Cardiovascular disease is among the leading causes of death in the US. It stems from the pathological buildup of plaque within the vasculature known as vascular calcification. Medial calcification, or arteriosclerosis is the buildup of plaque within the medial layer of the arteries resulting in artery wall stiffening and reduction of blood flow. Evidence suggests that the vascular smooth muscles cells (VSMCs) that line the medial layer of the arteries, undergo a phenotypic switch to osteoblast-like cells to deposit calcium while in this pathological state. The Wnt/BETA-catenin pathway could potentially play a role in the phenotypic modulation. Inhibition of the Wnt signaling pathway could be a promising approach to combat vascular calcification. Sclerostin (SOST) has been shown to be upregulated during arteriosclerosis in a manner that is indicative of the possible therapeutic potential of the protein. Therefore, we propose to confirm the role of Wnt signaling in vascular calcification and investigate the effects of SOST treatment on vascular calcification.

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Le principal objectif de notre projet de recherche était l’étude approfondie des mécanismes fondamentaux qui sous-tendent l’adaptation de l’os à la contrainte mécanique liée à la gravité, en évaluant les effets de deux modèles d’hypergravité sur le squelette de jeune souris saines. Des expériences de même durée, portant sur des animaux de même souche et de même âge, nous ont permis d’établir des comparaisons entre les différents types d’adaptation du squelette à une hypergravité continue, générée par centrifugation, et une hypergravité intermittente, obtenue par vibrations du corps entier (WBV). Nous avons observé que la centrifugation réduit la résorption et augmente la formation dans l’os trabéculaire alors que les WBV ne découplent pas les activités de résorption et formation qui sont, l’une et l’autre, stimulées. La centrifugation induit une réorganisation de la microarchitecture trabéculaire au niveau du fémur et des vertèbres mais n’a pas d’effet sur les paramètres de masse osseuse corticale. En revanche, les WBV stimulent l’expansion de l’os cortical et augmente sa densité minérale osseuse. Les deux modèles d’hypergravité induisent une diminution de l’expression de la sclérostine (inhibitrice de la formation) et une augmentation de celle de DMP1 (responsable de la minéralisation) dans le cortex fémoral. De plus, les deux modèles augmentent le nombre de vaisseaux sanguins dans la diaphyse fémorale. Sur le plan technique, nous avons développé avec succès une méthode d’IHC quantitative qui nous a permis de détecter et de valider statistiquement de faibles variations, induites par nos expérimentations, dans l’expression de protéines ostéocytaires. Nous pensons que les résultats obtenus en IHC devraient faire l’objet d’une analyse quantitative systématique et fournissons, à cet effet, un outil adapté aux échantillons murins ou humains inclus en paraffine ou MMA / The main focus of the research project was to further study fundamental mechanisms underlying bone adaptation to gravity-induced mechanical loading and to assess effects of two different hypergravity models on skeleton of young healthy mice. Same duration of the experiments and the use of animals of the same type and age allowed us to make comparisons between different skeleton adaptations to continuous hypergravity generated by centrifugation and to intermittent one generated by WBV. We observed that centrifugation reduced resorption and increased formation in trabecular bone, whereas WBV did not uncoupled resorption and formation activities and stimulated both of them simultaneously. Centrifugation resulted in reorganized trabecular microarchitecture in femur and vertebra but had no effect on cortical bone mass-structural parameters. In contrast, WBV stimulated cortical bone geometrical expansion in 3-week experiment and increased cortical mineral density in 9 weeks. Both hypergravity models resulted in lower Sclerostin and higher DMP1 expressions in femoral cortex. Also, both models resulted in higher number of blood vessels in femoral metaphysis, however only centrifugation increased vessels volume. In relation to technical objects of the research project, we successfully developed a method of quantitative IHC, which allowed us to detect and verify statistically even modest alterations of osteocyte protein expressions in our experiments. We believe that IHC results should always be quantitatively analyzed and we provide a tool for both mice and human bone samples embedded in paraffin or MMA

Stroke is now a well-recognised risk factor for hip fracture. The aim of this study was to elucidate the pathophysiological mechanisms by which hip bone loss occurs in hemiplegia and to test the efficacy of a novel pharmaceutical strategy for preserving bone in stroke patients. Patients who were admitted acutely with a first-ever stroke and who remained unable to walk one week later were studied prospectively for 12 months, with a series of bone mineral density measurements of the hips (dual energy X-ray absorptiometry) in the context of a randomised controlled trial. Untreated patients (n=13) experienced a decline in bone mineral density at the hemiplegic hip that was rapid, with the greatest losses in the trochanteric region of the affected side. This bone loss was prevented by the administration of a single 4 mg dose of the intravenous bisphosphonate, zoledronate (n=14) within 35 