scleroderma?
  1. Mirko Manetti1,
  2. Irene Rosa1,
  3. Anna Franca Milia1,
  4. Serena Guiducci2,
  5. Peter Carmeliet3,4,
  6. Lidia Ibba-Manneschi1,
  7. Marco Matucci-Cerinic2
  1. 1Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Florence, Italy
  2. 2Department of Experimental and Clinical Medicine, Section of Internal Medicine and Division of Rheumatology, Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy
  3. 3Laboratory of Angiogenesis and the Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium
  4. 4Laboratory of Angiogenesis and the Neurovascular Link, Vesalius Research Center, University of Leuven, Leuven, Belgium
  1. Correspondence to Dr Mirko Manetti, Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Largo Brambilla 3, Florence I-50134, Italy; mirkomanetti{at}yahoo.it, mirko.manetti{at}unifi.it

Abstract

Objective Urokinase-type plasminogen activator receptor (uPAR) is a key component of the fibrinolytic system involved in extracellular matrix remodelling and angiogenesis. The cleavage/inactivation of uPAR is a crucial step in fibroblast-to-myofibroblast transition and has been implicated in systemic sclerosis (SSc) microvasculopathy. In the present study, we investigated whether uPAR gene inactivation in mice could result in tissue fibrosis and peripheral microvasculopathy resembling human SSc.

Methods The expression of the native full-length form of uPAR in human skin biopsies was determined by immunohistochemistry. Skin and lung sections from uPAR-deficient (uPAR−/−) and wild-type (uPAR+/+) mice at 12 and 24 weeks of age were stained with haematoxylin-eosin, Masson's trichrome and Picrosirius red. Dermal thickness and hydroxyproline content in skin and lungs were quantified. Dermal myofibroblast and microvessel counts were determined by immunohistochemistry for α-smooth muscle actin and CD31, respectively. Endothelial cell apoptosis was assessed by TUNEL/CD31 immunofluorescence assay.

Results Full-length uPAR expression was significantly downregulated in SSc dermis, especially in fibroblasts and endothelial cells. Dermal thickness, collagen content and myofibroblast counts were significantly greater in uPAR−/− than in uPAR+/+ mice. In uPAR−/− mice, dermal fibrosis was paralleled by endothelial cell apoptosis and severe loss of microvessels. Lungs from uPAR−/− mice displayed non-specific interstitial pneumonia-like pathological features, both with inflammation and collagen deposition. Pulmonary pathology worsened significantly from 12 to 24 weeks, as shown by a significant increase in alveolar septal width and collagen content.

Conclusions uPAR−/− mice are a new animal model closely mimicking the histopathological features of SSc. This model warrants future studies.

  • Systemic Sclerosis
  • Pulmonary Fibrosis
  • Qualitative research
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Introduction

Systemic sclerosis (SSc, or scleroderma) is characterised by widespread peripheral microvascular damage, with endothelial cell apoptosis and loss of capillaries, and progressive fibrosis affecting the skin and internal organs, including the lungs, the gastrointestinal tract and the heart.1–4 In particular, pulmonary fibrosis, which manifests clinically as interstitial lung disease, is a leading cause of death among SSc patients.5

Despite substantial progress in managing complications that occur mostly as a result of organ failure, to date, there is still neither a cure nor a disease-specific treatment.6 Animal models are important for a better understanding of the mechanisms that trigger and sustain microvasculopathy and fibrosis in SSc and to exploit therapeutic interventions. However, preclinical testing of potential drugs is hampered by the low availability of animal models which recapitulate both the immune, vascular and fibrotic phenotypes of human SSc.7–9

Urokinase-type plasminogen activator receptor (uPAR, or CD87 antigen) is a key component of the fibrinolytic system, a well-characterised system of serine proteases which play an important role in the degradation of the extracellular matrix (ECM).10 ,11 Full-length uPAR is a glycosyl phosphatidylinositol-anchored 3-extracellular domain protein expressed by cells of several lineages, including lymphohaematopoietic cells (monocytes, neutrophils and activated T cells), endothelial cells, fibroblasts and myofibroblasts, that concentrates the serine protease activity of its ligand, uPA (or urokinase), at the cell-matrix interface.10 ,11 Receptor-bound uPA promotes ECM remodelling either directly through the conversion of plasminogen to plasmin or indirectly through the activation of matrix metalloproteinase zymogens and other proteins.10–12 Moreover, uPAR interacts with vitronectin and several integrin family members, and mediates multiple protease-independent effects, including cell differentiation, proliferation, adhesion and migration through intracellular signalling.10 ,13

