RESUMEN
Pseudoxanthoma elasticum is a heritable disease caused by ABCC6 deficiency. Patients develop ectopic calcification in skin, eyes, and vascular tissues. ABCC6, primarily found in liver and kidneys, mediates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi), a potent inhibitor of calcification. Pseudoxanthoma elasticum patients and Abcc6-/- mice display reduced PPi levels in plasma and peripheral tissues. Pseudoxanthoma elasticum is currently incurable, although some palliative treatments exist. In recent years, we have successfully developed therapeutic methodologies to compensate the PPi deficit in animal models and humans. Here, we inadvertently discovered that modulating dietary PPi can also be an effective approach to reducing calcification in Abcc6-/- mice. Our findings were prompted by a change in institutional rodent diet. The new chow was enriched in PPi, which increased plasma PPi, and significantly reduced mineralization in Abcc6-/- mice. We also found that dietary PPi is readily absorbed in humans. Our results suggest that the consumption of food naturally or artificially enriched in PPi represents a possible intervention to mitigate calcification progression in pseudoxanthoma elasticum, that dietary preferences of patients may explain pseudoxanthoma elasticum heterogeneous manifestations, and that animal chow has the potential to influence data reproducibility.
Asunto(s)
Calcinosis/tratamiento farmacológico , Suplementos Dietéticos , Seudoxantoma Elástico/tratamiento farmacológico , Seudoxantoma Elástico/patología , Pirofosfatasas/administración & dosificación , Animales , Biopsia con Aguja , Calcinosis/patología , Modelos Animales de Enfermedad , Femenino , Voluntarios Sanos , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Medición de Riesgo , Especificidad de la Especie , Resultado del TratamientoRESUMEN
Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.
Asunto(s)
Enfermedades del Recién Nacido/enzimología , Enfermedades del Recién Nacido/prevención & control , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Calcificación Vascular/enzimología , Calcificación Vascular/prevención & control , Animales , Arterias/enzimología , Arterias/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Recién Nacido , Enfermedades del Recién Nacido/genética , Enfermedades del Recién Nacido/mortalidad , Masculino , Ratones Endogámicos C57BL , Hidrolasas Diéster Fosfóricas/administración & dosificación , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/administración & dosificación , Pirofosfatasas/genética , Calcificación Vascular/genética , Calcificación Vascular/mortalidadRESUMEN
We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain â¿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice.
Asunto(s)
Pirofosfatasas/administración & dosificación , Trichinella spiralis/enzimología , Triquinelosis/inmunología , Vacunas/administración & dosificación , Administración Oral , Animales , Anticuerpos Antihelmínticos/inmunología , Citocinas/inmunología , Sistemas de Liberación de Medicamentos , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunidad Celular , Masculino , Ratones , Ratones Endogámicos BALB C , Pirofosfatasas/genética , Pirofosfatasas/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Células TH1/inmunología , Células Th2/inmunología , Trichinella spiralis/genética , Trichinella spiralis/inmunología , Triquinelosis/parasitología , Triquinelosis/prevención & control , Vacunación , Vacunas/genética , Vacunas/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Hidrolasas NudixRESUMEN
BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) calcification is a major cause of morbidity and mortality, yet the molecular mechanisms involved are poorly understood. Hence, an ex vivo model of calcification in intact AVs was developed in order to test the role of orthophosphate and pyrophosphate (PPi), both of which factors are known to influence vascular calcification. METHODS: Porcine AV leaflets were cultured in serum-free medium under static conditions for eight days, over which time leaflet architecture and viability were preserved. Calcification was measured as the incorporation of 45Ca, with confirmation by Alizarin Red staining. RESULTS: Calcification required both a high phosphate concentration (3.8 mM) and removal of PPi with alkaline phosphatase or inorganic pyrophosphatase. Calcification occurred predominantly on the fibrosa and was arrested by the bisphosphonate etidronate, a non-hydrolyzable analog of PPi. Leaflets released PPi into the medium, and this was enhanced by MLS38949, a specific inhibitor of tissue non-specific alkaline phosphatase (TNAP). Furthermore, leaflets synthesized PPi from extracellular ATP, which was reduced by ß,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1). CONCLUSION: The ex vivo AV calcification model developed in the present study showed that extracellular PPi, produced by valvular tissue, is a potent inhibitor of valvular calcification. In addition to synthesis, hydrolysis by TNAP also controls PPi levels and calcification. The results suggest that a decreased synthesis or increased hydrolysis of pyrophosphate may contribute to valvular calcification, and that bisphosphonates or inhibitors of TNAP are potential preventive strategies of the process. TNAP are potential preventive strategies.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/patología , Calcinosis/metabolismo , Calcinosis/patología , Difosfatos/metabolismo , Fosfatos/metabolismo , Fosfatasa Alcalina/administración & dosificación , Animales , Válvula Aórtica/metabolismo , Ácido Etidrónico/administración & dosificación , Femenino , Pirofosfatasas/administración & dosificación , Porcinos , Técnicas de Cultivo de TejidosRESUMEN
Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.
Asunto(s)
Células Endoteliales/metabolismo , Hígado/irrigación sanguínea , Complejos Multienzimáticos/farmacocinética , Metástasis de la Neoplasia/fisiopatología , Proteínas de Neoplasias/farmacocinética , Fosfodiesterasa I/farmacocinética , Hidrolasas Diéster Fosfóricas/farmacocinética , Pirofosfatasas/farmacocinética , Receptores Depuradores/metabolismo , Animales , Células Cultivadas/metabolismo , Formaldehído/farmacología , Humanos , Inyecciones Intravenosas , Masculino , Tasa de Depuración Metabólica , Ratones , Complejos Multienzimáticos/administración & dosificación , Complejos Multienzimáticos/sangre , Metástasis de la Neoplasia/prevención & control , Proteínas de Neoplasias/administración & dosificación , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/fisiología , Fosfodiesterasa I/administración & dosificación , Fosfodiesterasa I/sangre , Hidrolasas Diéster Fosfóricas/administración & dosificación , Hidrolasas Diéster Fosfóricas/sangre , Pirofosfatasas/administración & dosificación , Pirofosfatasas/sangre , Ratas , Ratas Wistar , Receptores Depuradores/antagonistas & inhibidores , Albúmina Sérica Bovina/farmacologíaRESUMEN
Autotaxin (ATX) was originally identified as a potent tumor cell motility-stimulating factor that displays multiple enzymatic activities including ATPase, Type I nucleotide pyrophosphatase/phosphodiesterase, and lysophospholipase D, depending on its substrates. We demonstrate herein that ATX is a key regulator of extracellular lysophosphatidic acid (LPA) that can act as survival factor, in addition to its mitogenic activity in mouse fibroblasts. Introduction of atx gene into NIH3T3 cells resulted in resistance to conditional apoptosis induced by serum-deprivation, and exogenous ATX protein prevented cells from death by starvation. Flow cytometric analysis showed that co-treatment of ATX with lysophosphatidylcholine as substrate rescued NIH3T3 cells from cellular apoptosis, and this survival activity of ATX was also demonstrated by caspase-3 degradation and PARP cleavage resulting from the enzymatic activity of extracellular ATX. Furthermore, the effect of ATX in preventing apoptosis appears to be mediated through the G-protein-coupled receptor pathway followed by the activation of phosphoinositide 3-kinase and Akt pathway leading to enhanced cell survival. These findings provide novel insights into understanding the functions of ATX as a key regulator of bioactive phospholipids and suggest interventions to correct dysfunction in conditions of tumor cell growth and metastasis.