Search

Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. Therapeutic aggrecanase inhibition for treatment of arthritic disease may thus not impede regenerative self-healing pathways based on chondrogenesis of local progenitor cells in the joint.

Human mesenchymal stem cells (MSC) have attracted much attention for tissue regeneration including repair of non-healing bone defects. Heterogeneity of MSC cultures and considerable donor variability however, still preclude standardised production of MSC and point on functional deficits for some human MSC populations. We aimed to identify functional correlates of donor-dependency of bone formation in order to develop a potency assay predicting the therapeutic capacity of human MSC before clinical transplantation. MSC from 29 donors were characterised in vitro and results were correlated to bone formation potency in a beta-tricalcium-phosphate (β-TCP)-scaffold after subcutaneous implantation into immunocompromised mice.In contrast to osteogenic in vitro differentiation parameters, a doubling time below 43.23 hours allowed to predict ectopic bone formation at high sensitivity (81.8%) and specificity (100%). Enriched conditions adapted from embryonic stem cell expansion rescued bone formation of inferior MSC populations while growth arrest of potent MSC by mitomycin C abolished bone formation, establishing a causal relationship between neo-bone formation and growth. Gene expression profiling confirmed a key role for proliferation status for the bone forming ability suggesting that a rate limiting anabolism and open chromatin determined and predicted the therapeutic potency of culture-expanded MSC. Proliferation-based potency testing and switch to enriched expansion conditions may pave the way for standardised production of MSC for bone repair.

Organ morphogenesis depends on mechanical interactions between cells and tissues. These interactions generate forces that can be sensed by cells and affect key cellular processes. However, how mechanical forces, together with biochemical signals, contribute to the shaping of complex organs is still largely unclear. We address this question using the seed of Arabidopsis as a model system. We show that seeds first experience a phase of rapid anisotropic growth that is dependent on the response of cortical microtubule (CMT) to forces, which guide cellulose deposition according to shape-driven stresses in the outermost layer of the seed coat. However, at later stages of development, we show that seed growth is isotropic and depends on the properties of an inner layer of the seed coat that stiffens its walls in response to tension but has isotropic material properties. Finally, we show that the transition from anisotropic to isotropic growth is due to the dampening of cortical microtubule responses to shape-driven stresses. Altogether, our work supports a model in which spatiotemporally distinct mechanical responses control the shape of developing seeds in Arabidopsis., (© 2024. The Author(s).)

Introduction: In France, 122 women were killed by their partner or ex-partner in 2021., Purpose of the Research: The principal objective of the AVIC-MG study, on women victims of domestic violence and their expectations of their general practitioner, was to observe whether the women in question, who visit specialist facilities for victims of domestic violence, would like to be questioned about domestic violence by their general practitioner (GP). The secondary objective was to describe this population of women and the characteristics of their GP visits during the last twelve months., Results: The study showed that more than 90 percent of these women had consulted a GP in the last twelve months and 65 percent of the mothers in the group had consulted a GP for their child(ren). The majority of these women (82 percent) wanted the GP to ask them about domestic violence. They had gone to the GP for specific reasons: fatigue, pain, psychological suffering (anxiety, sadness, difficulty sleeping)., Conclusion: The majority of women victims of domestic violence would like primary care practitioners to identify the abuse. Tools are available to help GPs with this complex identification, in particular the DECLICVIOLENCE.FR website.

During flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell-autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum-derived pollen wall components, while allowing metabolites such as sugars and amino acids to reach the developing pollen, remains unknown. Here, we show experimentally that in arabidopsis thaliana the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip, an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the peritapetal strip require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

