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This study explores the genetic factors associated with atypical femoral fractures (AFF), rare fractures associated with prolonged anti-resorptive therapy. AFF are fragility fractures that typically appear in the subtrochanteric or diaphyseal regions of the femur. While some cases resemble fractures in rare genetic bone disorders, the exact cause remains unclear. This study investigates 457 genes related to skeletal homeostasis in 13 AFF patients by exome sequencing, comparing the results with osteoporotic patients (n = 27) and Iberian samples from the 1000 Genomes Project (n = 107). Only one AFF case carried a pathogenic variant in the gene set, specifically in the ALPL gene. The study then examined variant accumulation in the gene set, revealing significantly more variants in AFF patients than in osteoporotic patients without AFF (p = 3.7 × 10-5), particularly in ACAN, AKAP13, ARHGEF3, P4HB, PITX2, and SUCO genes, all of them related to osteogenesis. This suggests that variant accumulation in bone-related genes may contribute to AFF risk. The polygenic nature of AFF implies that a complex interplay of genetic factors determines the susceptibility to AFF, with ACAN, SUCO, AKAP13, ARHGEF3, PITX2, and P4HB as potential genetic risk factors. Larger studies are needed to confirm the utility of gene set analysis in identifying patients at high risk of AFF during anti-resorptive therapy.
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Conservadores da Densidade Óssea , Doenças Ósseas , Fraturas do Fêmur , Humanos , Fraturas do Fêmur/genética , Fêmur/patologia , Diáfises , DifosfonatosRESUMO
PURPOSE: Human mesenchymal stem cells (MSCs) have the ability to differentiate into bone-forming osteoblasts. The aim of this study was to elucidate if MSCs from patients with OP show a senescent phenotype and explore their bone-forming ability in vivo. MATERIALS AND METHODS: MSCs from patients with OP and controls with osteoarthritis (OA) were implanted into the subcutaneous tissue of immunodeficient mice for histological analysis and expression of human genes by RT-PCR. The expression of senescence-associated phenotype (SASP) genes, as well as p16, p21, and galactosidase, was studied in cultures of MSCs. RESULTS: In vivo bone formation was evaluated in 103 implants (47 OP, 56 OA). New bone was observed in 45% of the implants with OP cells and 46% of those with OA cells (p = 0.99). The expression of several bone-related genes (collagen, osteocalcin, alkaline phosphatase, sialoprotein) was also similar in both groups. There were no differences between groups in SASP gene expression, p16, and p21 expression, or in senescence-associated galactosidase activity. CONCLUSION: Senescence markers and the osteogenic capacity in vivo of MSCs from patients with OP are not inferior to that of cells from controls of similar age with OA. This supports the interest of future studies to evaluate the potential use of autologous MSCs from OP patients in bone regeneration procedures.
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Fraturas do Quadril , Células-Tronco Mesenquimais , Animais , Diferenciação Celular/genética , Células Cultivadas , Fraturas do Quadril/metabolismo , Humanos , Camundongos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/genéticaRESUMO
Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.
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Reabsorção Óssea/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Osteócitos/metabolismo , Hormônio Paratireóideo/metabolismo , Ligante RANK/biossíntese , Transdução de Sinais/fisiologia , Animais , Reabsorção Óssea/genética , Células Cultivadas , Redes Reguladoras de Genes/fisiologia , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteócitos/citologia , Osteogênese/fisiologia , Hormônio Paratireóideo/genética , Ligante RANK/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.
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Linhagem Celular , Epigênese Genética , Osteoblastos/citologia , Osteogênese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica , Calcitriol/farmacologia , Diferenciação Celular , Metilação de DNA , Decitabina , Marcadores Genéticos/genética , Humanos , Camundongos , Camundongos Nus , Osteocalcina/genética , Osteocalcina/metabolismo , Alicerces TeciduaisRESUMO
Sclerostin, encoded by the SOST gene, works as an inhibitor of the Wnt pathway and therefore is an important regulator of bone homeostasis. Due to its potent action as an inhibitor of bone formation, blocking sclerostin activity is the purpose of recently developed anti-osteoporotic treatments. Two bone-specific transcription factors, RUNX2 and OSX, have been shown to interact and co-ordinately regulate the expression of bone-specific genes. Although it has been recently shown that sclerostin is targeted by OSX in mice, there is currently no information of whether this is also the case in human cells. We have identified SP-protein family and AML1 consensus binding sequences at the human SOST promoter and have shown that OSX, together with RUNX2, binds to a specific region close to the transcription start site. Furthermore, we show that OSX and RUNX2 activate SOST expression in a co-ordinated manner in vitro and that SOST expression levels show a significant positive correlation with OSX/RUNX2 expression levels in human bone. We also confirmed previous results showing an association of several SOST/RUNX2 polymorphisms with bone mineral density.