Previous studies have shown that uPAR cleavage and loss of function of the uPA/uPAR system in endothelial cells is implicated in SSc-related microvascular abnormalities and impaired angiogenesis.14 ,15 Indeed, uPAR is a central mediator of growth factor-induced endothelial cell migration, and experimental deficiency of uPAR leads to decreased angiogenic function and altered endothelial cell morphology both in vitro and in vivo.16–18 Evidence also suggests that uPAR gene deficiency may be involved in the pathogenesis and progression of dermal and renal fibrosis in mice.19 ,20 Additionally, the cleavage/inactivation of uPAR was shown to be a crucial step in the transdifferentiation of fibroblasts into myofibroblasts, which are most responsible for the excessive ECM production and deposition in SSc and other fibrotic disorders.21 ,22

The present study was designed to investigate whether inactivation of uPAR gene in mice could result in skin and lung fibrosis and peripheral microvasculopathy mimicking human SSc.

Materials and methods

Human skin biopsies

Paraffin-embedded sections of lesional forearm skin biopsies were obtained from 15 patients with SSc (13 women, 2 men) and 10 age-matched and sex-matched healthy donors as described elsewhere.23–25 The median age of SSc patients was 47 years (range 27–69 years) and their median disease duration calculated from the time of onset of the first clinical manifestation of SSc (other than Raynaud's phenomenon) was 6 years (range 1–16 years). Eight patients had the limited cutaneous subset, and seven had the diffuse cutaneous subset according to LeRoy et al.26 None of the patients was receiving immunosuppressive medication or other potentially disease-modifying drugs at the time of skin biopsy. All subjects signed an informed consent form approved by the local institutional review board.

Animals

Mice with a targeted deletion in the gene for uPAR, resulting in a complete deficiency of uPAR (uPAR−/−), were generated as previously described.27 Male uPAR−/− mice on a mixed C57BL/6 (75%)×129 (25%) background (developed by PC, Leuven, Belgium) were crossed with female wild-type (uPAR+/+) mice on a C57BL/6 background (Charles River Laboratories, Calco, Lecco, Italy). Heterozygous mice were then crossed to generate uPAR−/− and uPAR+/+ offspring from the same breeding groups. PCR of tail-tip genomic DNA was performed for determination of the absence or presence of a functional uPAR gene. Animals were given food and water ad libitum and were maintained on a 12 h light/12 h dark cycle schedule. Male uPAR−/− mice and wild-type uPAR+/+ littermates at 12 weeks of age (n=7 uPAR−/− mice, n=7 uPAR+/+ mice) and 24 weeks of age (n=7 uPAR−/− mice, n=6 uPAR+/+ mice) were used in the experiments. Mice were anaesthetised intraperitoneally with cloralium hydrate (400 mg/kg) and sacrificed by cervical dislocation. Upper back skin samples and lungs were rapidly removed and processed for histopathological and immunohistochemical analyses and hydroxyproline assay. All the animal experiments were performed in accordance with DL 116/92, and approved by the Institutional Animal Care and Use Committee of the University of Florence.

Histological analysis of mouse skin and lungs

For histological analysis, skin was excised from the upper back directly over the shoulder blades and spread onto a piece of filter paper prior to fixation. Skin and lung samples were fixed in 10% buffered formalin, dehydrated in graded alcohol series and embedded in paraffin. Tissue sections were cut (5 μm thick) using a Leica RM2255 rotary microtome (Leica Microsystems, Mannheim, Germany), deparaffinised in xylene and hydrated through graded alcohols to distilled water. For haematoxylin-eosin staining, sections were stained with Mayer's haematoxylin (Sigma–Aldrich, St Louis, Missouri, USA) for 15 min, rinsed in running tap water, counterstained with 1% Eosin Y aqueous solution (Bio-Optica, Milan, Italy) for 5 min, dehydrated through graded alcohols and cleared in xylene. Trichrome staining was performed using the Masson's trichrome with blue aniline staining kit (catalogue number 04-010802; Bio-Optica, Milan, Italy) according to the manufacturer's protocol. The stained sections were observed under a Leica DM4000 B microscope equipped with fully automated transmitted light and fluorescence axes (Leica Microsystems). Transmitted light images were captured using a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).

Evaluation of dermal thickness

For comparisons of dermal thickness, two skin samples were examined from every animal of each group, and 5 μm skin sections (three for each skin sample) were stained with haematoxylin-eosin. Dermal thickness was calculated at 10×microscopic magnification by measuring the distance between the dermal–epidermal junction and the dermal–subcutaneous fat junction (μm) in five randomly selected fields for each skin section. Two different examiners (MM, IR) performed the evaluation blindly.

