The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis.

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

Osteocalcin levels on oral glucose load in women being investigated for polycystic ovary syndrome.

OBJECTIVE: Osteocalcin (OC) might play a hormone-like role in energy metabolism and the regulatory circuit between the pancreas and osteoblasts. Effects of a 75-g oral glucose tolerance test (OGTT) on total OC, undercarboxylated (ucOC), and carboxylated osteocalcin (cOC) in insulin-resistant (IR) and noninsulin-resistant (nIR) premenopausal women was evaluated, and the relationships of changes in OC, ucOC, and cOC with area under the curve (AUC) insulin and the Matsuda index were examined. METHODS: In this cross-sectional study, 105 premenopausal women underwent OGTT; 18 were IR (homeostatic model assessment of insulin resistance [HOMA-IR] > 2.6; (2 with type 2 diabetes, 2 with impaired glucose tolerance), and 87 were nIR (3 with impaired glucose tolerance). Changes in total OC, ucOC, and cOC were evaluated 60 and 120 minutes after glucose loading. RESULTS: At baseline, IR subjects had significantly lower levels of total OC, cOC, and ucOC. In nIR women, total OC decreased by 19% from 18.0 ng/mL (14.5-24.7) at baseline to 14.6 ng/mL (10.9-17.8) after 120 minutes, ucOC decreased by 22% from 3.2 ng/mL (2.1-4.5) to 2.5 ng/mL (1.7-3.5), and cOC decreased by 26% from 14.9 ng/mL (12.1-20.4) to 11.1 ng/mL (9.0-14.5) (P < .001, respectively). No significant decreases were noted in IR subjects. The declines in OC and cOC predicted AUCinsulin (ΔOC: ß = 0.301, P = .001; ΔcOC: ß = 0.315, P < .001) and the Matsuda index (ΔOC: ß = -0.235, P = .003; ΔcOC: ß = -0.245, P = .002). CONCLUSIONS: Glucose intake lowers levels of OC, ucOC, and cOC in nIR women, the extent of which predicts IR and insulin sensitivity in premenopausal women. OC parameters seem suppressed in IR women. There might be a differential osteoblast response to oral glucose in IR and nIR women, with OC reflecting this finding.

Serum Col3-4: A new type III and IV collagen biochemical marker of synovial tissue turnover in patients with rheumatoid arthritis.

The objective of this study was to develop a serum biochemical marker of the degradation of type III and IV collagens, as an index of synovium turnover, and evaluate its performance in patients with rheumatoid arthritis (RA). An enzyme-linked immunosorbent assay for serum synovial collagen fragments (Col3-4) was developed using an antibody recognizing a specific sequence from human type III collagen, which shares 70% homology with type IV collagen. Immunohistochemistry was performed to localize Col3-4 and the matrix metalloprotease MMP-9 which is upregulated in RA synovial fibroblasts in the synovial tissue from a RA patient. Serum Col3-4 was measured in patients with RA (n = 66, 73% women, mean age 62 years, median disease activity score 28 with erythrocyte sedimentation rate (DAS28-ESR) 2.6) and in sex and age matched healthy controls (n = 70, 76% women, mean age 59 years). Col3-4 immunoassay demonstrated adequate analytical performances and recognized a circulating neoepitope resulting from the cleavage of type III and IV collagens. In RA synovium tissue, Col3-4 fragments were localized in the lining layer where destructive fibroblasts are present and around blood vessels rich in type IV collagen. MMP-9 colocalized with Col3-4 staining and efficiently released Col3-4 fragments from type III and type IV collagen digestion. Serum Col3-4 was markedly increased in patients with RA (+240% vs controls, p < 0.0001) and correlated with DAS28-ESR (r = 0.53, p < 0.0001). Patients with RA and active disease (DAS28-ESR > 3.2, n = 20) had 896% (p < 0.0001) higher levels than subjects with low activity (n = 46). Serum Col3-4 is a specific and sensitive biochemical marker reflecting MMP- mediated type III and IV collagen degradation from synovial tissue. Serum Col3-4 levels are markedly increased in patients with RA, particularly in those with active disease, suggesting that it may be useful for the clinical investigation of RA.

