Genome-Wide methylation and transcriptome analyses of bone marrow mesenchymal stem cells from osteoporotic patients

RESUMEN: La diferenciación de las células madre mesenquimales (MSCs) es esencial para el mantenimiento de la masa ósea. Nuestro objetivo fue caracterizar las marcas de metilación, la expresión génica (codificante y no codificante de proteína) y la capacidad de diferenciación de las MSC de médula ósea (BMSCs) de pacientes con fracturas de cadera osteoporóticas. Las BMSCs de pacientes con fracturas mostraron una mayor proliferación y una capacidad de diferenciación alterada. Los análisis de metilación de ADN revelaron que la mayoría los sitios diferencialmente metilados se dan en regiones genómicas con actividad potenciadora que a su vez se asociaron con genes expresados diferencialmente enriquecidos en vías relacionadas con la diferenciación osteogénica. Nuestros resultados sugieren que los mecanismos epigenéticos estudiados juegan un papel importante en la determinación del patrón de expresión génica de BMSCs derivadas de pacientes con osteoporosis. Y un mejor conocimiento de estas vías nos permitirá mejorar el metabolismo óseo en la osteoporosis.ABSTRACT: Mesenchymal stem cells (MSCs) osteogenic differentiation is essential for the maintenance of bone mass. The aim of this study was to characterize the DNA methylation marks, gene expression (coding and nonprotein-coding) and the ability to differentiate bone marrow stem cells (BMSCs) from patients with osteoporotic hip fractures. The BMSCs of patients with fractures showed greater proliferation and an altered differentiation capacity. DNA methylation analysis revealed that most differentially methylated sites are in genomic regions with enhancer activity. These enhancer regions were associated with differentially expressed genes, and these genes were enriched in bone related pathways, such as, osteogenic differentiation. Our results suggest that epigenetic mechanisms play an important role in the regulation of gene expression of BMSCs derived from patients with osteoporosis. A better knowledge of these pathways will permit us to improve bone metabolism in osteoporosis.Research carried out in this thesis was mainly developed in the Department of Medicine and Psychiatry of the Faculty of Medicine, University of Cantabria/ Mineral and lipid metabolism group of IDIVAL. The research was funded with grants from the Instituto de Salud Carlos III (PI12/615 and PI16/915). I have been funded by a predoctoral fellowship from the University of Cantabria and the Research Institute of Marques de Valdecilla Hospital (IDIVAL) (CVE-2016-11669)

Epigenetic related changes on air quality

The exposure to airborne particulate matter (PM) increases the risk of developing human diseases. Epigenetic mechanisms have been related to environmental exposures and human diseases. The present review is focused on current available studies, which show the relationship between epigenetic marks, exposure to air pollution and human's health. Air contaminants involved in epigenetic changes have been related to different specific mechanisms (DNA methylation, post-translational histone modifications and non-coding RNA transcripts), which are described in separate sections. Several studies describe how these epigenetic mechanisms are influenced by environmental factors including air pollution. This interaction between PM and epigenetic factors results in an altered profile of these marks, in both, globally and locus specific. Following this connection, specific epigenetic marks can be used as biomarkers, as well as, to find new therapeutic targets. For this purpose, some significant characteristics have been highlighted, such as, the spatiotemporal specificity of these marks, the relevance of the collected tissue and the specific changes stability. Air pollution has been related to a higher mortality rate due to non-accidental deaths. This exposure to particulate matter induces changes to the epigenome, which are increasing the susceptibility of human diseases. In conclusion, as several epigenetic change mechanisms remain unclear yet, further analyses derived from PM exposure must be performed to find new targets and disease biomarkers