Determination of collagen content in mouse skin and lung samples

Two independent methods, Picrosirius red staining and hydroxyproline assay, were used to evaluate the collagen content in skin and lung samples from uPAR−/− and uPAR+/+ mice. Picrosirius red staining accurately reflects the collagen content assessed with hydroxyproline assay and allows areas of localised collagen accumulation to be specifically evaluated. After deparaffinisation, the skin and lung sections (5 μm thick) were stained using the Picrosirius red staining kit (catalogue number 04-121873; Bio-Optica, Milan, Italy) according to the manufacturer's protocol. The stained sections were dehydrated through graded alcohols, cleared in xylene and observed under the Leica DM4000 B microscope. In each section, Picrosirius red-positive area was measured in five randomly chosen fields using the free-share ImageJ software (NIH, Bethesda, Maryland, USA; online at http://rsbweb.nih.gov/ij) and expressed as a percent of the observed in uPAR+/+ mice. Colourimetric quantification of hydroxyproline content was performed in small skin and lung biopsies (3 mm diameter) taken from every animal in each group. Briefly, frozen tissues were dehydrated, weighed and hydrolysed in 6 N HCl at 120°C for 3 h. After neutralisation in 6N NaOH, the samples were processed as described elsewhere.28 The absorbance was measured at 560 nm in duplicate with a microplate spectrophotometer.

Immunohistochemistry

Immunohistochemistry for the native full-length form of uPAR in human skin sections and α-smooth muscle actin (α-SMA) and CD31/platelet-endothelial cell adhesion molecule-1 (PECAM-1) in mouse skin sections was performed using an indirect immunoperoxidase method. 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 mouse monoclonal anti-uPAR/domain 1 (D1) (1:50 dilution; catalogue number 3931, American Diagnostica, Stamford, Connecticut, USA), rabbit polyclonal anti-α-SMA (1:100 dilution; catalogue number ab5694, Abcam, Cambridge, UK), or rabbit polyclonal anti-CD31/PECAM-1 (1:40 dilution; catalogue number ab28364, Abcam) antibodies in a humidified chamber overnight at 4°C. Skin sections were incubated sequentially with biotinylated secondary antibodies and the avidin-biotin-peroxidase complex (UltraVision Detection System). Immunoreactivity was developed using 3-amino-9-ethylcarbazole (AEC kit, LabVision) as chromogen. Parallel sections were incubated with isotype-matched and concentration-matched normal IgG (Sigma–Aldrich, St Louis, Missouri, USA) to replace the primary antibodies as negative staining controls. The sections were then examined under the Leica DM4000 B microscope and photographed by digital colour camera (Leica Microsystems).

Quantification of uPAR staining in human skin

uPAR staining was quantified in a semiquantitative manner, where (0) indicates no staining; (1) weak staining; (2) moderate staining and (3) strong staining of endothelial cells and fibroblasts at eight randomly chosen high-power fields (40× original magnification) per sample. Two different examiners (MM, IR) performed the evaluation blindly. When there was interobserver disagreement, the specimen was reviewed again by both observers and the disagreement resolved.

Quantification of myofibroblasts and microvessels in mouse skin

Myofibroblasts and microvessels were identified by staining for α-SMA and the pan-endothelial cell marker CD31, respectively. α-SMA-positive spindle-shaped cells (myofibroblasts) and CD31-positive microvessels were counted in five randomly chosen high-power fields (40× original magnification) of the dermis from each of three sections per sample. Counting was performed by two independent observers (MM, IR) in a blinded manner. The final result was the mean of the two different observations for each sample.

Immunofluorescence for profibrotic cytokines in mouse skin

Paraffin-embedded mouse skin sections (5 μm thick) were deparaffinised, washed three times in phosphate-buffered saline (PBS), incubated in 2 mg/ml glycine for 10 min to quench autofluorescence caused by free aldehydes, and then blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) in PBS. The slides were incubated overnight at 4°C with the following primary antibodies diluted in PBS with 1% BSA: mouse monoclonal anti-transforming growth factor-β (TGF-β) (1:100 dilution; catalogue number ab1279, Abcam), rabbit polyclonal anti-connective tissue growth factor (CTGF/CCN2) (1:200 dilution; catalogue number ab6992, Abcam) and mouse monoclonal anti-endothelin-1 (ET-1) (1:100 dilution; catalogue number ab2786, Abcam). After extensive washing in PBS, the sections were incubated with Alexa Fluor-488-conjugated goat antimouse or antirabbit IgG (1:200 dilution; Molecular Probes, Eugene, Oregon, USA) for 45 min at room temperature in the dark. Irrelevant isotype-matched and concentration-matched IgG (Sigma–Aldrich) were used as negative controls. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Chemicon International, Temecula, California, USA). The slides were then mounted with an antifade aqueous mounting medium (Biomeda Gel Mount, Electron Microscopy Sciences, Foster City, California, USA) and examined with the Leica DM4000 B microscope (Leica Microsystems). Densitometric analysis of the intensity of immunofluorescent staining was performed on digitised images using ImageJ software (NIH).

