RESUMEN
BACKGROUND: The 2022 multi-country outbreak of monkeypox (mpox) resulted in blood collection and public health agencies closely monitoring for changes in transmission dynamics that could pose a threat to the blood supply. While mpox virus (MPXV) is not known to be transfusion transmissible, there have been several studies demonstrating the detection of MPXV in blood. We evaluated the performance characteristics of a research use only (RUO) nucleic acid amplification test for MPXV. The assay was developed to detect MPXV DNA in plasma and serum specimens from human blood donors. METHODS AND MATERIALS: The sensitivity of the RUO MPXV Assay was determined using a synthetic DNA sequence, purified full-length genomic DNA, and a chemically inactivated virus. Specificity was determined using fresh plasma samples collected from blood donors during the outbreak. Plasma samples collected from donors considered at increased risk for exposure to mpox were also tested. RESULTS: For sensitivity, the 95% limit of detection (LOD) ranged from 0.26 copies/mL (inactivated virus) to 31.65 copies/mL (synthetic DNA) to 166.61 copies/mL (for full-length DNA). All donor samples tested with the RUO MPXV Assay were nonreactive, resulting in a specificity of 100% (95% CI, 99.93%-100.00%). DISCUSSION: The RUO MPXV Assay was developed as a potential blood donation screening assay in response to the outbreak. While not directly comparable, the 95% LOD fiducial limits obtained from partial- and full-length DNA analysis were similar to other manufacturers' MPXV assays. Additionally, this assay demonstrated high specificity for screening blood donors.
Asunto(s)
Mpox , Ácidos Nucleicos , Humanos , Donantes de Sangre , Mpox/diagnóstico , Mpox/epidemiología , Donación de Sangre , ADNRESUMEN
BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.
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Metabolismo Energético , Resistencia a la Insulina/genética , Obesidad/genética , Monoéster Fosfórico Hidrolasas/genética , Animales , Colina/metabolismo , Glucosa/metabolismo , Homeostasis , Masculino , Ratones , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
OBJECTIVE: Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium. APPROACH AND RESULTS: A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0µm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330µm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622µm2) and lipids (30754 vs. 77317µm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls. CONCLUSIONS: Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.
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Fosfatasa Alcalina/metabolismo , Enfermedad de la Arteria Coronaria/etiología , Hiperlipoproteinemia Tipo II/patología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Análisis Químico de la Sangre , Peso Corporal/efectos de los fármacos , Calcificación Fisiológica , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Citocinas/sangre , Dieta Aterogénica , Modelos Animales de Enfermedad , Ecocardiografía , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Inhibidores Enzimáticos/farmacología , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/enzimología , Miocardio/metabolismo , Miocardio/patología , Efecto Placebo , Receptores de LDL/genética , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.
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Calcificación Fisiológica , Vesículas Extracelulares/metabolismo , Odontoblastos/fisiología , Fosfatos/metabolismo , Animales , Biomarcadores/metabolismo , Calcio/metabolismo , Línea Celular , Matriz Extracelular/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , OsteogénesisRESUMEN
We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that â¼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to â¼50% for the Phospho1(-/-) MVs and â¼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.