Epigenetics of Skeletal Diseases

PURPOSE OF REVIEW: Epigenetic mechanisms modify gene activity in a stable manner without altering DNA sequence. They participate in the adaptation to the environment, as well as in the pathogenesis of common complex disorders. We provide an overview of the role of epigenetic mechanisms in bone biology and pathology. RECENT FINDINGS: Extensive evidence supports the involvement of epigenetic mechanisms (DNA methylation, post-translational modifications of histone tails, and non-coding RNAs) in the differentiation of bone cells and mechanotransduction. A variety of epigenetic abnormalities have been described in patients with osteoporosis, osteoarthritis, and skeletal cancers, but their actual pathogenetic roles are still unclear. A few drugs targeting epigenetic marks have been approved for neoplastic disorders, and many more are being actively investigated. Advances in the field of epigenetics underscore the complex interactions between genetic and environmental factors as determinants of osteoporosis and other common disorders. Likewise, they help to explain the mechanisms by which prenatal and post-natal external factors, from nutrition to psychological stress, impact our body and influence the risk of later disease.Acknowledgements: Supported by a grant from the Instituto de Salud Carlos III (PI 16/0915), which can be co-funded by the European Union (FEDER Funds)

Pharmacogenetics of osteoporosis: a pathway analysis of the genetic influence on the effects of antiresorptive drugs

Osteoporosis is a skeletal disorder defined by a decreased bone mineral density (BMD) and an increased susceptibility to fractures. Bisphosphonates and selective oestrogen receptor modulators (SERM) are among the most widely used drugs. They inhibit bone resorption by targeting the mevalonate and oestrogen pathways, respectively. The aim of this study was to determine if common variants of genes in those pathways influence drug responses. We studied 192 women treated with oral aminobisphosphonates and 51 with SERMs. Genotypes at 154 SNPs of the mevalonate pathway and 806 in the oestrogen pathway were analyzed. Several SNPs located in genes FDPS and FNTA were associated with the bisphosphonate-induced changes in hip bone mineral density (BMD), whereas polymorphisms of the PDSS1, CYP19A1, CYP1A1, and CYP1A2 genes were associated with SERM-induced changes in spine BMD. After multivariate analyses, genotypes combining genes FDPS and FNTA showed a stronger association with bisphosphonate response (r = 0.34; p = 0.00009), whereas the combination of CYP19A1 and PDSS1 genotypes was associated with the response to SERMs (r = 0.62, p = 0.0003). These results suggest that genotyping genes in these pathways may help predict the response to antiresorptive drugs and hence make personalized therapeutic choices.Funding: Supported by grants from Instituto de Salud Carlos III (PI18/00762; PI21/00532) that could be cofunded by European Union FEDER funds. Genotyping service was carried out at CEGEN-PRB3-ISCIII; it is supported by grant PT17/0019, of the PE I+D+i 2013–2016, funded by ISCIII and ERDF. Acknowledgments: Alvaro del Real received support by the postdoctoral grant Augusto Gonzalez de Linares of the University of Cantabria. We thank the skilful technical assistance of Carolina Sañudo and Alicia Martin-Rebollo

Exposure to particulate matter: direct and indirect role in the COVID-19 pandemic

Knowing the transmission factors and the natural environment that favor the spread of a viral infection is crucial to stop outbreaks and develop effective preventive strategies. This work aims to evaluate the role of Particulate Matter (PM) in the COVID-19 pandemic, focusing especially on that of PM as a vector for SARS-CoV-2. Exposure to PM has been related to new cases and to the clinical severity of people infected by SARS-CoV-2, which can be explained by the oxidative stress and the inflammatory response generated by these particles when entering the respiratory system, as well as by the role of PM in the expression of ACE-2 in respiratory cells in human hosts. In addition, different authors have detected SARS-CoV-2 RNA in PM sampled both in outdoor and indoor environments. The results of various studies lead to the hypothesis that the aerosols emitted by an infected person could be deposited in other suspended particles, sometimes of natural but especially of anthropogenic origin, that form the basal PM. However, the viability of the virus in PM has not yet been demonstrated. Should PM be confirmed as a vector of transmission, prevention strategies ought to be adapted, and PM sampling in outdoor environments could become an indicator of viral load in a specific area.“This work has been carried out within the framework of the project “Air pollution and COVID-19: what can we learn from this pandemic?” of the Call for Grants from the BBVA Foundation to Scientific Research Teams in SARS-CoV-2 and COVID-19, in the area of Ecology and Veterinary Medicine