Detection of endothelial cell apoptosis by a combined TUNEL/CD31 immunofluorescence assay

For immunohistochemical detection and quantification of apoptotic endothelial cells in mouse skin sections we used the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technology (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany) in combination with immunofluorescence for CD31. Skin sections were deparaffinised and boiled for 10 min in 10 mM sodium citrate buffer (pH 6.0). The slides were rinsed twice with PBS and incubated with TUNEL reaction mixture for 1 h at 37°C in a humidified chamber in the dark. After extensive washing in PBS, the sections were blocked for 30 min at room temperature with 1% BSA in PBS, and subsequently incubated with a rabbit polyclonal anti-CD31 antibody (1:40 dilution; Abcam) overnight at 4°C. The CD31 immunoreaction was revealed using Rhodamine Red-X-conjugated goat anti-rabbit IgG (1:200 dilution; Molecular Probes). Nuclei were counterstained with DAPI (Chemicon International). The percentage of apoptotic endothelial cells was calculated semiquantitatively as TUNEL/DAPI/CD31-positive cells in proportion to all DAPI/CD31-positive cells. Cell counting was performed on five randomly chosen microscopic high-power fields (40× original magnification) of the dermis from each of three sections per sample by two independent blinded observers (MM, IR), and the mean of the two different observations was used for analysis.

Statistical analysis

Data are represented as mean±SD. The Student t test for non-related samples was used for statistical analysis. A p value of less than 0.05 was considered statistically significant.

Results

Decreased expression of full-length uPAR in the skin of SSc patients

Previous studies have shown that uPAR cleavage/inactivation is crucially involved in fibroblast-to-myofibroblast transition and SSc endothelial cell-impaired angiogenic function.14 ,15 ,21 However, to propose uPAR−/− mice as an animal model for both fibrosis and peripheral microvasculopathy of SSc, full-length uPAR should also be downregulated in human SSc skin. Therefore, we used immunohistochemistry and immunofluorescence to analyse the expression of full-length uPAR in skin biopsies from SSc patients and healthy subjects (figure 1A–D). Full-length uPAR immunostaining was significantly decreased in the affected dermis of SSc patients compared with healthy controls, especially in fibroblasts and microvascular endothelial cells (p<0.001 for both) (figure 1A–E). Full-length uPAR was downregulated throughout different disease stages without obvious differences between the limited and diffuse cutaneous SSc subsets (data not shown).

Figure 1

Decreased expression of the native full-length form of urokinase-type plasminogen activator receptor (uPAR) in the skin of patients with systemic sclerosis (SSc). (A–D) Representative microphotographs of skin sections from healthy controls (A and B) and SSc patients (C and D) immunostained with mouse monoclonal anti-uPAR/domain 1 antibodies. (A and C) Immunoperoxidase staining. (B and D) Immunofluorescent staining. Full-length uPAR immunostaining is decreased in dermal microvascular endothelial cells (A and C insets; B and D arrows) and fibroblasts (B and D arrowheads) of SSc skin compared with healthy control skin. Original magnification ×20 (A and C), ×40 (A and C insets), ×63 (B and D). (E) Semiquantitative analysis of uPAR immunostaining in dermal endothelial cells and fibroblasts. Data are mean±SD of immunostaining score performed on skin sections from 15 SSc patients and 10 healthy controls. *p<0.001 vs control.

uPAR−/− mice display increased dermal thickness, collagen content, myofibroblast counts and expression of profibrotic cytokines

The degree of mouse skin fibrosis was quantified by measurement of the dermal thickness and collagen content. Histological examination of haematoxylin-eosin-stained skin sections demonstrated a considerable increase in the dermal thickness of uPAR−/− mice as compared with age-matched uPAR+/+ mice (figure 2A). At both 12 and 24 weeks of age, quantitative analysis showed that the dermal thickness of uPAR−/− mice was significantly greater than that of uPAR+/+ mice (p<0.001 for both) (figure 2B). No significant difference in dermal thickness was observed between uPAR−/− mice at 12 and 24 weeks of age (figure 2B). Beyond dermal thickening, Masson's trichrome staining revealed an accumulation of ECM components in the skin of uPAR−/− mice (figure 2C–E). Moreover, in uPAR-/- mice the subcutaneous fat tissue was partly replaced by connective tissue with severe perivascular fibrosis (figure 2D,E). Perivascular inflammatory cell infiltrates and clusters of degranulating mast cells were often observed in the deeper dermal layers of uPAR−/− mice, especially at 12 weeks of age (figure 2E).