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Densidad Ósea/fisiología , Calcificación Fisiológica/fisiología , Condrocitos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Animales , Ratones , Ratones Noqueados , Monoéster Fosfórico Hidrolasas/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genéticaRESUMEN
BACKGROUND: Ectopic vascular calcification is a common condition associated with aging, atherosclerosis, diabetes, and/or chronic kidney disease. Smooth muscle cells are the best characterized source of osteogenic progenitors in the vasculature; however, recent studies suggest that cells of endothelial origin can also promote calcification. To test this, we sought to increase the osteogenic potential of endothelial cells by overexpressing tissue-nonspecific alkaline phosphatase (TNAP), a key enzyme that regulates biomineralization, and to determine the pathophysiological effect of endothelial TNAP on vascular calcification and cardiovascular function. METHODS AND RESULTS: We demonstrated previously that mice transgenic for ALPL (gene encoding human TNAP) develop severe arterial medial calcification and reduced viability when TNAP is overexpressed in smooth muscle cells. In this study, we expressed the ALPL transgene in endothelial cells following endothelial-specific Tie2-Cre recombination. Mice with endothelial TNAP overexpression survived well into adulthood and displayed generalized arterial calcification. Genes associated with osteochondrogenesis (Runx2, Bglap, Spp1, Opg, and Col2a1) were upregulated in the aortas of endothelial TNAP animals compared with controls. Lesions in coronary arteries of endothelial TNAP mice showed immunoreactivity to Runx2, osteocalcin, osteopontin, and collagen II as well as increased deposition of sialoproteins revealed by lectin staining. By 23 weeks of age, endothelial TNAP mice developed elevated blood pressure and compensatory left ventricular hypertrophy with preserved ejection fraction. CONCLUSIONS: This study presented a novel genetic model demonstrating the osteogenic potential of TNAP-positive endothelial cells in promoting pathophysiological vascular calcification.
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Fosfatasa Alcalina/metabolismo , Calcinosis/metabolismo , Endotelio Vascular/metabolismo , Enfermedad Arterial Periférica/metabolismo , Animales , Calcinosis/etiología , Calcinosis/patología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Femenino , Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Enfermedad Arterial Periférica/etiología , Enfermedad Arterial Periférica/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Newts have the ability to repeatedly regenerate their lens even during ageing. However, it is unclear whether this regeneration reflects an undisturbed genetic activity. To answer this question, we compared the transcriptomes of lenses, irises and tails from aged newts that had undergone lens regeneration 19 times with the equivalent tissues from young newts that had never experienced lens regeneration. Our analysis indicates that repeatedly regenerated lenses showed a robust transcriptional program comparable to young never-regenerated lenses. In contrast, the tail, which was never regenerated, showed gene expression signatures of ageing. Our analysis strongly suggests that, with respect to gene expression, the regenerated lenses have not deviated from a robust transcriptional program even after multiple events of regeneration throughout the life of the newt. In addition, our study provides a new paradigm in biology, and establishes the newt as a key model for the study of regeneration in relation to ageing.
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Cristalino/fisiología , Regeneración , Salamandridae/genética , Transcripción Genética , AnimalesRESUMEN
Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl(-/-) mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, vertebral and tooth mineralization defects in Alpl(-/-) mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl(-/-) mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microscopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl(-/-) mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl(-/-) calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.
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Fosfatasa Alcalina/genética , Fosfatasa Alcalina/uso terapéutico , Craneosinostosis/tratamiento farmacológico , Terapia de Reemplazo Enzimático , Terapia Enzimática , Hipofosfatasia/tratamiento farmacológico , Inmunoglobulina G/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Células 3T3 , Animales , Huesos/patología , Calcificación Fisiológica , Proliferación Celular , Anomalías Craneofaciales/genética , Craneosinostosis/fisiopatología , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Hipofosfatasia/genética , Hipofosfatasia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatos/química , Microtomografía por Rayos XRESUMEN
Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl(-/-)) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl(-/-) mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl(-/-) mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, µCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl(-/-) mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.
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Fosfatasa Alcalina/genética , Hipofosfatasia/genética , Animales , Calcificación Fisiológica , Cemento Dental , Esmalte Dental/patología , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Odontogénesis/fisiología , Osteomalacia/patología , Fenotipo , Raquitismo/patología , Microtomografía por Rayos XRESUMEN
Medial vascular calcification (MVC) is a pathological phenomenon that causes vascular stiffening and can lead to heart failure; it is common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases. These conditions share the common feature of tissue-nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X-linked manner. Hemizygous overexpressor male mice (Tagln-Cre(+/-) ; Hprt(ALPL) (/Y) or TNAP-OE) show extensive vascular calcification, high blood pressure, and cardiac hypertrophy, and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln-Cre(+/-) ; Hprt(ALPL) (/-) ) female TNAP-OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP-OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug-like pharmacokinetic characteristics. TNAP-OE mice were treated with the prototypical TNAP inhibitor SBI-425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle-treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target.
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Fosfatasa Alcalina/metabolismo , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiopatología , Calcificación Vascular/enzimología , Calcificación Vascular/fisiopatología , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Aorta/enzimología , Aorta/patología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Masculino , Ratones Transgénicos , Músculo Liso Vascular/efectos de los fármacos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Resultado del Tratamiento , Ultrasonografía , Calcificación Vascular/sangre , Calcificación Vascular/diagnóstico por imagenRESUMEN
TRPS1 (tricho-rhino-phalangeal syndrome) is a unique GATA-type transcription factor that acts as a transcriptional repressor. TRPS1 deficiency and dysregulated TRPS1 expression result in skeletal and dental abnormalities implicating TRPS1 in endochondral bone formation and tooth development. Moreover, patients with tricho-rhino-phalangeal syndrome frequently present with low bone mass indicating TRPS1 involvement in bone homeostasis. In addition, our previous data demonstrated accelerated mineralization of the perichondrium in Trps1 mutant mice and impaired dentin mineralization in Col1a1-Trps1 transgenic mice, implicating Trps1 in the mineralization process. To understand the role of Trps1 in the differentiation and function of cells producing mineralized matrix, we used a preodontoblastic cell line as a model of dentin mineralization. We generated both Trps1-deficient and Trps1-overexpressing stable cell lines and analyzed the progression of mineralization by alkaline phosphatase and alizarin red staining. As predicted, based on our previous in vivo data, delayed and decreased mineralization of Trps1-overexpressing odontoblastic cells was observed when compared with control cells. This was associated with down-regulation of genes regulating phosphate homeostasis. Interestingly, Trps1-deficient cells lost the ability to mineralize and demonstrated decreased expression of several genes critical for initiating the mineralization process, including Alpl and Phospho1. Based on these data, we have concluded that Trps1 serves two critical and context-dependent functions in odontoblast-regulated mineralization as follows: 1) Trps1 is required for odontoblast maturation by supporting expression of genes crucial for initiating the mineralization process, and 2) Trps1 represses the function of mature cells and, consequently, restricts the extent of extracellular matrix mineralization.
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Proteínas de Unión al ADN/metabolismo , Dentina/crecimiento & desarrollo , Dentina/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Proteínas de Unión al ADN/genética , Dentinogénesis , Humanos , Odontoblastos/citología , Odontoblastos/metabolismo , Proteínas Represoras , Factores de Transcripción/genéticaRESUMEN
PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) have nonredundant functions during skeletal mineralization. Although TNAP deficiency (Alpl(-/-) mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PPi ), comparably elevated levels of PPi in Phospho1(-/-) mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PPi in Alpl(-/-) mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1(-/-) mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p-OPN) peptides in Phospho1(-/-) mice, suggesting that accumulation of p-OPN causes the skeletal abnormalities in Phospho1(-/-) mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1(-/-) as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1(-/-) mice at 3 months of age. Mineralization assays comparing [Phospho1(-/-) ; Spp1(-/-) ], Phospho1(-/-) , and Spp1(-/-) chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1(-/-) ; Spp1(-/-) ] mice. Thus, although Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PPi and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PPi and elevated p-OPN levels causing the respective skeletal phenotypes in Alpl(-/-) and Phospho1(-/-) mice.
