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Overexpression of the nucleotide-binding leucine-rich repeat protein 3 (NLRP3) inflammasome in chronic auto-immune diseases leads to skeletal anomalies, with severe osteopenia due to the activation of osteoclasts. Reproducing this phenotype in Nlrp3 knock-in mice has provided insights into the role of NLRP3 in bone metabolism. We studied the role of NLRP3 in physiological bone development using a complete Nlrp3 knock-out mouse model. We found impaired skeletal development in Nlrp3-/- mice, resulting in a shorter stature than that of Nlrp3+/+ mice. These growth defects were associated with altered femur bone growth, characterized by a deficient growth plate and an osteopenic profile of the trabeculae. No differences in osteoclast recruitment or activity were observed. Instead, Nlrp3-/- femurs showed a less mineralized matrix in the trabeculae than those of Nlrp3+/+ mice, as well as less bone sialoprotein (BSP) expressing hypertrophic chondrocytes. In vitro, primary osteoblasts lacking NLRP3 expression showed defective mineralization, together with the downregulation of BSP expression. Finally, follow-up by micro-CT highlighted the role of NLPR3 in bone growth, occurring early in living mice, as the osteopenic phenotype diminishes over time. Overall, our data suggest that NLRP3 is involved in bone edification via the regulation of hypertrophic chondrocyte maturation and osteoblast activity. Furthermore, the defect appeared to be transitory, as the skeleton recovered with aging.

International audience; BACKGROUND: Markers of exposure to environmental toxicants are urgently needed. Tooth enamel, with its unique properties, is able to record certain environmental conditions during its formation. Enamel formation and quality are dependent on hormonal regulation and environmental conditions, including exposure to endocrine disrupting chemicals (EDCs). Among EDCs, phthalates such as di-(2-ethylhexyl) phthalate (DEHP) raise concerns about their contribution to various pathologies, including those of mineralized tissues. OBJECTIVES: The effects of exposure to low-doses of DEHP on the continually growing incisors were analyzed in mouse males and females. METHODS: Adult male and female C57BL/6J mice were exposed daily to 0.5, 5, and 50 lg=kg per day DEHP for 12 wk and their incisors clinically examined. Incisors of males were further analyzed by scanning electron microscopy (SEM), micro X-ray computed tomography (micro-computed tomography; lCT), and nanoindentation for the enamel, histology and real-time quantitative polymerase chain reaction (RT-qPCR) for the dental epithelium. RESULTS: Clinical macroscopic observations of incisors showed various dose-dependent dental lesions such as opacities, scratches, and enamel breakdown in 30.5% of males (10 of 34 total incisors across three independent experiments), and 15.6% of females (7 of 46 incisors) at the highest dose, among which 18.1% (6 of 34 total incisors across three independent experiments) and 8.9% (4 of 46 incisors), respectively, had broken incisors. SEM showed an altered enamel surface and ultrastructure in DEHP-exposed male mice. Further characterization of the enamel defects in males by lCT showed a lower mineral density than controls, and nanoindentation showed a lower enamel hardness during all stages of enamel mineralization, with more pronounced alterations in the external part of the enamel. A delay in enamel mineralization was shown by several approaches (lCT, histology, and RT-qPCR). DISCUSSION: We conclude that DEHP disrupted enamel development in mice by directly acting on dental cells with higher prevalence and severity in males than in females. The time window of DEHP effects on mouse tooth development led to typical alterations of structural, biochemical, and mechanical properties of enamel comparable to other EDCs, such as bisphenol A. The future characterization of dental defects in humans and animals due to environmental toxicants might be helpful in proposing them as early markers of exposure to such molecules.

Description, A new therapeutic approach for the treatment of a skeletal disorder with AAVs bypasses bone resistance to gene transfer., Adeno-associated virus (AAV) vectors are a well-established gene transfer approach for rare genetic diseases. Nonetheless, some tissues, such as bone, remain refractory to AAV. X-linked hypophosphatemia (XLH) is a rare skeletal disorder associated with increased levels of fibroblast growth factor 23 (FGF23), resulting in skeletal deformities and short stature. The conventional treatment for XLH, lifelong phosphate and active vitamin D analogs supplementation, partially improves quality of life and is associated with severe long-term side effects. Recently, a monoclonal antibody against FGF23 has been approved for XLH but remains a high-cost lifelong therapy. We developed a liver-targeting AAV vector to inhibit FGF23 signaling. We showed that hepatic expression of the C-terminal tail of FGF23 corrected skeletal manifestations and osteomalacia in a XLH mouse model. Our data provide proof of concept for AAV gene transfer to treat XLH, a prototypical bone disease, further expanding the use of this modality to treat skeletal disorders.