Figure 2

Mice deficient for urokinase-type plasminogen activator receptor (uPAR−/−) display increased dermal thickness and accumulation of extracellular matrix. (A) Representative microphotographs of haematoxylin-eosin-stained skin sections from uPAR−/− mice and wild-type (uPAR+/+) mice at 12 weeks of age (n=7 uPAR−/− mice, n=7 uPAR+/+ mice) and 24 weeks of age (n=7 uPAR−/− mice, n=6 uPAR+/+ mice). The blue lines indicate the distance between the dermal–epidermal junction and the dermal–subcutaneous fat junction (dermal thickness). Original magnification ×10. Scale bars are indicated in each panel. (B) Quantification of dermal thickness (μm). Data are mean±SD. *p<0.001 vs uPAR+/+ mice. (C–E) Representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and uPAR+/+ mice (n=7) stained with Masson's trichrome. The extracellular matrix is stained blue, nuclei are stained dark red/purple and cytoplasm is stained red. An accumulation of extracellular matrix is evident in the skin of uPAR−/− mice (C–E). (D and E) In uPAR−/− mice the subcutaneous fat tissue is partly replaced by connective tissue. (D) A severe perivascular fibrosis is also evident (arrows). (E) Perivascular inflammatory cell infiltrates and clusters of degranulating mast cells are observed in the deeper dermal layers of uPAR−/− mice (arrowheads). The inset shows a higher magnification view of degranulating mast cells and perivascular inflammatory cells. Original magnification ×10 (C), ×20 (D), ×40 (E), ×63 (E inset). Scale bars are indicated in each panel.

To specifically investigate the effect of uPAR deficiency on dermal collagen deposition, the collagen content in the skin of uPAR−/− and uPAR+/+ mice was measured using two independent methods, Picrosirius red staining and hydroxyproline assay. Picrosirius red staining findings paralleled those obtained with Masson's trichrome staining, showing dense accumulation of thickened and closely packed collagen bundles and abundant perivascular collagen deposition in the skin of uPAR−/− mice (figure 3A–C). Image analysis revealed that Picrosirius red-positive area was significantly greater in the dermis of uPAR−/− mice than in wild-type littermates (p<0.001) (figure 3D). Similarly, the hydroxyproline content was significantly increased in the skin of uPAR−/− mice compared with uPAR+/+ mice (p<0.001) (figure 3E). Moreover, in uPAR−/− mice there was a significant increase in the number of α-SMA-positive dermal myofibroblasts compared with wild-type mice (p<0.001) (figure 3F,G). A strong expression of TGF-β, CTGF/CCN2 and ET-1 profibrotic cytokines was observed in the dermis of uPAR−/− mice (figure 4). Conversely, the expression of these cytokines was very weak or even undetectable in the skin of uPAR+/+ mice (figure 4). Semiquantitative analysis of immunofluorescent staining intensity on skin sections showed that the expression of the three profibrotic molecules was significantly increased in the dermis of uPAR−/− mice compared with uPAR+/+ mice (all p<0.001) (figure 4).

Figure 3

Increased collagen content and myofibroblast counts in the skin of mice deficient for urokinase-type plasminogen activator receptor (uPAR−/−). (A–C) Representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and wild-type (uPAR+/+) mice (n=7) stained with Picrosirius red. Collagen is stained red. Thickened and closely packed collagen bundles (B) and abundant perivascular collagen deposition (C, arrows) are observed in the skin of uPAR−/− mice. Original magnification ×10 (A), ×40 (B and C). Scale bars are indicated in each panel. (D and E) Quantification of Picrosirius red-positive area (D) and hydroxyproline content (E) both expressed as a percent of the observed in uPAR+/+ mice. Data are mean±SD. *p<0.001 vs uPAR+/+ mice. (F) Representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and wild-type (uPAR+/+) mice (n=7) immunostained with rabbit polyclonal anti-α-smooth muscle actin (α-SMA) antibodies. Myofibroblasts were identified as α-SMA-positive spindle-shaped cells (F, arrows). Original magnification ×40. Scale bars are indicated in each panel. (G) Quantification of myofibroblasts in the dermis of uPAR−/− and uPAR+/+ mice. Data are mean±SD of α-SMA-positive myofibroblast counts per high-power field. *p<0.001 vs uPAR+/+ mice.