This review aims to assess the benefits and adverse effects of sacubitril/valsartan in heart failure, with a focus on important patient outcomes. A systematic review was conducted of double-blind randomized controlled trials (RCTs) comparing sacubitril/valsartan versus a reference drug, in heart failure patients with reduced (HFrEF) and preserved (HFpEF) ejection fraction, published in French or English. Searches were undertaken of Medline, Cochrane Central, and Embase. The primary outcomes were all-cause mortality and adverse events. From 2 082 articles analyzed, 5 were included. For all-cause mortality, the absolute numbers for HFrEF (2 RCTs, 4627 patients) were 16% on sacubitril/valsartan and 18% on enalapril, with a risk ratio (RR) of 0.85 [CI = 0.78, 0.93], and 13% vs 14% in with HFpEF (2 RCTs, 5097 patients), with no statistical difference. Under the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, the evidence for HFrEF patients was of moderate quality. For HFrEF patients, an increased risk of symptomatic hypotension and angioedema (low quality of evidence) was shown. There was no statistical difference for the risk of hyperkalemia or worsening renal function. There was a protective RR (0.50 [0.34, 0.75]) for worsening renal function for patients with HFpEF, with a high quality of evidence despite similar absolute numbers (1.4% vs. 2.8%). To keep in mind for shared decision-making, sacubitril/valsartan reduces all-cause mortality in HFrEF patients but for HFpEF further data are needed. Take into consideration the small number of studies to date to assess the risks., (© 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Purpose: Angiotensin-converting enzyme (ACE) inhibitors are part of first-line treatment for reduced ejection fraction heart failure (HFrEF). The aim was to assess the benefits and adverse effects of ACE inhibitors in HFrEF with a focus on important patient outcomes., Methods: A systematic review of double-blind randomized clinical trials (RCTs) and comparison of ACE inhibitors versus placebo, in HFrEF patients published in French or English. Searches were undertaken of Medline, Cochrane Central, and Embase. The primary outcomes were all-cause mortality and adverse events., Results: From 636 articles analysed, 11 were included (13,882 patients). For all-cause mortality (5 RCTs, 9277 patients), the number needed to treat (NNT) to avoid one death at 6 months was 50 (33-107). The NNT to prevent one death at 12 months (6 RCTs, 13,016 patients) was 63 (35-314). Under the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, the evidence was of moderate quality. The number needed to harm was 12 (10-15) for cough, 20 (14-31) for hypotension, 23 (17-36) for dizziness, 31 (23-47) for hyperkalaemia, and 49 (30-121) for increased creatinine levels. The quality of evidence was moderate for these criteria except for cough (low quality of evidence)., Conclusion: This review focuses on clinical elements necessary in a shared decision-making process. In practice, general practitioners will be able to use these data to discuss ACE inhibitor treatment with HFrEF patients. This study was registered in the PROSPERO registry under the reference number CRD42018096930.

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an "auto-immune" type interaction., Competing Interests: The authors have declared that no competing interests exist.

Background: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo., Methods: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated., Results: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice., Conclusions: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

Aim of the Study: To obtain a consensus from a panel of experts (GP and cardiologists) on the elements to appear on the correspondence sent by GP at the patient's first consultation with the cardiologist and on the response of the cardiologist., Method: A list of proposals concerning the content of the exchanges between the GP and the cardiologist was established by a scientific council of three GPs and one cardiologist, based on a review of the literature and their practices. This list was submitted for evaluation to a panel of GP and cardiologists experts using the modified RAND/UCLA Delphi method., Results: Twenty nine experts (16 MG and 13 cardiologists) participated in the two evaluation rounds. For the contents of the letter written by the GP, 11 themes have reached consensus: administrative data, reason for consultation, history of the disease, recent constants, current treatments, current or previous pathologies and cardiovascular risk factors, physical activity, psychosocial context, test results, question asked to the cardiologist, cardiologist's perimeter of action. For the contents of the letter of the cardiologist's response, 11 themes were agreed: administrative data, reason for consultation, previous information, clinical examination, ECG, ultrasound, other complementary examinations, answer to the question asked by the GP, dietary treatments, proposed treatments, proposal for follow-up and management., Conclusion: This study have reached consensus on the elements to appear on the letters exchanged between the GP and the cardiologist., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma., Methods: Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis., Results: The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other., Conclusions: The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells.

Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1β and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. Therapeutic aggrecanase inhibition for treatment of arthritic disease may thus not impede regenerative self-healing pathways based on chondrogenesis of local progenitor cells in the joint.

Objective: Although several systemic and local factors are known to impair fracture healing, there is still no explanation, why some patients with sufficient fracture stability, showing none of the existing risk factors, still fail to heal normally. An investigation of local gene expression patterns in the fracture gap of patients with non-unions could decisively contribute to a better understanding of the pathophysiology of impaired fracture healing. For the first time, this study compares the expression of a large variety of osteogenic and chondrogenic genes in patients with regular and failed fracture healing., Methods: Between March 2006 and May 2007, a total of 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. Tissue samples of patients with normal and failed fracture healing were collected intraoperatively. Patients were divided into groups depending on the fracture date, and only patients with fractures two to four weeks old and patients with non-unions more than 9 months old were included in the final analysis. For the gene expression analysis, a customised cDNA array - containing 226 genes involved in osteo- and chondrogenesis - was used., Results: In the cDNA array analysis, the expression of eight genes was significantly elevated two-fold or more in the group with failed fracture healing relative to the normal controls. Conversely, no genes were found to be expressed at a higher level in the control group. The identified genes are supposed to be involved in extracellular matrix assembly, cytoskeletal structure, and differentiative and proliferative processes., Conclusions: The differences in gene expression pattern indicate a change in the composition and structure of the extracellular matrix, and a possible turn in the healing programme towards fibrous scar tissue formation, leading to non-union., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hydrophobins are fungal proteins with the ability to form immunologically inert membranes of high stability, properties that makes them attractive candidates for orthopaedic implant coatings. Cell adhesion on the surface of such implants is necessary for better integration with the neighbouring tissue; however, hydrophobin surfaces do not mediate cell adhesion. The aim of this project was therefore to investigate whether the class I hydrophobin DewA from Aspergillus nidulans can be functionalized for use on orthopaedic implant surfaces. DewA variants bearing either one RGD sequence or the laminin globular domain LG3 binding motif were engineered. The surfaces of both variants showed significantly increased adhesion of mesenchymal stem cells (MSCs), osteoblasts, fibroblasts and chondrocytes; in contrast, the insertion of binding motifs RGD and LG3 in DewA did not increase Staphylococcus aureus adhesion to the hydrophobin surfaces. Proliferation of MSCs and their osteogenic, chondrogenic and adipogenic differentiation potential were not affected on these surfaces. The engineered surfaces therefore enhanced MSC adhesion without interfering with their functionality or leading to increased risk of bacterial infection., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Human mesenchymal stem cells (MSC) have attracted much attention for tissue regeneration including repair of non-healing bone defects. Heterogeneity of MSC cultures and considerable donor variability however, still preclude standardised production of MSC and point on functional deficits for some human MSC populations. We aimed to identify functional correlates of donor-dependency of bone formation in order to develop a potency assay predicting the therapeutic capacity of human MSC before clinical transplantation. MSC from 29 donors were characterised in vitro and results were correlated to bone formation potency in a beta-tricalcium-phosphate (β-TCP)-scaffold after subcutaneous implantation into immunocompromised mice. In contrast to osteogenic in vitro differentiation parameters, a doubling time below 43.23 hours allowed to predict ectopic bone formation at high sensitivity (81.8%) and specificity (100%). Enriched conditions adapted from embryonic stem cell expansion rescued bone formation of inferior MSC populations while growth arrest of potent MSC by mitomycin C abolished bone formation, establishing a causal relationship between neo-bone formation and growth. Gene expression profiling confirmed a key role for proliferation status for the bone forming ability suggesting that a rate limiting anabolism and open chromatin determined and predicted the therapeutic potency of culture-expanded MSC. Proliferation-based potency testing and switch to enriched expansion conditions may pave the way for standardised production of MSC for bone repair.

