Comparative proteomic analysis of hypertrophic chondrocytes in osteoarthritis

Background

Osteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis.

Methods

Chondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures.

Results

The proteomic analysis led to the identification of a total of ~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10⁻⁵). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes.

Conclusion

In this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9085-6) contains supplementary material, which is available to authorized users.     

Profiling of the eye aqueous humor in exfoliation syndrome by high-performance liquid chromatographic analysis of hyaluronan and galactosaminoglycans

The concentrations of hyaluronan and galactosaminoglycans - i.e., chondroitin sulfate and dermatan sulfate - were measured in the aqueous humor of the eye from patients with exfoliation syndrome and from healthy persons. The glycosaminoglycans/proteoglycans were almost completely precipitated (>97%) with ethanol in the presence of dextran as carrier and, following enzymic digestion, hyaluronan and galactosaminoglycans, were quantitatively converted to Δ^4,5-disaccharides. Non-degraded heparan sulfate and proteins/glycoproteins were removed by ultrafiltration using a Centricon 3 membrane. Separation and determination of hyaluronan- and galactosaminoglycan-derived Δ-disaccharides were performed by ion-suppression HPLC. For an accurate analysis in triplicate, as little as 50 μl of aqueous humor is required. Application of this method to the analysis of samples from six patients with exfoliation syndrome and three healthy persons showed that hyaluronan levels in patients (6.65–16.15 μg ml⁻¹) were significantly higher (3–8 times) than in healthy persons (2.0–2.24 μg ml⁻¹). There was no significant alteration in the galactosaminoglycan concentration. The obtained data open a new area in the deeper understanding of the exfoliation syndrome pathophysiology and in establishing a highly sensitivity and accurate HPLC method for its diagnosis and patient's follow-up.     

Denial of reward in the neonate shapes sociability and serotonergic activity in the adult rat

Background

Manipulations of the early environment are linked to long-lasting alterations of emotionality and social capabilities. Denial of rewarding mother-pup interactions in early life of rats could serve as model for child neglect. Negative consequences for social competence in later life, accompanied by changes in the serotonergic system would be expected. In contrast, rewarding mother-pup contact should promote adequate social abilities.

Methodology/Principal Findings

Male Wistar rats trained in a T-maze during postnatal days 10–13 under denial (DER) or permission (RER) of maternal contact were tested for play behavior in adolescence and for coping with defeat in adulthood. We estimated serotonin (5-HT) levels in the brain under basal conditions and following defeat, as well as serotonin receptor 1A (5-HT1A) and serotonin transporter (SERT) expression. DER rats exhibited increased aggressive-like play behavior in adolescence (i.e. increased nape attacks, p<0.0001) and selected a proactive coping style during defeat in adulthood (higher sum of proactive behaviors: number of attacks, flights, rearings and defensive upright posture; p = 0.011, p<0.05 vs RER, non-handled-NH). In adulthood, they had lower 5-HT levels in both the prefrontal cortex (p<0.05 vs RER) and the amygdala (p<0.05 vs NH), increased 5-HT levels following defeat (PFC p<0.0001) and decreased serotonin turnover (amygdala p = 0.008). The number of 5-HT1A immunopositive cells in the CA1 hippocampal area was increased (p<0.05 DER, vs RER, NH); SERT levels in the amygdala were elevated (p<0.05 vs RER, NH), but were lower in the prefrontal cortex (p<0.05 vs NH).

Conclusions/Significance

Denial of expected maternal reward early in life negatively affects sociability and the serotonergic system in a complex manner. We propose that our animal model could contribute to the identification of the neurobiological correlates of early neglect effects on social behavior and coping with challenges, but also in parallel with the effects of a rewarding early-life environment.     

Genetic polymorphisms within tumor necrosis factor gene promoter region: a role for susceptibility to ventilator-associated pneumonia

Debatable findings exist among various studies regarding the impact of single nucleotide polymorphisms (SNPs) within the promoter region of the tumor necrosis factor (TNF) gene for susceptibility to infections. Their impact was investigated in a cohort of mechanically ventilated patients who developed ventilator-associated pneumonia (VAP). Two-hundred and thirteen mechanically ventilated patients who developed VAP were enrolled. Genomic DNA was extracted and SNPs at the -376, -308 and -238 position of the promoter region of the TNF gene were assessed by restriction fragment length polymorphisms. Monocytes were isolated from 47 patients when they developed sepsis and stimulated by bacterial endotoxin for the production of TNFα and of interleukin-6 (IL-6). Patients were divided into two groups; 166 patients bearing only wild-type alleles of all three studied polymorphisms; and 47 patients carrying at least one A allele of the three studied SNPs. Time between start of mechanical ventilation and advent of VAP was significantly shorter in the second group than in the first group (log-rank: 4.416, p: 0.041). When VAP supervened, disease severity did not differ between groups. Stimulation of TNFα and of IL-6 was much greater by monocytes for patients carrying A alleles. Carriage of at least one A allele of the three studied SNPs at the promoter region of the TNF-gene is associated with shorter time to development of VAP but it is not associated with disease severity. Findings may be related with a role of the studied SNPs in the production of pro-inflammatory cytokines.     

Influence of common preanalytical variations on the metabolic profile of serum samples in biobanks

A blood pre-centrifugation delay of 24 h at room temperature influenced the proton NMR spectroscopic profiles of human serum. A blood pre-centrifugation delay of 24 h at 4°C did not influence the spectroscopic profile as compared with 4 h delays at either room temperature or 4°C. Five or ten serum freeze–thaw cycles also influenced the proton NMR spectroscopic profiles. Certain common in vitro preanalytical variations occurring in biobanks may impact the metabolic profile of human serum.     

Association of systemic lupus erythematosus clinical features with European population genetic substructure

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8×10⁻⁴), oral ulcers (P = 6.9×10⁻⁴) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested.     

Sex steroid receptors in skeletal differentiation and epithelial neoplasia: is tissue‐specific intervention possible?

Sex steroids, through their receptors, have potent effects on the signal pathways involved in osteogenic or myogenic differentiation. However, a considerable segment of those signal pathways has a prominent role in epithelial neoplastic transformation. The capability to intervene locally has focused on specific ligands for the receptors. Nevertheless, many signals are mapped to interactions of steroid receptor motifs with heterologous regulatory proteins. Some of those proteins interact with the glucocorticoid receptor and other factors essential to cell fate. Interactions of steroid receptor domain motifs with heterologous proteins affect specific target pathways; consequently, manipulation of specified protein modules complexed with steroid receptors may be a next major step for enhancing molecular targeted therapeutics. In the future, intervention at specific sections of receptor primary sequence may prove therapeutically more efficient in targeting pathways of choice than ligand selectivity can be.     

Comprehensive research synopsis and systematic meta-analyses in Parkinson's disease genetics: The PDGene database

More than 800 published genetic association studies have implicated dozens of potential risk loci in Parkinson's disease (PD). To facilitate the interpretation of these findings, we have created a dedicated online resource, PDGene, that comprehensively collects and meta-analyzes all published studies in the field. A systematic literature screen of -27,000 articles yielded 828 eligible articles from which relevant data were extracted. In addition, individual-level data from three publicly available genome-wide association studies (GWAS) were obtained and subjected to genotype imputation and analysis. Overall, we performed meta-analyses on more than seven million polymorphisms originating either from GWAS datasets and/or from smaller scale PD association studies. Meta-analyses on 147 SNPs were supplemented by unpublished GWAS data from up to 16,452 PD cases and 48,810 controls. Eleven loci showed genome-wide significant (P < 5 × 10(-8)) association with disease risk: BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT, MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25. In addition, we identified novel evidence for genome-wide significant association with a polymorphism in ITGA8 (rs7077361, OR 0.88, P = 1.3 × 10(-8)). All meta-analysis results are freely available on a dedicated online database (www.pdgene.org), which is cross-linked with a customized track on the UCSC Genome Browser. Our study provides an exhaustive and up-to-date summary of the status of PD genetics research that can be readily scaled to include the results of future large-scale genetics projects, including next-generation sequencing studies.     

Conformational dynamics of the EGFR kinase domain reveals structural features involved in activation

The epidermal growth factor receptor (EGFR) has been the focus of intensive studies because of its importance in cancer research. Thus, a broader understanding of the molecular mechanism of activation of the EGFR kinase will have profound significance for the development of novel therapeutics. Numerous crystal structures of EGFR kinase, including the structure of the activating‐kinase dimer, have provided snapshots of the specific pathway. Herein, we performed unrestrained‐, as well as targeted‐molecular dynamics simulations based on these data, to gain further insight into the conformational changes responsible for activation. Comparison of the monomer‐ versus activating‐EGFR‐dimer simulations indicates that the dimerization is stabilizing structural elements associated with the activated state and predicts new salt‐bridge interactions involving activation‐loop residues that may also be associated with that state. Targeted molecular dynamics simulations of the inactive‐to‐active EGFR transition, as well as the reverse pathway, confirm the formation of conserved structural features of functional importance for the activity or stabilization of either conformation. Interestingly, simulations of the L834R mutant, which is associated with cancer, suggest that the structural basis of the activation induced by that mutation might be the ability of the mutated R834 residue to consecutively form salt bridges with neighboring acidic residues and cause destabilization of a hydrophobic cluster in the inactive state. Proteins 2009. © 2008 Wiley‐Liss, Inc.