Identification of TGFß signatures in six murine models mimicking different osteoarthritis clinical phenotypes.

OBJECTIVE: TGFß is a key player in cartilage homeostasis and OA pathology. However, few data are available on the role of TGFß signalling in the different OA phenotypes. Here, we analysed the TGFß pathway by transcriptomic analysis in six mouse models of OA. METHOD: We have brought together seven expert laboratories in OA pathophysiology and, used inter-laboratories standard operating procedures and quality controls to increase experimental reproducibility and decrease bias. As none of the available OA models covers the complexity and heterogeneity of the human disease, we used six different murine models of knee OA: from post-traumatic/mechanical models (meniscectomy (MNX), MNX and hypergravity (HG-MNX), MNX and high fat diet (HF-MNX), MNX and seipin knock-out (SP-MNX)) to aging-related OA and inflammatory OA (collagenase-induced OA (CIOA)). Four controls (MNX-sham, young, SP-sham, CIOA-sham) were added. OsteoArthritis Research Society International (OARSI)-based scoring of femoral condyles and ribonucleic acid (RNA) extraction from tibial plateau samples were done by single operators as well as the transcriptomic analysis of the TGFß family pathway by Custom TaqMan® Array Microfluidic Cards. RESULTS: The transcriptomic analysis revealed specific gene signatures in each of the six models; however, no gene was deregulated in all six OA models. Of interest, we found that the combinatorial Gdf5-Cd36-Ltbp4 signature might discriminate distinct subgroups of OA: Cd36 upregulation is a hallmark of MNX-related OA while Gdf5 and Ltbp4 upregulation is related to MNX-induced OA and CIOA. CONCLUSION: These findings stress the OA animal model heterogeneity and the need of caution when extrapolating results from one model to another.

TGFßi is involved in the chondrogenic differentiation of mesenchymal stem cells and is dysregulated in osteoarthritis.

OBJECTIVE: Transforming growth factor-ß (TGFß) is a major regulator of cartilage homeostasis and its deregulation has been associated with osteoarthritis (OA). Deregulation of the TGFß pathway in mesenchymal stem cells (MSCs) has been proposed to be at the onset of OA. Using a secretome analysis, we identified a member of the TGFß family, TGFß-induced protein (TGFßi or ßIGH3), expressed in MSCs and we investigated its function and regulation during OA. DESIGN: Cartilage, bone, synovium, infrapatellar fat pad and bone marrow-MSCs were isolated from patients with OA or healthy subjects. Chondrogenesis of BM-MSCs was induced by TGFß3 in micropellet culture. Expression of TGFßi was quantified by RT-qPCR, ELISA or immunohistochemistry. Role of TGFßi was investigated in gain and loss of function experiments in BM-MSCs and chondrocytes. RESULTS: TGFßi was up-regulated in early stages of chondrogenesis and its knock-down in BM-MSCs resulted in the down-regulation of mature and hypertrophic chondrocyte markers. It likely occurred through the modulation of adhesion molecules including integrin (ITG)ß1, ITGß5 and N-cadherin. We also showed that TGFßi was upregulated in vitro in a model of OA chondrocytes, and its silencing enhanced the hypertrophic marker type X collagen. In addition, TGFßi was up-regulated in bone and cartilage from OA patients while its expression was reduced in BM-MSCs. Similar findings were observed in a murine model of OA. CONCLUSIONS: Our results revealed a dual role of TGFßi during chondrogenesis and pointed its deregulation in OA joint tissues. Modulating TGFßi in BM-MSCs might be of interest in cartilage regenerative medicine.

Lack of anti-inflammatory and anti-catabolic effects on basal inflamed osteoarthritic chondrocytes or synoviocytes by adipose stem cell-conditioned medium.

OBJECTIVE: To define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes. METHODS: OA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed. RESULTS: Most ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors. CONCLUSIONS: These data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.

Native human adipose stromal cells: localization, morphology and phenotype.

