Endoglin haploinsufficiency is associated with differential regulation of extracellular matrix production during skin fibrosis and cartilage repair in mice.

Transforming growth factor (TGF)-ß is a multifunctional growth factor with potent pro-fibrotic effects. Endoglin is a TGF-ß co-receptor that strongly regulates TGF-ß signaling in a variety of cell types. Although aberrant regulation of TGF-ß signaling is known to play a key role in fibrotic diseases such as scleroderma and impaired cartilage repair, the significance of endoglin function in regulating these processes is poorly understood. Here we examined whether endoglin haploinsufficiency regulates extracellular (ECM) protein expression and fibrotic responses during bleomycin induced skin fibrosis and surgically induced osteoarthritis, using endoglin-heterozygous (Eng+/-) mice and wild-type (Eng+/+) littermates. Skin fibrosis was induced by injecting mice intradermally with bleomycin or vehicle. Osteoarthritis was induced surgically by destabilization of medial meniscus. Dermal thickness, cartilage integrity and ECM protein expression were then determined. Eng+/- mice subjected to bleomycin challenge show a marked decrease in dermal thickness (P < 0.005) and reduced collagen content and decreased collagen I, fibronectin, alpha-smooth muscle actin levels as compared to Eng+/+ mice, both under basal and bleomycin treated conditions. Eng+/- mice undergoing surgically induced osteoarthritis show no differences in the degree of cartilage degradation, as compared to Eng+/+ mice, although chondrocytes isolated from Eng+/- display markedly enhanced collagen II levels. Our findings suggest that endoglin haploinsufficiency in mice ameliorates bleomycin-induced skin fibrosis suggesting that endoglin represents a pro-fibrotic factor in the mouse skin. However, endoglin haploinsufficiency does not protect these mice from surgically indiced cartilage degradation, demonstrating differential regulation of endoglin action during skin and cartilage repair.

Corrigendum: ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis.

This corrects the article DOI: 10.1038/nm.4419.

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-ß1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-ß1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10-sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

PPARγ deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage.

OBJECTIVES: We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. METHODS: Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. RESULTS: Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. SIGNIFICANCE: PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis.

OBJECTIVES: Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo. METHODS: Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling. RESULTS: mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage. CONCLUSIONS: This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

Adult cartilage-specific peroxisome proliferator-activated receptor gamma knockout mice exhibit the spontaneous osteoarthritis phenotype.

Osteoarthritis (OA) is an age-related progressive degenerative joint disease. Peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is suggested as an attractive therapeutic target to counteract degradative mechanisms associated with OA. Studies suggest that activation of PPARγ by its agonists can reduce the synthesis of OA catabolic and inflammatory factors and the development of cartilage lesions in OA animal models. Because these agonists impart several PPARγ-independent effects, the specific in vivo function of PPARγ in cartilage homeostasis and OA remains largely unknown. Herein, we describe the in vivo role of PPARγ in OA using cartilage-specific PPARγ knockout (KO) mice generated using the Cre-lox system. Adult PPARγ KO mice exhibited a spontaneous OA phenotype associated with enhanced cartilage degradation, hypocellularity, synovial and cartilage fibrosis, synovial inflammation, mononuclear cell influx in the synovium, and increased expression of catabolic factors, including matrix metalloproteinase-13, accompanied by an increase in staining for matrix metalloproteinase-generated aggrecan and type II collagen neoepitopes (VDIPEN and C1-2C). We demonstrate that PPARγ-deficient articular cartilage exhibits elevated expression of the additional catabolic factors hypoxia-inducible factor-2α, syndecan-4, and a disintegrin and metalloproteinase with thrombospondin motifs 5 and of the inflammatory factors cyclooxygenase-2 and inducible nitric oxide synthase. In conclusion, PPARγ is a critical regulator of cartilage health, the lack of which leads to an accelerated spontaneous OA phenotype.

Association of cartilage-specific deletion of peroxisome proliferator-activated receptor γ with abnormal endochondral ossification and impaired cartilage growth and development in a murine model.

OBJECTIVE: Long bones develop through the strictly regulated process of endochondral ossification within the growth plate, resulting in the replacement of cartilage by bone. Defects in this process can result in skeletal abnormalities and a predisposition to degenerative joint diseases such as osteoarthritis (OA). Studies suggest that activation of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is an important therapeutic target in OA. To devise PPARγ-related therapies in OA, it is critical to identify the role of this transcription factor in cartilage biology. Therefore, this study sought to determine the in vivo role of PPARγ in endochondral ossification and cartilage development, using cartilage-specific PPARγ-knockout (KO) mice. METHODS: Cartilage-specific PPARγ-KO mice were generated using the Cre/loxP system. Histomorphometric and immunohistochemical analyses were performed to assess the patterns of ossification, proliferation, differentiation, and hypertrophy of chondrocytes, skeletal organization, bone density, and calcium deposition in the KO mice. RESULTS: PPARγ-KO mice exhibited reductions in body length, body weight, length of the long bones, skeletal growth, cellularity, bone density, calcium deposition, and trabecular bone thickness, abnormal organization of the growth plate, loss of columnar organization, shorter hypertrophic zones, and delayed primary and secondary ossification. Immunohistochemical analyses for Sox9, 5-bromo-2'-deoxyuridine, p57, type X collagen, and platelet endothelial cell adhesion molecule 1 revealed reductions in the differentiation, proliferation, and hypertrophy of chondrocytes and in vascularization of the growth plate in mutant mice. Isolated chondrocytes and cartilage explants from mutant mice showed aberrant expression of Sox9 and extracellular matrix markers, including aggrecan, type II collagen, and matrix metalloproteinase 13. In addition, chondrocytes from mutant mice exhibited enhanced phosphorylation of p38 and decreased expression of Indian hedgehog. CONCLUSION: The presence of PPARγ is required for normal endochondral ossification and cartilage development in vivo.