Tsk Mouse Research Articles

Translational engagement of lysophosphatidic acid receptor 1 in skin fibrosis: from dermal fibroblasts of patients with scleroderma to tight skin 1 mouse.

Background and PurposeGenetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA1) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA1 receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis.Experimental ApproachDermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model.Key ResultsSAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA1 receptor. The LPA functional response (Ca2+) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice.Conclusion and ImplicationsThe effects of SAR100842 on LPA‐induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA1 receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA1 receptor antagonists in systemic sclerosis.

Suppressive Regulation by MFG-E8 of Latent Transforming Growth Factor β-Induced Fibrosis via Binding to αv Integrin: Significance in the Pathogenesis of Fibrosis in Systemic Sclerosis.

Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule-associated protein with epidermal growth factor- and factor VIII-like domains (MFG-E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG-E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG-E8 in skin fibrosis in SSc. We assessed expression of MFG-E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG-E8 (rMFG-E8) on latent transforming growth factor β (TGFβ)-induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG-E8 on fibrosis mouse models. We demonstrated that MFG-E8 expression around dermal blood vessels and the serum MFG-E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG-E8 significantly inhibited latent TGFβ-induced expression of type I collagen, α-smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3-8), which suggested that MFG-E8 inhibited activation of latent TGFβ as well as TGFβ signaling via binding to αv integrin. In a mouse model of bleomycin-induced fibrosis (n = 5-8) and in a TSK mouse model (a genetic model of SSc) (n = 5-10), deficient expression of MFG-E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG-E8 significantly inhibited bleomycin-induced dermal fibrosis. These results suggest that vasculopathy-induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG-E8, may be associated with the decreased expression of MFG-E8 in SSc and that the deficient inhibitory regulation of latent TGFβ-induced skin fibrosis by MFG-E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients.

Fibrillin-1 mutant mice (tight skin) show increased spontaneous and trauma-induced osteoarthritis development

Purpose: Osteoarthritis development is a major chronic disorder affected >8million British people. Despite the high prevalence of OA, there are currently no therapies to slow disease progression. Fibrillin-1 is an extracellular matrix protein found in elastic fibres. One of its main roles is to control growth factor bioavailability, which play vital functions in joint homeostasis. Fibrillin-1 mutations, as those found in Tight Skin mice (TSK), can increase TGFß signalling, and leads to tight skin, myocardial hypertrophy, marfan-like skeletal phenotype and lung emphysema. The aim of this study is to assess OA development in TSK mice in spontaneous and in trauma-induced models. Methods: Immunohistochemistry for Fibrillin-1 was performed in CBA and Str/ort mice (spontaneous OA), and in trauma-induced OA. For the ageing model, knees from 35wk-old TSK and littermate control male mice were collected, microCT scanned and used for histology. The non-invasive loading model was used and the right knee of male TSK and WT mice were loaded i) a single time at 7 or 9N loads and joints analysed 1 weeks later; or ii) repetitively for 2 weeks to induce OA progression, and joints assessed 6 weeks after the last loading episode. Histology was performed on these joints after decalcification (10% formic acid), serial sections cut across the whole joint at 6μm, and AC degradation severity assessed (OARSI grading system). Results: Fibrillin-1 was found in the pericellular matrix of chondrocytes in both the growth plate (resting and hypertrophic cells) and in the uncalcified articular cartilage in healthy CBA mice. During the development of OA in Str/ort mice and in response to mechanical trauma, Fibrillin-1 immunolabelling was decreased. TSK mice showed spontaneous increased joint space mineralised tissue volume (including meniscus and ligament calcification) and increased AC degradation compared to WT at 35wks of age. In contrast, no significant differences were seen in tibial epiphyseal bone (medial and lateral; BV/TV, Trabecular Thickness, Trabecular Separation, Trabecular number). A single loading episode at both 9 and 7N produced AC lesions in the lateral femur in both WT and TSK mice, however severity of lesions created were more severe in TSK mice (preliminary data). Repetitive loading episodes lead to slight increase in mineralised joint space tissue between TSK and litter mate WT controls (preliminary data), which from our previous studies has shown to be correlated with OA severity. Conclusions: This study shows that Fibrillin-1 is decreased from the pericellular matrix during OA development in spontaneous and trauma-induced OA, suggesting increased Fibrillin-1 degradation. In addition, mutations in Fibrillin-1 in the TSK mouse lead to increased spontaneous and trauma-induced OA development. These data suggest that normal Fibrillin-1 expression and function may play an important role in joint homeostasis and that abnormal expression or mutations promote OA development initiation and progression.

Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model.

ObjectiveSystemic sclerosis (SSc) is an autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Cadherin-11 (CDH11) expression is increased in fibrotic skin and lung tissue. Targeting CDH11 may be an effective approach to treating fibrosis. We hypothesize that targeting CDH11 will decrease fibrosis in the tight skin-1 (Tsk-1) mouse model.MethodsCDH11 expression was determined in the Tsk-1 mouse model using quantitative real time PCR and immunofluorescence (IF). Inhibitory anti- CDH11 monoclonal antibodies were tested in Tsk-1 mice for their ability to decrease hypodermal fibrosis.ResultsExpression of CDH11 was increased in fibrotic skin from Tsk-1 mice compared to pallid controls. IF staining demonstrated that CDH11 expression localized to fibroblasts within the hypodermis of fibrotic skin. Treatment with inhibitory anti-CDH11 monoclonal antibodies decreased hypodermal thickness and fibrotic mediators in Tsk-1 mice compared to control antibodies.ConclusionsThese data demonstrate an important role for CDH11 in the development of skin fibrosis in Tsk-1 mice. These data add to the growing evidence for the important role of CDH11 in tissue fibrosis and fibrotic disease such as systemic sclerosis.

Paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis

BackgroundSystemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. ObjectiveIn this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. MethodsSeven weeks old female B6.Cg-Fbn1Tsk/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. ResultsThe results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFβ-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. ConclusionsPaquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

FRI0516 Paquinimod (ABR-215757), an Immunomodulatory Compound, Reduces Fibrosis in the Tight Skin-1 (TSK-1) Model for Systemic Sclerosis

BackgroundPaquinimod (ABR-215757), a new oral small molecular drug, belongs to the quinoline-3-carboxamide derivatives, a class of structurally related compounds (1). It targets the S100A9 protein and disrupts its binding to...

S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

ObjectivesS100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we...

OP0015 Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis

BackgroundFibrosis of the skin and internal organs is a cardinal feature of systemic sclerosis (SSc). Since it disrupts the physiologic tissue architecture and causes organ failure, fibrosis leads to a...

Inactivation of the cannabinoid receptor CB1 prevents leukocyte infiltration and experimental fibrosis

Cannabinoids are derivates of the marijuana component Δ(9) -tetrahydrocannabinol that exert their effects on mesenchymal cells and immune cells via CB1 and CB2 receptors. The aim of the present study was to evaluate the role of CB1 in systemic sclerosis. CB1-deficient (CB1(-/-) ) mice and wild-type littermates (CB1(+/+) mice) were injected with bleomycin. CB1 signaling was activated in vivo with the selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA). Bone marrow transplantation experiments were performed to investigate whether the phenotype of CB1(-/-) mice was mediated by leukocytes or mesenchymal cells. The role of CB1 was also investigated in the TSK-1 mouse model. CB1(-/-) mice were protected from bleomycin-induced dermal fibrosis, with reduced dermal thickening, hydroxyproline content, and myofibroblast counts. Inactivation of CB1 decreased the number of infiltrating T cells and macrophages in lesional skin. In contrast, activation of CB1 with ACEA increased leukocyte infiltration and enhanced the fibrotic response to bleomycin. The phenotype of CB1(-/-) mice was mimicked by transplantation of CB1(-/-) mouse bone marrow into CB1(+/+) mice, demonstrating that CB1 exerts its profibrotic effects indirectly by regulating leukocyte infiltration. Consistently, knockdown of CB1 did not prevent fibrosis in the inflammation-independent TSK-1 mouse model. We demonstrate that the cannabinoid receptor CB1 is crucial for leukocyte infiltration and secondary fibroblast activation and that inactivation of CB1 exerts potent antifibrotic effects in inflammation-driven models of fibrosis.

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.