OBJECTIVES: Beside having roles in energy homeostasis and endocrine modulation, adipose tissue (AT) is now considered a promising source of mesenchymal stromal cells (adipose-derived stromal cells or ASCs) for regenerative medicine. Despite numerous studies on cultured ASCs, native human ASCs are rarely investigated. Indeed, the phenotype of ASCs in their native state, their localization within AT and comparison with bone marrow-derived mesenchymal stromal cells (BM-MSCs) has been poorly investigated. DESIGN: To address these issues, the stroma vascular fraction (SVF) of human AT was extracted and native cell subtypes were isolated by immunoselection to study their clonogenic potential in culture. Immunohistology on samples of human AT in combination with reconstruction of confocal sections were performed in order to localize ASCs. RESULTS: Compared with BM-MNCs, all native ASCs were found in the CD34(+) cell fraction of the AT-SVF. Native ASCs expressed classical mesenchymal markers described for BM-MSCs. Interestingly, CD34 expression decreased during ASC cell culture and was negatively correlated with cell proliferation rate. Immunohistological analysis revealed that native ASCs exhibited specific morphological features with protrusions. They were found scattered in AT stroma and did not express in vivo pericytic markers such as NG2, CD140b or alpha-smooth muscle actin, which appeared during the culture process. Finally, ASCs spontaneous commitment to adipocytic lineage was enhanced in AT from obese humans. CONCLUSIONS: The use of complementary methodological approaches to study native human ASCs revealed their immunophenotype, their specific morphology, their location within AT and their stemness. Furthermore, our data strongly suggest that human ASCs participate in adipogenesis during AT development.

Validation of a new fluidic device for mechanical stimulation and characterization of microspheres: A first step towards cartilage characterization.

Articular cartilage is made of chondrocytes surrounded by their extracellular matrix that can both sense and respond to various mechanical stimuli. One of the most widely used in vitro model to study cartilage growth is the model of mesenchymal stromal cells-derived cartilage micropellet. However, mechanical stimulation of micropellets has never been reported probably because of their small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of cartilage micropellets. The fluidic-based device was designed for the concomitant stimulation or characterization of six microspheres placed into the conical wells of a tank. In the present study, the device was validated using alginate-, collagen- and crosslinked collagen-based microspheres. Different types and ranges of pressure signals (square, sinusoidal and constant) were applied. The mechanical properties of microspheres were equivalent to those determined by a conventional compression test. Accuracy, repeatability and reproducibility of all types of pressure signals were demonstrated even though square signals were less accurate and sinusoidal signals were less reproducible than the others. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with diameters in the range of 900 to 1500 µm. Mechanical stimulation can be performed on six microspheres in parallel allowing the mechanical and molecular characterization of the same group of cartilage micropellets. The device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli.

Mesenchymal stem cells-derived cartilage micropellets: A relevant in vitro model for biomechanical and mechanobiological studies of cartilage growth.

The prevalence of diseases that affect the articular cartilage is increasing due to population ageing, but the current treatments are only palliative. One innovative approach to repair cartilage defects is tissue engineering and the use of mesenchymal stem/stromal cells (MSCs). Although the combination of MSCs with biocompatible scaffolds has been extensively investigated, no product is commercially available yet. This could be explained by the lack of mechanical stimulation during in vitro culture and the absence of proper and stable cartilage matrix formation, leading to poor integration after implantation. The objective of the present study was to investigate the biomechanical behaviour of MSC differentiation in micropellets, a well-defined 3D in vitro model of cartilage differentiation and growth, in view of tissue engineering applications. MSC micropellet chondrogenic differentiation was induced by exposure to TGFß3. At different time points during differentiation (35 days of culture), their global mechanical properties were assessed using a very sensitive compression device coupled to an identification procedure based on a finite element parametric model. Micropellets displayed both a non-linear strain-induced stiffening behaviour and a dissipative behaviour that increased from day 14 to day 29, with a maximum instantaneous Young's modulus of 179.9 ± 18.8 kPa. Moreover, chondrocyte gene expression levels were strongly correlated with the observed mechanical properties. This study indicates that cartilage micropellets display the biochemical and biomechanical characteristics required for investigating and recapitulating the different stages of cartilage development.