days of stroke onset. Computed tomography of the hips in 8 untreated patients more than a year after stroke confirmed that the greatest difference between sides was in the trochanteric region. Serum vitamin D measurements in 44 patients with acute stroke were substantially lower than healthy elderly controls, with 77% of patients in the insufficient range, suggesting that vitamin D insufficiency preceded stroke. Histomorphometric analysis of iliac bone biopsies from hemiplegic patients 10 weeks following stroke showed normal erosion parameters, but a striking decrease in the surface extent of osteoid when compared with healthy reference values. Unexpectedly, treatment with zoledronate was associated with a significantly higher osteoid surface compared with placebo treated subjects in cancellous, endocortical and cortical bone. Sclerostin, a newly discovered osteocyte-derived protein was studied using immunohistochemical staining of the bone biopsies. Sclerostin is known to be an inhibitor of active osteoblasts, which led to the hypothesis that in stroke, the proportion of osteocytes expressing sclerostin would be inversely associated with the surface extent of bone formation. Histological analysis revealed widespread expression of sclerostin in osteocytes and their canaliculi in all subjects. However, examining individual osteocytes in relation to bone forming surfaces revealed that newly embedded osteocytes did not express sclerostin until after primary mineralisation. It is proposed that this precise pattern and timing of sclerostin expression by osteocytes allows bone formation to continue locally (during remodelling), but prevents excessive new bone formation elsewhere, as seen in the single gene disorder sclerosteosis.

Sclerosteosis is a severe autosomal recessive sclerosing skeletal dysplasia with no available treatment. It is characterised by excessive bone formation and is caused by mutations in the SOST gene that lead to loss of expression of sclerostin, a protein that acts as a negative regulator of bone formation by binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) Wnt co-receptors to inhibit the canonical Wnt/β-catenin signalling pathway. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. Recombinant wild type mouse sclerostin (mScl) and two novel mScl fusion proteins containing a C-terminal human immunoglobulin G1 (IgG1) antibody fraction crystallisable (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), to increase serum half-life and promote localisation to bone, respectively, were produced and purified using mammalian expression and standard chromatography techniques. These recombinant mScl proteins bound to LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in an in vitro bone nodule formation assay. Pharmacokinetic assessment following a single dose administered to wild type (WT) or SOST knock out (SOST-/-) mice indicated that the presence of the hFc increased protein half-life from less than 5 minutes to at least 1.5 days. The effect of a 6-week treatment with these proteins on the skeletal phenotype of young SOST-/- mice revealed that mScl hFc PD treatment resulted in a modest but significant reduction in trabecular bone volume compared with the vehicle control. There was no marked effect on cortical bone indices assessed by μCT, whole body areal bone mineral density by DXA, or terminal levels of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in any of the SOST-/- or WT treatment groups, possibly due to insufficient exposure. Administration of recombinant mScl hFc PD protein partially corrected the high bone mass phenotype of the SOST-/- mouse, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. However, the modest efficacy indicates that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored. / Thesis (PhD)--University of Pretoria, 2020. / UCB Pharma (Slough, UK) / National Research Foundation (NRF) / University of Pretoria (Pretoria, South Africa) / Paraclinical Sciences / PhD / Unrestricted

In the human genome, the SOST gene codes for a protein sclerostin. Sclerostin is an osteocyte-expressed negative regulator of bone formation. When the SOST gene is not coded, bone formation is reduced in individuals during skeletal maturation. This study utilizes nanoindentation methods to test for the mechanical properties of bones that both express and do not express the SOST gene. 100 transgenic murine femurs were obtained from Lawrence Livermore Labs in the form of 6 and 8 month SOST transgenic mice, 6 and 12 month SOST knockout mice, and wild type control littermates for each of the 4 age groups. Prior to nanoindentation the bones were broken in a previous experiment under three-point bending tests. Samples were embedded in epoxy and polished to a 0.05 micron level before indentation. Results showed significant difference amongst the treatment group effects for maximum load, hardness and elastic modulus. SOST KO mice had significantly higher values for these properties in comparison to the transgenic and wild type littermates. Additionally, side by side limb differences were examined in which there was a significant difference found amongst the treatment groups. Indentations were conducted in the 4 anatomical regions of each femur in expectation of examining any differences amongst them which resulted with no significant findings amongst them. Data from this study will support research which may result in potential new gene therapies targeted for the treatment of bone diseases such as osteoporosis.

Injuries of the hands and wrist are prevalent in many occupations requiring repetitive tasks and may be further aggravated by advancing age; these injuries are termed work related musculoskeletal disorders (WMSDs). Prior studies using an innovative operant rat model of reaching and grasping as a model of WMSDs demonstrated exposure dependent changes in forelimb bones of young adult rats performing repetitive tasks ≤ 3 months. No one has yet to examine if aging enhances forelimb bone degradative changes occurring with WMSDs, or if forelimb bones adapt or degrade further in response to moderate versus high demand repetitive tasks performed for prolonged time periods (up to 24 months). Bone remodeling is a normal biological process that allows bones to adjust to strains. Unfortunately, both aging and inflammation can deregulate the balance between bone resorption and formation. Aging mammals display increased baseline inflammatory-cytokine levels, both systemically and at the tissue level. Several inflammatory cytokines have been shown to stimulate osteoclastogenesis leading to bone resorption and reduced bone formation. We have reported increased production of inflammatory cytokines in serum and musculotendinous tissues of aged animals performing a repetitive reaching and grasping tasks for up to 12 weeks, warranting further examination of whether aged rats performing these tasks have increased bone resorptive changes, compared to young adult rats. We hypothesized that aging would enhance bone degradative changes in our model as a consequence of increased bone inflammatory responses to a moderate demand repetitive task. Therefore, our first aim was to examine forearm grip strength, trabecular and cortical bone quality, and inflammatory cytokine levels in radii of mature (14-18 mo of age) and young adult (2.5-6.5 mo of age) female Sprague Dawley rats after performance of a high repetition low force (HRLF) task for 12 weeks, compared to each other and age-matched controls. We found that mature rats performing a moderate demand repetitive task for 12 weeks had decreased bone formation and quality, particularly cortical bone quality, compared to young adult rats performing the same task, with increased inflammatory and decreased anti-inflammatory responses, and perhaps lower grip strength, as likely contributors. An adaptive bone response was observed in young adult animals performing a moderate level task of high repetition low force for 12 weeks. In contrast, a previous study showed bone degradative changes in young adult rats performing a high demand task of high repetition high force task for 12 weeks. Osteocytes are the mechanosensing cells of bones, and disruption or changes to their environment can lead to apoptosis or molecular changes. In models of forced bone loading to bone fatigue, osteocyte apoptosis increases sclerostin levels and osteoclast recruitment. Increased sclerostin also leads to increased RANKL production. In contrast, low level loading for a short period reduces sclerostin levels and encourages bone formation. We hypothesized that long-term muscle loading at high repetition low force loads would induce further bone adaptation, but that long-term high repetition high force muscle loading would result in detrimental bone loss, as well as alterations in these two bone remodeling proteins, RANKL and sclerostin. Therefore, our second aim was to determine if prolonged performance of a moderate demand upper extremity reaching and grasping task by young adult rats would continue to enhance forelimb bone formation and quality. We hypothesized that continued performance of a high repetition low force (HRLF) task for 24 weeks would lead to increased bone formation. We also hypothesized that RANKL and sclerostin, two proteins that have not been investigated in our rat model of WMSDs, would be reduced in rats performing a HRLF task for 24 weeks, as the bones reach adaptation. We found that 24 week HRLF rats showed several indices of bone formation and adaptation to the task; as well as reduced sclerostin immunoexpression, compared to controls, a reduction that likely contributed to the enhanced bone formation. To expand on this investigation, in our third aim, we investigated the impact of performance of a high repetition high force (HRHF) task for 18 weeks on young adult rat forelimb bones, and on sclerostin and RANKL levels. We observed detrimental trabecular bone remodeling in the radius, including decreased trabeculae bone volume, number and thickness, increased trabecular separation and anisotropy, and a transition to rod-shaped trabeculae in 18-week HRHF task