Increased Expression of Wnt2 and SFRP4 in Tsk Mouse Skin: Role of Wnt Signaling in Altered Dermal Fibrillin Deposition and Systemic Sclerosis

Systemic sclerosis (SSc) is a complex human disorder characterized by progressive skin fibrosis. To better understand the molecular basis of dermal fibrosis in SSc, we analyzed microarray gene expression in skin of the Tight-skin (Tsk) mouse, an animal model where skin fibrosis is caused by an in-frame duplication in fibrillin-1 (Fbn-1). Tsk skin showed increased mRNA levels of several genes involved in Wnt signaling, including Wnt2, Wnt9a, Wnt10b and Wnt11; Dapper homolog antagonist of beta-catenin (DACT1) and DACT2; Wnt-induced secreted protein 2; and secreted frizzled-related protein (SFRP)2 and SFRP4. RNase protection and northern blot confirmed microarray results. Furthermore, Wnt3a markedly stimulated matrix assembly of microfibrillar proteins, including Fbn-1, by cultured fibroblasts, suggesting that Wnts contribute to increased microfibrillar matrices in Tsk skin. Further analysis showed that SFRP4 expression is specifically increased in tissues expressing Tsk-Fbn-1, such as skeletal muscle and skin. The increase in SFRP4 mRNA in Tsk skin started 2 weeks after birth, following the increase in Wnt2 mRNA that occurred at birth. This suggests that SFRP4 may modulate Wnt functions in Tsk skin fibrosis. Lesional skin from SSc patients also showed large increases in SFRP4 mRNA and protein levels in the deep dermis compared to healthy skin, suggesting that the Wnt pathway might regulate skin fibrosis in SSc.

Increased expression of type I collagen induced by microfibril‐associated glycoprotein 2: Novel mechanistic insights into the molecular basis of dermal fibrosis in scleroderma

Mutations in fibrillin 1, a key component of extracellular microfibrils, are associated with connective tissue disorders such as Marfan's syndrome or skin fibrosis in the tight skin mouse model of scleroderma. Previous studies have suggested that fibrillin 1 mediates skin fibrosis via its interface with associated microfibrillar proteins and type I collagen; in particular, microfibril-associated glycoprotein 2 (MAGP-2), an extracellular matrix protein that binds to fibrillins and the alphavbeta3 integrin, is increased in TSK mouse and human scleroderma skin. Because the function of MAGP-2 in the biologic processes of the matrix remains unknown, this study investigated whether MAGP-2 regulates type I collagen. Fibroblast cultures conditionally overexpressing MAGP-2 were developed. Cells were analyzed by Western blotting, Northern blotting, pulse-chase analysis, and immunofluorescence to assess the effect of MAGP-2 on type I collagen. Cells overexpressing MAGP-2 formed increased MAGP-2 matrix and showed a 3-fold increase in intracellular type I procollagen. This increase was associated with increased levels of type I collagen in the medium and matrix. Increased type I collagen colocalized with the MAGP-2 matrix. MAGP-2 overexpression had no effect on type I procollagen messenger RNA, but markedly increased the half-life of type I procollagen. MAGP-2 induced type I collagen even under conditions in which no MAGP-2 matrix was detectable, and did not require the presence of the RGD motif of MAGP-2 in its integrin-binding site. This study shows that MAGP-2 stabilizes type I procollagen, identifying an important function of MAGP-2 in extracellular matrix homeostasis. It also suggests that MAGP-2 might mediate skin fibrosis in TSK mice and in patients with scleroderma.

Mutant fibrillin 1 from tight skin mice increases extracellular matrix incorporation of microfibril-associated glycoprotein 2 and type I collagen.

Skin fibrosis in the TSK mouse, a model of skin fibrosis seen in systemic sclerosis (SSc), is caused by a large in-frame duplication in the Fbn1 gene, tsk-Fbn1. We investigated whether tsk-Fbn1 might cause dermal fibrosis by affecting Fbn1 and associated extracellular matrices. We also studied whether deposition of microfibril-associated glycoprotein 2 (MAGP-2), a protein that is associated with fibrillin 1, was altered in the skin of patients with SSc. An in vitro model of the TSK mouse was created by conditionally expressing tsk-Fbn1 in mouse embryonic fibroblasts (MEFs). Cell cultures were examined by immunofluorescence and Western and Northern blotting to determine the effect of tsk-Fbn1 on the structure, expression, and deposition of fibrillin 1 (Fbn-1), type I collagen, and MAGP-2. The skin of TSK mice and SSc patients was analyzed by immunohistochemistry for MAGP-2 expression. Expression of tsk-Fbn1 in cultured MEF cells altered the morphology of Fbn-1 fibers and increased the deposition of type I collagen into the extracellular matrix (ECM) without concomitantly changing messenger RNA expression, secretion, or processing of type I procollagen. Moreover, MEF cells expressing tsk-Fbn1 showed increased MAGP-2 matrix. MAGP-2 was increased in the dermis of TSK mice. Fibrotic SSc skin also showed higher levels of MAGP-2 in the dermis than nonfibrotic SSc skin and normal skin. Tsk-Fbn1 altered ECM organization and caused fibrosis by affecting the deposition of MAGP-2 or other Fbn-1-associated proteins. Alterations in microfibril structure or deposition might contribute to fibrosis in SSc.

Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight‐skin mouse

To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP-3) in Tsk1 mouse and human systemic sclerosis (SSc) skin. Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non-Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP-3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP-3 was explored in transgenic mouse fibroblasts and by transient transfection assays. MCP-3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early-stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP-3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proalpha2(I) collagen promoter-reporter gene constructs were activated by MCP-3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP-3 could be diminished by a neutralizing antibody to transforming growth factor beta. We demonstrate, for the first time, overexpression of MCP-3 in early-stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.

Association of 5′-untranslated region of the Fibrillin-1 gene with Japanese scleroderma

Excessive production of extracellular matrix (ECM) constituents is a hallmark scleroderma or systemic sclerosis (SSc). Fibrillin-1, a major component of microfibrils in the ECM, may play a role in the pathogenesis of SSc. The TSK1 mouse model of SSc bears an in-frame duplication of the Fibrillin-1 gene ( FBN1) which results in a larger than normal protein that is more susceptible to proteolysis. Metabolic labeling studies of Fibrillin-1 in human SSc dermal fibroblasts demonstrated that while normal amounts of Fibrillin-1 are synthesized, the protein itself appears to be unstable. Moreover, autoantibodies specific for Fibrillin-1 have been demonstrated in serum from SSc patients and TSK1 mice. In particular, a high frequency of anti-Fibrillin-1 was observed in Japanese patients with diffuse and limited scleroderma or CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) syndrome. Genetic studies in a Native American population with high prevalence of using microsatellite marker showed strong association between FBN1 haplotypes and SSc. Subsequently, studies of FBN1 single nucleotide polymorphisms (SNPs) demonstrated that certain FBN1 haplotypes were associated with SSc in both Native American and Japanese patients with limited scleroderma. Thus, FBN1 was sequenced in 22 Japanese SSc patients to ascertain the presence of any relevant mutations or SNPs. Sequence analysis revealed eight coding and 14 non-coding SNPs and other polymorphisms. Among them, a CT insertion in the 5′-untranslated region of exon A had a significant negative association with disease.

Autoantibodies to the Extracellular Matrix Microfibrillar Protein, Fibrillin-1, in Patients with Scleroderma and Other Connective Tissue Diseases

A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sjögren's syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.

Effect of Targeted Mutation in Collagen V α2 Gene on Development of Cutaneous Hyperplasia in Tight Skin Mice

Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.

Serum antielastase deficiency in tight-skin mice with genetic emphysema

The tight-skin (Tsk) mouse is a model of genetically determined emphysema. The cause for the development of the lung lesion is unknown. In the present study we investigated the lung morphometry and the serum elastase inhibitory capacity (EIC) of Tsk mice. Mean interalveolar distance was significantly greater (+60%) in Tsk mice than in C57 B1 6J , NMRI, and Balb c mice, which have similar values. Serum of Tsk mice against mouse leukocyte elastase (MLE) has significantly lower EIC values than that of NMRI, Balb c (−64%), and C57 B1 6J (−50%) mice. Similar results were obtained when porcine pancreatic elastase (PPE) was used. Against human leukocyte elastase (HLE), however, there was no difference among the strains, all of which had high EIC values. Preincubation of mouse (C57 B1 6J ) serum with chloramine-T (CT) resulted in an almost complete inhibition of EIC against MLE and PPE but only in a 20% inhibition against HLE using a synthetic substrate. Using elastin Congo Red as substrate, CT inhibited EIC against MLE and PPE by approximately 70% but did not affect the EIC against HLE. These results indicate that (1) the Tsk mouse can be considered a model of severe inborn deficiency of serum antielastase activity which is associated with emphysema; and (2) MLE and PPE can be considered interchangeable in studies of serum EIC in the mouse. On the other hand, the differences between MLE and HLE preclude the use of HLE for EIC determination in this species.

Mast Cells and Their Degranulation in the Tsk Mouse Model of Scleroderma

The Tsk mouse is a genetically transmitted example of cutaneous fibrosis which has been compared with human scleroderma. During a systematic histopathological study of the Tsk mouse, both an increased number and an increased proportion of degranulated mast cells were observed. The consistent association of mast cells and fibrosis in scleroderma, graft-vs-host reactions (GVHR), and now the Tsk mouse raises the question of a pathogenetic role for mast cells in fibrotic disorders in general.