The Baka pygmies are known for their short stature resulting from a reduced growth rate during infancy. They are peculiar also for their teeth erupt earlier than in any other African population, and their posterior dentition is larger than in non-pygmies. However, the Baka’s dental morphology, as several other aspects of their biology, is still understudied. In this study we explore the Baka’s upper and lower second deciduous molars variation in comparison to a human sample including Africans, Asians, Europeans, and South Americans by means of 3D geometric morphometrics. We analyzed both the outer and inner aspect of the dental crowns, as well as their general size based on the natural logarithm of centroid sizes. Our results show largely overlapping morphologies between populations, especially for the lower molars. The distal region of the second molars is the most variable, with the Baka’s lower second molars showing the most extreme range of variation for this aspect and in terms of crown height. Upper and lower second molars showed high morphological covariation (RV=0.82). The Baka and South Americans’ second molars were confirmed among the largest in our sample. Despite of the Baka’s particular growth pattern, long-lasting isolation and extreme dental variation, it is not possible to distinguish them from other populations on the basis of their second molar morphology only.

International audience; Blood perfusion of grafted tissue constructs is a hindrance to the success of stem cell-based therapies by limiting cell survival and tissue regeneration. Implantation of a pre-vascularized network engineered in vitro has thus emerged as a promising strategy for promoting blood supply deep into the construct, relying on inosculation with the host vasculature. We aimed to fabricate in vitro tissue constructs with mature microvascular networks, displaying perivascular recruitment and basement membrane, taking advantage of the angiogenic properties of dental pulp stem cells and self-assembly of endothelial cells into capillaries. Using digital scanned light-sheet microscopy, we characterized the generation of dense microvascular networks in collagen hydrogels and established parameters for quantification of perivascular recruitment. We also performed original time-lapse analysis of stem cell recruitment. These experiments demonstrated that perivascular recruitment of dental pulp stem cells is driven by PDGF-BB. Recruited stem cells participated in deposition of vascular basement membrane and vessel maturation. Mature microvascular networks thus generated were then compared to those lacking perivascular coverage generated using stem cell conditioned medium. Implantation in athymic nude mice demonstrated that in vitro maturation of microvascular networks improved blood perfusion and cell survival within the construct. Taken together, these data demonstrate the strong potential of in vitro production of mature microvasculature for improving cell-based therapies.

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The Baka Pygmies are known for their short stature resulting from a reduced growth rate during infancy. They are peculiar also for their teeth erupt earlier than in any other African population, and their posterior dentition is larger than in non-Pygmy populations. However, the Baka’s dental morphology, like several other aspects of their biology, is still understudied. Here, we explore the variation of the Baka’s deciduous upper and lower second molars (dm2s) in comparison to a geographically heterogeneous human sample by means of 3D geometric morphometrics and analysis of dental traits. Our results show that the different populations largely overlap based on the shape of their dm2s, especially the lower ones. Their distal region and the height of the dentinal crown differ the most, with the Baka showing the most extreme range of variation. Upper and lower dm2s covary to a great extent (RV=0.82). The Baka’s and South Americans’ dm2s were confirmed among the largest in our sample. Despite the Baka’s unique growth pattern, long-lasting isolation, and extreme dental variation, it is not possible to distinguish them from other populations based on their dm2s’ morphology only.

ObjectiveTo evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).MethodsIVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.ResultsIVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF.ConclusionWe demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements.

International audience; INTRODUCTION:Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biological functions, the contribution of endothelial TXNIP has not been well-defined in regards to endothelial and vascular function or in post-ischemic revascularisation. We postulated that inhibition of endothelial TXNIP with siRNA or in a Cre-LoxP system could be involved in protection from high fat, high protein, low carbohydrate (HFHPLC) diet-induced oxidative stress and endothelial dysfunction, leading to vascular damage and impaired revascularisation in vivo.METHODS AND RESULTS:To investigate the role of endothelial TXNIP, the TXNIP gene was deleted in endothelial cells using anti-TXNIP siRNA treatment or the Cre-LoxP system. Murine models were fed a HFHPLC diet, known to induce metabolic disorders. Endothelial TXNIP targeting resulted in protection against metabolic disorder-related endothelial oxidative stress and endothelial dysfunction. This protective effect mitigates media cell loss induced by metabolic disorders and hampered metabolic disorder-related vascular dysfunction assessed by aortic reactivity and distensibility. In aortic ring cultures, metabolic disorders impaired vessel sprouting and this alteration was alleviated by deletion of endothelial TXNIP. When subjected to ischemia, mice fed a HFHPLC diet exhibited defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. However, reducing endothelial TXNIP rescued metabolic disorder-related impairment of ischemia-induced revascularisation.CONCLUSION:Collectively, these results show that targeting endothelial TXNIP in metabolic disorders is essential to maintaining endothelial function, vascular function and improving ischemia-induced revascularisation, making TXNIP a potential therapeutic target for therapy of vascular complications related to metabolic disorders.

