Signature of microRNA expression during osteogenic differentiation of bone marrow MSCs reveals a putative role of miR-335-5p in osteoarthritis.

BACKGROUND: The aim of this study was to evaluate, the existence of a signature of differentially expressed microRNAs (miRNAs) during osteogenic differentiation of bone marrow MSCs from OA and healthy donors and to describe their possible implication in joint regeneration through modulation of molecular mechanisms involved in homeostatic control in OA pathophysiology. METHODS: Following phenotypic assessment of BM-MSCs obtained from OA diagnosed patients (n = 10) and non-OA (n = 10), total small RNA was isolated after osteogenic induction for 1, 10 and 21 days, miRNA profiles were generated using a commercial expression array of 754 well-characterized miRNAs. MiRNAs, with consistent differential expression were selected for further validation by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: A total of 246 miRNAs were differentially expressed (fold change ≥ ± 2, P ≤0.05) between OA and non-OA BM-MSC samples; these miRNAs showed variable interactions depending on the cell and differentiation status. Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis. In particular hsa-miR-335-5p, a critical regulator in bone homeostasis, was further studied. hsa-miR-335-5p downregulation in OA-MSCs, as well as their host coding gene, MEST, were also assessed. CONCLUSIONS: To our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation. We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features. These findings, may contribute to our understanding of the molecular mechanisms involved in MSCs mediated homeostatic control in OA pathophysiology that could be applicable in future therapeutic approaches.

Influence of Mesenchymal Stem Cell Sources on Their Regenerative Capacities on Different Surfaces.

Current gold-standard strategies for bone regeneration do not achieve the optimal recovery of bone biomechanical properties. To bypass these limitations, tissue engineering techniques based on hybrid materials made up of osteoprogenitor cells-such as mesenchymal stem cells (MSCs)-and bioactive ceramic scaffolds-such as calcium phosphate-based (CaPs) bioceramics-seem promising. The biological properties of MSCs are influenced by the tissue source. This study aims to define the optimal MSC source and construct (i.e., the MSC-CaP combination) for clinical application in bone regeneration. A previous iTRAQ analysis generated the hypothesis that anatomical proximity to bone has a direct effect on MSC phenotype. MSCs were isolated from adipose tissue, bone marrow, and dental pulp, then cultured both on a plastic surface and on CaPs (hydroxyapatite and ß-tricalcium phosphate), to compare their biological features. On plastic, MSCs isolated from dental pulp (DPSCs) presented the highest proliferation capacity and the greatest osteogenic potential. On both CaPs, DPSCs demonstrated the greatest capacity to colonise the bioceramics. Furthermore, the results demonstrated a trend that DPSCs had the most robust increase in ALP activity. Regarding CaPs, ß-tricalcium phosphate obtained the best viability results, while hydroxyapatite had the highest ALP activity values. Therefore, we propose DPSCs as suitable MSCs for cell-based bone regeneration strategies.

Publisher Correction: A predominant involvement of the triple seropositive patients and others with rheumatoid factor in the association of smoking with rheumatoid arthritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A predominant involvement of the triple seropositive patients and others with rheumatoid factor in the association of smoking with rheumatoid arthritis.

The major environmental risk factor for rheumatoid arthritis (RA) is smoking, which according to a widely accepted model induces protein citrullination in the lungs, triggering the production of anti-citrullinated protein antibodies (ACPA) and RA development. Nevertheless, some research findings do not fit this model. Therefore, we obtained six independent cohorts with 2253 RA patients for a detailed analysis of the association between smoking and RA autoantibodies. Our results showed a predominant association of smoking with the concurrent presence of the three antibodies: rheumatoid factor (RF), ACPA and anti-carbamylated protein antibodies (ACarPA) (3 Ab vs. 0 Ab: OR = 1.99, p = 2.5 × 10-8). Meta-analysis with previous data (4491 patients) confirmed the predominant association with the concurrent presence of the three antibodies (3 Ab vs. 0 Ab: OR = 2.00, p = 4.4 ×10-16) and revealed that smoking was exclusively associated with the presence of RF in patients with one or two antibodies (RF+1+2 vs. RF-0+1+2: OR = 1.32, p = 0.0002). In contrast, no specific association with ACPA or ACarPA was found. Therefore, these results showed the need to understand how smoking favors the concordance of RA specific antibodies and RF triggering, perhaps involving smoking-induced epitope spreading and other hypothesized mechanisms.

Functional implications of single nucleotide polymorphisms rs662 and rs854860 on the antioxidative activity of paraoxonase1 (PON1) in patients with rheumatoid arthritis.