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Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição Sp7/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Densidade Óssea/fisiologia , Regulação da Expressão Gênica/genética , Marcadores Genéticos , Humanos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Regiões Promotoras Genéticas/genéticaRESUMO
Osteoporosis causes important morbidity among elderly individuals. Fragility fractures, and especially hip fractures, have a particularly negative impact on the patients' quality of life. The role of epigenetic mechanisms in the pathogenesis of many disorders is increasingly recognized, yet little is known about their role in non-malignant bone disorders such as osteoporosis. The aim of this study was to explore the expression of miRNAs in patients with osteoporotic hip fractures. Trabecular bone samples were obtained from the femoral heads of patients undergoing replacement surgery for osteoporotic hip fractures and non-fracture controls with hip osteoarthritis. Levels of 760 miRNA were analyzed by real-time PCR. Thirteen miRNAs showed nominally significant (p < 0.05) differences between both groups. Six miRNAs (miR-187, miR-193a-3p, miR-214, miR518f, miR-636, and miR-210) were selected for the replication stage. These miRNAs were individually analyzed in a larger group of 38 bone samples. At this stage, we confirmed statistically significant differences across groups for mir-187 and miR-518f. The median relative expression levels of miR-187 were 5.3-fold higher in the non-fracture group (p = 0.002). On the contrary, miR-518f was preferentially expressed in bones from osteoporotic patients (8.6-fold higher in fractures; p = 0.046). In this first hypothesis-free study of the bone microRNome we found two miRNAs, miR-187, and miR-518f, differentially regulated in osteoporotic bone. Further studies are needed to elucidate the mechanisms involved in the association of these miRNAs with fractures.
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Osso e Ossos/metabolismo , Fraturas do Quadril/metabolismo , MicroRNAs/metabolismo , Osteoporose/metabolismo , Fraturas por Osteoporose/metabolismo , Idoso , Idoso de 80 Anos ou mais , Densidade Óssea/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Qualidade de VidaRESUMO
Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.
Assuntos
Proteínas Morfogenéticas Ósseas/biossíntese , Linhagem Celular Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Marcadores Genéticos , Humanos , Immunoblotting , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese/fisiologia , Osteossarcoma , TranscriptomaRESUMO
OBJECTIVE: To determine genome-wide methylation profiles of bone from patients with hip osteoarthritis (OA) and those with osteoporotic (OP) hip fractures. METHODS: Trabecular bone pieces were obtained from the central part of the femoral head of 27 patients with hip fractures and 26 patients with hip OA. DNA was isolated, and methylation was explored with Illumina methylation arrays. RNA was extracted, pooled, and deep-sequenced to obtain the whole transcriptome. Differentially methylated regions were identified, and connections between genes with differentially methylated regions were explored by pathway and text-mining analyses. RESULTS: After quality control, methylation of 23,367 CpG sites (13,463 genes) was analyzed. There was a genome-wide inverse relationship between methylation and gene expression in both patient groups. Comparison of OP and OA bones revealed 241 CpG sites, located in 228 genes, with significant differences in methylation (false discovery rate<0.05). Of them, 217 were less methylated in OP than in OA. The absolute methylation differences were >5% in 128 CpG sites and >10% in 45 CpG sites. The differentially methylated genes were enriched for association with bone traits in the genome-wide association study catalog. Pathway analysis and text-mining analysis with Gene Relationships Across Implicated Loci software revealed enrichment in genes participating in glycoprotein metabolism or cell differentiation, and particularly in the homeobox superfamily of transcription factors. CONCLUSION: Genome-wide methylation profiling of bone samples revealed differentially methylated regions in OP and OA. These regions were enriched in genes associated with cell differentiation and skeletal embryogenesis, such as those in the homeobox superfamily, suggesting the existence of a developmental component in the predisposition to these disorders.