International audience; Background:Genetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands.Methods:We describe the generation of a CF rat model (F508del) homozygous for the p.Phe508del mutation in the transmembrane conductance regulator (Cftr) gene. This model was compared to new Cftr -/- rats (CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro-computed tomography. The activity of CFTR was evaluated by nasal potential difference (NPD) and short-circuit current measurements. The effect of VX-809 and VX-770 was analyzed on nasal epithelial primary cell cultures from F508del rats.Results:Both newborn F508del and Knock out (KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short-circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508del rats. Exposure of F508del CFTR nasal primary cultures to a combination of VX-809 and VX-770 improved CFTR-mediated Cl- transport.Conclusions:The F508del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics.

Nowadays, the preservation of dental pulp vitality is an integral part of our daily therapies. The success of these treatments depends on the clinical situation as well as the biomaterials used. Mineral Trioxide aggregate and BiodentineTM are commonly used as pulp capping materials. One objective of vital pulp therapy is the repair/regeneration of the pulp. In addition to the initial inflammatory status of the pulp, the nature and quality of the new mineralized tissue obtained after pulp capping directly influence the success of the treatment. In order to characterize the reparative dentin, in the current study, the chemical composition and microstructure of the dentin bridge after direct pulp capping using Biodentine&trade, and mineral trioxide aggregate (MTA) was studied by using Raman microspectroscopy and scanning electron microscopy, respectively. The results showed that the reparative dentin bridge observed in both groups presented dentin tubules and chemical composition similar to primary dentin. With the limitations of this study, the calcium-silicate-based cements used as pulp capping materials provide an optimal environment for pulp healing, resulting in a reparative dentin resembling on certain points of the primary dentin and the regeneration of the pulp.

FASL (CD178) is known for its role in triggering apoptosis, mostly in relation with immune cells but additional functions have been reported more recently, including those in bone development. Examination of postnatal FasL-deficient mice (gld) showed an increased bone deposition in adult mice when compared with wild types. However, a different phenotype was observed prenatally, when the gld bone was underdeveloped. The aim of the following investigation was to evaluate this indication for an growth-dependent bone phenotype of gld mice and to search for the 'switch point'. This study focused on the mandibular/alveolar bone as an important structure for tooth anchorage. In vivo micro-computed tomography (CT) analysis was performed at different stages during the first month (6, 12 and 24 days) of postnatal bone development. In 6-day-old gld mice, a decrease in bone volume/tissue volume (BV/TV), trabecular thickness and trabecular number was revealed. In contrast, the 12-day-old gld mice showed an increased BV/TV and trabecular thickness in the alveolar bone. The same observation applied for bone status in 24-day-old gld mice. Therefore, changes in the bone phenotype occurred between day 6 and 12 of the postnatal development. The switch point is likely related to the changing proportion of bone cells at these stages of development, when the number of osteocytes increases. Indeed, the immunohistochemical analysis of FASL localized this protein in osteoblasts, whereas osteocytes were mostly negative at examined stages. The impact of FASL particularly on osteoblasts would agree with an earlier in vivo observed effect of FASL deficiency on expression of Mmp2, typical for osteoblasts, in the gld mandibular/alveolar bone. Notably, an age-dependent bone phenotype was reported in Mmp2-deficient mice.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue-engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor-2 (FGF-2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF-2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF-2 priming. After implantation in immunodeficient mice, the tissue-engineered constructs seeded with FGF-2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF-2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844–857

