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Inactivation of urokinase-type plasminogen activator receptor (uPAR) gene induces dermal and pulmonary fibrosis and... Article in Annals of the rheumatic diseases · July 2013 DOI: 10.1136/annrheumdis-2013-203706 · Source: PubMed
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ARD Online First, published on July 12, 2013 as 10.1136/annrheumdis-2013-203706 Basic and translational research
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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? Mirko Manetti,1 Irene Rosa,1 Anna Franca Milia,1 Serena Guiducci,2 Peter Carmeliet,3,4 Lidia Ibba-Manneschi,1 Marco Matucci-Cerinic2 Handling editor Tore K Kvien 1
Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Florence, Italy 2 Department of Experimental and Clinical Medicine, Section of Internal Medicine and Division of Rheumatology, Azienda OspedalieroUniversitaria Careggi, University of Florence, Florence, Italy 3 Laboratory of Angiogenesis and the Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium 4 Laboratory of Angiogenesis and the Neurovascular Link, Vesalius Research Center, University of Leuven, Leuven, Belgium 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;
[email protected], mirko.manetti@unifi.it LI-M and MM-C contributed equally. Received 30 March 2013 Revised 25 May 2013 Accepted 29 June 2013
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.
INTRODUCTION To cite: Manetti M, Rosa I, Milia AF, et al. Ann Rheum Dis Published Online First: [please include Day Month Year] doi:10.1136/ annrheumdis-2013-203706
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 phosphatidylinositolanchored 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
Manetti M,Article et al. Ann author Rheum Dis (or 2013;0:1–10. doi:10.1136/annrheumdis-2013-203706 1 Copyright their employer) 2013. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Basic and translational research 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 haematoxylineosin 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 2
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 Manetti M, et al. Ann Rheum Dis 2013;0:1–10. doi:10.1136/annrheumdis-2013-203706
Basic and translational research (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.
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