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Osso e Ossos/metabolismo , Metilação de DNA , Osteoartrite do Quadril/genética , Osteoporose/genética , Fraturas por Osteoporose/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Osteoartrite do Quadril/metabolismo , Osteoporose/metabolismo , Fraturas por Osteoporose/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
BACKGROUND: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. METHODS: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments. RESULTS: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. CONCLUSION: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.
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Araquidonato 5-Lipoxigenase , Mutação de Sentido Incorreto , Ligante RANK , Feminino , Humanos , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteosclerose/genética , Osteosclerose/patologia , Osteosclerose/metabolismo , Ligante RANK/metabolismo , Ligante RANK/genética , Transdução de Sinais , Pessoa de Meia-IdadeRESUMO
We hypothesized that miRNAs present in vitreous humor could be a sort of "biological black box," storing information about physiological and environmental circumstances at death. As a proof of concept, we analyzed the vitreous humor miRNA signature to explore its forensic potential applications, such as determining the time of the day at death. The miRNAs present in vitreous humor from individuals who died at daytime or at nighttime were analyzed by quantitative real-time polymerase chain reaction (qPCR) array. Target miRNAs showing significant differences between groups were studied in a larger sample by individual qPCR assays. After array analysis of miRNAs in seven samples, significant expression differences were detected between individuals who died at daytime and at nighttime regarding mir-34c, mir-541, mir-888, mir-484, and mir-142-5p. miR-222 appeared as the best reference gene. The results were replicated in 34 vitreous humor samples, and the day-night differences were confirmed for miR-142-5p and miR-541, suggesting that miRNA levels may be related to either the ambient light or the circadian clock at the time of death. There was no correlation between miRNA levels and the time elapsed after death, suggesting that they were stable at least for 24 h. In conclusion, this report supports the potential forensic utility of the analysis of miRNAs in the vitreous humor in applications such as determining the time of death.
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Autopsia/métodos , Ritmo Circadiano/genética , Genética Forense/métodos , MicroRNAs/análise , Corpo Vítreo/química , Feminino , Perfilação da Expressão Gênica , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Fatores de TempoRESUMO
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.
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The purpose of this study was to analyze the regenerative capacity of mesenchymal stem cells (MSCs) in the treatment of fractures. MSCs extracted from patients with osteoporotic hip fractures or hip osteoarthritis undergoing hip replacement surgeries were cultured and injected into mice with femoral fracture. Two experimental models were established, one for the systemic administration of MSCs (n = 29) and another one for local administration (n = 30). Fracture consolidation was assessed by micro-CT and histology. The degree of radiological consolidation and corticalization was better with MSCs from osteoporosis than from osteoarthritis, being significant after systemic administration (p = 0.0302 consolidation; p = 0.0243 corticalization). The histological degree of consolidation was also better with MSCs from osteoporosis than from osteoarthritis. Differences in histological scores after systemic infusion were as follows: Allen, p = 0.0278; Huo, p = 0.3471; and Bone Bridge, p = 0.0935. After local administration at the fracture site, differences in histological scores were as follows: Allen, p = 0.0764; Huo, p = 0.0256; and Bone Bridge, p = 0.0012. As osteoporosis and control groups were similar, those differences depended on an inhibitory influence by MSCs from patients with osteoarthritis. In conclusion, we found an unexpected impairment of consolidation induced by MSCs from patients with osteoarthritis. However, MSCs from patients with osteoporosis compared favorably with cells from patients with osteoarthritis. In other words, based on this study and previous studies, MSCs from patients with osteoporosis do not appear to have worse bone-regenerating capabilities than MSCs from non-osteoporotic individuals of similar age.
Assuntos
Fraturas do Fêmur , Células-Tronco Mesenquimais , Osteoartrite , Osteoporose , Fraturas por Osteoporose , Animais , Modelos Animais de Doenças , Fraturas do Fêmur/terapia , Consolidação da Fratura , Humanos , CamundongosRESUMO
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-κB 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-α (HIF1-α), 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-1α 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.
