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1.
J Biol Chem ; 299(5): 104635, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36963490

RESUMEN

Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate, or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mechanistic target of rapamycin complex 1 is activated by purine accumulation, mechanistic target of rapamycin complex 1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation.


Asunto(s)
Adipogénesis , Metabolismo de los Lípidos , Nucleótidos , Animales , Ratones , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Nucleótidos/biosíntesis , Purinas/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Transducción de Señal
2.
Infect Immun ; 91(4): e0052922, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-36877063

RESUMEN

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia on S. aureus virulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing to S. aureus pathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential for S. aureus survival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential for S. aureus survival in hyperglycemic mice was the gene encoding superoxide dismutase A (sodA), one of two S. aureus superoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that a sodA mutant exhibits attenuated survival in vitro in high glucose and in vivo during osteomyelitis in hyperglycemic mice. SodA therefore plays an important role during growth in high glucose and promotes S. aureus survival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing to S. aureus survival during hyperglycemic infection.


Asunto(s)
Hiperglucemia , Osteomielitis , Infecciones Estafilocócicas , Animales , Ratones , Staphylococcus aureus/genética , Genes Bacterianos , Ratones Obesos , Hiperglucemia/genética , Glucosa , Infecciones Estafilocócicas/microbiología , Osteomielitis/microbiología
3.
Dev Biol ; 470: 136-146, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33217406

RESUMEN

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.


Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Articulaciones/embriología , Miositis Osificante/embriología , Miositis Osificante/metabolismo , Dedos del Pie/embriología , Animales , Tipificación del Cuerpo , Condrogénesis , Modelos Animales de Enfermedad , Miembro Anterior/anomalías , Miembro Anterior/embriología , Factor 5 de Diferenciación de Crecimiento/metabolismo , Placa de Crecimiento/embriología , Miembro Posterior/anomalías , Miembro Posterior/embriología , Articulaciones/anomalías , Articulaciones/metabolismo , Ratones , Osteogénesis , Transducción de Señal , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Células Madre/fisiología , Dedos del Pie/anomalías
4.
Infect Immun ; 90(11): e0041722, 2022 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-36226943

RESUMEN

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9-/-) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9-/- mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.


Asunto(s)
Osteomielitis , Infecciones Estafilocócicas , Ratones , Animales , Staphylococcus aureus , Receptor Toll-Like 2/genética , Receptor Toll-Like 9 , Infecciones Estafilocócicas/microbiología , Osteomielitis/microbiología , Receptores Toll-Like , Ratones Noqueados , Ratones Endogámicos C57BL
5.
Mol Genet Metab ; 133(4): 378-385, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34154922

RESUMEN

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.


Asunto(s)
Enfermedades Óseas/fisiopatología , Progresión de la Enfermedad , Mucopolisacaridosis VII/complicaciones , Columna Vertebral/patología , Animales , Animales Recién Nacidos , Enfermedades Óseas/genética , Huesos/patología , Perros , Femenino , Crecimiento y Desarrollo , Masculino , Mucopolisacaridosis VII/genética , Osteogénesis
6.
Mol Cell ; 42(2): 250-60, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21458342

RESUMEN

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.


Asunto(s)
Diseño Asistido por Computadora , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas/química , Sitios de Unión , Técnicas de Química Analítica , Modelos Moleculares , Peso Molecular , Mutación , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas/genética , Proteínas/metabolismo , Propiedades de Superficie
7.
Mol Genet Metab ; 118(4): 232-43, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27296532

RESUMEN

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Skeletal disease is common in MPS patients, with the severity varying both within and between subtypes. Within the spectrum of skeletal disease, spinal manifestations are particularly prevalent. Developmental and degenerative abnormalities affecting the substructures of the spine can result in compression of the spinal cord and associated neural elements. Resulting neurological complications, including pain and paralysis, significantly reduce patient quality of life and life expectancy. Systemic therapies for MPS, such as hematopoietic stem cell transplantation and enzyme replacement therapy, have shown limited efficacy for improving spinal manifestations in patients and animal models. Therefore, there is a pressing need for new therapeutic approaches that specifically target this debilitating aspect of the disease. In this review, we examine how pathological abnormalities affecting the key substructures of the spine - the discs, vertebrae, odontoid process and dura - contribute to the progression of spinal deformity and symptomatic compression of neural elements. Specifically, we review current understanding of the underlying pathophysiology of spine disease in MPS, how the tissues of the spine respond to current clinical and experimental treatments, and discuss future strategies for improving the efficacy of these treatments.


