Encapsulation of allogeneic mesenchymal stem cells in alginate extends local presence and therapeutic function.

Bone marrow derived mesenchymal stem cells (MSCs) have immunomodulatory and trophic capacities. For therapeutic application in local chronic inflammatory diseases, MSCs, preferably of allogeneic origin, have to retain immunomodulatory properties. This might be achieved by encapsulation of MSCs in a biomaterial that protects them from the host immune system. Most studies investigating the properties of MSCs for therapeutic application use short term cultures of cells in monolayer. Since the physical environment of MSCs can influence their functionality, we evaluated the feasibility of preserving the immunomodulatory properties of MSCs encapsulated in a three-dimensional alginate construct. After 5 weeks of implantation in immunocompetent rats, active allogeneic MSCs encapsulated in alginate were still detectable by Bio Luminescence Imaging and Magnetic Resonance Imaging of luciferase transduced and superparamagnetic iron oxide labelled MSCs. MSCs injected in saline were only detectable up to 1 week after injection. Moreover, the MSCs encapsulated in alginate responded to inflammatory stimuli similarly to MSCs in monolayer culture. In addition, MSC-alginate beads secreted immunomodulatory and trophic factors and inhibited T-cell proliferation after 30 d of in vitro culture. Our data indicate that allogeneic MSCs encapsulated in alginate persist locally and could act as an interactive immunomodulatory or trophic factor release system for several weeks, making this an interesting system to investigate for application in inflammatory disease conditions.

JC virus reactivation in patients with autoimmune rheumatic diseases treated with rituximab.

OBJECTIVES: To evaluate the presence of John Cunningham virus (JCV) DNA in patients with autoimmune rheumatic diseases (ARDs) treated with rituximab (RTX). METHOD: We assessed the JCV DNA levels in peripheral blood mononuclear cell (PBMC), serum, and urine samples by quantitative real-time polymerase chain reaction (qPCR) in a cohort of 42 ARD patients (20 of whom were being treated with RTX) and 42 healthy donors. Approximately 1 year later, we collected further samples from 32 of these 42 ARD patients, all of whom were being treated with RTX. We studied the association between these viral DNA prevalences and various clinical and demographic variables. RESULTS: The viral prevalence in PBMC, serum, and urine samples was 2.4% (1/42), 0%, and 50% (21/42), respectively, in the healthy donors, and 26% (8/31), 16% (5/31), and 86% (25/29), respectively, in the patients treated with RTX. The viral prevalences were not associated with any of the demographic or clinical variables included in the study. CONCLUSIONS: We detected a viral reactivation in PBMCs and serum during the RTX treatment that did not seem to be influenced by either use of immunosuppressive drugs or the length of treatment with the monoclonal antibody. Although this reactivation was asymptomatic, the viral presence in blood could increase the probability of the appearance of a neurotrophic variant of JCV.

A genome-wide association study identifies a new locus associated with the response to anti-TNF therapy in rheumatoid arthritis.

Anti-Tumor Necrosis Factor (anti-TNF) drugs are biologic agents commonly used to treat rheumatoid arthritis (RA). However, anti-TNFs are not effective in approximately one out of four treated patients. We conducted a Genome-Wide Association Study (GWAS) to identify the genetic variation associated with the response to anti-TNF therapy in RA. In the discovery stage, 372 RA patients treated with an anti-TNF agent (infliximab, adalimumab or etanercept) were analyzed and treatment response was defined at 12 weeks of therapy. We found a genome-wide significant association in the MED15 gene with the response to etanercept (P<1.5e-8). Using an independent cohort of 245 RA patients, we performed a replication study of the most significant GWAS associations. We replicated the association at the MED15 locus and found suggestive evidence of association in the previously associated MAFB locus. The results of this study suggest novel mechanisms associated with the response to anti-TNF therapies.

Association of valine and leucine at HLA-DRB1 position 11 with radiographic progression in rheumatoid arthritis, independent of the shared epitope alleles but not independent of anti-citrullinated protein antibodies.