Remodeling phenotype of human subcutaneous adipose tissue macrophages.

BACKGROUND: Adipose tissue macrophages (ATMs) have become a focus of attention recently because they have been shown to accumulate with an increase in fat mass and to be involved in the genesis of insulin resistance in obese mice. However, the phenotype and functions of human ATMs are still to be defined. METHODS AND RESULTS: The present study, performed on human subcutaneous AT, showed that ATMs from lean to overweight individuals are composed of distinct macrophage subsets based on the expression of several cell surface markers: CD45, CD14, CD31, CD44, HLA-DR, CD206, and CD16, as assessed by flow cytometry. ATMs isolated by an immunoselection protocol showed a mixed expression of proinflammatory (tumor necrosis factor-alpha, interleukin-6 [IL-6], IL-23, monocyte chemoattractant protein-1, IL-8, cyclooxygenase-2) and antiinflammatory (IL-10, transforming growth factor-beta, alternative macrophage activation-associated cc chemokine-1, cyclooxygenase-1) factors. Fat mass enlargement is associated with accumulation of the CD206+/CD16- macrophage subset that exhibits an M2 remodeling phenotype characterized by decreased expression of proinflammatory IL-8 and cyclooxygenase-2 and increased expression of lymphatic vessel endothelial hyaluronan receptor-1. ATMs specifically produced and released matrix metalloproteinase-9 compared with adipocytes and capillary endothelial cells, and secretion of matrix metalloproteinase-9 from human AT in vivo, assessed by arteriovenous difference measurement, was correlated with body mass index. Finally, ATMs exerted a marked proangiogenic effect on AT-derived endothelial and progenitor cells. CONCLUSIONS: The present results showed that the ATMs that accumulate with fat mass development exhibit a particular M2 remodeling phenotype. ATMs may be active players in the process of AT development through the extension of the capillary network and in the genesis of obesity-associated cardiovascular pathologies.

[Benign fibrolipomyomatous tumor of the perinephric capsule. A report on one case]. / Les tumerus fibro-lipo-myomateuses bénignes de la capsule rénale. Une observation.

The authors describe a case of perinephric capsule tumor and emphasize the difficult in establishing diagnosis by radiological and especially angiographic investigations. The exact nature of the tumor can be determined only by histological examination. They review the published literature which demonstrates the rare nature of capsular fibrolipomyomata having clinical manifestations, the difficulty in making a differential diagnosis from hamartoma, and the possibility of malignant changes occurring in the tumor.

Somatostatin, gastrin, and cholinergic muscarinic binding sites in rat gastric, duodenal, and jejunal mucosa.

Clinical and experimental data indicate that the concentration of gastrin-I and somatostatin binding sites in human and rat gastric and duodenal mucosa may be changed in several pathologic conditions, including human peptic ulcer and cancer diseases. There are no data, however, indicating the distribution of receptor binding sites in the normal upper gastrointestinal tract. We studied the regional distribution of somatostatin-14, gastrin-I, and cholinergic muscarinic binding sites in membrane preparations from rat gastric corporeal and antral mucosa and in mucosa obtained from the duodenum and jejunum. The corporeal mucosa contained the most high-affinity gastrin binding sites (Bmax = 39.1 +/- 6.5 fmol/mg protein; Kd = 1.1 +/- 0.4 nM). The antral mucosa contained the most somatostatin and cholinergic muscarinic binding sites (Bmax = 65.7 +/- 6.6 fmol/mg protein and 460.3 +/- 101.8 fmol/mg protein, respectively). The duodenal and jejunal mucosal membranes contained somatostatin, gastrin, and cholinergic muscarinic binding sites in decreasing concentrations. Concentrations of binding sites are characteristic for particular gut regions and may help in analyzing their abnormalities.