A computer controlled dynamic bioreactor for continuous ultra-slow uniaxial distraction of a scaffold-free three-dimensional (3D) mesenchymal stem cell pellet culture was designed to investigate the influence of stepless tensile strain on behavior of distinct primary cells like osteoblasts, chondroblasts, or stem cells without the influence of an artificial culture matrix. The main advantages of this device include the following capabilities: (1) Application of uniaxial ultra-slow stepless distraction within a range of 0.5-250 μm/h and real-time control of the distraction distance with high accuracy (mean error -3.4%); (2) tension strain can be applied on a 3D cell culture within a standard CO(2) -incubator without use of an artificial culture matrix; (3) possibility of histological investigation without loss of distraction; (4) feasibility of molecular analysis on RNA and protein level. This is the first report on a distraction device capable of applying continuous tensile strain to a scaffold-free 3D cell culture within physiological ranges of motion comparable to distraction ostegenesis in vivo. We expect the newly designed microdistraction device to increase our understanding on the regulatory mechanisms of mechanical strains on the metabolism of stem cells., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

Multipotent mesenchymal stromal cells (MSCs) are an attractive cell source for cell therapy in cartilage. Although their therapeutic potential is clear, the requirements and conditions for effective induction of chondrogenesis in MSCs and for the production of a stable cartilaginous tissue by these cells are far from being understood. Different sources of MSCs have been considered for cartilage tissue engineering, mainly based on criteria of availability, as for adipose tissue, or of proximity to cartilage and the joint environment in vivo, as for bone marrow and synovial tissues. Focussing on human MSCs, this review will provide an overview of studies featuring comparative analysis of the chondrogenic differentiation of MSCs from different sources. In particular, it will examine the influence of the cells' origin on the requirements for the induction of chondrogenesis and on the phenotype achieved by the cells after differentiation.

Aims: The molecular mechanisms underlying the progression of central chondrosarcoma are so far poorly understood. The aim of this study was to identify genes involved in the progression of these tumours by comparison of gene expression and correlation of expression profiles to histological grade and clinical outcome., Methods and Results: Array-based gene expression profiling of 19 chondrosarcoma samples was performed. Beside differences in the expression of cartilage matrix molecules, high-grade chondrosarcoma showed enhanced expression of the matrix metalloproteinase MMP-2 and of the hypoxia-inducible molecule galectin 1. Immunohistochemical analysis of galectin 1 and of further hypoxia-associated proteins was performed on 68 central and peripheral tumour samples. Hypoxia-inducible factor 1alpha (HIF-1alpha) activation was significantly elevated in high-grade central chondrosarcoma. A negative correlation of carbonic anhydrase IX expression to metastasis-free survival was independent of histological grade., Conclusions: The expression patterns identified in this study point towards a substantial role for angiogenic and hypoxic signalling in chondrosarcoma progression. The constitutive activation of the transcription factor HIF-1alpha in high-grade chondrosarcoma could play a central role in the regulation of cell metabolism and vascularization in these tumours and may, for this reason, represent a potential target for chondrosarcoma therapy.

Mesenchymal stem cells (MSC) from bone marrow or adipose tissue (ASC) are broadly discussed as a cell population able to support cartilage regeneration and thus represent interesting candidates for cell-based tissue engineering in cartilage. ASC could represent an easily accessible and therefore particularly suitable source of cells. Their chondrogenic differentiation potential is, however, lower than that of MSC. The aim of this work was to characterise ASC in comparison to MSC in order to identify genes which may be involved in mechanisms causing the altered chondrogenic potential of ASC. Representational difference analysis was used to identify genes with higher expression in undifferentiated ASC than in MSC. Expression levels of identified genes were confirmed by real-time RT-PCR. Integral membrane protein 2A (ITM2A) was higher expressed in expanded ASC than in MSC in a donor-independent manner. During early chondrogenic differentiation in spheroid cultures ITM2A levels remained low in MSC and a transient down-regulation occurred in ASC correlating with successful chondrogenesis. Persisting ITM2A levels were found in non-differentiating ASC. Consistent with this finding, forced expression of ITM2A in the mouse mesenchymal stem cell line C3H10T1/2 prevented chondrogenic induction. In conclusion, ITM2A may in early stages of differentiation be associated with an inhibition of the initiation of chondrogenesis and elevated expression of ITM2A in ASC may therefore be linked to the poorer chondrogenic differentiation potential of these cells., (2009 International Society of Differentiation. Published by Elsevier Ltd.)