OBJECTIVE: For decades it has been known that the HLA-DRB1 shared epitope (SE) alleles are associated with an increased risk of development and progression of rheumatoid arthritis (RA). Recently, the following variations in the peptide-binding grooves of HLA molecules that predispose to RA development have been identified: Val and Leu at HLA-DRB1 position 11, Asp at HLA-B position 9, and Phe at HLA-DPB1 position 9. This study was undertaken to investigate whether these variants are also associated with radiographic progression in RA, independent of SE and anti-citrullinated protein antibody (ACPA) status. METHODS: A total of 4,911 radiograph sets from 1,878 RA patients included in the Leiden Early Arthritis Clinic (The Netherlands), Umeå (Sweden), Hospital Clinico San Carlos-Rheumatoid Arthritis (Spain), and National Data Bank for Rheumatic Diseases (US) cohorts were studied. HLA was imputed using single-nucleotide polymorphism data from an Immunochip, and the amino acids listed above were tested in relation to radiographic progression per cohort using an additive model. Results from the 4 cohorts were combined in inverse-variance weighted meta-analyses using a fixed-effects model. Analyses were conditioned on SE and ACPA status. RESULTS: Val and Leu at HLA-DRB1 position 11 were associated with more radiographic progression (meta-analysis P = 5.11 × 10(-7)); this effect was independent of SE status (meta-analysis P = 0.022) but not independent of ACPA status. Phe at HLA-DPB1 position 9 was associated with more severe radiographic progression (meta-analysis P = 0.024), though not independent of SE status. Asp at HLA-B position 9 was not associated with radiographic progression. CONCLUSION: Val and Leu at HLA-DRB1 position 11 conferred a risk of a higher rate of radiographic progression independent of SE status but not independent of ACPA status. These findings support the relevance of these amino acids at position 11.

Estudio del papel de los miARN en células madre mesenquimales aisladas de pacientes artrósicos / Study of the role of miRNA in mesenchymal stem cells isolated from osteoarthritis patients

Objetivo: Los miARN actúan como silenciadores génicos que están implicados en la regulación de funciones celulares esenciales. El miR-335 participa regulando los procesos de diferenciación celular en células progenitoras. Las células madre mesenquimales (MSC) son células progenitoras de los condrocitos y osteoblastos encargados del mantenimiento homeostático del cartílago y hueso. El objetivo de este estudio era determinar una posible asociación entre la expresión de miR-335 y la enfermedad artrósica. Metodología: Las MSC obtenidas de la médula ósea de 3 pacientes artrósicos y 3 controles sin signos clínicos de artrosis ni osteoporosis se cultivaron y caracterizaron fenotípica y funcionalmente en el pase 3 de cultivo. Así mismo, mediante PCR cuantitativa se determinaron los niveles de expresión de miR-335 y del gen mesoderm-specific transcript ---MEST---, que controla su expresión. Resultados: Se detectaron diferencias entre las MSC aisladas de pacientes con artrosis y los controles en los niveles de expresión de miR-335 y de MEST (mediana [rango intercuartílico]: 1,69 [0,85-1,74]; 3,85 [3,20-5,67]). Aunque las diferencias detectadas no alcanzaron una significación estadística (p = 0,1), sí se apreció una clara tendencia a una menor expresión de miR-335 en las MSC de pacientes artrósicos. Conclusiones: Teniendo en cuenta que miR-335 tiene como dianas potenciales diferentes genes que participan en la vía de se˜nalización de la Wnt, la tendencia observada podría determinar, al menos en parte, algunas de las alteraciones que determinan el inicio o progresión de la artrosis, y puede, por lo tanto, servir en el diseño de futuras dianas terapéuticas para el tratamiento de esta enfermedad (AU)

Objective: MiRNAs act as gene silencers that are involved in the regulation of essential cell functions. miR-335 is involved in regulating cell differentiation processes in progenitor cells. Mesenchymal stem cells (MSCs) are progenitor cells of chondrocytes and osteoblasts responsible for homeostatic maintenance of cartilage and bone. The aim of this study was to determine a possible relationship between the expression of miR-335 and osteoarthritis. Methods: MSCs obtained from the bone marrow of 3 osteoarthritic patients and 3 controls with no clinical signs of osteoarthritis or osteoporosis were cultured and phenotypically and functionally characterised in a 3-step culture. Expression levels of miR-335 and the mesodermspecific transcript gene ---MEST--- that controls its expression were determined by quantitative PCR. Results: Differences in the expression levels of miR-335 and MEST (median [interquartile range]: 1.69 [0.85-1.74], and 3.85 [3.20-5.67] were detected between MSCs isolated from patients with osteoarthritis and controls. Although the differences detected did not reach statistical significance (P = .1), a clear trend towards lower expression of miR-335 in osteoarthritis MSCs was observed. Conclusions: Given that miR-335 has the different genes involved in the Wnt signalling pathway as potential targets, the observed trend may help to ascertain, at least partially, some of the alterations which determine the onset or progression of osteoarthritis, and can therefore serve for the design of future therapeutic targets for the treatment of this disease (AU)

[Study of the role of miRNA in mesenchymal stem cells isolated from osteoarthritis patients]. / Estudio del papel de los miARN en células madre mesenquimales aisladas de pacientes artrósicos.

