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1.
Kidney Int ; 94(4): 716-727, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30041812

RESUMEN

PiT-2, a type III sodium-dependent phosphate transporter, is a causative gene for the brain arteriolar calcification in people with familial basal ganglion calcification. Here we examined the effect of PiT-2 haploinsufficiency on vascular calcification in uremic mice using wild-type and global PiT-2 heterozygous knockout mice. PiT-2 haploinsufficiency enhanced the development of vascular calcification in mice with chronic kidney disease fed a high-phosphate diet. No differences were observed in the serum mineral biomarkers and kidney function between the wild-type and PiT-2 heterozygous knockout groups. Micro computed tomography analyses of femurs showed that haploinsufficiency of PiT-2 decreased trabecular bone mineral density in uremia. In vitro, sodium-dependent phosphate uptake was decreased in cultured vascular smooth muscle cells isolated from PiT-2 heterozygous knockout mice compared with those from wild-type mice. PiT-2 haploinsufficiency increased phosphate-induced calcification of cultured vascular smooth muscle cells compared to the wild-type. Furthermore, compared to wild-type vascular smooth muscle cells, PiT-2 deficient vascular smooth muscle cells had lower osteoprotegerin levels and increased matrix calcification, which was attenuated by osteoprotegerin supplementation. Thus, PiT-2 in vascular smooth muscle cells protects against phosphate-induced vascular calcification and may be a therapeutic target in the chronic kidney disease population.


Asunto(s)
Fosfatos/metabolismo , Insuficiencia Renal Crónica/complicaciones , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Calcificación Vascular/genética , Animales , Biomarcadores/sangre , Densidad Ósea/genética , Femenino , Haploinsuficiencia , Heterocigoto , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Osteoprotegerina/metabolismo , Fosfatos/administración & dosificación , Insuficiencia Renal Crónica/sangre , Uremia/complicaciones , Calcificación Vascular/sangre
2.
Am J Physiol Renal Physiol ; 307(8): F891-900, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-25143458

RESUMEN

Patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) have significant cardiovascular morbidity and mortality that is in part due to the development of vascular calcification. Vascular calcification is an active, highly regulated process that shares many similarities with normal bone formation. New discoveries related to extracellular vesicles, microRNAs, and calciprotein particles continue to reveal the mechanisms that are involved in the initiation and progression of vascular calcification in CKD. Further innovations in these fields are critical for the development of biomarkers and therapeutic options for patients with CKD and ESRD.


Asunto(s)
Fallo Renal Crónico/fisiopatología , Insuficiencia Renal Crónica/fisiopatología , Calcificación Vascular/fisiopatología , Calcio/metabolismo , Progresión de la Enfermedad , Elastina/metabolismo , Humanos , Músculo Liso Vascular/citología , Fósforo/metabolismo , Factores de Riesgo , Calcificación Vascular/patología
3.
Biomaterials ; 30(27): 4513-21, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19501906

RESUMEN

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PP(i)), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (P(i)), an inducer of hydroxyapatite formation. P(i) is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with beta-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Huesos/fisiología , Enzimas Inmovilizadas/metabolismo , Fibrina/metabolismo , Nanoestructuras/química , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Biomarcadores/metabolismo , Regeneración Ósea , Calcificación Fisiológica , Bovinos , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Medios de Cultivo , Regulación de la Expresión Génica , Ratones , Modelos Biológicos , Nanoestructuras/ultraestructura , Osteoblastos/citología , Osteoblastos/ultraestructura , Fósforo/metabolismo , Porosidad , Cráneo/patología , Cráneo/ultraestructura , Microtomografía por Rayos X
4.
Kidney Int ; 75(12): 1297-1307, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19322138

RESUMEN

Arterial medial calcification is a major complication in patients with chronic kidney disease and is a strong predictor of cardiovascular and all-cause mortality. We sought to determine the role of dietary phosphorus and the severity of uremia on vascular calcification in calcification-prone DBA/2 mice. Severe and moderate uremia was induced by renal ablation of varying magnitudes. Extensive arterial-medial calcification developed only when the uremic mice were placed on a high-phosphate diet. Arterial calcification in the severely uremic mice fed a high-phosphate diet was significantly associated with hyperphosphatemia. Moderately uremic mice on this diet were not hyperphosphatemic but had a significant rise in their serum levels of fibroblast growth factor 23 (FGF-23) and osteopontin that significantly correlated with arterial medial calcification. Although there was widespread arterial medial calcification, there was no histological evidence of atherosclerosis. At early stages of calcification, the osteochondrogenic markers Runx2 and osteopontin were upregulated, but the smooth muscle cell marker SM22alpha decreased in medial cells, as did the number of smooth muscle cells in extensively calcified regions. These findings suggest that phosphate loading and the severity of uremia play critical roles in controlling arterial medial calcification in mice. Further, FGF-23 and osteopontin may be markers and/or inducers of this process.