Mesenchymal stromal cells (MSC) are an attractive source for cell therapy and tissue engineering of joint cartilage. Common chondrogenic in vitro protocols, however, induce hypertrophic markers like COL10A1, matrix metalloproteinase 13 (MMP13), and alkaline phosphatase (ALP) reminiscent of endochondral bone formation. To direct MSC toward articular chondrocytes more specifically, a better understanding of the regulatory steps is desirable. Proteases are important players in matrix remodeling, display inhibitory effects on growth plate development and MMP13 inhibition prevented hypertrophy of bovine chondrocytes. The aim of this study was to evaluate whether the activity of proteases and MMPs, especially MMP13, is crucial for the transition of MSC toward mature chondrocytes and could allow to selectively influence aspects of early and late chondrogenic differentiation. Protease inhibitors were added during MSC chondrogenesis and stage-specific markers were assessed by histology, qPCR, and ALP quantification. Chondrogenesis was little affected by leupeptin, pepstatin, or aprotinin. In contrast, broad spectrum pan-MMP inhibitors dose dependently suppressed proteoglycan deposition, collagen type II and type X staining, ALP activity, and reduced SOX9 and COL2A1 expression. A selective MMP13 inhibitor allowed chondrogenesis and showed only weak effects on ALP activity. In conclusion, transition of MSC toward mature chondrocytes in vitro depended on molecules suppressed by pan-MMP inhibitors identifying chondrogenic differentiation of MSC as a sophistically regulated process in which catabolic enzymes are capable to directly influence cellular fate. In future therapeutic applications of diseased joints, the tested MMP13-specific inhibitor promises suppression of collagen type II degradation without imposing a risk to impair MSC-driven regeneration processes.

Phenotypic and molecular parallels between the development of chondrosarcoma and the differentiation of chondrocytes in normal growth plate suggest that chondrosarcoma may arise from mesenchymal precursor cells driven towards chondrogenesis. We hypothesized that a comparison between cartilaginous tumours and their possible physiological cells of origin, mesenchymal stem cells (MSCs), might have biological and clinical relevance. MSCs from eight donors were submitted to chondrogenic differentiation in spheroid cultures. Expression profiles of MSCs at days 0, 7, 14, 28 and 42 of chondrogenesis and of 18 chondrosarcomas with different histological grades were studied using a customized cDNA array. Hierarchical clustering of MSC gene expression during chondrogenesis allowed the classification of samples in a pre-chondrogenic and a chondrogenic cluster corresponding to the phenotypes of early and late differentiation stages. The 74 genes differentially expressed between the two clusters were defined as chondrogenesis-relevant genes. Gene expression profiles of chondrosarcoma were submitted to hierarchical clustering on the basis of these chondrogenesis-relevant genes. This analysis allowed clear distinction between grade I and grade III chondrosarcoma and separated grade II chondrosarcoma into two groups. All grade II chondrosarcomas with occurrence of metastasis were found together with the grade III chondrosarcomas in the pre-chondrogenic cluster. This analysis shows that a molecular approach based on the comparison of tumour samples to an in vitro model for chondrogenic differentiation allows a new classification of chondrosarcoma in two clusters. These data suggest that the identification of a pre-chondrogenic and a chondrogenic phenotype for chondrosarcoma by gene expression profiling could develop into a useful tool to predict the clinical behaviour of chondrosarcoma.

Objective: Human articular chondrocytes do not express COL10A1 and do not undergo hypertrophy except in close vicinity to subchondral bone. In contrast, chondrocytes produced in vitro from mesenchymal stem cells (MSCs) show premature COL10A1 expression and cannot form stable ectopic cartilage transplants, which indicates that they may be phenotypically unstable and not suitable for treatment of articular cartilage lesions. CpG methylation established during natural development may play a role in suppression of COL10A1 expression and hypertrophy in human articular chondrocytes. This study was undertaken to compare gene methylation patterns and expression of COL10A1 and COL2A1 in chondrocyte and MSC populations, in order to determine whether failed genomic methylation patterns correlate with an unstable chondrocyte phenotype after chondrogenesis of MSCs., Methods: COL10A1 and COL2A1 regulatory gene regions were computationally searched for CpG-rich regions. CpG methylation of genomic DNA from human articular chondrocytes, MSCs, and MSC-derived chondrocytes was analyzed by Combined Bisulfite Restriction Analysis and by sequencing of polymerase chain reaction fragments amplified from bisulfite-treated genomic DNA., Results: The CpG island around the transcription start site of COL2A1 was unmethylated in all cell groups independent of COL2A1 expression, while 9 tested CpG sites in the sparse CpG promoter of COL10A1 were consistently methylated in human articular chondrocytes. Induction of COL10A1 expression during chondrogenesis of MSCs correlated with demethylation of 2 CpG sites in the COL10A1 promoter., Conclusion: Our findings indicate that methylation-based COL10A1 gene silencing is established in cartilage tissue and human articular chondrocytes. Altered methylation levels at 2 CpG sites of COL10A1 in MSCs and their demethylation during chondrogenesis may facilitate induction of COL10A1 as observed during in vitro chondrogenesis of MSCs.

Objective: Monolayer expansion of human articular chondrocytes (HACs) is known to result in progressive dedifferentiation of the chondrocytes and loss of their stable cartilage formation capacity in vivo. For an optimal outcome of chondrocyte-based repair strategies, HACs capable of ectopic cartilage formation may be required. This study was undertaken to identify secreted candidate molecules, in supernatants of cultured HACs, that could serve as predictors of the ectopic cartilage formation capacity of cells., Methods: Standardized medium supernatants (n = 5 knee cartilage samples) of freshly isolated HACs (PD0) and of HACs expanded for 2 or 6 population doublings (PD2 and PD6, respectively) were screened by a multiplexed immunoassay for 15 distinct interleukins, 8 matrix metalloproteinases (MMPs), and 11 miscellaneous soluble factors. Cartilage differentiation markers such as cartilage oligomeric matrix protein and YKL-40 were determined by enzyme-linked immunosorbent assay. HACs from each culture were subcutaneously transplanted into SCID mice, and the capacity of the chondrocytes to form stable cartilage was examined histologically 4 weeks later., Results: Whereas freshly isolated (PD0) HACs generated stable ectopic cartilage that was positive for type II collagen, none of the cell transplants at PD6 formed cartilaginous matrix. Loss of the ectopic cartilage formation capacity between PD0 and PD6 correlated with a drop in the secretion of MMP-3 to <10% of initial levels, whereas changes in the other investigated molecules were not predictive. Chondrocytes with MMP-3 levels of >or=20% of initial levels synthesized cartilaginous matrix, whereas those with low MMP-3 levels (<10% of initial levels) at PD2 failed to regenerate ectopic cartilage., Conclusion: Loss of the capacity for stable ectopic cartilage formation in the course of HAC dedifferentiation can be predicted by determining the relative levels of MMP-3, demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.

Objective: Mesenchymal stem cells (MSCs) are a population of cells broadly discussed to support cartilage repair. The differentiation of MSCs into articular chondrocytes is, however, still poorly understood on the molecular level. The aim of this study was to perform an almost genome-wide screen for genes differentially expressed between cartilage and MSCs and to extract new markers useful to define chondrocyte differentiation stages., Methods: Gene expression profiles of MSCs (n=8) and articular cartilage from OA patients (n=7) were compared on a 30,000 cDNA-fragment array and differentially expressed genes were extracted by subtraction. Expression of selected genes was assessed during in vitro chondrogenic differentiation of MSCs and during dedifferentiation of expanded chondrocytes using quantitative and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Protein secretion was measured by enzyme-linked immunosorbent assay., Results: Eighty-seven genes were differentially expressed between MSCs and cartilage with a more than three-fold difference. Sixty-seven of them were higher expressed in cartilage and among them 15 genes were previously not detected in cartilage. Differential expression was confirmed for 69% of 26 reanalysed genes by RT-PCR. The profiles of three unknown transcripts and six protease-related molecules were characterised during differentiation. SERPINA1 and SERPINA3 mRNA expression correlated with chondrogenic differentiation of MSCs and dedifferentiation of chondrocytes, and SERPINA1 protein levels in culture supernatants could be correlated alike., Conclusions: cDNA-array analysis identified SERPINA1 and A3 as new differentiation-relevant genes for cartilage. Since SERPINA1 secretion correlated with both chondrogenesis of MSCs and dedifferentiation during chondrocyte expansion, it represents an attractive marker for refinement of chondrocyte differentiation.

We explored whether CLA isomers and other C18 FA affect (i) lipid content and FA concentrations in total adipocyte lipids, (ii) FA synthesis from glucose in TAG and phospholipids of primary brown (BAT) and white adipocytes (WAT), and (iii) mRNA expression of uncoupling protein 1 (UCP1) in primary brown adipocytes of Djungarian hamsters (Phodopus sungorus). c9,t11-CLA, oleic, linoleic, and alpha-linolenic acid increased whereas t10,c12-CLA decreased lipid accumulation in both adipocyte types. t10,c12-CLA treatment affected FA composition mainly in BAT cells. CLA incorporation into lipids, in particular c9,t11-CLA, was higher in BAT. In both cell types, t10,c12-CLA treatment reduced the incorporation of glucose 13C carbon into FA of TAG and phospholipids, whereas c9,t11-CLA, linoleic, and alpha-linolenic acid either did not influence or dose-dependently increased glucose carbon incorporation into FA. UCP1 mRNA expression was inhibited by t10,c12-CLA but increased by c9,t11-CLA, linoleic, and alpha-linolenic acid. It is concluded that c9,t11-CLA and t10,c12-CLA have distinctly different effects on lipid metabolism in primary adipocytes. The effects of c9,t11-CLA are similar to those of other unsaturated C18 FA. The opposite effects of c9,t11-CLA and t10,c12-CLA are evident in both WAT and BAT cultures; however, brown adipocytes seem to be more susceptible to CLA treatment.

Gene expression profiling through the application of microarrays provides comprehensive assessment of gene expression levels in a given tissue or cell population, as well as information on changes of gene expression in altered physiological or pathological situations. Microarrays are particularly suited to study interactions in the regulation of large numbers of different genes, since their expression is analyzed simultaneously. For improved understanding of the physiology of adipose tissue, and consequently obesity and diabetes, identification of covariability in gene expression was attempted by analysis of the individual variability of gene expression in subcutaneous white and brown fat of the Siberian dwarf hamster using microarrays containing approximately 300 cDNA fragments of adipose genes. No sex-dependant variability in gene expression could be found, and overall individual variability was rather low, with more than 80% of clones showing a coefficient of variation lower than 30%. Uncoupling protein 1 (UCP1) displayed a high variability of gene expression in brown fat, which was negatively correlated with the gene expression of complement factor B (FactB), implying a possible functional relationship.

White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and delta-6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, alpha-actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinoma-associated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon beta-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferator-activated receptor-gamma (PPARgamma), uncoupling protein 1 (UCP1), cytochrome c oxidase.

We investigated the effect of the specific beta(3)-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture. Proliferation of both white and brown preadipocytes was stimulated by a general beta-adrenergic agonist (isoproterenol) but not by CL. Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM. Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity. UCP-1 content was largely increased by CL in BAT but not in WAT cultures. Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol. COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by beta-adrenergic stimulation. We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via beta1 or beta(2), but not beta(3)-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro beta-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes.