Beneficial Impact of Eicosapentaenoic Acid on the Adverse Effects Induced by Palmitate and Hyperglycemia on Healthy Rat Chondrocyte.

Osteoarthritis (OA) is the most prevalent form of arthritis and a major cause of pain and disability. The pathology of OA involves the whole joint in an inflammatory and degenerative process, especially in articular cartilage. OA may be divided into distinguishable phenotypes including one associated with the metabolic syndrome (MetS) of which dyslipidemia and hyperglycemia have been individually linked to OA. Since their combined role in OA pathogenesis remains to be elucidated, we investigated the chondrocyte response to these metabolic stresses, and determined whether a n-3 polyunsaturated fatty acid (PUFA), i.e., eicosapentaenoic acid (EPA), may preserve chondrocyte functions. Rat chondrocytes were cultured with palmitic acid (PA) and/or EPA in normal or high glucose conditions. The expression of genes encoding proteins found in cartilage matrix (type 2 collagen and aggrecan) or involved in degenerative (metalloproteinases, MMPs) or in inflammatory (cyclooxygenase-2, COX-2 and microsomal prostaglandin E synthase, mPGES) processes was analyzed by qPCR. Prostaglandin E2 (PGE2) release was also evaluated by an enzyme-linked immunosorbent assay. Our data indicated that PA dose-dependently up-regulated the mRNA expression of MMP-3 and -13. PA also induced the expression of COX-2 and mPGES and promoted the synthesis of PGE2. Glucose at high concentrations further increased the chondrocyte response to PA. Interestingly, EPA suppressed the inflammatory effects of PA and glucose, and strongly reduced MMP-13 expression. Among the free fatty acid receptors (FFARs), FFAR4 partly mediated the EPA effects and the activation of FFAR1 markedly reduced the inflammatory effects of PA in high glucose conditions. Our findings demonstrate that dyslipidemia associated with hyperglycemia may contribute to OA pathogenesis and explains why an excess of saturated fatty acids and a low level in n-3 PUFAs may disrupt cartilage homeostasis.

Efficient TGF-ß1 Delivery to Articular Chondrocytes In Vitro Using Agro-Based Liposomes.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-ß1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-ß1 to rat chondrocytes. Results showed that TGF-ß1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-ß1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Nanoliposomes from Agro-Resources as Promising Delivery Systems for Chondrocytes.

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH.

The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

Promotion of avian endothelial cell differentiation by GATA transcription factors.

In the avian embryo, endothelial cells originate from several sources, including the lateral plate and somite mesoderm. In this study, we show that Gata transcription factors are expressed in the lateral plate and in vasculogenic regions of the avian somite and are able to promote a vascular endothelial fate when ectopically expressed in somite precursors. A fusion of GATA4 to the transcriptional activator VP16 promoted endothelium formation, indicating that GATA transcription factors promote vasculogenesis via activation of downstream targets, while a fusion of GATA4 to the transcriptional repressor engrailed repressed expression of Vascular Endothelial Growth Factor Receptor 2, a marker of endothelial precursors. These findings indicate a role for GATA transcription factors in the differentiation of the endothelium.

A preliminary study exploring the mechanical properties of normal and Mgp-deficient mouse femurs during early growth.

Matrix Gla protein (MGP) is mostly known to be a calcification inhibitor, as its absence leads to ectopic calcification of different tissues such as cartilage or arteries. MGP deficiency also leads to low bone mass and delayed bone growth. In the present contribution, we investigate the effect of MGP deficiency on the structural and material mechanical bone properties by focusing on the elastic response of femurs undergoing three-points bending. To this aim, biomechanical tests are performed on femurs issued from Mgp-deficient mice at 14, 21, 28, and 35 days of postnatal life and compared to healthy control femurs. µCT acquisitions enable to reconstruct bone geometries and are used to construct subject-specific finite element models avoiding some of the reported limitations concerning the use of beam-like assumptions for small bone samples. Our results indicate that MGP deficiency may be associated to differences in both structural and material properties of femurs during early stages of development. MGP deficiency appears to be related to a decrease in bone dimensions, compensated by higher material properties resulting in similar structural bone properties at P35. The search for a unique density-elasticity relationship based on calibrated bone mineral density (BMD) indicates that MGP deficiency may affect bone tissue in several ways, that may not be represented uniquely from the quantification of BMD. Despite of its limitation to elastic response, the present preliminary study reports for the very first time the mechanical skeletal properties of Mgp-deficient mice at early stages of development.

Keutel Syndrome, a Review of 50 Years of Literature.

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp -/- mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology.

Targeting bone morphogenetic protein signaling on renal and vascular diseases.

PURPOSE OF REVIEW: Normal development and adult physiology of the kidney and vasculature rely heavily on bone morphogenetic proteins (BMPs). Here we compile evidence that favors the notion that BMPs are also critically involved in the process of generation and maintenance of renal and vascular diseases. RECENT FINDINGS: Molecular manipulation of BMP signaling in vivo and in vitro has been instrumental in showing the protective role of BMPs on renal fibrosis and diabetic nephropathy. Similarly, activation of those pathways produces phenotypic changes in vascular smooth muscle and endothelial cells, tightly linked to the pathogenesis of vascular calcification, hypertrophy and atherosclerosis. SUMMARY: Gain-of-function and loss-of-function experiments targeting BMP pathway agonists and inhibitors lead to significant progress in the comprehension of renal and vascular normal and altered behavior. The demonstration that BMP signaling plays an important part in pathological conditions of the vasculature and the kidney opens up possibilities for the development of diagnostic and therapeutic tools.

ATDC5 cells as a model of cartilage extracellular matrix neosynthesis, maturation and assembly.

Fibrillar collagens and proteoglycans (PGs) are quantitatively the major constituents of extracellular matrices (ECM). They carry numerous crucial post-translational modifications (PTMs) that tune the resulting biomechanical properties of the corresponding tissues. The mechanisms determining these PTMs remain largely unknown, notably because available established cell lines do not recapitulate much of the complexity of the machineries involved. ATDC5 cells are a model of chondrogenesis widely used for decades, but it remains described mostly at histological and transcriptional levels. Here, we asked to what extent this model recapitulates the events of ECM synthesis and processing occurring in cartilage. Insulin-stimulated ATDC5 cells exhibit up- or down-regulation of more than one-hundred proteins, including a number of known participants in chondrogenesis and major markers thereof. However, they also lack several ECM components considered of significant, yet more subtle, function in cartilage. Still, they assemble the large PG aggrecan and type II collagen, both carrying most of their in vivo PTMs, into an ECM. Remarkably, collagen crosslinking is fully lysyl oxidase (LOX)-dependent. The ATDC5 model recapitulates critical aspects of the cartilage ECM-processing machinery and should be useful to decipher the mechanisms involved. Proteomics data are available via ProteomeXchange with identifier PXD014121. SIGNIFICANCE: The present work provides the first proteome characterization of the ATDC5 chondrogenesis model, which has been used for decades in the field of cartilage biology. The results demonstrate the up- and down-regulation of more than one hundred proteins. Overall, specific drawbacks of the model are pointed out, that will be important to take into consideration for future studies. However, major cartilage components are massively assembled into an extracellular matrix and carry most of their post-translational modifications occurring in cartilage tissue. Unlike other available established cell lines, the ATDC5 model recapitulates major aspects of cartilage biosynthesis and should be useful in investigating the mechanisms that regulate collagen maturation events.

Endogenous Calcification Inhibitors in the Prevention of Vascular Calcification: A Consensus Statement From the COST Action EuroSoftCalcNet.

The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PPi). The importance of PPi for inhibiting arterial calcification has been reinforced by the protective effects of PPi in various mouse models displaying ectopic calcifications. Besides PPi, Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp-deficient mice develop spontaneous calcification of the arterial media. Whereas PPi and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.

Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells.

OBJECTIVES: Fibroblast Growth Factor 23 (FGF23) is well documented as a crucial player in the systemic regulation of phosphate homeostasis. Moreover, loss-of-function experiments have revealed that FGF23 also has a phosphate-independent and local impact on skeletogenesis. Here, we used ATDC5 cell line to investigate the expression of FGF23 and the role it may play locally during the differentiation of these cells. METHODS: ATDC5 cells were differentiated in the presence of insulin, and treated with recombinant FGF23 (rFGF23), inorganic phosphate (Pi) and/or PD173074, an inhibitor of FGF receptors (FGFRs). The mRNA expressions of FGF23, FGFRs and markers of hypertophy, Col X and MMP13, were determined by qPCR analysis and FGF23 production was assessed by ELISA. FGFR activation was determined by immunoprecipitation and immunoblotting. RESULTS: FGF23 mRNA expression and production were increased during ATDC5 differentiation. At D28 in particular, rFGF23 stimulation increased hypertrophic markers expression, as Col X and MMP13, and mineralization. A synergic effect of Pi and rFGF23 stimulation was observed on these markers and on the mineralization process. The use of PD173074, a pan-FGFR inhibitor, decreased terminal differentiation of ATDC5 by preventing rFGF23 pro-hypertrophic effects. CONCLUSIONS: Altogether, our results provide evidence that FGF23 plays an important role during differentiation of ATDC5 cell line, by promoting both hypertrophy and mineralization.

New perspectives on rare connective tissue calcifying diseases.

Connective tissue calcifying diseases (CTCs) are characterized by abnormal calcium deposition in connective tissues. CTCs are caused by multiple factors including chronic diseases (Type II diabetes mellitus, chronic kidney disease), the use of pharmaceuticals (e.g. warfarin, glucocorticoids) and inherited rare genetic diseases such as pseudoxanthoma elasticum (PXE), generalized arterial calcification in infancy (GACI) and Keutel syndrome (KTLS). This review explores our current knowledge of these rare inherited CTCs, and highlights the most promising avenues for pharmaceutical intervention. Advancing our understanding of rare inherited forms of CTC is not only essential for the development of therapeutic strategies for patients suffering from these diseases, but also fundamental to delineating the mechanisms underpinning acquired chronic forms of CTC.

Expression of endothelins in human cardiogenesis.

Genetic disruption of endothelin (ET) 1, endothelin-converting enzyme (ECE) 1, and endothelin receptor A (ET(A)) in "knockout" or mutant mouse models result in defects in branchial arch derived craniofacial tissues and in cardiac outflow and great vessel structures. Interestingly, certain types of human congenital cardiovascular malformations such as Catch 22 syndrome and type B interruption of the aortic arch strongly resemble defects seen in knockout animal models. To better address the exact involvement of the ET system in heart formation we explored the spatiotemporal pattern of expression of the components of the ET system during critical phases of cardiogenesis in the human embryo (3-6 weeks of development; Carnegie stages 10-17) by in situ hybridization. We detected high ET-1 mRNA expression in endocardial cells lining the heart outflow tract in the region where the future aortic valves will form. No hybridization signal corresponding to pre-pro-ET-3 was observed in the heart. At the same location, the underlying myocytes express ET(A) mRNA. Whereas a functional role of ET in the valve formation can be proposed because of the simultaneous presence of all the components of the endothelin system (ET-1/ECE-1/ET(A)), this seems not to be the case for the formation of the ventricular septum where endocardial cells do not express ET-1, and only a weak ET(A) hybridization signal was detected in the surrounding myocardium. An abnormal hemodynamism indirectly due to valve malformation may be the indirect cause of this septal defect. The results of this study suggest an important role for the ET system in the formation of certain anatomical structures of the developing human heart.

A chicken model of pharmacologically-induced Hirschsprung disease reveals an unexpected role of glucocorticoids in enteric aganglionosis.