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Envejecimiento , Densidad Ósea/genética , Calcificación Fisiológica/genética , Condrocitos/metabolismo , Osteopontina/deficiencia , Fenotipo , Monoéster Fosfórico Hidrolasas/deficiencia , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Antígenos de Diferenciación , Condrocitos/patología , Ratones , Ratones NoqueadosRESUMEN
Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E) hydrolyze extracellular AMP to adenosine in dorsal root ganglia (DRG) neurons and in the dorsal spinal cord. Previously, we found that adenosine production was reduced, but not eliminated, in Papâ»/â»/Nt5eâ»/â» double knock-out (dKO) mice, suggesting that a third AMP ectonucleotidase was present in these tissues. Here, we found that tissue-nonspecific alkaline phosphatase (TNAP, encoded by the Alpl gene) is expressed and functional in DRG neurons and spinal neurons. Using a cell-based assay, we found that TNAP rapidly hydrolyzed extracellular AMP and activated adenosine receptors. This activity was eliminated by MLS-0038949, a selective pharmacological inhibitor of TNAP. In addition, MLS-0038949 eliminated AMP hydrolysis in DRG and spinal lamina II of dKO mice. Using fast-scan-cyclic voltammetry, we found that adenosine was rapidly produced from AMP in spinal cord slices from dKO mice, but virtually no adenosine was produced in spinal cord slices from dKO mice treated with MLS-0038949. Last, we found that AMP inhibited excitatory neurotransmission via adenosine A1 receptor activation in spinal cord slices from wild-type, Papâ»/â», Nt5eâ»/â», and dKO mice, but failed to inhibit neurotransmission in slices from dKO mice treated with MLS-0038949. These data suggest that triple elimination of TNAP, PAP, and NT5E is required to block AMP hydrolysis to adenosine in DRG neurons and dorsal spinal cord. Moreover, our data reveal that TNAP, PAP, and NT5E are the main AMP ectonucleotidases in primary somatosensory neurons and regulate physiology by metabolizing extracellular purine nucleotides.
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5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Fosfatasa Alcalina/metabolismo , Ganglios Espinales/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Fosfatasa Ácida , Animales , Proteínas Ligadas a GPI/metabolismo , Ganglios Espinales/química , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Médula Espinal/química , Médula Espinal/metabolismo , Distribución Tisular/fisiologíaRESUMEN
Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alplâ»/â» mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alplâ»/â» mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alplâ»/â» mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10 to 20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by micro-computed tomography (µCT) analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alplâ»/â» mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than nontransgenic controls and osteoblasts from [Col1a1-Tnapâº/â»; Alplâ»/â»] mice are able to mineralize to the level of Alplâº/â» heterozygous osteoblasts, whereas Alplâ»/â» osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alplâ»/â» mice are comprised of phosphorylated forms of OPN whereas wild-type (WT) and [Col1a1-Tnapâº/â»; Alplâ»/â»] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alplâ»/â» bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹74FQVS¹78DEQY¹8²PDAT¹86DEDLT¹9¹)SHMK and FRIp(S²99HELES³°4S³°5S³°6S³°7)EVN, with one nonlocalized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the promineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Calcificación Fisiológica , Regulación Enzimológica de la Expresión Génica , Osteoblastos/metabolismo , Osteopontina/metabolismo , Fosfatasa Alcalina/genética , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Humanos , Ratones , Ratones Noqueados , Osteopontina/genética , Fosforilación/genéticaRESUMEN
Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro-osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles-mediated initiation of mineralization during endochondral ossification. Wild-type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1(-/-) VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high-throughput screening and mechanistic analysis and two of these inhibitors, designated MLS-0390838 and MLS-0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue-nonspecific alkaline phosphatase (TNAP) inhibitor MLS-0038949. PHOSPHO1 inhibition by MLS-0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS-0263839 and TNAP by MLS-0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization-related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that "phosphatase inhibition" may be a useful therapeutic strategy to reduce MVC.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Calcificación Vascular/enzimología , Calcificación Vascular/patología , 2-Piridinilmetilsulfinilbencimidazoles/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Animales , Calcificación Fisiológica/efectos de los fármacos , Dominio Catalítico , Simulación por Computador , Inhibidores Enzimáticos/química , Regulación de la Expresión Génica/efectos de los fármacos , Concentración 50 Inhibidora , Lansoprazol , Ratones , Modelos Moleculares , Miocitos del Músculo Liso/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl(-/-), aka Akp2(-/-)) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl(-/-) mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl(-/-) mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP.