Circulating autotaxin levels in healthy teenagers: Data from the Vitados cohort.

Autotaxin (ATX) is a secreted enzyme with a lysophospholipase D activity, mainly secreted by adipocytes and widely expressed. Its major function is to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), an essential bioactive lipid involved in multiple cell processes. The ATX-LPA axis is increasingly studied because of its involvement in numerous pathological conditions, more specifically in inflammatory or neoplastic diseases, and in obesity. Circulating ATX levels gradually increase with the stage of some pathologies, such as liver fibrosis, thus making them a potentially interesting non-invasive marker for fibrosis estimation. Normal circulating levels of ATX have been established in healthy adults, but no data exist at the pediatric age. The aim of our study is to describe the physiological concentrations of circulating ATX levels in healthy teenagers through a secondary analysis of the VITADOS cohort. Our study included 38 teenagers of Caucasian origin (12 males, 26 females). Their median age was 13 years for males and 14 years for females, ranging from Tanner 1 to 5. BMI was at the 25th percentile for males and 54th percentile for females, and median blood pressure was normal. ATX median levels were 1,049 (450-2201) ng/ml. There was no difference in ATX levels between sexes in teenagers, which was in contrast to the male and female differences described in the adult population. ATX levels significantly decreased with age and pubertal status, reaching adult levels at the end of puberty. Our study also suggested positive correlations between ATX levels and blood pressure (BP), lipid metabolism, and bone biomarkers. However, except for LDL cholesterol, these factors were also significantly correlated with age, which might be a confounding factor. Still, a correlation between ATX and diastolic BP was described in obese adult patients. No correlation was found between ATX levels and inflammatory marker C-reactive protein (CRP), Body Mass Index (BMI), and biomarkers of phosphate/calcium metabolism. In conclusion, our study is the first to describe the decline in ATX levels with puberty and the physiological concentrations of ATX levels in healthy teenagers. It will be of utmost importance when performing clinical studies in children with chronic diseases to keep these kinetics in mind, as circulating ATX might become a non-invasive prognostic biomarker in pediatric chronic diseases.

Dysregulation of MicroRNAs in Adult Osteogenesis Imperfecta: The miROI Study.

As epigenetic regulators of gene expression, circulating micro-RiboNucleic Acids (miRNAs) have been described in several bone diseases as potential prognostic markers. The aim of our study was to identify circulating miRNAs potentially associated with the severity of osteogenesis imperfecta (OI) in three steps. We have screened by RNA sequencing for the miRNAs that were differentially expressed in sera of a small group of OI patients versus controls and then conducted a validation phase by RT-qPCR analysis of sera of a larger patient population. In the first phase of miROI, we found 79 miRNAs that were significantly differentially expressed. We therefore selected 19 of them as the most relevant. In the second phase, we were able to validate the significant overexpression of 8 miRNAs in the larger OI group. Finally, we looked for a relationship between the level of variation of the validated miRNAs and the clinical characteristics of OI. We found a significant difference in the expression of two microRNAs in those patients with dentinogenesis imperfecta. After reviewing the literature, we found 6 of the 8 miRNAs already known to have a direct action on bone homeostasis. Furthermore, the use of a miRNA-gene interaction prediction model revealed a 100% probability of interaction between 2 of the 8 confirmed miRNAs and COL1A1 and/or COL1A2. This is the first study to establish the miRNA signature in OI, showing a significant modification of miRNA expression potentially involved in the regulation of genes involved in the physiopathology of OI. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Cathepsin K preferentially solubilizes matured bone matrix.

Bone collagen undergoes a series of enzymatic and nonenzymatic posttranslational modifications with maturation. The aim of this study was to analyze the collagenolytic efficiency of cathepsin K in relation to the extent of bone collagen age. Bone collagen posttranslational maturation was induced in vitro by preincubating bovine fetal cortical bone specimens at 37 °C for different times. The collagen enzymatic cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), the advanced glycation end product pentosidine (PEN), and the native (α) and ß-isomerized C-telopeptide (CTX) isomers were measured in each bone specimen. After extraction, bone collagen was incubated with human recombinant cathepsin K at different concentrations and its collagenolytic activity was measured by the release of hydroxyproline. To assess the affinity of cathepsin K for isomerized and nonisomerized CTX isomers, incubation with cathepsin K was also performed in the presence of various concentrations of a specific inhibitor. We showed that preincubation of bone collagen at 37 °C induces a marked increase in the bone concentration of PYD, DPD, and PEN and of CTX isomerization as reflected by the ratio of α-/ßCTX. This increase was associated with a parallel increase in the efficiency of cathepsin K to solubilize bone collagen. When cathepsin K was incubated in the presence of an inhibitor, the ß-isomerized form of collagen from 3-month- and 8-year-old bovine bone was more susceptible to degradation than the native α form. These results suggest that the collagenolytic activity of cathepsin K may be increased toward more matured bone collagen.

Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study.

OBJECTIVES: In the context of the scarcity of biomarkers for knee osteoarthritis (OA), we examined the associations of prevalent and incident OA with the expression levels of serum miRNAs in subjects with and without OA. METHODS: With a next-generation sequencing approach, we compared the miRome expression of 10 women with knee OA and 10 age-matched healthy subjects. By real-time qPCR, we analyzed the expression levels of 19 miRNAs at baseline selecting 43 women with prevalent knee OA (Kellgren Lawrence score of 2/3), 23 women with incident knee OA over a 4-year follow-up and 67 healthy subjects without prevalent or incident OA matched for age and body mass index. RESULTS: Serum miR-146a-5p was significantly increased in the group of prevalent knee OA compared with controls (relative quantification (RQ); median [Interquartile range] 1.12 [0.73; 1.46] vs 0.85 [0.62; 1.03], p = 0.015). The likelihood of prevalent knee OA was significantly increased (odds ratio [95% confidence interval (CI)] 1.83 [1.21-2.77], p = 0.004) for each quartile increase in serum miR-146a-5p. The women with miR-146a-5p levels above the median (0.851) had a higher risk of prevalent knee OA compared to those below the median [95% CI] 4.62 [1.85-11.5], p = 0.001. Moreover, we found a significant association between the baseline level of serum miR-186-5p and the risk of incident knee OA (Q4 vs Q1-3; odds ratio [95% CI] 6.13 [1.14-32.9], p = 0.034). CONCLUSION: We showed for the first time that miR-146a-5p and miR-186-5p are significantly associated with prevalent and incident knee OA, respectively.

A Signature of Circulating miRNAs Associated With Fibrous Dysplasia of Bone: the mirDys Study.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research.

Selected serum microRNA, abdominal aortic calcification and risk of osteoporotic fracture.

CONTEXT: MicroRNA (miRNA) regulate post-transcriptionally the expression of osteogenesis and angiogenesis associated genes and emerge as potential non-invasive biomarkers in vascular and bone diseases. Severe abdominal aortic calcification (AAC) is associated with higher risk of cardiovascular event and of fragility fracture. OBJECTIVE: To identify miRNA linked to the aggravation of AAC and to incident osteoporotic fracture. DESIGN: Postmenopausal women (>50 years) with available serum at inclusion and data for each outcome (Kauppila score and incident fracture) were selected from the OFELY prospective cohort. We conducted a case-control study in 434 age-matched women, 50% with incident osteoporotic fracture over 20 years of follow-up and a second study in 183 women to explore AAC over 17 years. METHODS: Serum expression of three miRNA involved in vascular calcification and bone turnover regulation (miRs-26a-5p,-34a-5p, and -223-5p) was quantified at baseline by TaqMan Advanced miRNA technology and expressed by relative quantification. Outcomes were the association of miRNA levels with (1) incident osteoporotic fractures during 20 years, (2) AAC aggravation during 17 years. RESULTS: MiRNA level was not associated with incident fractures (miR-26a-5p: 1.06 vs 0.99, p = 0.07; miR-34a-5p: 1.15 vs 1.26, p = 0.35; miR-223a-5p: 1.01 vs 1.05, p = 0.32). 93 women had an increase in Kauppila score over 17 years while 90 did not. None of the miRNAs was associated with an aggravation in AAC (miR-26a-5p: 1.09 vs 1.10, p = 0.95; miR-34a-5p: 0.78 vs 0.73, p = 0.90; miR-223-5p: 0.97 vs 0.78, p = 0.11). CONCLUSIONS: Circulating miR-26a-5p, -34a-5p and -223-5p are not significantly associated with incident fracture and AAC aggravation.