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Introduction: Cell-free DNA (cfDNA) methylation is an important molecular biomarker, which provides information about the regulation of gene expression in the tissue of origin. There is an inverse correlation between SOST gene methylation and expression levels. Methods: We analyzed SOST promoter methylation in cfDNA from serum, and compared it with DNA from blood and bone cells from patients undergoing hip replacement surgery. We also measured cfDNA methylation in 28 osteoporotic patients at baseline and after 6 months of antiosteoporotic therapy (alendronate, teriparatide, or denosumab). Results: SOST gene promoter methylation levels in serum cfDNA were very similar to those of bone-derived DNA (79% ± 12% and 82% ± 7%, respectively), but lower than methylation levels in blood cell DNA (87% ± 10%). Furthermore, there was a positive correlation between an individual's SOST DNA methylation values in serum and bone. No differences in either serum sclerostin levels or SOST methylation were found after 6-months of therapy with antiosteoporotic drugs. Conclusions: Our results suggest that serum cfDNA does not originate from blood cells, but rather from bone. However, since we did not confirm changes in this marker after therapy with bone-active drugs, further studies examining the correlation between bone changes of SOST expression and SOST methylation in cfDNA are needed to confirm its potential role as a bone biomarker.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Artroplastia de Quadril , Ácidos Nucleicos Livres/metabolismo , Metilação de DNA , Osteoporose/sangue , Regiões Promotoras Genéticas , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Long noncoding RNAs (lncRNAs) contribute toward regulating gene expression and cell differentiation and may be involved in the pathogenesis of several diseases. The objective of this study was to determine the expression patterns of lncRNAs in bone marrow mesenchymal stem cells (BMSCs) derived from patients with osteoporotic fractures and their relevance to osteogenic function. The BMSCs were isolated from the femoral head of patients with hip fractures (FRX) and controls with osteoarthritis (OA). We found 74 differentially expressed genes between FRX and OA, of which 33 were of the lncRNA type. Among them, 52 genes (20 lncRNAs) were replicated in another independent dataset. The differentially expressed lncRNAs were over-represented among those correlated with differentially expressed protein-coding genes. In addition, the comparison of pre- and post-differentiated paired samples revealed 163 differentially expressed genes, of which 99 were of the lncRNA type. Among them, the overexpression of LINC00341 induced an upregulation of typical osteoblastic genes. In conclusion, the analysis of lncRNA expression in BMSCs shows specific patterns in patients with osteoporotic fractures, as well as changes associated with osteogenic differentiation. The regulation of bone genes through lncRNAs might bring new opportunities for designing bone anabolic therapies in systemic and localized bone disorders.
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Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoporose/genética , RNA Longo não Codificante/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Osteogênese , Osteoporose/patologia , RNA Longo não Codificante/genética , TranscriptomaRESUMO
Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
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Transgenes/genética , Animais , Linhagem Celular , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução Genética , Transgenes/fisiologiaRESUMO
CONTEXT: Osteoporosis has a significant genetic component. The aromatase-dependent conversion of androgenic precursors is the main source of estrogens in postmenopausal women. OBJECTIVE: The objective of the investigation was to study the relationship of a set of single nucleotide polymorphisms (SNPs) of the aromatase gene with osteoporosis and determine their functional influence on gene transcription. DESIGN, PARTICIPANTS, AND METHODS: This was a case-control study including 135 women with vertebral fractures due to postmenopausal osteoporosis and 312 controls. Alleles at four SNPs situated between exons I.2 and 3 were determined by Taqman assays. Total aromatase RNA and differential allelic-specific expression were studied by RT-real time PCR in adipose tissue samples taken from 50 individuals. RESULTS: The SNPs studied were in strong linkage disequilibrium. A common haplotype, present in about half of the population, was identified as being associated with an increased risk of fractures (odds ratio 1.8, 95% confidence interval 1.2-2.8, P = 0.006). There was evidence of differential allelic expression. In heterozygous individuals, transcripts bearing T alleles at rs700518 SNP (which were included in the risk haplotype) were less abundant than those with the alternative C alleles (P < 0.001). Total aromatase expression was four times lower in fat samples from individuals who were homozygotes for the unfavorable alleles than in the opposite homozygotes (P = 0.007). CONCLUSIONS: A common haplotype of aromatase associated with gene expression is also associated with the risk of osteoporotic vertebral fractures in postmenopausal women. These data are in line with the hypothesis that the aromatase-dependent synthesis of estrogens plays an important role in bone homeostasis in postmenopausal women.