Asunto(s)
Glicosaminoglicanos/metabolismo , Mucopolisacaridosis/fisiopatología , Enfermedades de la Columna Vertebral/fisiopatología , Terapia de Reemplazo Enzimático , Humanos , Mucopolisacaridosis/terapia , Calidad de Vida , Enfermedades de la Columna Vertebral/terapia , Columna Vertebral/fisiopatología
8.
Mol Genet Metab ; 116(3): 195-203, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26422116

RESUMEN

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.


Asunto(s)
Condrocitos/citología , Epífisis/citología , Mucopolisacaridosis VII/complicaciones , Mucopolisacaridosis VII/fisiopatología , Osteogénesis , Animales , Enfermedades Óseas/etiología , Enfermedades Óseas/fisiopatología , Diferenciación Celular , Perros , Glicosaminoglicanos/metabolismo , Humanos , Hipertrofia , Columna Vertebral/fisiología , Microtomografía por Rayos X
9.
Heliyon ; 10(15): e35103, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39170274

RESUMEN

Perfusion of porous scaffolds transports cells to the surface to yield cellular constructs for 3D models of disease and for tissue engineering applications. While ceramic scaffolds mimic the structure and composition of trabecular bone, their opacity and tortuous pores limit the penetration of light into the interior. Scaffolds that are both perfusable and amenable to fluorescence microscopy are therefore needed to visualize the spatiotemporal dynamics of cells in the bone microenvironment. In this study, a hybrid injection molding approach was designed to enable rapid prototyping of collector arrays with variable configurations that are amenable to longitudinal imaging of attached human mesenchymal stem cells (hMSCs) using fluorescence microscopy. Cylindrical collectors were arranged in an array that is permeable to perfusion in the xy-plane and to light in the z-direction for imaging from below. The effects of the collector radius, number, and spacing on the collection efficiency of perfused hMSCs was simulated using computational fluid dynamics (CFD) and measured experimentally using fluorescence microscopy. The effect of collector diameter on simulated and experimental cell collection efficiencies followed a trend similar to that predicted by interception theory corrected for intermolecular and hydrodynamic forces for the arrays with constant collector spacing. In contrast, arrays designed with constant collector number yielded collection efficiencies that poorly fit the trend with collector radius predicted by interception theory. CFD simulations of collection efficiency agreed with experimental measurements within a factor of two. These findings highlight the utility of CFD simulations and hybrid injection molding for rapid prototyping of collector arrays to optimize the longitudinal imaging of cells without the need for expensive and time-consuming tooling.

10.
Cell Host Microbe ; 31(10): 1604-1619.e10, 2023 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-37794592

RESUMEN

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.


Asunto(s)
Microbiota , Obesidad Infantil , Humanos , Niño , Animales , Ratones , Metabolismo de los Lípidos , Dieta Alta en Grasa/efectos adversos , Antibacterianos , Poliésteres , Ratones Endogámicos C57BL
11.
Front Cell Dev Biol ; 10: 841831, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35359439

RESUMEN

The intervertebral disc (IVD) is the fibrocartilaginous joint located between each vertebral body that confers flexibility and weight bearing capabilities to the spine. The IVD plays an important role in absorbing shock and stress applied to the spine, which helps to protect not only the vertebral bones, but also the brain and the rest of the central nervous system. Degeneration of the IVD is correlated with back pain, which can be debilitating and severely affects quality of life. Indeed, back pain results in substantial socioeconomic losses and healthcare costs globally each year, with about 85% of the world population experiencing back pain at some point in their lifetimes. Currently, therapeutic strategies for treating IVD degeneration are limited, and as such, there is great interest in advancing treatments for back pain. Ideally, treatments for back pain would restore native structure and thereby function to the degenerated IVD. However, the complex developmental origin and tissue composition of the IVD along with the avascular nature of the mature disc makes regeneration of the IVD a uniquely challenging task. Investigators across the field of IVD research have been working to elucidate the mechanisms behind the formation of this multifaceted structure, which may identify new therapeutic targets and inform development of novel regenerative strategies. This review summarizes current knowledge base on IVD development, degeneration, and regenerative strategies taken from traditional genetic approaches and omics studies and discusses the future landscape of investigations in IVD research and advancement of clinical therapies.

