Age- and Gender-Related Differences in the Geometric Properties and Biomechanical Significance of Intracortical Porosity in the Distal Radius and Tibia

Cortical bone contributes the majority of overall bone mass and bears the bulk of axial loads in the peripheral skeleton. Bone metabolic disorders often are manifested by cortical microstructural changes via osteonal remodeling and endocortical trabecularization. The goal of this study was to characterize intracortical porosity in a cross-sectional patient cohort using novel quantitative computational methods applied to high-resolution peripheral quantitative computed tomography (HR-pQCT) images of the distal radius and tibia. The distal radius and tibia of 151 subjects (57 male, 94 female; 47 ± 16 years of age, range 20 to 78 years) were imaged using HR-pQCT. Intracortical porosity (Ct.Po) was calculated as the pore volume normalized by the sum of the pore and cortical bone volume. Micro–finite element analysis (µFE) was used to simulate 1% uniaxial compression for two scenarios per data set: (1) the original structure and (2) the structure with intracortical porosity artificially occluded. Differential biomechanical indices for stiffness (ΔK), modulus (ΔE), failure load (ΔF), and cortical load fraction (ΔCt.LF) were calculated as the difference between original and occluded values. Regression analysis revealed that cortical porosity, as depicted by HR-pQCT, exhibited moderate but significant age-related dependence for both male and female cohorts (radius ρ = 0.7; tibia ρ = 0.5; p < .001). In contrast, standard cortical metrics (Ct.Th, Ct.Ar, and Ct.vBMD) were more weakly correlated or not significantly correlated with age in this population. Furthermore, differential µFE analysis revealed that the biomechanical deficit (ΔK) associated with cortical porosity was significantly higher for postmenopausal women than for premenopausal women (p < .001). Finally, porosity-related measures provided the only significant decade-wise discrimination in the radius for females in their fifties versus females in their sixties (p < .01). Several important conclusions can be drawn from these results. Age-related differences in cortical porosity, as detected by HR-pQCT, are more pronounced than differences in standard cortical metrics. The biomechanical significance of these structural differences increases with age for men and women and provides discriminatory information for menopause-related bone quality effects. © 2010 American Society for Bone and Mineral Research

miR-125b Promotes Early Germ Layer Specification through Lin28/let-7d and Preferential Differentiation of Mesoderm in Human Embryonic Stem Cells

Unlike other essential organs, the heart does not undergo tissue repair following injury. Human embryonic stem cells (hESCs) grow indefinitely in culture while maintaining the ability to differentiate into many tissues of the body. As such, they provide a unique opportunity to explore the mechanisms that control human tissue development, as well as treat diseases characterized by tissue loss, including heart failure. MicroRNAs are small, non-coding RNAs that are known to play critical roles in the regulation of gene expression. We profiled the expression of microRNAs during hESC differentiation into myocardial precursors and cardiomyocytes (CMs), and determined clusters of human microRNAs that are specifically regulated during this process. We determined that miR-125b overexpression results in upregulation of the early cardiac transcription factors, GATA4 and Nkx2-5, and accelerated progression of hESC-derived myocardial precursors to an embryonic CM phenotype. We used an in silico approach to identify Lin28 as a target of miR-125b, and validated this interaction using miR-125b knockdown. Anti-miR-125b inhibitor experiments also showed that miR-125b controls the expression of miRNA let-7d, likely through the negative regulatory effects of Lin28 on let-7. We then determined that miR-125b overexpression inhibits the expression of Nanog and Oct4 and promotes the onset of Brachyury expression, suggesting that miR-125b controls the early events of human CM differentiation by inhibiting hESC pluripotency and promoting mesodermal differentiation. These studies identified miR-125b as an important regulator of hESC differentiation in general, and the development of hESC-derived mesoderm and cardiac muscle in particular. Manipulation of miR-125b-mediated pathways may provide a novel approach to directing the differentiation of hESC-derived CMs for cell therapy applications

Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease

<div><p>Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.</p></div

miR129-5p expression in hESC-derived cardiomyocytes with hypoxia.

<p>A) hESC-derived cardiomyocytes were cultured in 1% O₂ and relative expression of vascular endothelial growth factor A (VEGFA) and miR129-5p were assessed by qPCR. Upregulation of hypoxia-mediated VEGFA and downregulation of miR129-5p was seen. Data shown are mean±SEM (n = 3); *p<0.05. B) To demonstrate viability of hESC-derived cardiomyocytes grown in 1% O2, cells were stained with trypan blue and percent live cells (trypan blue-excluding) were calculated. Hypoxia did not significantly affect cell viability. Data shown are mean±SEM (n = 3).</p

Bone morphogenetic protein receptor 2 is a target of miR129-5p in HL1 cells.

<p>A) <i>In silico</i> analysis of potential miR129-5p targets using Target Scan Human identified bone morphogenetic protein receptor 2 (BMPR2) as a potential target. Conservation among mammals of the miR129-5p binding site in the 3’UTR of BMPR2 is shown below. Bta, cow; Cfa, dog; Cpo, guinea pig; Eca, horse; Fca, cat; Hsa, human; Laf, elephant; Mml, rhesus; Mmu, mouse; Ocu, rabbit; Ptr, chimpanzee; Rno, rat; Sar, shrew. B) HL1 cells transfected with BMPR2 3’UTR reporter (REP) show increased luciferase activity in 1% O₂ and 100 μM H₂O₂. Co-transfection with miR129-5p (mimic) downregulates luciferase activity. Reporter plasmid expressing luciferase with scrambled BMPR2 3’UTR sequence (REP_scr) was used as control. Data shown are mean±SEM (n = 3); *p < 0.05; **p<0.01. C) HL1 cells were grown in 1% O₂ or 100 μM H₂O₂ and relative expression of BMPR2 in the presence and absence of transfected miR129-5p (mimic) was assessed by qPCR. An increase in BMPR2 expression was seen under both conditions of oxidative stress, and this increase was suppressed by overexpression of miR129-5p. Data shown are mean±SEM (n = 3); *p<0.05; **p<0.01.</p

miR129-5p expression in HL1 cells with oxidative stress.

<p>A) HL1 cells were grown in 1% O₂ or 100 μM H₂O₂ and relative expression of vascular endothelial growth factor A (VEGFA), glucose transporter 1 (GLUT1), and β2-adrenergic receptor (ADBR2) were assessed by qPCR. An increase in expression of all three hypoxia-mediated genes was seen under both conditions. Data shown are mean±SEM (n = 3); *p<0.05; **p<0.01. B) To demonstrate viability of HL1 cells grown in 1% O₂ or H₂O₂, cells were stained with trypan blue and percent live cells (trypan blue-excluding) were calculated. Neither condition significantly affected HL1 cell viability. Data shown are mean±SEM (n = 3). C) miR129-5p expression in HL1 cells grown in 1% O₂ or H₂O₂ was analyzed by qPCR. Under both conditions, miR129-5p expression was downregulated. Data shown are mean±SEM (n = 3); **p<0.01.</p

Serial miR129-5p expression in univentricular heart failure patients.

<p>Serial plasma samples from three patients were evaluated for miR129-5p expression and plotted relative to Ross score at the time of sample collection. Two patients had undergone partial cavopulmonary connection surgery (Stage II), one with morphological RV and one with morphological LV. One patient had undergone completion of cavopulmonary connection (i.e., extracardiac Fontan; Stage III) and had a morphological RV. In each patient, miR129-5p expression was downregulated with increasing Ross score. Data shown are mean ± SEM (n = 3).</p

miRNA expression in pooled samples from univentricular heart failure patients.

<p>Pooled sera from patients with Ross scores <3, 3–5, and >5 were evaluated for expression of miRNAs previously implicated as biomarkers of HF in adult patients. miR129-5p showed a decrease in expression with increasing Ross score. Data shown are mean ± SEM (n = 4–6); **p<0.01.</p

miR129-5p expression by ventricular morphology and stage of palliation.

<p>Sera from individual patients were evaluated for miR129-5p expression and plotted relative to Ross score. Individual plots of patients stratified by ventricular morphology (ALL, all patients; RV, right ventricular morphology; LV, left ventricular morphology) and stage of surgical palliation (Stage I, stabilization of aortic and pulmonary blood flow; Stage II, establishment of partial cavopulmonary circulation between the superior vena cava and pulmonary arteries; Stage III, completion of cavopulmonary circulation) are shown. miR129-5p expression was downregulated with increasing Ross score independent of ventricular morphology or stage of palliation. Mean data are shown (n = 3).</p

Lin28 expression is controlled by miR-125b in differentiating hESCs.

<p>Untransfected hESCs (Cntl) or hESCs transfected with anti-miR-125b inhibitor (anti-125b) were cultured in differentiation medium for 2 or 8 days. <b>A</b>) Cells were analyzed for expression of miR-125b by qPCR. Appropriate downregulation of mature miR-125b was seen in both undifferentiated and differentiating hESCs. Data shown are mean±s.e.m. (N = 3). **, p<0.01; ***, p<0.001. <b>B</b>) Cell lysates were assayed for Lin28 by immunoblot analysis compared to undifferentiated cells (Undiff). Lin28 protein expression was noticeably decreased in hESCs differentiated for 2 or 8 days compared to undifferentiated cells, while transfection with anti-125b induced Lin28 expression in undifferentiated as well as differentiating cells (TOP). Actin was used as a loading control. Representative results are shown. Quantitation of fluorescent signals is shown (BOTTOM). Data shown are mean±s.e.m. (N = 3). *, p<0.05; **, p<0.01; ***, p<0.001.</p