BACKGROUND: Atherosclerosis leading to cardiovascular disease (CVD) is the main cause of mortality and morbidity in patients with rheumatoid arthritis (RA). Paraoxonase1 (PON1) is the best understood member of plasma paraoxonases with anti-atherogenic properties. PATIENTS AND METHODS: Spanish RA (n = 549) consecutively recruited from 1 single center and 477 ethnically matched healthy controls were included in a case-control study. The concentration of PON1 was evaluated by means of an enzyme-linked immunosorbent sssay (ELISA). An arylesterase/paraoxonase assay kit was used to evaluate PON1 activity. Sample genotyping was performed by using TaqMan assays-on-demand. All results were expressed as medians ± interquartile range. One-way ANOVA comparisons were done using a nonparametric Kruskall-Wallis test. P values under 0.05 were considered to be significant. RESULTS: The concentration of PON1 in the RA group was higher than in control group (p = 0.0003), although the differences were not significant when PON1 activities were compared between both groups. No significant differences were found related to distributions of rs662 genotypes in RA patients compared to healthy controls. Among rs854860 polymorphisms, overall genotype was widely distributed between RA patients and controls. Overall PON1 concentration in plasma was not significantly different between individuals carrying any of rs662 (p = 0.8501) or rs854860 (p = 0.2741) polymorphisms. Although PON1 levels were not associated with any of the SNPs in the study, differences appear when enzyme activities are compared for each SNP separately. CVD in RA patients correlate with increased PON1 levels and lower PON1 activity. CONCLUSIONS: Although protective role of PON1 against oxidative damage in vivo could be related to other activities, in our study arylesterase activity was useful to identify phenotypic differences with emphasis placed on two SNPs coding for nonconservative amino acid changes in the functional protein.

Specific Association of HLA-DRB1*03 With Anti-Carbamylated Protein Antibodies in Patients With Rheumatoid Arthritis.

OBJECTIVE: Recognition of a new type of rheumatoid arthritis (RA)-specific autoantibody, the anti-carbamylated protein antibodies (anti-CarP), has provided an opportunity to improve the management and understanding of RA. The current study was undertaken to assess the relationship between anti-CarP antibodies and HLA-DRB1 alleles in RA. METHODS: Serum samples were obtained from 3 different collections, comprising a total of 1,126 RA patients. Serum reactivity against in vitro carbamylated fetal calf serum proteins was determined by enzyme-linked immunosorbent assay. HLA-DRB1 alleles were determined using either hybridization techniques or imputation from HLA-dense genotypes. Results of these analyses were combined in a meta-analysis with data from 3 previously reported cohorts. The carrier frequencies of the common HLA-DRB1 alleles were compared between the antibody-positive RA subgroups and the double-negative subgroup of RA patients stratified by anti-citrullinated protein antibody (ACPA)/anti-CarP antibody status, and also between the 4 RA patient strata and healthy controls. RESULTS: Meta-analysis was conducted with 3,709 RA patients and 2,305 healthy control subjects. Results revealed a significant increase in frequency of HLA-DRB1*03 carriers in the ACPA-/anti-CarP+ subgroup as compared to ACPA-/anti-CarP- RA patients and healthy controls; this was consistently found across the 6 sample collections. This association of HLA-DRB1*03 with ACPA-/anti-CarP+ RA was independent of the presence of the shared allele (SE) and any other confounders analyzed. No other allele was specifically associated with the ACPA-/anti-CarP+ RA patient subgroup. In contrast, frequency of the SE was significantly increased in the ACPA+/anti-CarP- and ACPA+/anti-CarP+ RA patient subgroups, without a significant distinction between them. Furthermore, some alleles (including HLA-DRB1*03) were associated with protection from ACPA+ RA. CONCLUSION: These findings indicate a specific association of HLA-DRB1*03 with ACPA-/anti-CarP+ RA, suggesting that preferential presentation of carbamylated peptides could be a new mechanism underlying the contribution of HLA alleles to RA susceptibility.

Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: implications for multiple sclerosis.

Axonal damage is a correlate for increasing disability in multiple sclerosis. Animal models such as experimental autoimmune encephalomyelitis (EAE) may help to develop better therapeutical neuroprotective strategies for the human disease. Here we investigate the pattern of axonal injury in murine myelin oligodendrocyte glycoprotein peptide 35-55 (MOG) induced EAE. Inflammatory infiltration, axonal densities and expression of amyloid precursor protein (APP), neurofilaments (SMI31 and 32) as well as expression of sodium channels were quantified in lesions, the perilesional area and normal appearing white matter (NAWM). Quantification of T cells and macrophages revealed a significant reduction of inflammatory infiltration at later disease stages despite an increase of demyelinated areas and persistent clinical disability. In lesions, axonal density was already significantly reduced early and throughout all investigated disease stages. A significant axonal loss was also seen in the grey matter and at later time points in the perilesion as well as NAWM. Numbers of axons characterized by non-phosphorylated neurofilaments and re-distribution of sodium channels 1.2 and 1.6 increased over the course of MOG-EAE whilst APP positive axons peaked at the maximum of disease. Finally, double-labeling experiments revealed a strong colocalization of sodium channels with APP, neurofilaments and the axonal nodal protein Caspr, but not glial and myelin markers in actively demyelinating lesions. In summary, progressive axonal loss distant from lesions is mainly associated with changes in neurofilament phosphorylation, re-distribution of sodium channels and demyelination. This axonal loss is dissociated from acute inflammatory infiltration and markedly correlates with clinical impairment. Consequently, therapeutic intervention may be promising at early stages of EAE focusing on inflammation, or later in disease targeting degenerative mechanisms.