Analysis of serum proteome after treatment of osteoporosis with anabolic or antiresorptive drugs

The aim of the study was to explore new markers in serum proteome associated with the response to antiosteoporosis drugs, namely teriparatide and denosumab. We obtained serum samples from 14 patients with osteoporosis, both at baseline and after 6 months of treatment with teriparatide (n = 10) or denosumab (n = 4). Samples were analyzed by nanoliquid chromatography coupled to high-resolution mass spectrometry on a QTOF 5600 (SCIEX) apparatus. The spectrometry data were analyzed with Mascot against the UniProtKB base and then several quality-control filters were applied for the identification of peptides (false discovery rate, FDR q < 0.02) and their quantification (FDR q < 0.05). In the group treated with teriparatide, 28 proteins were identified with significant differences before and after treatment. A pathway analysis by using the Reactome database revealed significant enrichment in the Insulin Like Growth Factor 1 (IGF-I) (FDR q 4 × 10-²) and innate immune system (FDR q 2 × 10-³) pathways. Among patients treated with denosumab, we observed significant differences in the levels of 10 proteins, which were also enriched in the pathways related to the innate immune system (FDR q 3 × 10-²). These results suggest that the innate immune system may be involved in the response to antiosteoporosis drugs.Funding: Supported by grants from FEIOMM (Grant No.17/0025) and ProteoRed-ISCIII. Acknowledgments: Alvaro del Real received support by the postdoctoral grant Augusto Gonzalez de Linares of the University of Cantabria

Hyperbaric Oxygen Therapy Does Not Have a Negative Impact on Bone Signaling Pathways in Humans

Introduction: Oxygen is emerging as an important factor in the local regulation of bone remodeling. Some preclinical data suggest that hyperoxia may have deleterious effects on bone cells. However, its clinical relevance is unclear. Hence, we studied the effect of hyperbaric oxygen therapy (HBOT) on serum biomarkers reflecting the status of the Wnt and receptor activator of NF-?B ligand (RANKL) pathways, two core pathways for bone homeostasis. Materials and methods: This was a prospective study of 20 patients undergoing HBOT (mean age 58 yrs., range 35?82 yrs.) because of complications of radiotherapy or chronic anal fissure. Patients were subjected to HBOT (100% oxygen; 2.4 atmospheres absolute for 90 min). The average number of HBOT sessions was 20 ± 5 (range 8?31). Serum hypoxia-inducible factor 1-? (HIF1-?), osteoprotegerin (OPG), RANKL, and the Wnt inhibitors sclerostin and dickkopf-1 (DKK1) were measured at baseline and after HBOT by using specific immunoassays. Results: HIF-1? in eight patients with measurable serum levels increased from 0.084 (0.098) ng/mL at baseline to 0.146 (0.130) ng/mL after HBOT (p = 0.028). However, HBOT did not induce any significant changes in the serum levels of OPG, RANKL, sclerostin or DKK1. This was independent of the patients? diagnosis, either neoplasia or benign. Conclusion: Despite the potential concerns about hyperoxia, we found no evidence that HBOT has any detrimental effect on bone homeostasis

Cribado de riesgo poligénico de fracturas óseas en mujeres de España con osteoporosis

Dado que la osteoporosis tiene un importante componente hereditario, el conocimiento de las variantes genéticas implicadas puede permitir la identificación precoz de los sujetos en riesgo y la instauración de medidas preventivas no farmacológicas. Por otro lado, puede ayudar a seleccionar más eficientemente los grupos de población en los que aplicar medios diagnósticos y terapéuticos más sofisticados. La osteoporosis tiene un carácter poligénico, por lo que se están intentando elaborar puntuaciones de riesgo basadas en el análisis de múltiples genes ("polygenic risk scores", PRS). En esta línea, recientemente se ha propuesto un PRS generado a partir de los datos genotípicos y de ultrasonografía de calcáneo como ayuda en la predicción de fracturas (Forgetta V, PLoS Med. 2020 ;17(7): e1003152). El objetivo de este estudio fue analizar la capacidad de ese índice para predecir osteoporosis en la población española. Estudiamos 1.747 mujeres, de las cuales 307 (65±9 años) fueron previamente diagnosticadas con osteoporosis y 1.440 (49±17 años) eran controles de población general. En todas ellas se hizo un análisis genómico con el Spanish Biobank Array. Tras solapar las variantes genotipadas con las del estudio referido, analizamos finalmente un total de 10.319 marcadores. Las puntuaciones del PRS fueron significativamente menores en el grupo de osteoporosis que en el grupo control (-2,40 versus -1,75; p=5,68x10-12). Esas diferencias persistían después de incluir la edad como covariable (p=2,78x10-11). Con el modelo ajustado por la edad, el PRS mostró un poder predictivo bastante alto, con un área bajo la curva de 0,875 (95% IC 0,775-0,925). En comparación con las mujeres de los otros cuartiles, aquellas con puntuación PRS en el primer cuartil tenían un riesgo de osteoporosis significativamente mayor (OR=2,1; 95% IC 1,6-2,7; p=9,01x10-8). Sin embargo, dentro del grupo de osteoporosis, no hemos encontrado asociación del PRS con variables como la DMO basal de región lumbar, cadera total o cuello de fémur. En resumen, la aplicación de este PRS muestra diferencias significativas entre la población general española y las pacientes con osteoporosis, lo que sugiere su utilidad dentro de estrategias de identificación de sujetos en riesgo basado en criterios clínicos-genéticos

Analysis of Bone Histomorphometry in Rat and Guinea Pig Animal Models Subject to Hypoxia

Hypoxia may be associated with alterations in bone remodeling, but the published results are contradictory. The aim of this study was to characterize the bone morphometry changes subject to hypoxia for a better understanding of the bone response to hypoxia and its possible clinical consequences on the bone metabolism. This study analyzed the bone morphometry parameters by micro-computed tomography (?CT) in rat and guinea pig normobaric hypoxia models. Adult male and female Wistar rats were exposed to chronic hypoxia for 7 and 15 days. Additionally, adult male guinea pigs were exposed to chronic hypoxia for 15 days. The results showed that rats exposed to chronic constant and intermittent hypoxic conditions had a worse trabecular and cortical bone health than control rats (under a normoxic condition). Rats under chronic constant hypoxia were associated with a more deteriorated cortical tibia thickness, trabecular femur and tibia bone volume over the total volume (BV/TV), tibia trabecular number (Tb.N), and trabecular femur and tibia bone mineral density (BMD). In the case of chronic intermittent hypoxia, rats subjected to intermittent hypoxia had a lower cortical femur tissue mineral density (TMD), lower trabecular tibia BV/TV, and lower trabecular thickness (Tb.Th) of the tibia and lower tibia Tb.N. The results also showed that obese rats under a hypoxic condition had worse values for the femur and tibia BV/TV, tibia trabecular separation (Tb.Sp), femur and tibia Tb.N, and BMD for the femur and tibia than normoweight rats under a hypoxic condition. In conclusion, hypoxia and obesity may modify bone remodeling, and thus bone microarchitecture, and they might lead to reductions in the bone strength and therefore increase the risk of fragility fracture.Funding: The present study was supported by grant reference BFU2015-70616-R from MINECOFEDER (Spain Government) and Programa Estratégico IBGM, Escalera de Excelencia, Ref. CCVC8485, Consejería de Educacion, JCyL (Spain)