OBJECTIVE: MiRNAs act as gene silencers that are involved in the regulation of essential cell functions. miR-335 is involved in regulating cell differentiation processes in progenitor cells. Mesenchymal stem cells (MSCs) are progenitor cells of chondrocytes and osteoblasts responsible for homeostatic maintenance of cartilage and bone. The aim of this study was to determine a possible relationship between the expression of miR-335 and osteoarthritis. METHODS: MSCs obtained from the bone marrow of 3 osteoarthritic patients and 3 controls with no clinical signs of osteoarthritis or osteoporosis were cultured and phenotypically and functionally characterised in a 3-step culture. Expression levels of miR-335 and the mesoderm-specific transcript gene -MEST- that controls its expression were determined by quantitative PCR. RESULTS: Differences in the expression levels of miR-335 and MEST (median [interquartile range]: 1.69 [0.85-1.74], and 3.85 [3.20-5.67] were detected between MSCs isolated from patients with osteoarthritis and controls. Although the differences detected did not reach statistical significance (P=.1), a clear trend towards lower expression of miR-335 in osteoarthritis MSCs was observed. CONCLUSIONS: Given that miR-335 has the different genes involved in the Wnt signalling pathway as potential targets, the observed trend may help to ascertain, at least partially, some of the alterations which determine the onset or progression of osteoarthritis, and can therefore serve for the design of future therapeutic targets for the treatment of this disease.

The 11q23.3 genomic region-rs964184-is associated with cardiovascular disease in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory disease associated with high risk of cardiovascular (CV) events. Recently, the rs964184 polymorphism has been associated with coronary artery disease in nonrheumatic Caucasian individuals. 2160 Spanish RA patients were genotyped for the rs964184 polymorphism. Sex, age at diagnosis and traditional CV risk factors (diabetes mellitus, dyslipidemia and smoking habit) were associated with increased risk of CV events. Interestingly, RA patients carrying the rs964184 GG genotype had significantly higher risk of CV events than those with CC genotype [hazard ratio (HR) = 2.91, 95% confidence interval (CI): 1.36-6.26, P = 0.006] after adjusting the results for sex, age at diagnosis and traditional CV risk factors. Our results indicate that rs964184 polymorphism is associated with CV disease in RA.

Leflunomide discontinuation in rheumatoid arthritis and influence of associated disease-modifying anti-rheumatic drugs: a survival analysis.

OBJECTIVES: The aims of this study were to describe the rate of leflunomide discontinuation in rheumatoid arthritis (RA) patients, in standard clinical practice, and to analyse which factors could influence this rate, paying particular attention to the concomitant treatment with other disease-modifying anti-rheumatic drugs (DMARDs). METHOD: We selected RA patients, diagnosed according to the 1987 American College of Rheumatology (ACR) criteria, attending the rheumatology outpatient clinic of the San Carlos Clinical Hospital (Madrid, Spain), who had started treatment with leflunomide between 1 January 2006 and 1 January 2011. Clinical records were examined until withdrawal of the drug, loss of follow-up, or 1 October 2011. Kaplan-Meier curves were set to account for leflunomide withdrawal. Cox bivariate and multivariate regression models were conducted to examine risk factors for leflunomide discontinuation. RESULTS: The incident rate (IR) for leflunomide discontinuation, regardless of the cause, was 27 per 100 patient-years [95% confidence interval (CI) 22-31]. We observed, in both the bivariate and multivariate regression analysis, that those aged > 75 years at the start of the leflunomide treatment and undergoing concurrent treatment with methotrexate (MTX) and/or hydroxychloroquine (HC) had a significantly higher risk of leflunomide discontinuation. CONCLUSIONS: An older age at the start of the treatment with leflunomide, or concomitant treatment with MTX and/or HC, could be associated with a higher risk of leflunomide discontinuation, regardless of the cause. Therefore, when taking MTX or HC, patients receiving leflunomide should be closely monitored early to detect the occurrence of adverse reactions, and hence prevent their aggravation.

Expression of human endogenous retrovirus HERV-K18 is associated with clinical severity in osteoarthritis patients.

OBJECTIVES: The aim of this study was to evaluate the involvement of human endogenous retrovirus K18 (HERV-K18) in osteoarthritis (OA), by genotyping the HERV-K18 env locus in OA patients and controls, and analysing HERV-K18 RNA expression and its association with OA risk and clinical variables. METHOD: We recruited 558 patients with symptomatic OA and 600 controls. We performed the genotyping by TaqMan assays and the analysis of expression by quantitative real-time polymerase chain reaction (qRT-PCR). Scores on the Western Ontario and McMasters Universities Osteoarthritis Index (WOMAC), the Lequesne index, and the Stanford Health Assessment Questionnaire (HAQ) were analysed with regard to the expression levels of HERV-K18. RESULTS: The 18.3 haplotype tended towards an association with OA risk and concordantly this haplotype was associated with a higher HERV-K18 expression (p = 0.05). We found statistically significant differences when we compared the scores on the WOMAC, the Lequesne index for knee and hip, and the HAQ between OA patients with higher expression [normalization ratio (NR) > 10] and OA patients without HERV-K18 expression (p = 0.0003, 0.0005, 0.002, and 0.05, respectively), and also when the comparison was made between OA patients with higher expression (NR > 10) and OA patients with low expression of HERV-K18 (NR = 1) for the WOMAC and the Lequesne index for knee and hip (p = 0.002, 0.013, and 0.006, respectively). CONCLUSIONS: We found an association between health status measurement systems and severity index for OA and the levels of expression of HERV-K18. These results suggest the possible involvement of HERV-K18 in the aetiopathogenesis of the disease.

Single-nucleotide polymorphisms at the 9p21.3 genomic region not associated with the risk of cardiovascular disease in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic polygenic inflammatory disease associated with accelerated atherosclerosis and high risk of cardiovascular disease (CVD). In this study, we evaluated the potential association of 9p21.3 single-nucleotide polymorphisms (SNPs) - previously linked to coronary artery disease - and CVD risk in 2001 Spanish RA patients genotyped for 9p21.3 SNPs using TaqMan™ assays. Carotid intima media thickness (cIMT) and presence of carotid plaques were also analyzed. Cox regression model did not disclose significant differences between patients who experienced CVD and those who did not. Neither association was found between cIMT or carotid plaques and SNPs allele distribution. In conclusion, results do not support a role of rs10116277 or rs1537375 SNPs in CVD risk in Spanish RA patients.

Association of the methionine sulfoxide reductase A rs10903323 gene polymorphism with cardiovascular disease in patients with rheumatoid arthritis.

OBJECTIVE: The methionine sulfoxide reductase A (MSRA) gene is related to oxidative stress that has been involved in the susceptibility to rheumatoid arthritis (RA) in genome-wide pathway analysis and replication studies. The aim of the present study was to determine whether the MSRA gene is implicated in susceptibility to cardiovascular (CV) disease in RA patients. METHODS: A total of 1302 patients fulfilling the 1987 American College of Rheumatism classification criteria for RA were genotyped for the MSRA rs10903323 (G/A) polymorphism. Two hundred and thirty-three (17.9%) patients experienced CV events. Human leucocyte antigen (HLA)-DRB1 genotyping was performed using molecular-based methods. Multiple logistic regression models were constructed with adjustments for gender, age at RA diagnosis, follow-up, rheumatoid shared epitope, and traditional CV risk as potential confounders. RESULTS: There were no statistically significant differences in the allele or genotype frequencies for the MSRA rs10903323 polymorphism between RA patients who experienced CV events and those who did not. However, an adjusted logistic regression model disclosed that the minor allele G yielded a marginally significant increased risk of CV events in this series of patients with RA [p = 0.05, odds ratio (OR) 1.68, 95% confidence interval (CI) 1.00-2.85]. When the logistic regression model was adjusted for anti-cyclic citrullinated peptide (anti-CCP) antibody status instead of for shared epitope, an increased risk of having ischaemic heart disease was found in patients carrying the minor allele G (p = 0.04, OR 2.00, 95% CI 1.03-3.88). CONCLUSION: The MSRA rs10903323 gene polymorphism may be implicated in the increased risk to develop CV events, in particular ischaemic heart disease, observed in RA patients.

Influence of MHCIITA rs3087456 and rs4774 polymorphisms in the susceptibility to cardiovascular disease of patients with rheumatoid arthritis.

OBJECTIVES: MHCIITA is a major regulator of MHC expression that has been reported to be involved in the susceptibility to rheumatoid arthritis (RA) and myocardial infarction. In this study we investigated the potential association of two MHCIITA gene polymorphisms with cardiovascular (CV) risk in patients with RA. METHODS: 1302 patients fulfilling the 1987 ACR classification criteria for RA were genotyped for the MHCIITA rs3087456 and rs4774 gene polymorphisms to determine the influence of MHCIITA variants in the development of CV events. The potential influence of these polymorphisms in the development of subclinical atherosclerosis was also analysed in a subgroup of patients with no history of CV events by the assessment of two surrogate markers of atherosclerosis; brachial and carotid ultrasonography to determine endothelial function and carotid artery intima-media thickness, respectively. RESULTS: No statistically significant differences in the allele or genotype frequencies for each individual MHCIITA gene polymorphism between RA patients who experienced CV events, or not, were found. This was also the case when each polymorphism was assessed according to results obtained from surrogate markers of atherosclerosis. Also, in assessing the combined influence of both MHCIITA gene polymorphisms in the risk of CV disease after adjustment for gender, age at time of disease diagnosis, follow-up time, traditional CV risk factors, and shared epitope status, patients with CV events only showed a marginally decreased frequency of the MHCIITA rs3087456-rs4774 G-G allele combination (p=0.08; odds ratio: 0.63 [95% confidence interval: 0.37-1.05]). CONCLUSIONS: Our data do not support an influence of MHCIITA rs3087456 and rs4774 polymorphisms in the increased risk of CV events of patients with RA.

Tratamiento comparado de las lesiones del manguito rotador mediante el uso de células madre mesenquimales en combinación con membranas de colágeno tipo I / Rotator cuff injury comparative treatment between isolated mesenchymal stem cells or combined with collagen I membranes

Objetivo: Estudiar, en rata, la rotura completa del tendón del m. supraespinoso (SE) y su reparación mediante una membrana de colágeno tipo I con células mesenquimales pluripotenciales (MSCs). Material y método: Seccionamos unilateralmente el tendón SE, en ratas Sprague-Dawley de nueve meses de edad. Un mes después de la intervención, se realizó una segunda cirugía para reparar el tendón según tres grupos de tratamiento: cirugía convencional usando una sutura (n=30); con membrana de colágeno tipo I sin células (n=30) y con membrana de colágeno tipo I con 1x106 MSCs alogénicas (n=30; grupo MSC-Membrana). La reparación se evaluó al mes (n=30), a los dos2 meses (n=30) y a los nueve meses (n=30) mediante criterios biomecánicos e histológicos. Resultados: Las matrices de colágeno con MSC en los defectos creados mejoraron la resistencia y la rigidez del tendón SE a los tres meses comparado con las otras dos cirugías reparadoras. Ningún tratamiento logró un patrón histológicamente organizado. Conclusión: El tratamiento con MSC es seguro en los desgarros del manguito rotador (AU)

Objective: To study, in a rat model, the supraspinatus tendon (SE) rupture with isolated MSCs or combined with collagen membranes Material and method: A chronic rotator cuff tear injury model was developed by unilaterally detaching the SE tendon of aged Sprague-Dawley rats (9 months). One month post-injury, a second surgery was then performed to repair the tears by: 1. single suture (n=30), 2. suture and type I collagen membranes (n=30) and 3. suture and type I collagen membranes with 1x106 allogeneic MSCs; (n=30). Lesion restoration was evaluated at one month (n=30), two months (n=30) and three months (n=30) post-injury by biomechanical, histological and inmunohistological criterias. Results: All experimental conditions were well tolerated and no adverse effects were observed. Implantation of collagen type I membrane with MSCs into surgically created tendon defects improved strength and stiffness of SE tendon three months after treatment compared with the other two repair surgeries. No treatment achieved a histological organized pattern. Conclusion: The therapeutic use of allogenic MSCs in collagen type I membrane in the rotator cuff tears seems to be the most promising treatment in our experimental model (AU)

Lack of association of IL6R rs2228145 and IL6ST/gp130 rs2228044 gene polymorphisms with cardiovascular disease in patients with rheumatoid arthritis.

Interleukin-6 (IL-6) is a key mediator of inflammation in rheumatoid arthritis (RA) and its actions may be controlled by the IL-6 receptor (IL-6R). IL-6 transducer (IL-6ST/ gp130) is the signal transducing subunit of the IL-6R. We assessed the influence of the IL6R and the IL6ST/gp130 genes in the risk of cardiovascular (CV) disease in RA. For this purpose, 1250 Spanish patients with RA were genotyped for the IL6R rs2228145 and IL6ST/gp130 rs2228044 functional gene polymorphisms. Patients were stratified according to the presence or absence of CV events. Also, a subgroup of patients without CV events was assessed for the presence of subclinical atherosclerosis using two surrogate markers of atherosclerosis (flow-mediated endothelium-dependent vasodilatation and carotid intima-media thickness). No significant differences in the genotype and allele frequencies for both gene polymorphisms between patients with and without CV events were observed. It was also the case when values of surrogate markers of atherosclerosis were compared according to IL6R and IL6ST genotype frequencies. In conclusion, our results do not confirm an association of IL6R rs2228145 and IL6ST/gp130 rs2228044 polymorphisms with CV disease in RA.

Lack of association of NAMPT rs9770242 and rs59744560 polymorphisms with disease susceptibility and cardiovascular risk in patients with rheumatoid arthritis.

OBJECTIVES: Visfatin is an adipokine encoded by the NAMPT (PBEF1) gene. In this study we assessed the potential association of two NAMPT gene polymorphisms with disease susceptibility and cardiovascular (CV) risk in patients with rheumatoid arthritis (RA). METHODS: A total of 1,395 patients fulfilling the 1987 ACR classification criteria for RA and 1,230 matched controls, were genotyped for the NAMPT rs9770242 and rs59744560 gene polymorphisms, located within the proximal promoter, using predesigned TaqMan single nucleotide polymorphism genotyping assay. Also, HLA-DRB1 genotyping was performed using molecular based methods. In a second step, 1,196 patients in whom full information was available were assessed to determine the influence of NAMPT rs9770242 and rs59744560 polymorphisms in the development of CV events. Also, the potential influence of these polymorphisms in the development of subclinical atherosclerosis was assessed in a subgroup of patients with no history of CV events by brachial artery reactivity to determine flow-mediated endothelium-dependent and endothelium-independent vasodilatation (n=125) and by B-mode ultrasonography to determine the carotid artery intima-media thickness (n=105). RESULTS: No statistically significant differences in the allele or genotype frequencies for the NAMPT gene polymorphisms between RA patients and controls were found. A modest non significant lower frequency of the minor allele G of rs9770242 polymorphism was observed among patients with CV disease (20.62%) compared to those without CV disease (22.83%) (p=0.39). Also, a slight nonsignificant lower frequency of the minor allele T of rs59744560 polymorphism in patients with CV events (9.81%) compared with those RA patients who did not experience CV disease (13.07%) (p=0.11) was observed. Likewise, no significant association between the NAMPT polymorphisms with surrogate markers of subclinical atherosclerosis was found in patients with RA. CONCLUSIONS: NAMPT rs9770242 and rs59744560 polymorphisms are not markers of disease susceptibility and CV disease in RA.

Vascular endothelial growth factor A and cardiovascular disease in rheumatoid arthritis patients.

To determine the contribution of the vascular endothelial growth factor A (VEGFA) rs2010963 (-634 G>C) and rs1570360 (-1154 G>A) polymorphisms to the risk of cardiovascular (CV) disease in a series of patients with rheumatoid arthritis (RA). Six hundred sixty-one patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of the Hospital Xeral-Calde, Lugo, and the Hospital San Carlos, Madrid, Spain, were studied. Patients were genotyped for the VEGFA rs2010963 (-634 G>C) and rs1570360 (-1154 G>A) polymorphisms using predesigned TaqMan single nucleotide polymorphism (SNP) genotyping assay (Applied Biosystems, Foster City, CA). Also, human leukocyte antigen (HLA) DRB1 genotyping was performed using molecular-based methods. Clinical histories of the patients were reviewed for the presence of CV events that were considered to be present if the patient had ischemic heart disease, heart failure, cerebrovascular accident, or peripheral arteriopathy. Also, a subgroup of patients without the history of CV events was assessed for the presence of subclinical atherosclerosis manifested by the presence of endothelial dysfunction by brachial artery reactivity (n = 126) and increased carotid artery intima-media thickness (n = 105) using high resolution Doppler ultrasonography. No significant association between the VEGFA rs2010963 and the rs1570360 polymorphisms (neither isolated nor joined as allelic combinations) with clinically evident CV disease was found in this series of patients with RA. It was also the case when we examined the contribution of these polymorphisms to the development of subclinical atherosclerosis. VEGFA polymorphisms do not seem to exert a significant influence on the risk of CV disease in patients with RA.