Differential Expression of HOX Genes in Mesenchymal Stem Cells from Osteoarthritic Patients Is Independent of Their Promoter Methylation.

Skeletogenesis, remodeling, and maintenance in adult tissues are regulated by sequential activation of genes coding for specific transcription factors. The conserved Homeobox genes (HOX, in humans) are involved in several skeletal pathologies. Osteoarthritis (OA) is characterized by homeostatic alterations of cartilage and bone synthesis, resulting in cartilage destruction and increased bone formation. We postulate that alterations in HOX expression in Mesenchymal Stem cells (MSCs) are likely one of the causes explaining the homeostatic alterations in OA and that this altered expression could be the result of epigenetic regulation. The expression of HOX genes in osteoarthritic-derived MSCs was screened using PCR arrays. Epigenetic regulation of HOX was analyzed measuring the degree of DNA methylation in their promoters. We demonstrate the downregulated expression of HOXA9 and HOXC8 in OA-MSCs. However, their expression does not correlate with promoter methylation status, suggesting that other epigenetic mechanisms could be implicated in the regulation of HOX expression. Studies on the role of these genes under active differentiation conditions need to be addressed for a better knowledge of the mechanisms regulating the expression of HOX, to allow a better understanding of OA pathology and to define possible biomarkers for therapeutic treatment.

Altered Expression of Wnt Signaling Pathway Components in Osteogenesis of Mesenchymal Stem Cells in Osteoarthritis Patients.

INTRODUCTION: Osteoarthritis (OA) is characterized by altered homeostasis of joint cartilage and bone, whose functional properties rely on chondrocytes and osteoblasts, belonging to mesenchymal stem cells (MSCs). WNT signaling acts as a hub integrating and crosstalking with other signaling pathways leading to the regulation of MSC functions. The aim of this study was to evaluate the existence of a differential signaling between Healthy and OA-MSCs during osteogenesis. METHODS: MSCs of seven OA patients and six healthy controls were isolated, characterised and expanded. During in vitro osteogenesis, cells were recovered at days 1, 10 and 21. RNA and protein content was obtained. Expression of WNT pathway genes was evaluated using RT-qPCR. Functional studies were also performed to study the MSC osteogenic commitment and functional and post-traslational status of ß-catenin and several receptor tyrosine kinases. RESULTS: Several genes were downregulated in OA-MSCs during osteogenesis in vitro. These included soluble Wnts, inhibitors, receptors, co-receptors, several kinases and transcription factors. Basal levels of ß-catenin were higher in OA-MSCs, but calcium deposition and expression of osteogenic genes was similar between Healthy and OA-MSCs. Interestingly an increased phosphorylation of p44/42 MAPK (ERK1/2) signaling node was present in OA-MSCs. CONCLUSION: Our results point to the existence in OA-MSCs of alterations in expression of Wnt pathway components during in vitro osteogenesis that are partially compensated by post-translational mechanisms modulating the function of other pathways. We also point the relevance of other signaling pathways in OA pathophysiology suggesting their role in the maintenance of joint homeostasis through modulation of MSC osteogenic potential.

Re-expression of a developmentally restricted potassium channel in autoimmune demyelination: Kv1.4 is implicated in oligodendroglial proliferation.

Mechanisms of lesion repair in multiple sclerosis are incompletely understood. To some degree, remyelination can occur, associated with an increase of proliferating oligodendroglial cells. Recently, the expression of potassium channels has been implicated in the control of oligodendrocyte precursor cell proliferation in vitro. We investigated the expression of Kv1.4 potassium channels in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Confocal microscopy revealed expression of Kv1.4 in AN2-positive oligodendrocyte precursor cells and premyelinating oligodendrocytes in vitro but neither in mature oligodendrocytes nor in the spinal cords of healthy adult mice. After induction of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, Kv1.4 immunoreactivity was detected in or around lesions already during disease onset with a peak early and a subsequent decrease in the late phase of the disease. Kv1.4 expression was confined to 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendroglial cells, which were actively proliferating and ensheathed naked axons. After a demyelinating episode, the number of Kv1.4 and 2',3'-cyclic nucleotide 3'-phosphodiesterase double-positive cells was greatly reduced in ciliary neurotrophic factor knockout mice, a model with impaired lesion repair. In summary, the re-expression of an oligodendroglial potassium channel may have a functional implication on oligodendroglial cell cycle progression, thus influencing tissue repair in experimental autoimmune encephalomyelitis and multiple sclerosis.