Figure 4

Increased expression of transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF/CCN2) and endothelin-1 (ET-1) profibrotic cytokines in the dermis of mice deficient for urokinase-type plasminogen activator receptor (uPAR−/−). Left panels, representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and wild-type (uPAR+/+) mice (n=7) immunostained for TGF-β, CTGF/CCN2 and ET-1 (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Original magnification ×20. Scale bars are indicated in each panel. Right panels, densitometric analysis of immunofluorescent staining. Data are mean±SD of optical density in arbitrary units. *p<0.001 vs uPAR+/+ mice.

No relevant differences in any of the assessed parameters could be found between uPAR−/− mice aged 12 and 24 weeks (data not shown).

uPAR−/− mice display a reduced number of dermal microvessels and increased apoptosis of endothelial cells

The reduction in dermal microvessels is a hallmark of the peripheral microangiopathy in human SSc.3 Therefore, we next assessed the microvessel density in skin sections of uPAR−/− mice compared with wild-type mice. A significant decrease in the number of CD31-positive dermal capillaries could be observed in uPAR−/− mice (p<0.001) (figure 5A).

Figure 5

Decreased microvessel density and increased apoptosis of endothelial cells in the dermis of mice deficient for urokinase-type plasminogen activator receptor (uPAR−/−). (A) Representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and wild-type (uPAR+/+) mice (n=7) immunostained for the pan-endothelial cell marker CD31 are shown. CD31-positive microvessels are indicated by arrows. Original magnification ×40. Scale bars are indicated in each panel. Data are mean±SD of CD31-positive microvessel counts per high-power field. *p<0.001 vs uPAR+/+ mice. (B) Representative microphotographs of skin sections from 12-week-old uPAR−/− mice (n=7) and wild-type (uPAR+/+) mice (n=7) subjected to combined TUNEL (green)/CD31 (red) immunofluorescence assay and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Apoptotic endothelial cell nuclei are green/blue double stained (arrows). Original magnification ×40. Scale bars are indicated in each panel. Semiquantitative analysis revealed a significantly higher percentage of TUNEL-positive endothelial cells in the dermis of uPAR−/− mice compared with wild-type mice. Data are mean±SD. *p<0.001 vs uPAR+/+ mice.

Apoptosis of endothelial cells is considered one of the earliest phenomena in the development of the microangiopathy in human SSc.3 Therefore, we evaluated whether apoptosis of endothelial cells could play a role in the observed peripheral microangiopathy in uPAR−/− mice. Analysis of skin sections by a combined TUNEL/CD31 immunofluorescence assay demonstrated a significantly higher number of apoptotic endothelial cells in the dermis of uPAR−/− mice compared with wild-type littermates (p<0.001) (figure 5B).

No significant difference in the number of either dermal microvessels or apoptotic endothelial cells was found between uPAR−/− mice at 12 and 24 weeks of age (data not shown).

No intima-media proliferation was observed in the dermal vessels of uPAR−/− mice.

uPAR−/− mice develop progressive pulmonary fibrosis

Lung specimens from 12-week-old and 24-week-old uPAR−/− mice exhibited SSc-like features of non-specific interstitial pneumonia29 with large patchy areas of lung parenchyma displaying a uniform interstitial involvement characterised by both diffuse cellular inflammation and collagen deposition (figure 6A,B). At both 12 and 24 weeks of age, alveolar septal width and hydroxyproline content were significantly increased compared with age-matched uPAR+/+ mice (p<0.001 for both) (figure 6C,D). Moreover, in uPAR−/− mice, pulmonary pathology worsened significantly from 12 to 24 weeks of age, as shown by a significant increase in the thickness of alveolar septa and collagen content (p<0.001 for both) (figure 6A–D). No intima-media proliferation was observed in the pulmonary vessels of uPAR−/− mice.