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (μCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease characterized by fibroblast proliferation, extracellular matrix deposition, destruction of pulmonary alveolar architecture and vascular remodeling. Apart pirfenidone or nintendanib that only slow down the fibrotic process, there is no curative treatment other than lung transplantation. Because cell therapy approaches have been proposed in IPF, we hypothesized that injection of endothelial colony-forming cells (ECFCs), the vasculogenic subtype of endothelial progenitor cells, could modulate fibrosis in a Nude mouse model of bleomycin induced-pulmonary fibrosis. Mice were injected with ECFCs isolated from cord blood and from peripheral blood of adult IPF patients at two time-points: during the development of the fibrosis or once the fibrosis was constituted. We assessed morbidity, weight variation, collagen deposition, lung imaging by microCT, Fulton score and microvascular density. Neither ECFCs isolated from cord blood nor from IPF patients were able to modulate fibrosis or vascular density during fibrogenesis or when fibrosis was constituted. These findings indicate that human ECFCs do not promote an adaptive regenerative response in the lung upon fibrosis or angiogenic process in the setting of bleomycin-induced pulmonary fibrosis in Nude mice.

FasL is a well-known actor in the apoptotic pathways but recent reports have pointed to its important novel roles beyond cell death, as observed also for bone cells. This is supported by non-apoptotic appearance of FasL during osteogenesis and by significant bone alterations unrelated to apoptosis in FasL deficient (gld) mice. The molecular mechanism behind this novel role has not yet been revealed. In this report, intramembranous bone, where osteoblasts differentiate directly from mesenchymal precursors without intermediary chondrogenic step, was investigated. Mouse mandibular bone surrounding the first lower molar was used as a model. The stage where a complex set of bone cells (osteoblasts, osteocytes, osteoclasts) is first present during development was selected for an initial examination. Immunohistochemical staining detected FasL in non-apoptotic cells at this stage. Further, FasL deficient vs. wild type samples subjected to osteogenic PCR Array analysis displayed a significantly decreased expression of Mmp2 in gld bone. To examine the possibility of this novel FasL-Mmp2 relationship, intramembranous bone-derived osteoblastic cells (MC3T3-E1) were treated with anti-FasL antibody or rmFasL. Indeed, the FasL neutralization caused a decreased expression of Mmp2 and rmFasL added to the cells resulted in the opposite effect. Since Mmp2-/- mice display age-dependent alterations in the intramembranous bone, early stages of gld mandibular bone were examined and age-dependent phenotype was was confirmed also in gld mice. Taken together, the present in vivo and in vitro findings point to a new non-apoptotic function of FasL in bone development associated with Mmp2 expression.

Background We aimed to investigate the efficacy of abatacept in preclinical mouse models of digestive involvement, pulmonary fibrosis, and related pulmonary hypertension (PH), mimicking internal organ involvement in systemic sclerosis (SSc). Methods Abatacept has been evaluated in the chronic graft-versus-host disease (cGvHD) mouse model (abatacept 1 mg/mL for 6 weeks), characterized by liver and intestinal fibrosis and in the Fra-2 mouse model (1 mg/mL or 10 mg/mL for 4 weeks), characterized by interstitial lung disease (ILD) and pulmonary vascular remodeling leading to PH. Results In the cGvHD model, abatacept significantly decreased liver transaminase levels and markedly improved colon inflammation. In the Fra-2 model, abatacept alleviated ILD, with a significant reduction in lung density on chest microcomputed tomography (CT), fibrosis histological score, and lung biochemical markers. Moreover, abatacept reversed PH in Fra-2 mice by improving vessel remodeling and related cardiac hemodynamic impairment. Abatacept significantly reduced fibrogenic marker levels, T-cell proliferation, and M1/M2 macrophage infiltration in lesional lungs of Fra-2 mice. Conclusion Abatacept improves digestive involvement, prevents lung fibrosis, and attenuates PH. These findings suggest that abatacept might be an appealing therapeutic approach beyond skin fibrosis for organ involvement in SSc. Electronic supplementary material The online version of this article (10.1186/s13075-018-1694-9) contains supplementary material, which is available to authorized users.