The enteric nervous system originates from neural crest cells that migrate in chains as they colonize the embryonic gut, eventually forming the myenteric and submucosal plexus. Failure of the neural crest cells to colonize the gut leads to aganglionosis in the terminal gut, a pathological condition called Hirschsprung disease (HSCR) in humans, also known as congenital megacolon or intestinal aganglionosis. One of the characteristics of the human HSCR is its variable penetrance, which may be attributable to the interaction between genetic factors, such as the endothelin-3/endothelin receptor B pathway, and non-genetic modulators, although the role of the latter has not well been established. We have created a novel HSCR model in the chick embryo allowing to test the ability of non-genetic modifiers to alter the HSCR phenotype. Chick embryos treated by phosphoramidon, which blocks the generation of endothelin-3, failed to develop enteric ganglia in the very distal bowel, characteristic of an HSCR-like phenotype. Administration of dexamethasone influenced the phenotype, suggesting that glucocorticoids may be environmental modulators of the penetrance of the aganglionosis in HSCR disease.

Phosphate-induced mineralization of tracheal smooth muscle and cartilage cells.

BACKGROUND: During aging or various diseases, pathologic mineralization may occur in joints or in the vascular wall. This is due to the deposition of phosphate (Pi)-containing crystals into the extracellular matrix of articular chondrocytes or vascular smooth muscle cells. The mineralization ability of chondrocytes and smooth muscle cells of other tissue has not been investigated. OBJECTIVE: In this context, our work seeks to study the response induced by Pi on cartilage and smooth muscle cells from tracheal origin. METHODS: We have established a dissection procedure to harvest and isolate chondrocyte and smooth muscle cells from adult mouse trachea. Both cell types were then exposed to different concentrations of Pi (1, 3 or 5 mM) up to 14 days. Mineralization was evaluated by alizarin red staining, which identifies calcium deposition. The expression of genes characterizing the phenotypic identity of the cells and involved in the mineralization process was assessed by RT-qPCR. RESULTS: Treatment of tracheal chondrocytes and smooth muscle cells with increasing concentrations of Pi (3 and 5 mM) induced mineralization as revealed by positive alizarin red staining as early as 7 days of culture. Moreover, gene expression analysis revealed profound phenotypic changes in both cell types and suggested they mineralize through TNAP-independent or -dependent mechanisms, respectively. CONCLUSIONS: Our data indicate that, comparably to articular chondrocytes or vascular smooth muscles, chondrocyte and smooth muscle cells from the trachea are prone to mineralize under high-phosphate conditions.

Angiopoietin 2 alters pancreatic vascularization in diabetic conditions.

AIMS/HYPOTHESIS: Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. METHODS: 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1-10 twice/week. RESULTS: In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1-10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. CONCLUSIONS: This work highlighted an important role of Ang2 in pancreatic vascular defects induced by hyperglycaemia.

Pathologic calcification of adult vascular smooth muscle cells differs on their crest or mesodermal embryonic origin.

Vascular calcifications can occur in the elderly and in patients suffering from various diseases. Interestingly, depending on the pathology, different regions of the arterial system can be affected. Embryonic observations have clearly indicated that vascular smooth muscle cell (VSMC) origin is notably heterogeneous. For instance, in the aorta, VSMCs colonizing the aortic arch region derive from cardiac neural crest cells, whereas those populating the descending aorta derive from the mesoderm. We examined here whether the embryonic origin of aortic VSMCs would correlate with their ability to mineralize. Under hyperphosphatemic conditions that induce vascular calcifications, we performed ex vivo aortic explant cultures as well as in vitro VSMC cultures from wild-type mice. Our data showed that VSMC embryonic origin affects their ability to mineralize. Indeed, the aortic arch media made up of VSMCs of neural crest origin calcifies significantly earlier than the descending aorta composed of VSMCs, which are mesoderm-derived. Similar results were obtained with cultured VSMCs harvested from both aortic regions. We also demonstrated that in a mouse model deficient in matrix Gla protein, a potent calcification inhibitor, developing extensive and spontaneous medial calcifications of the aorta, lesions initiate in the aortic arch. Subsequently, calcifications progress outside the aortic arch region and ultimately spread all over the entire arterial tree, including the descending aorta. Altogether, our results support an unsuspected correlation between VSMCs of embryonic origin and the timing of appearance of calcifications.