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Aromatase/genética , Regulação Enzimológica da Expressão Gênica , Osteoporose Pós-Menopausa/genética , Pós-Menopausa/genética , Adulto , Idoso , Aromatase/metabolismo , Estrogênios/metabolismo , Feminino , Fraturas Espontâneas/epidemiologia , Fraturas Espontâneas/genética , Fraturas Espontâneas/metabolismo , Predisposição Genética para Doença/epidemiologia , Haplótipos , Humanos , Desequilíbrio de Ligação , Osteoporose Pós-Menopausa/epidemiologia , Osteoporose Pós-Menopausa/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
The influence of the orientation of rat bones on their mechanical response is analyzed in this research. 28 femora obtained from 14 Sprague-Dawley rats were subjected to three-point bending tests, comparing the anteroposterior and posteroanterior orientations. The results show that the whole-bone loading capacity of the femora tested in the posteroanterior orientation clearly exceeds that of the anteroposterior oriented bones. Likewise, the intrinsic (tissue-level) loading capacity of the bones tested in the posteroanterior orientation is manifestly higher than that of the bones tested in the opposite direction. The analysis carried out shows that applying beam theory for symmetric cross-sections leads to underestimating the stress state in the cross-section. In this sense, it is generally recommendable to use the non-symmetric beam theory in order to obtain the normal stresses during bending tests. The geometric, intrinsic and global changes resulting from the orientation of the bones was assessed, finding out that it is the variation in the intrinsic properties which explains the change measured in the whole-bone properties. The experimental scope was increased, including 8 additional femora on which a series of Vickers tests were carried out in the anterior and posterior regions of the cross-section. In all cases the hardness obtained in the anterior region is larger than in the posterior region. This result confirms that the mechanical properties of the bone tissue depend on its position in the cross-section and provides a reliable explanation to understand the response of the bones when subjected to bending tests. These results stress the importance of reporting the orientation of the bones in any scientific paper because, otherwise, it would be impossible to properly assess its impact and relevance.
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Osso e Ossos/fisiologia , Estresse Mecânico , Animais , Fenômenos Biomecânicos , Ratos , Ratos Sprague-DawleyRESUMO
Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation.
Assuntos
Metilação de DNA , Células-Tronco Mesenquimais/metabolismo , Fraturas por Osteoporose/genética , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Feminino , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Fraturas por Osteoporose/metabolismoRESUMO
OBJECTIVE: The aromatization of androgenic precursors is the main source of estrogens in postmenopausal women. We tested the hypothesis that allelic variants of the genes coding for aromatase and estrogen receptors (ER) could interact to determine the estrogenic signals on the bone tissue and, consequently, bone mineral density (BMD). DESIGN: Cross-sectional study including 331 postmenopausal women. METHODS: BMD was measured by dual energy x-ray absorptiometry. A CG polymorphism of the aromatase gene as well as three polymorphisms of ERalpha (a TA repeat in the promoter region, a C T single nucleotide polymorphism (SNP) in intron 1 and an AG SNP in exon 8) and a CA repeat polymorphism of ERbeta were studied. RESULTS: Age, body weight and the aromatase genotype were associated with BMD. Allelic variants of ERbeta and the exon 8 of ERalpha did not show a significant association with BMD. The polymorphisms located on the promoter and intron 1 of ERalpha interacted strongly with aromatase. Thus, in women TT homozygous for the ERalpha gene, there was a marked influence of aromatase genotypes on BMD: spine BMD was 0.724 +/- 0.027 g/cm2 in women with CC aromatase alleles and 0.926 +/- 0.032 g/cm2 in those with GG alleles (P < 0.001). Hip BMD in women with CC and GG aromatase genotypes was 0.722 +/- 0.020 and 0.842 +/- 0.026 g/cm2 respectively (P = 0.002). On the contrary, there were no aromatase-related differences in BMD in women with CT/CC alleles of ERalpha. Similarly, aromatase-related differences in BMD were found in women with short alleles at the promoter region of ERalpha, but not in those with long alleles. Both ERalpha polymorphisms were in strong linkage disequilibrium (P < 0.001). CONCLUSION: These results suggest that the interaction between polymorphisms of genes involved in estrogen synthesis and estrogen signaling exerts an important influence on BMD in postmenopausal women, thus helping to explain, in part, its heritable component. Nevertheless, further studies are warranted to confirm this gene-to-gene interaction in other populations.