Background Cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) is an immunoregulatory membrane receptor resulting in the down-regulation of T-cell responses. A previous report showed that abatacept (CTLA4-Ig) prevented and induced regression of inflammation-driven dermal fibrosis in two different mouse models of systemic sclerosis (SSc). 1 Objectives We aimed to assess the effects of abatacept in two complementary mouse models reflecting digestive involvement, lung fibrosis and pulmonary hypertension (PH), mimicking severe SSc organ damage. Methods Abatacept was given in the chronic graft-versus-host disease (cGvHD) mouse model, characterised by digestive involvement, and in the Fra-2 mouse model which is characterised by non-specific interstitial pneumonia and pulmonary vascular remodelling leading to PH. Mice were treated by intraperitoneal injections of abatacept (1 mg/mL in the cGvHD model for 6 weeks; 1 mg/mL or 10 mg/mL in the Fra-2 mouse model for 4 weeks) or human IgG1 (100 mg) used as a negative control. Results In the cGvHD model, treatment of allogeneically mice with abatacept led to a significant reduction of alanine aminotransferase (24%, p=0.014) and aspartate aminotransferase levels (61%, p When assessed by chest micro-CT imaging, Fra-2 mice treated with abatacept displayed a 12% decrease in lung density (10 mg/ml, p=0.037) as well as an increase in functional residual capacity as compared to IgG1-treated mice (16% for 1 mg/ml, p=0.001% and 14% for 10 mg/ml, p=0.005). Consistent with these results, abatacept 10 mg/L decreased histological fibrosis score (Ashcroft score) as well as hydroxyproline content by 79% (p=0.009) and 31% (p=0.044) respectively, as compared to IgG1-treated mice. Treatment with abatacept 10 mg/mL markedly reduced protein levels in the lesional lungs of Fra-2 mice of the fibrogenic markers MCP1 by 79% (p=0.043) and osteopontin by 87% (p=0.039). Levels of TGF-b were also reduced with abatacept (61% for 1 mg/mL, p=0.037% and 69% for 10 mg/mL, p=0.013). Further, abatacept decreased M1 and M2 macrophages infiltration as well as T-cell proliferation in the lesional lungs of Fra-2 mice. Upon treatment with abatacept a reduction of right ventricular systolic pressure (28.1±1.5 mmHg vs 36.0±5.1 mmHg, p=0.037 for 10 mg/mL) and right ventricular hypertrophy (0.29±0.01 vs 0.33±0.010, p=0.037 and 29±0.01% vs 0.33±0.01% for 10 mg/mL, p=0.037) was observed compared to IgG1-treated mice. Consistent with these findings, abatacept 10 mg/mL was associated with significant decrease in percent medial wall thickness and numbers of muscularized distal pulmonary arteries. Conclusions We demonstrate that treatment with abatacept improves digestive involvement, prevents lung fibrosis and attenuates PH in SSc pre-clinical mice models. These findings suggest that abatacept might be an appealing therapeutic approach for severe internal organ involvement in SSc beyond its already demonstrated effects on skin fibrosis. Reference [1] Ponsoye M, et al. Ann Rheum Dis2016Dec;75(12):2142–9. Disclosure of Interest None declared

CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.

Wnt proteins are lipid-modified, short-range signals that control stem cell self-renewal and tissue regeneration. We identified a population of Wnt responsive cells in the pulp cavity, characterized their function, and then created a pulp injury. The repair response was evaluated over time using molecular, cellular, and quantitative assays. We tested how healing was impacted by wound environments in which Wnt signaling was amplified. We found that a Wnt-amplified environment was associated with superior pulp healing. Although cell death was still rampant, the number of cells undergoing apoptosis was significantly reduced. This resulted in significantly better survival of injured pulp cells, and resulted in the formation of more tertiary dentin. We engineered a liposome-reconstituted form of WNT3A then tested whether this biomimetic compound could activate cells in the injured tooth pulp and stimulate dentin regeneration. Pulp cells responded to the elevated Wnt stimulus by differentiating into secretory odontoblasts. Thus, transiently amplifying the body's natural Wnt response resulted in improved pulp vitality. These data have direct clinical implications for treating dental caries, the most prevalent disease affecting mankind. © 2015 American Society for Bone and Mineral Research.

X-linked hypophosphatemia (XLH) is a dento-osseous disorder caused by inactivating mutations in the PHEX gene, leading to renal phosphate wasting and hypophosphatemia, and impaired mineralization of bones and teeth. In the oral cavity, recent reports suggest a higher susceptibility of XLH patients to periodontitis, where patients present with impaired tooth cementum - a bone-like tissue involved in tooth attachment to the jaw bones and post-eruption tooth positioning - and a higher frequency of intrabony defects. In the present study, the pathobiology of alveolar bone and tooth cementum was investigated in the Hyp mouse, the murine analog of XLH. PHEX deficiency in XLH/Hyp dramatically alters the periodontal phenotype, with hypoplasia of tooth root cementum associated with a lack of periodontal ligament attachment and the presence of an immature apatitic mineral phase of all periodontal mineralized tissues. Challenging the Hyp periodontium in two surgical experimental models - ligature-induced periodontal breakdown and repair, and a model of tooth movement adaptation inducing cementum formation - we show that bone and cementum formation, and their healing, are altered. Bone and cementum mineralization appear similarly disturbed, where hypomineralized pericellular matrix surrounds cells, and where the protein osteopontin (OPN, a mineralization inhibitor) accumulates in a tissue-specific manner, most notably in the perilacunar matrix surrounding osteocytes. Although the pathobiology is different between XLH/Hyp bone and cementum, our results show a major XLH phenotype in oral mineralized tissues consistent with variations in patient susceptibility to periodontal disorders.