animals, compared to food restricted control rats. In the 18-week HRHF rats, osteoclast numbers increased and osteoblast numbers decreased, concomitant with increased osteocyte apoptosis and empty lacunae, compared to control rats. Also, mRNA and protein levels of RANKL increased and sclerostin decreased in the 18-week HRHF rats, compared to to control rats. Thus, prolonged performance of a high demand task of high repetition high force induced detrimental trabecular bone changes. The increased RANKL likely contributed to these changes, and although sclerostin level decreased, a change that should contribute to enhanced osteoblast activity, bone formation was not rescued. In conclusion, prolonged performance of a HRLF task by young adult rats leads to reduced sclerostin levels and increased bone formation and bone quality. Aged rats performing the same HRLF task showed increased bone degradative changes that might increase fracture risk. In contrast, prolonged performance of a HRHF task by young adult rats leads to increased bone resorption and degradation, changes associated with RANKL expression. Sclerostin levels were reduced by the HRHF task, but failed to rescue bone formation. / Cell Biology

Le groupe des KDIGO a défini un nouveau syndrome regroupant les troubles minéraux et osseux et les calcifications vasculaires au cours de la maladie rénale chronique {TMO-MRC). Les TMO-MRC sont associés à une augmentation du risque de fracture, de calcification vasculaire et à une surmortalité. La quête d'un biomarqueur fiable de la maladie osseuse et des calcifications vasculaires reste le défi du néphrologue. Au cours des dernières années, de nombreuses protéines de l'os ont été associées aux calcifications vasculaires chez les patients urémiques, tels que les phosphatases alcalines osseuses et, plus récemment, la sclérostine. Cette petite protéine est secrétée par l'ostéocyte et inhibe l'ostéoformation en bloquant la voie de signalisation Wnt dans l'ostéoblaste. Il a récemment été suggéré que la sclérostine aurait une activité catabolique sur l'os et serait impliquée dans la déminéralisation du squelette. L'objectif de ce travail était d'étudier la sclérostine au cours de la MRC. Nous avons tout d'abord montré que les concentrations sériques de sclérostine augmentaient avec la baisse du débit de filtration glomérulaire mesurée par la clairance de l'inuline et ce dès le stade 3 de la MRC, indépendamment de l'âge. De plus, cette étude nous a permis de montrer pour la première fois que la phosphorémie était indépendamment et positivement associée à la sclérostine sérique. Ensuite, nous avons retrouvé une association positive et forte entre la concentration sérique de sclérostine et les calcifications vasculaires chez des patients en hémodialyse chronique. Enfin, nous avons montré que les calcifications artérielles étaient significativement associées à une qualité osseuse corticale altérée étudiée en scanner quantitatif de haute résolution. Ces résultats suggèrent que la sclérostine pourrait constituer un messager important dans la relation entre l'os et la paroi vasculaire calcifiée des patients atteints d'une insuffisance rénale chronique terminale / The KDlGO group identified a new syndrome involving mineral and bone disorders and vascular calcification in chronic kidney disease (CKD-MBD}. CKD-MBD are associated with an increased risk of fracture, vascular calcification and increased mortality. The search for a reliable biomarker of bone disease and vascular calcification remains the challenge of the nephrologist. ln recent years, many bone proteins have been associated with vascular calcification in uremic patients, such as bone alkaline phosphatase and more recently sclerostin. This small protein is secreted by the osteocyte and is an inhibitor of bone formation by blocking the Wnt signaling in osteoblasts. lt has recently been suggested that sclerostin has a catabolic activity on bone and is involved in the demineralization of the skeleton. The aim of this research was to study the sclerostin during the course of CKD. We first showed that serum levels of sclerostin increased with the decrease of glomerular filtration rate measured by inulin clearance already from stage 3 chronic kidney disease, regardless of age. Furthermore, in this study, we showed for the first time that serum phosphorus was independently and positively associated with serum sclerostin. Subsequently we found a positive and strong association between serum sclerostin and vascular calcification in maintenance hemodialysis patients. Finally, we have shown that arterial calcification were significantly associated with an altered cortical bone quality studied by high resolution peripheral quantitative computed tomography. These results suggest that sclerostin could be an important messenger in the cross-talk between the bone and the calcified vascular wall in end stage renal disease

Astronauts at are risk of losing 1.0-1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs and diets high in nutrients, potassium, calcium, and vitamin D, all designed to preserve the skeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth factor-1 and its receptor insulin-like growth factor-1 receptor (GFR1) play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

INTRODUÇÃO: O hiperparatireoidismo secundário (HPTS) é uma complicação da doença renal crônica que compromete a integridade do esqueleto. Pacientes com HPS submetidos à paratireoidectomia (PTX) passam de uma condição de níveis séricos de paratormônio (PTH) muito elevados para outra, onde esses níveis hormonais caem drasticamente. Os efeitos da PTX no tecido ósseo são mal compreendidos, especialmente no que se refere às proteínas expressas por osteócitos, como o fator de crescimento de fibroblastos 23 (FGF23), dentin matrix protein 1 (DMP-1), fosfoglicoproteína de matriz extracelular (MEPE), esclerostina, Fator nuclear Kappa beta ligante (RANKL) e osteoprotegerina (OPG), que regulam a remodelação e a mineralização óssea. OBJETIVOS: Caracterizar a expressão óssea dessas proteínas por imuno-histoquímica e estabelecer relações com os dados da histomorfometria do tecido ósseo em pacientes com HPS, antes e após a PTX. MÉTODOS: Estudamos biópsias ósseas obtidas de um banco de biópsias de 23 pacientes com DRC e HPTS, que foram realizadas antes e 12 meses após a PTX. RESULTADOS: A avaliação dos parâmetros histomorfométricos demonstrou uma melhora da microarquitetura óssea, porém com um maior retardo em sua mineralização após a PTX. A análise da expressão das proteínas osteocíticas revelou um aumento significativo na expressão da esclerostina e da OPG e uma diminuição da relação RANKL/OPG após a PTX, sugerindo a participação dessas proteínas na melhora das lesões ósseas decorrentes do HPTS. Observamos um aumento significativo na expressão da OPG no grupo de pacientes que evoluiu com defeito de mineralização somente após a cirurgia, sugerindo a participação dessa proteína no retardo de mineralização óssea desses pacientes. A expressão das proteínas osteocíticas que participam da formação e mineralização óssea apresentou correlação com parâmetros envolvidos na remodelação óssea. CONCLUSÕES: Mudanças significativas na expressão óssea de proteínas osteocíticas que podem potencialmente regular a remodelação e a mineralização óssea foram observadas após a PTX / INTRODUCTION: Secondary hyperparathyroidism (SHPT) is a complication of chronic kidney disease that compromises skeletal integrity. Patients with SHPT undergoing parathyroidectomy (PTX) go from a very high serum parathyroid hormone (PTH) condition to another, where these hormonal levels dramatically fall. The effects of PTX on bone tissue are poorly understood, especially as regards proteins expressed by osteocytes, such as fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP-1), extracellular matrix phosphoglycoprotein (MEPE), sclerostin, Kappa beta ligand nuclear factor (RANKL) and osteoprotegerin (OPG), which regulate bone remodeling and mineralization. OBJECTIVES: Characterize bone expression of these proteins by immunohistochemistry and establish relations with bone tissue histomorphometry data in SHPT patients, before and after PTX. METHODS: We studied bone biopsies obtained from a biopsy database of 23 patients with CKD and SHPT, which were performed before PTX and 12 months after PTX. RESULTS: Evaluation of histomorphometric parameters showed improvement of bone microarchitecture, but with longer delay in mineralization after PTX. Analysis of osteocyte protein expression revealed significant increase in sclerostin and OPG expression and decrease in RANKL/OPG ratio after PTX, suggesting participation of these proteins in improvement of bone lesions due to SHPT. We observed significant increase in OPG expression in the group of patients who evolved with mineralization defect only after surgery, suggesting participation of this protein in bone mineralization delay of these patients. Expression of osteocyte proteins that participate in bone formation and mineralization correlated with parameters involved in bone remodeling. CONCLUSIONS: Significant changes in bone expression of osteocyte proteins that can potentially regulate bone remodeling and mineralization were observed after PTX