12.
Front Cell Infect Microbiol ; 12: 985467, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36204648

RESUMEN

Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. Staphylococcus aureus is the most frequently isolated pathogen in osteomyelitis and triggers significant bone loss. Hypoxia-inducible factor (HIF) signaling has been implicated in antibacterial immune responses as well as bone development and repair. In this study, the impact of bone cell HIF signaling on antibacterial responses and pathologic changes in bone architecture was explored using genetic models with knockout of either Hif1a or a negative regulator of HIF-1α, Vhl. Deletion of Hif1a in osteoblast-lineage cells via Osx-Cre (Hif1aΔOB ) had no impact on bacterial clearance or pathologic changes in bone architecture in a model of post-traumatic osteomyelitis. Knockout of Vhl in osteoblast-lineage cells via Osx-Cre (VhlΔOB ) caused expected increases in trabecular bone volume per total volume (BV/TV) at baseline and, intriguingly, did not exhibit an infection-mediated decline in trabecular BV/TV, unlike control mice. Despite this phenotype, bacterial burdens were not affected by loss of Vhl. In vitro studies demonstrated that transcriptional regulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG) is altered in osteoblast-lineage cells with knockout of Vhl. After observing no impact on bacterial clearance with osteoblast-lineage conditional knockouts, a LysM-Cre model was used to generate Hif1aΔMyeloid and VhlΔMyeloid mouse models to explore the impact of myeloid cell HIF signaling. In both Hif1aΔMyeloid and VhlΔMyeloid models, bacterial clearance was not impacted. Moreover, minimal impacts on bone architecture were observed. Thus, skeletal HIF signaling was not found to impact bacterial clearance in our mouse model of post-traumatic osteomyelitis, but Vhl deletion in the osteoblast lineage was found to limit infection-mediated trabecular bone loss, possibly via altered regulation of RANKL-OPG gene transcription.


Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Osteomielitis , Animales , Antibacterianos , Hueso Esponjoso , Citocinas , Ligandos , Ratones , Ratones Noqueados , Osteoprotegerina/genética , Receptor Activador del Factor Nuclear kappa-B , Staphylococcus aureus/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética
13.
Bone ; 154: 116237, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34695616

RESUMEN

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14 days to 6 months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6 months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.


Asunto(s)
Enfermedades Óseas , Mucopolisacaridosis I , Animales , Enfermedades Óseas/terapia , Modelos Animales de Enfermedad , Perros , Humanos , Litio/uso terapéutico , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/patología , Vértebras Torácicas/patología , Microtomografía por Rayos X
14.
Cartilage ; 12(4): 512-525, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-30971109

RESUMEN

OBJECTIVE: Skeletal tissues such as intervertebral disc and articular cartilage possess limited innate potential to regenerate, in part due to their avascularity and low cell density. Despite recent advances in mesenchymal stem cell (MSC)-based disc and cartilage regeneration, key challenges remain, including the sensitivity of these cells to in vivo microenvironmental stress such as low oxygen and limited nutrition. The objective of this study was to investigate whether preconditioning with hypoxia and/or transforming growth factor-ß 3 (TGF-ß3) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment. DESIGN: MSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-ß3, and the global effects on gene expression were examined using microarrays. Subsequently, the effects of preconditioning on MSC survival and extracellular matrix production were examined using low oxygen and nutrient-limited pellet culture experiments. RESULTS: Hypoxic preconditioning resulted in upregulation of genes associated with growth, cell-cell signaling, metabolism, and cell stress response pathways, and significantly enhanced MSC survival for all donors in low oxygen and nutrient-limited pellet culture. In contrast, TGF-ß3 preconditioning diminished survival. The nature and magnitude of the effects of preconditioning with either hypoxia or TGF-ß3 on glycosaminoglycan production were donor dependent. CONCLUSIONS: These results strongly support the use of hypoxic preconditioning to improve postimplantation MSC survival in avascular tissues such as disc and cartilage.


Asunto(s)
Cartílago Articular , Células Madre Mesenquimatosas , Animales , Médula Ósea , Bovinos , Hipoxia , Nutrientes , Oxígeno , Factor de Crecimiento Transformador beta3
15.
Acta Biomater ; 130: 485-496, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34129957

RESUMEN

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.


Asunto(s)
Durapatita , Osteogénesis , Animales , Regeneración Ósea , Cartílago , Poliuretanos , Ratas , Ratas Sprague-Dawley , Ingeniería de Tejidos , Andamios del Tejido
16.
Biochim Biophys Acta Mol Basis Dis ; 1865(2): 322-328, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30453012

RESUMEN

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3ß, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.


Asunto(s)
Ceramidas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Lipofuscinosis Ceroideas Neuronales/metabolismo , Proteína Fosfatasa 2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Glucosilceramidasa/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/deficiencia , Modelos Biológicos , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingolípidos/metabolismo
17.
Bone ; 128: 115042, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31442675

RESUMEN

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ß-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/ß-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.


Asunto(s)
Enfermedades Óseas/metabolismo , Mucopolisacaridosis VII/metabolismo , Osteogénesis/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Condrocitos/metabolismo , Perros , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Mutación Missense/genética , Osteogénesis/genética , Análisis de Componente Principal , Secuenciación del Exoma
18.
J Orthop Res ; 36(10): 2771-2779, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29687490

RESUMEN

Low back pain due to disc degeneration is a major cause of morbidity and health care expenditures worldwide. While stem cell-based therapies hold promise for disc regeneration, there is an urgent need to develop improved in vivo animal models to further develop and validate these potential treatments. The objectives of this study were to characterize a percutaneous needle injury model of intervertebral disc degeneration in the mouse caudal spine, and compare two non-invasive quantitative imaging techniques, microcomputed tomography and magnetic resonance imaging (MRI), as effective measures of disc degeneration in this model. Percutaneous needle injury of mouse caudal discs was undertaken using different needle sizes and injury types (unilateral or bilateral annulus fibrosus (AF) puncture). Mice were euthanized 4 weeks post-injury, and MRI and microcomputed tomography were used to determine T2 relaxation time of the NP and disc height index, respectively. Disc condition was then further assessed using semi-quantitative histological grading. Bilateral AF puncture with either 27 or 29G needles resulted in significantly lower T2 relaxation times compared to uninjured controls, while disc height index was not significantly affected by any injury type. There was a strong, inverse linear relationship between histological grade and NP T2 relaxation time. In this study, we demonstrated that quantitative MRI can detect disc degeneration in the mouse caudal spine 4 weeks following percutaneous needle injury, and may therefore serve as a surrogate for histology in longitudinal studies of both disc degeneration and cell-based therapies for disc regeneration using this model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2771-2779, 2018.


Asunto(s)
Modelos Animales de Enfermedad , Degeneración del Disco Intervertebral/diagnóstico por imagen , Imagen por Resonancia Magnética , Microtomografía por Rayos X , Animales , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/patología , Masculino , Ratones Endogámicos C57BL
19.
J Orthop Res ; 36(5): 1356-1369, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29227558

RESUMEN

Degenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. CLINICAL SIGNIFICANCE: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356-1369, 2018.


Asunto(s)
Matriz Extracelular/metabolismo , Redes Reguladoras de Genes , Disco Intervertebral/metabolismo , Análisis de Secuencia de ARN , Adulto , Anciano , Anillo Fibroso/metabolismo , Humanos , Persona de Mediana Edad
20.
Sci Rep ; 7(1): 10504, 2017 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-28874804

RESUMEN

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.


Asunto(s)
Perfilación de la Expresión Génica , Notocorda/citología , Núcleo Pulposo/embriología , Núcleo Pulposo/metabolismo , Transcriptoma , Animales , Embrión de Mamíferos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Transgénicos , ARN Mensajero , Reproducibilidad de los Resultados , Transducción de Señal , Columna Vertebral/embriología , Columna Vertebral/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
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