Background Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors known to modulate fibrosis. The pan-PPAR agonist IVA337 recently demonstrated efficacy in prevention and treatment of experimental skin fibrosis (1). Objectives Our objective was to evaluate the antifibrotic effects of IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH). Methods IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterized by non-specific interstitial pneumonia and severe vascular remodeling of pulmonary arteries leading to PH. Mice received 2 doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. Results Both 30 mg/kg and 100 mg/kg doses of IVA337 were well tolerated in all mouse models. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis induced by bleomycin compared to mice receiving 30 mg/kg of IVA337 or vehicle. Indeed, IVA337 (100 mg/kg) strongly reduced by 61% and 28% tissue density on histological measurements and total lung hydroxyproline concentrations, respectively, as compared to vehicle. IVA337 at 100 mg/kg also significantly decreased col1, col3 and fibronectin in lesional lungs. Similarly, Fra-2 transgenic mice treated with 100 mg/kg of IVA337 displayed reduced lung density (20% vs. vehicle) and significant increase of functional residual capacity (30% vs. vehicle) when assessed by chest micro-CT imaging. These results were emphasized by a 50% reduction of the Ascroft fibrosis score (Figure 1A) and by a 48% reduction of hydroxyproline concentrations upon IVA337 (100 mg/kg) compared to vehicle treated mice. Successful targeting of the TGF-b signaling axis was observed in both mouse models upon treatment with 100 mg/kg of IVA337. 100 mg/kg IVA337 also significantly reduced T cell and B cell infiltration in lesional lungs of Fra-2 transgenic mice. Regarding vessel remodeling and related pulmonary hypertension, treatment with 100 mg/kg of IVA337 led to a substantial attenuation of right ventricular systolic pressure and right ventricular hypertrophy compared to mice receiving the vehicle (Figure 1B and 1C). Furthermore, IVA337 given at 100 mg/kg markedly reduced medial wall thickness (Figure 1D) and the number of muscularized distal pulmonary arteries. In vitro in primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner TGFβ-mediated fibroblasts to myofibroblasts transition and PDGF-mediated proliferation. Conclusions We demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for systemic sclerosis both, for skin and key cardiovascular complications. References Ruzehaji et al, Ann Rheum Dis 2016;75(12):2175–2183. Disclosure of Interest J. Avouac: None declared, I. Konstantinova Employee of: INVENTIVA, C. Guignabert: None declared, J. Sadoine: None declared, G. Thomas: None declared, S. Pezet: None declared, A. Cauvet: None declared, L. Tu: None declared, J. M. Luccarini Employee of: INVENTIVA, J. L. Junien Consultant for: INVENTIVA, P. Broqua Employee of: INVENTIVA, Y. Allanore Grant/research support from: Inventiva

Introduction La fibrose pulmonaire idiopathique (FPI) est une pathologie pulmonaire severe caracterisee par une proliferation de fibroblastes, un depot de matrice extracellulaire entrainant la destruction de l’architecture alveolaire pulmonaire et un important remodelage vasculaire. A l’exception de la pirfenidone et du nintedanib qui ralentissent le processus fibrotique, le seul traitement curatif est la transplantation pulmonaire. Les therapies cellulaires ayant deja ete envisagees dans la FPI, nous avons suppose que l’injection d’« Endothelial colony-forming cells » (ECFCs), le sous-type vasculogene des progeniteurs endothelio-circulants, pourrait moduler la fibrose dans un modele de fibrose pulmonaire induite par injection intratracheale de bleomycine chez la souris immunodeprimee Nude. Methodes Apres administration intratracheale de bleomycine, les souris Nude ont recu une injection intraveineuse (IV) d’ECFCs isoles de sang de cordon (CB-ECFC) ou de prelevements sanguins de patients FPI (FPI-ECFCs) a deux stades differents : durant la periode de developpement de la fibrose et une fois cette derniere constituee. Nous avons evalue la morbidite, la variation de poids, les depots de collagene, le score de Fulton, la fibrose pulmonaire par TDM et histomorphometrie et la densite microvasculaire. Nous avons egalement recherche la presence des ECFCs dans les poumons de souris par RT-qPCR specifique de l’ARN humain ou par hybridation in situ. Resultats Ni les CB-ECFCs ni les IPF-ECFCs ne modulent la fibrose ou la densite vasculaire que ce soit au cours de la fibrogenese ou une fois la fibrose constituee. De plus, les ECFCs ne sont pas detectables au sein du tissu pulmonaire 10 a 15 jours apres leur injection. Conclusion Ces resultats montrent que l’injection ECFCs humaines n’entraine pas de reponse adaptative regerative que ce soit au niveau de la fibrose ou de la vascularisation pulmonaire dans le modele de fibrose pulmonaire induite par la bleomycine chez la souris Nude.

The 'Competing interests' statement of this Article has been updated; see accompanying Amendment for further details.

To elucidate the role of gene candidates involved in pulmonary hypertension (PH) associated with systemic sclerosis (SSc).Gene candidates were identified through microarray experiments performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in endothelial progenitor cell (EPC)-derived endothelial cells (ECs) obtained from patients with SSc-associated PH, patients with SSc without PH, and healthy control subjects. Expression of identified gene candidates was assessed by quantitative sandwich enzyme-linked immunosorbent assay in the serum, and by immunohistochemistry in lesional lung tissue. The functional importance of the identified gene candidates was then evaluated in fos-related antigen 2-transgenic (Fra-2-Tg) mice that spontaneously develop SSc-like features associated with an intense pulmonary vascular remodeling.Microarray experiments revealed that the matrix metalloproteinase 10 (MMP-10) gene was the top up-regulated gene in SSc-associated PH EPC-derived ECs. Circulating serum proMMP10 concentrations were markedly increased in patients with SSc-associated PH compared to SSc patients without PH and healthy controls. Consistent with these observations, a strong MMP10 staining of the thickened wall of distal pulmonary arteries was found both in the lungs of patients with SSc-associated PH and in the lungs of Fra-2-Tg mice. Daily treatment of Fra-2-Tg mice with neutralizing anti-MMP10 antibodies did not significantly affect the development and severity of pulmonary fibrosis, but did reverse established PH and markedly reduced pulmonary vascular remodeling by reducing cell proliferation, cell survival, and the platelet-derived growth factor signaling axis.Gene expression profiling of EPC-derived ECs identified MMP10 as a novel candidate gene in SSc-associated PH. MMP10 is overexpressed in the serum and pulmonary arteries of patients with SSc-associated PH, and its blockade alleviates PH in the Fra-2-Tg mouse model. MMP10 appears to be a prospective treatment target for this devastating disorder.

The authors would like to thank all the people who contributed to the ovariectomy procedures, the diet delivery, and the tissue sampling. They also thank Vincent Poitout and AMGEN for providing them the GPR40 mouse strain. They also express gratitude to Lisa Nucke for English grammar and syntax improvement; International audience; SCOPE: The stimulation of the free fatty acid receptor G-protein coupled receptor (GPR) 40 by GW9508 prevents bone loss by inhibiting osteoclast activity, both in vitro and in vivo. Here, we questioned whether the stimulation of the GPR40 receptor by dietary fatty acids may lead to the same beneficial effect on bone.METHODS AND RESULTS: We investigated (i) the impact of a fatty acid enriched diet (high-fat diet [HFD]) on bone health in C57/BL6 female mice depending on (ii) the estrogen status (ovariectomy) and (iii) the genotype (GPR40(+/+) or GPR40(-/-) ). Bone mineral density (BMD), body composition, weight, inflammation and bone remodeling parameters were monitored. HFD decreased BMD in HFD-SH-GPR40(+/+) mice but OVX failed to further impact BMD in HFD-OVX-GPR40(+/+) mice, while additional bone loss was observed in HFD-OVX-GPR40(-/-) animals. These data suggest that when stimulated by fatty acid enriched diets GPR40 contributes to counteract ovariectomy-induced bone alteration. The sparing effect is supported by the modulation of both the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio in blood stream and the expression level of inflammatory markers in adipose tissues. Bone preservation by GPR40 stimulation is dependent on the presence of long-chain saturated fatty acids.CONCLUSION: GPR40 contributes to counter ovariectomy-induced bone loss in a context of saturated fatty acid enrichment

International audience; Background and aimsThe arterial wall calcium score and circulating free DNA levels are now used in clinical practice as biomarkers of cardiovascular risk. Calcium phosphate apatite retention in the arterial wall necessitates precipitation on an anionic platform. Here, we explore the role of tissue-free DNA as such a platform.MethodsThe first step consisted of histological observation of samples from human and rat calcified arteries. Various stains were used to evaluate colocalization of free DNA with calcified tissue (alizarin red, fluorescent Hoechst, DNA immunostaining and TUNEL assay). Sections were treated by EDTA to reveal calcification background. Secondly, a rat model of vascular calcifications induced by intra-aortic infusions of free DNA and elastase + free DNA was developed. Rat aortas underwent a micro-CT for calcium score calculation at 3 weeks. Rat and human calcifications were qualitatively characterized using μFourier Transform Infrared Spectroscopy (μFTIR) and Field Emission-Scanning Electron Microscopy (FE-SEM).ResultsOur histological study shows colocalization of calcified arterial plaques with free DNA. In the intra-aortic infusion model, free DNA was able to penetrate into the arterial wall and induce calcifications whereas no microscopic calcification was seen in control aortas. The calcification score in the elastase + free DNA group was significantly higher than in the control groups. Qualitative evaluation with μFTIR and FE-SEM demonstrated typical calcium phosphate retention in human and rat arterial specimens.ConclusionsThis translational study demonstrates that free DNA could be involved in arterial calcification formation by precipitating calcium phosphate apatite crystals in the vessel wall.

Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (μCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF., info:eu-repo/semantics/published

Wnt proteins are lipid-modified, short-range signals that control stem cell self-renewal and tissue regeneration. We identified a population of Wnt responsive cells in the pulp cavity, characterized their function, and then created a pulp injury. The repair response was evaluated over time using molecular, cellular, and quantitative assays. We tested how healing was impacted by wound environments in which Wnt signaling was amplified. We found that a Wnt-amplified environment was associated with superior pulp healing. Although cell death was still rampant, the number of cells undergoing apoptosis was significantly reduced. This resulted in significantly better survival of injured pulp cells, and resulted in the formation of more tertiary dentin. We engineered a liposome-reconstituted form of WNT3A then tested whether this biomimetic compound could activate cells in the injured tooth pulp and stimulate dentin regeneration. Pulp cells responded to the elevated Wnt stimulus by differentiating into secretory odontoblasts. Thus, transiently amplifying the body's natural Wnt response resulted in improved pulp vitality. These data have direct clinical implications for treating dental caries, the most prevalent disease affecting mankind.

Background Systemic sclerosis (SSc) is a severe fibrotic disease with no effective treatment. The OX40-OX40L axis is emerging as a new potential target in autoimmune diseases offering the advantage of a targeted approach on co-stimulatory signals with limited impact on the whole immune response and very preliminary data suggested its involvement in SSc. Objectives To investigate the contribution of OX40L in SSc Methods We assessed the expression of OX40L in fibrotic skin and in the serum of SSc-patients. The efficacy of a targeted therapy against OX40L in vivo was evaluated using complementary experimental mouse models. Results Expression of OX40L protein was higher in fibrotic skin of SSc-patients as compared to healthy controls (p=0.003), particularly in diffuse cutaneous forms. Soluble OX40 levels were significantly higher in SSc-patients (n=177) than in healthy controls (n=100). Longitudinal analyses of our cohort showed that high level of serum OX40L at baseline was highly predictive of worsening of dermal and lung fibrosis during the follow-up (HR: 8.28 [2.11–32.50] and 4.60 [1.52–12.18], P Invalidation of OX40L prevented dermal fibrosis in the bleomycin mouse model with a decrease in dermal thickness, hydroxyprolin content and myofibroblasts count. OX40L invalidation was associated with a decrease in T cell, B cell and macrophage infiltrates and in cytokine release (IL-6, TNF-α) in lesional skin of mice. A transcriptomic approach identified two main pathways regulated by OX40L blockade: NF kappa B and AP 1 signaling. Then pharmacologic approaches, using a monoclonal anti-OX40L antibody, demonstrated that the blockade of OX40L not only prevented dermal fibrosis, but also induced regression of established fibrosis. Thereafter, the use of this monoclonal antibody demonstrated in the fra-2 transgenic mouse model marked protection from fibrosing alveolitis (measured by CT-scan (Figure) and histology) and vessel remodeling leading to pulmonary arterial hypertension. Conclusions Altogether, our results show that OX40L is as an attractive target in inflammation-driven fibrosis. OX40L appears as a very promising biomarker in SSc and an appealing potential therapy to reduce both skin and lung fibrosis. Disclosure of Interest None declared