Lack of association between LEP rs2167270 (19 G>A) polymorphism and disease susceptibility and cardiovascular disease in patients with rheumatoid arthritis.

OBJECTIVES: To assess the potential association between LEP rs2167270 (19 G>A) gene polymorphism and disease susceptibility and cardiovascular disease (CV) in patients with rheumatoid arthritis (RA). METHODS: 773 patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo, and Hospital San Carlos, Madrid, Spain, and 957 matched controls, were studied. Patients were genotyped for the LEP rs2167270 (19G>A) polymorphism, located within the 5´UTR, using predesigned TaqMan single nucleotide polymorphism genotyping assay. Also, HLA-DRB1 genotyping was performed using molecular based methods. Subclinical atherosclerosis was assessed in a subgroup of patients with no history of CV events by brachial artery reactivity to determine flow-mediated endothelium-dependent and endothelium-independent vasodilatation (n=133) and by B-mode ultrasonography of the carotid artery intima-media thickness (n=113). RESULTS: No statistically significant differences in the genotype or allele frequencies of the LEP rs2167270 gene polymorphism between patients with RA and controls were seen. Likewise, LEP rs2167270 polymorphism did not influence the development of CV events. Also, no significant differences in LEP rs2167270 genotype or allele distribution were seen when results of surrogate markers of subclinical atherosclerosis were assessed. CONCLUSIONS: LEP rs2167270 polymorphism does not seem to be a genetic risk factor for disease susceptibility or clinically evident CV disease and subclinical atherosclerosis in patients with RA.

Lack of association between ADIPOQ rs266729 and ADIPOQ rs1501299 polymorphisms and cardiovascular disease in rheumatoid arthritis patients.

To assess the potential association between ADIPOQ rs266729 and rs1501299 gene polymorphisms, either isolated or in combination, and cardiovascular disease in patients with rheumatoid arthritis (RA), 674 patients seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo, and Hospital San Carlos, Madrid, Spain, were analyzed. Genotyping was performed using predesigned TaqMan assays (Applied Biosystems, Foster City, CA). Carotid intima-media thickness, flow-mediated endothelium-dependent and endothelium-independent post-nitroglycerin vasodilatation, which are used as surrogate markers of subclinical atherosclerosis, were measured in a subsample. No significant differences in the genotype, allele or allele combination frequencies of both polymorphisms were found between RA patients with or without cardiovascular events or subclinical atherosclerosis. Therefore, ADIPOQ rs266729 and rs1501299 polymorphisms do not seem to be associated with cardiovascular disease in RA.

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells.

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.

Lack of association of PTPN22, STAT4 and TRAF1/C5 gene polymorphisms with cardiovascular risk in rheumatoid arthritis.

OBJECTIVES: To determine whether the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms may be implicated in the development of cardiovascular (CV) events and subclinical atherosclerosis manifested by the presence of endothelial dysfunction or increased carotid intima-media thickness (IMT) in a series of Spanish patients with rheumatoid arthritis (RA). METHODS: Six hundred and twelve patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo, and Hospital San Carlos, Madrid, were studied. Patients were genotyped using predesigned TaqMan single nucleotide polymorphism genotyping assays. Moreover, between March and December 2007, a subgroup of unselected RA patients with no history of CV events was studied for the presence of subclinical atherosclerosis by the assessment of the endothelial function (n=126) and the carotid artery IMT (n=110) by ultrasonography studies. RESULTS: No significant differences in the allele or genotype frequencies for the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms between RA patients with or without CV events were found. It was also the case when we analysed the potential influence of the genotypes in the presence of endothelial dysfunction or increased carotid artery IMT of patients with RA. CONCLUSIONS: Our results do not show that the PTPN22, STAT4 and TRAF1/C5 gene polymorphisms may confer a direct risk of CV disease in patients with RA.