Asunto(s)
Fosfatasa Alcalina/uso terapéutico , Esmalte Dental/patología , Terapia de Reemplazo Enzimático , Hipofosfatasia/prevención & control , Hipofosfatasia/terapia , Inmunoglobulina G/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Fosfatasa Alcalina/deficiencia , Fosfatasa Alcalina/metabolismo , Animales , Esmalte Dental/diagnóstico por imagen , Esmalte Dental/embriología , Esmalte Dental/enzimología , Humanos , Inmunohistoquímica , Incisivo/diagnóstico por imagen , Incisivo/patología , Incisivo/ultraestructura , Ratones , Minerales/metabolismo , Diente Molar/diagnóstico por imagen , Diente Molar/patología , Diente Molar/ultraestructura , Transporte de Proteínas , Microtomografía por Rayos XRESUMEN
The extent to which adult newts retain regenerative capability remains one of the greatest unanswered questions in the regeneration field. Here we report a long-term lens regeneration project spanning 16 years that was undertaken to address this question. Over that time, the lens was removed 18 times from the same animals, and by the time of the last tissue collection, specimens were at least 30 years old. Regenerated lens tissues number 18 and number 17, from the last and the second to the last extraction, respectively, were analysed structurally and in terms of gene expression. Both exhibited structural properties identical to lenses from younger animals that had never experienced lens regeneration. Expression of mRNAs encoding key lens structural proteins or transcription factors was very similar to that of controls. Thus, contrary to the belief that regeneration becomes less efficient with time or repetition, repeated regeneration, even at old age, does not alter newt regenerative capacity.
Asunto(s)
Envejecimiento/fisiología , Proteínas del Ojo/metabolismo , Cristalino/fisiología , Regeneración/fisiología , Salamandridae/fisiología , Animales , Cartilla de ADN/genética , ADN Complementario/genética , Técnicas Histológicas , Japón , Cristalino/anatomía & histología , Cristalino/metabolismo , Cristalino/cirugía , Masculino , Reacción en Cadena de la Polimerasa , Salamandridae/metabolismoRESUMEN
Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2mg/kg for 43days. Radiographs, µCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED(80) (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PP(i) concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP in patients, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP to mice. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Hipofosfatasia/tratamiento farmacológico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Fosfatasa Alcalina/genética , Animales , Terapia de Reemplazo Enzimático/métodos , Ratones , Ratones Noqueados , Osteomalacia/tratamiento farmacológico , Proteínas Recombinantes/genéticaRESUMEN
Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1(-/-) mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1(-/-) mice did not fracture during 3-point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1(-/-) tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1(-/-) tibia. The altered dihydroxylysinonorleucine/hydroxylysinonorleucine and pyridinoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1(-/-) mice and this we associated with increased bone remodeling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1(-/-) bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone.
Asunto(s)
Calcificación Fisiológica/fisiología , Fracturas Espontáneas/prevención & control , Fracturas Espontáneas/fisiopatología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Biomarcadores/metabolismo , Fenómenos Biomecánicos/fisiología , Remodelación Ósea/fisiología , Colágeno/metabolismo , Elasticidad , Fémur/patología , Fémur/fisiopatología , Fracturas Espontáneas/enzimología , Dureza , Ratones , Monoéster Fosfórico Hidrolasas/deficiencia , Tibia/patología , Tibia/fisiopatología , ViscosidadRESUMEN
Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP(i)). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1(-/-) mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1(-/-) tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1(-/-) mice show reduced levels of TNAP and elevated plasma PP(i) concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1(-/-) mice despite normalization of their plasma PP(i) levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P(i) influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization.