RESUMEN
Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Calcinosis/prevención & control , Difosfatos/uso terapéutico , Seudoxantoma Elástico/prevención & control , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Enfermedad Aguda , Animales , Calcinosis/metabolismo , Calcinosis/patología , Enfermedad Crónica , Difosfatos/administración & dosificación , Difosfatos/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Ácido Etidrónico/uso terapéutico , Femenino , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fenotipo , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , TransgenesRESUMEN
Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 µM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine.
Asunto(s)
Aporfinas/farmacología , Bilis/metabolismo , Colagogos y Coleréticos/farmacología , Hígado/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/agonistas , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Aporfinas/administración & dosificación , Aporfinas/metabolismo , Colagogos y Coleréticos/administración & dosificación , Colagogos y Coleréticos/metabolismo , Perros , Etinilestradiol/farmacología , Femenino , Glutatión/metabolismo , Células Hep G2 , Eliminación Hepatobiliar , Humanos , Infusiones Intravenosas , Cinética , Hígado/metabolismo , Células de Riñón Canino Madin Darby , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ósmosis , Ratas Endogámicas Lew , Ratas Transgénicas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transfección , Regulación hacia ArribaRESUMEN
Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, demonstrates progressive build-up of calcium phosphate and proteoglycans in the skin, eye, and arteries, and is associated to myocardial infarctions, stroke, blindness, and elevated carotid intima-media thickness (CIMT). Although CIMT reduction with magnesium (Mg) has been documented in a mouse model for PXE (Abcc6(-/-) ), it is not clear if Mg is effective in humans with PXE to reduce CIMT. To examine this, we calculated the rate of change of CIMT (washout) in 15- and 12-month-old Abcc6(-/-) mice fed standard rodent diet with or without Mg supplementation for 2 months. Using means in untreated 15- and 12-month-old Abcc6(-/-) mice (145 and 120 µm, respectively), the rate of change was 8.3 µm/month. Using means in treated 15- and 12-month-old Abcc6(-/-) mice (118 and 104.6 µm, respectively), the rate of change was 4.5 µm. Compared to normal progression of CIMT in humans without PXE, PXE has advanced atherosclerosis and possibly a higher CIMT rate of change. This experiment may portend, at least in PXE, the rationale for a 1-year oral Mg CIMT clinical trial and may be useful for application in other progressive mineralizing disorders like atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/deficiencia , Arterias Carótidas/efectos de los fármacos , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Grosor Intima-Media Carotídeo , Óxido de Magnesio/farmacología , Seudoxantoma Elástico/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Factores de TiempoRESUMEN
A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.
Asunto(s)
Inhibidores de Adenilato Ciclasa , Agonistas alfa-Adrenérgicos/uso terapéutico , Antagonistas Adrenérgicos alfa/uso terapéutico , Degeneración Macular/congénito , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/biosíntesis , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Receptor de Serotonina 5-HT2A/biosíntesis , Receptores Adrenérgicos alfa 2/biosíntesis , Antagonistas de la Serotonina/uso terapéutico , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Oxidorreductasas de Alcohol/deficiencia , Oxidorreductasas de Alcohol/genética , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Doxazosina/farmacología , Doxazosina/uso terapéutico , Evaluación Preclínica de Medicamentos , Guanabenzo/farmacología , Guanabenzo/uso terapéutico , Humanos , Luz/efectos adversos , Macaca fascicularis , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/genética , Degeneración Macular/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/fisiología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Especies Reactivas de Oxígeno , Receptor de Serotonina 5-HT2A/genética , Receptores Adrenérgicos alfa 2/genética , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Antagonistas de la Serotonina/farmacología , Transducción de Señal , Enfermedad de StargardtRESUMEN
Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by mutations in the ABCC6 gene. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis, we developed a model in which Abcc6(-/-) mice, which recapitulate features of PXE, were fed a diet supplemented with warfarin and vitamin K1. Warfarin action was confirmed by significantly increased serum levels of oxidized vitamin K. For mice placed on a warfarin-containing diet, quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits in a number of tissues. Mice fed a warfarin-containing diet were also shown to have abundant uncarboxylated form of matrix Gla protein, which allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, 1747 patients with PXE from the approximately 4000 patients in the PXE International database were surveyed about the use of warfarin. Of the 539 respondents, 2.6% reported past or present use of warfarin. Based on the prevalence of PXE (approximately 1:50,000), thousands of patients with PXE worldwide may be at risk for worsening of PXE as a result of warfarin therapy.