Asunto(s)
Arterias/patología , Calcinosis/sangre , Calcinosis/etiología , Fosfatos/administración & dosificación , Uremia/sangre , Uremia/complicaciones , Enfermedades Vasculares/sangre , Enfermedades Vasculares/etiología , Animales , Arterias/metabolismo , Calcinosis/metabolismo , Calcinosis/patología , Calcio/sangre , Calcio/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Humanos , Ratones , Ratones Endogámicos DBA , Osteopontina/sangre , Osteopontina/metabolismo , Fosfatos/toxicidad , Fósforo/sangre , Uremia/metabolismo , Uremia/patología , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patología
5.
Cardiovasc Res ; 66(2): 324-33, 2005 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-15820201

RESUMEN

OBJECTIVE: Vascular calcification is an actively regulated process, correlating with cardiovascular morbidity and mortality especially in patients with diabetes and chronic renal diseases. Osteopontin (OPN) is abundantly expressed in human calcified arteries and inhibits vascular calcification in vitro and in vivo. How OPN functions in vascular calcification, however, is less clear. METHODS: Smooth muscle cells (SMCs) were isolated from aortas of OPN knock-out (OPN-/-) and wild type (OPN+/+) mice. RESULTS: OPN-/- SMCs were identical to OPN+/+ SMCs in morphology and stained positively for SM lineage proteins, desmin, smooth muscle alpha-actin and SM22alpha. No spontaneous calcification was observed in OPN-/- SMCs under normal culture conditions or in medium containing 1%, 3%, or 5% fetal bovine serum. However, when cultured in medium containing elevated concentrations of inorganic phosphate, an inducer of vascular calcification, a significantly higher calcification was observed in OPN-/- SMCs compared to OPN+/+ SMCs that, in response to elevated phosphate, synthesized and secreted OPN into the culture. Finally, retroviral transduction of mouse OPN cDNA into OPN-/- SMCs rescued the calcification phenotype of the cells. CONCLUSION: These results are the first to demonstrate an inhibitory role of endogenously produced OPN on SMC calcification, suggesting a novel feedback mechanism where OPN produced locally by the SMCs may serve as an important inducible inhibitor of vascular calcification.


Asunto(s)
Calcinosis/etiología , Músculo Liso Vascular/metabolismo , Sialoglicoproteínas/deficiencia , Animales , Aorta , Calcinosis/metabolismo , Técnicas de Cultivo de Célula , ADN Complementario/administración & dosificación , Susceptibilidad a Enfermedades , Vectores Genéticos/administración & dosificación , Ratones , Ratones Noqueados , Osteopontina , Fosfatos/farmacología , Retroviridae/genética , Sialoglicoproteínas/genética , Transducción Genética/métodos
6.
Kidney Int ; 66(6): 2293-9, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15569318

RESUMEN

BACKGROUND: Hyperphosphatemia, elevated calcium x phosphorus product (Ca x P), and calcium burden, major causes of vascular calcification, are correlated with increased cardiovascular morbidity and mortality in dialysis patients. METHODS: To address the underlying mechanisms responsible for these findings, we have utilized an in vitro human smooth muscle cell (HSMC) model of vascular calcification. Previous studies using this system demonstrated enhanced calcification of HSMC cultures treated with phosphorus levels in the hyperphosphatemic range, and implicated a sodium-dependent phosphate cotransport-dependent mechanism in this effect. In the present study, we examine the effect of increasing calcium concentrations on HSMC calcification in vitro. RESULTS: Increasing calcium to levels observed in hypercalcemic individuals increased mineralization of HSMC cultures under normal phosphorus conditions. Importantly, at these total calcium concentrations, ionized calcium levels increased from 1.2 mmol/L to 1.7 mmol/L, consistent with levels observed physiologically in normocalcemic and hypercalcemic individuals, respectively. Furthermore, increasing both calcium and phosphorus levels led to accelerated and increased mineralization in the cultures. Calcium-induced mineralization was dependent on the function of a sodium-dependent phosphate cotransporter, since it was inhibited by phosphonoformic acid (PFA). While elevated calcium did not affect short-term phosphorus transport kinetics, long-term elevated calcium treatment of HSMCs induced expression of the sodium-dependent phosphate cotransporter, Pit-1. CONCLUSION: These studies suggest that elevated calcium may stimulate HSMC mineralization by elevating Ca x P product and enhancing the sodium-dependent phosphate cotransporter-dependent mineralization pathway previously observed in HSMCs.


Asunto(s)
Calcinosis/metabolismo , Calcio/metabolismo , Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Aorta/citología , Calcificación Fisiológica/fisiología , Calcio/farmacología , Células Cultivadas , Espacio Extracelular/metabolismo , Humanos , Hipercalcemia/metabolismo , Técnicas In Vitro , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Fósforo/metabolismo , Fósforo/farmacología , Transducción de Señal/fisiología
7.
J Biomed Mater Res A ; 67(1): 334-43, 2003 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-14517893

RESUMEN

A wide array of technologies exist for the characterization and quantification of molecules present at surfaces. We have used several of these experimental and instrumental techniques for the analysis of a novel biomaterial surface. Osteopontin, an extracellular matrix molecule involved in wound-healing processes, has been chosen as a relevant model protein to immobilize onto poly(2-hydroxyethyl methacrylate) [poly(HEMA)]. Electron spectroscopy for chemical analysis and time-of-flight secondary ion mass spectrometry were used to verify the surface chemistry and the presence of protein. Iodination of osteopontin yielded quantitative data supportive of dose-dependent immobilization. Enzyme-linked immunosorbent assay was also used to investigate the presence of osteopontin on poly(HEMA). Finally, the cell adhesive properties of immobilized osteopontin were confirmed by using a bovine aortic endothelial cell adhesion assay. The use of multiple tools to characterize the many facets of a biomaterial surface will undoubtedly improve our understanding of the surface and facilitate the amelioration of in vivo performance.


Asunto(s)
Materiales Biocompatibles/metabolismo , Polihidroxietil Metacrilato/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Aorta/metabolismo , Materiales Biocompatibles/química , Bovinos , Adhesión Celular/fisiología , ADN Complementario , Células Endoteliales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Radioisótopos de Yodo , Lisina/metabolismo , Osteopontina , Polihidroxietil Metacrilato/química , Ratas , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Espectrometría de Masa de Ion Secundario
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