Figure 6

Mice deficient for urokinase-type plasminogen activator receptor (uPAR−/−) develop progressive pulmonary fibrosis. (A and B) Representative microphotographs of lung sections from uPAR−/− mice and wild-type (uPAR+/+) mice at (A) 12 weeks of age (n=7 uPAR−/− mice, n=7 uPAR+/+ mice) and (B) 24 weeks of age (n=7 uPAR−/− mice, n=6 uPAR+/+ mice) stained with haematoxylin-eosin (upper panels) and Picrosirius red (lower panels). In uPAR−/− mice, large patchy areas of lung parenchyma display the typical features of non-specific interstitial pneumonia with interstitial cellular inflammation and collagen accumulation. The insets show higher magnification views of thickened alveolar septa. Original magnification ×20, ×40 (insets). Scale bars=100 μm. (C, D) Quantification of alveolar septal width (C) and hydroxyproline content (D) both expressed as a percent of the observed in 12-week-old uPAR+/+ mice. Data are mean±SD. *p<0.001 vs uPAR+/+ mice. #p<0.001 vs 12-week-old uPAR−/− mice.

Discussion

Our data provide evidence that uPAR−/− mice display some of the most important histopathological hallmarks of human SSc, such as dermal fibrosis, severe reduction in the number of dermal microvessels and pulmonary fibrosis.

Despite some recent advances in uncovering the cellular and molecular pathogenetic mechanisms of SSc, its aetiology still remains unclear. As a consequence, treatments including disease-modifying therapies are available for specific organ-based complications, but there is a need for further therapies able to stop or reverse fibrosis and vasculopathy and, thus, to substantially modify the natural progression of the disease.6 ,30 The limited progress in the understanding of disease pathomechanisms and developing innovative therapies is caused in part by the lack of animal models characterised by both the fibrotic and vascular phenotypes resembling human SSc. Indeed, several models have been proposed to study different aspects of the disease, but none of these models can fully encompass all the features of SSc.7 ,8 In particular, all currently available animal models of scleroderma display severe dermal fibrosis, while only few of them are suitable to study peripheral small-vessel vasculopathy and internal organ involvement, such as pulmonary arterial hypertension, interstitial lung disease and gut fibrosis.7–9 31–33

uPAR together with uPA constitutes a key protease system that promotes ECM remodelling, growth factor activation and cell migration. uPA is an extracellular serine protease that binds to cell surface uPAR and generates plasmin from plasminogen at the cell–matrix interface. Plasmin is a broad-spectrum protease that cleaves fibrin and other ECM proteins and also promotes cell migration by activating matrix-sequestered metalloproteinases and growth factors.10 In addition to the generation of plasmin, uPA binding to uPAR promotes the interaction of uPAR with integral membrane proteins, such as integrins, which signal intracellularly to promote cytoskeletal reorganisation and cell migration.10 ,13 uPAR has three extracellular domains, termed D1D2D3. The D1 domain represents the principal uPA binding site,11 which can be cleaved from the full-length protein by multiple proteases such as plasmin, uPA, trypsin, chymotrypsin and matrix metalloproteinases.34 Full-length uPAR binds to uPA, integrins and vitronectin, whereas cleaved uPAR (D2D3) cannot. Therefore, the downstream effects of uPAR cleavage are (1) the inhibition of uPA protease activity and uPA-induced intracellular signalling; (2) the inhibition of the uPAR-mediated regulation of integrin functions which results in an integrin-dependent increase in cell adhesion on collagen, fibronectin and laminin and (3) a decrease in uPA-stimulated cell migration.10 ,35 It is well known that enhanced cell adhesion stimulates myofibroblast differentiation, because the stabilisation of cell attachment to ECM generates the cell tension required for assembly of α-SMA into stress fibres, which are characteristic of the myofibroblast.22 ,36 Indeed, it has been demonstrated that uPAR cleavage/inactivation is necessary for TGF-β-induced fibroblast-to-myofibroblast transition, and that downregulation of uPA/uPAR results in increased cell surface integrin expression and adhesion to collagen, promoting the persistent myofibroblast phenotype that triggers fibrosis.21 ,22 ,37 Besides fibroblasts, the uPA/uPAR system also regulates important endothelial cell activities. In fact, uPAR provides endothelial cells with a proper cell surface enzymatic machinery and organises the appropriate phases of adhesion and ECM degradation critical to endothelial cell migration and angiogenesis.16–18 In this context, our group has previously demonstrated that in SSc dermal microvascular endothelial cells, uPAR undergoes a matrix metalloproteinase-12-dependent cleavage between D1 and D2 which results in impaired cytoskeletal rearrangement, cell migration, invasion, proliferation and angiogenesis.14 ,15 Interestingly, neutralisation of matrix metalloproteinase-12 by RNA silencing prevented uPAR cleavage and could, in part, restore the ability of SSc endothelial cells to produce capillary structures in vitro.38 Moreover, we also reported the association of an uPAR gene promoter genetic variant with the vascular complications of SSc.39 Here, we also provide the first ex vivo evidence that full-length uPAR is downregulated in SSc dermis, especially in microvascular endothelial cells and fibroblasts.