Extracellular post-translational modifications of collagen are major determinants of biomechanical properties of fetal bovine cortical bone.

Mechanical behavior of bone depends on its mass and architecture, and on the material properties of the matrix, which is composed of a mineral phase and an organic component mainly constituted of type I collagen. Mineral accounts largely for the stiffness of bone, whereas type I collagen provides bone its ductility and toughness, i.e., its ability to undergo deformation and absorb energy after it begins to yield. The molecular mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are unclear. We used an in vitro model of fetal bovine cortical bone specimens (n = 44), where the extent of type I collagen cross-linking was modified by incubation at 37 degrees C for 0, 60, 90 and 120 days, keeping constant the architecture and the mineral content. At each incubation time, the following parameters were determined: (1) the bone concentration of enzymatic (pyridinoline; PYD and deoxypyridinoline, DPD) and non-enzymatic (pentosidine) crosslinks by HPLC, (2) the extent of aspartic acid isomerization of the type I collagen C-telopeptide (CTX) by ELISA of native (alpha CTX) and isomerized (beta CTX) forms, (3) the mineral density by DXA, (4) the porosity by micro-computed tomography and (5) the bending and compressive mechanical properties. Incubation of bone specimens at 37 degrees C for 60 days increased the level (per molecule of collagen) of PYD (+98%, P = 0.005), DPD (+42%, P = 0.013), pentosidine (+55-fold, P = 0.005), and the degree of type I collagen C-telopeptide isomerization (+4.9-fold, P = 0.005). These biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption (P < 0.02 for all), with no significant change of bone stiffness. In multivariate analyses, the level of collagen cross-linking was associated with yield stress and post-yield energy absorption independently of bone mineral density, explaining up to 25% of their variance. We conclude that the extent and nature of collagen cross-linking contribute to the mechanical properties of fetal bovine cortical bone independently of bone mineral density.

Lower fracture risk in older men with higher sclerostin concentration: a prospective analysis from the MINOS study.

Sclerostin is synthesized by osteocytes and inhibits bone formation. We measured serum sclerostin levels in 710 men aged 50 years and older. Bone mineral density (BMD) was measured at the lumbar spine, hip, and distal forearm. Serum sclerostin increased with age (unadjusted r = 0.30, p < 0.001). After adjustment for age, weight, and bioavailable 17ß-estradiol, serum sclerostin correlated positively with BMD (r = 0.24 to 0.35, p < 0.001) and negatively with the levels of bone turnover markers (r = - 0.09 to - 0.23, p < 0.05 to 0.001). During a 10-year follow-up, 75 men sustained fragility fractures. Fracture risk was lower in the two upper quintiles of sclerostin combined versus three lower quintiles combined (6.1 versus 13.5%, p < 0.01). We compared fracture risk in the two highest quintiles combined versus three lower quintiles combined using the Cox model adjusted for age, weight, leisure physical activity, BMD, bone width (tubular bones), prevalent fracture, prevalent falls, ischemic heart disease, and severe abdominal aortic calcification. Men with higher sclerostin concentration had lower fracture risk (adjusted for hip BMD, hazard ratio [HR] = 0.55, 95% confidence interval [CI] 0.31 to 0.96, p < 0.05). The results were similar in 47 men with major fragility fractures (adjusted for lumbar spine BMD: HR = 0.39, 95% CI 0.17 to 0.90, p < 0.05). Men who had higher sclerostin and higher BMD (two highest quintiles) had lower risk of fracture compared with men who had lower BMD and lower sclerostin levels (three lower quintiles) (HR = 0.24, 95% CI 0.10 to 0.62, p < 0.005). Circulating sclerostin was not associated with mortality rate or the incidence of major cardiovascular events. Thus, in older men, higher serum sclerostin levels are associated with lower risk of fracture, higher BMD, and lower bone turnover rate.