On this background, we herein investigated whether uPAR gene knockout in mice could result in fibrosis and peripheral microvasculopathy resembling human SSc. Our results confirm and extend recent findings by Kanno et al.20 showing that the absence of uPAR is associated with the development of dermal fibrosis in mice and allow us to propose the uPAR−/− mice as a novel animal model of SSc. Indeed, the skin of uPAR−/− mice displayed most of the typical pathological features of SSc. Dermal thickness, collagen content and myofibroblast counts were significantly greater in uPAR−/− mice than in wild-type mice. Similar to SSc, the skin of uPAR−/− mice was characterised by the presence of thickened, closely packed and irregularly distributed collagen bundles, abundant perivascular fibrosis and partial replacement of subcutaneous fat with connective tissue. Moreover, in the dermis of uPAR−/− mice we also found a marked overexpression of the SSc-related profibrotic factors TGFβ, CTGF/CCN2 and ET-1. TGF-β appears to play a crucial role in the pathogenesis of skin and lung fibrosis in SSc, activating fibroblasts/myofibroblasts and causing a potent stimulation of profibrotic CTGF/CCN2 synthesis in fibroblasts and endothelial cells.40 ,41 ET-1 is a potent activator of fibroblast proliferation and ECM synthesis, and its enhanced production has been well described in SSc.42–44 Additionally, ET-1 is required for the ability of TGFβ to induce fibrogenic effects both in vitro and in vivo.44

Besides SSc-like fibrotic skin changes, here we show for the first time that uPAR−/− mice can also be proposed as an animal model for the peripheral microvasculopathy of SSc. In our uPAR−/− mice, dermal fibrosis was paralleled by endothelial cell apoptosis and severe loss of microvessels closely mimicking those in the skin of SSc patients. In fact, endothelial cell apoptosis has been proposed as a primary pathogenetic event underlying skin lesions in human SSc.45 Consistent with our findings, it has been shown that uPA mediates endothelial cell survival, and that its antiapoptotic activity is dependent on the presence of uPAR.46 Additionally, the findings of dermal capillary rarefaction in uPAR−/− mice are in agreement with our previous observations that uPAR cleavage/inactivation is largely responsible for the impaired angiogenic function of SSc microvascular endothelial cells,3 ,14 ,15 and with a study reporting that uPAR−/− murine endothelial cells are unable to form mature capillary-like lumen structures both in vitro and in vascular endothelial growth factor-enriched Matrigel implants.18

Finally, we could also show for the first time that the lungs of uPAR−/− mice exhibit SSc-like pathological features. We observed large patchy areas of lung parenchyma displaying a uniform interstitial involvement with both diffuse cellular inflammation and collagen deposition closely resembling the non-specific interstitial pneumonia pattern of SSc. However, we could not find any histological evidence of vessel intima-media proliferation resembling SSc-related pulmonary arterial hypertension. Further studies will be required to ascertain whether uPAR−/− mice may also develop SSc-like features in other internal organs, such as the heart, kidneys and gastrointestinal tract. In this regard, it was reported that uPAR deficiency accelerates renal fibrosis in obstructive nephropathy.19

In conclusion, we provide the evidence that the absence of uPAR is associated with the development of dermal and pulmonary fibrosis and peripheral microvasculopathy in mice mimicking human SSc. The pathogenetic features observed in uPAR−/− mice, such as the increased numbers of dermal myofibroblasts, enhanced expression of profibrotic factors, endothelial cell apoptosis and reduction in capillary density, are particularly interesting since they may help to elucidate similar processes occurring during SSc. We believe that uPAR−/− mice might be a promising preclinical model to study the pathogenetic mechanisms of human SSc and enable testing of antifibrotic agents and drugs targeting small-vessel vasculopathy simultaneously. Because we also show that full-length uPAR is downregulated in human SSc, and previous studies have implicated uPAR cleavage/inactivation in fibroblast-to-myofibroblast transition21 and SSc-impaired angiogenesis,14 ,15 blocking uPAR cleavage/inactivation might represent a promising future strategy to treat both fibrosis and microvasculopathy in SSc patients.

References

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Footnotes

  • Handling editor Tore K Kvien

  • LI-M and MM-C contributed equally.

  • Contributors All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Study conception and design: MM, IR, LI-M and MM-C. Acquisition of data: MM, IR, AFM, SG, PC, LI-M and MM-C. Analysis and interpretation of data: MM, IR, AFM, LI-M and MM-C.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval The study was approved by the local institutional review board.

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

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