Critical parameters of the in vitro method of vascular smooth muscle cell calcification

[Background]: Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo. [Experimental Procedures and Results]: Rat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca2+ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO2 vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¿. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO43--containing ACP. [Conclusions]: The precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.Funding was provided by a research grant SAF2012-33898 to VS, http://www.mineco.gob.es/portal/site/mineco/idi, Spanish Ministry of Economy and Competitiveness; research grant MAT2014-54975-R to AM, http://www.mineco.gob.es/portal/site/mineco/idi, Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Several phosphate transport processes are present in vascular smooth muscle cells

We have studied Pi handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting in 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride-dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under non-precipitating conditions. This work not only shows that the Pi handling by cells is highly complex, but also that the transport systems are shared with other ions such as bicarbonate or sulfate

Mecanismos de calcificación vascular medial en uremia e hiperfosfatemia

La calcificación vascular medial (CVM) es un proceso de esclerosis de la pared de las arterias asociada a la enfermedad renal crónica (ERC) entre otras. En esta tesis se ha estudiado los cambios tempranos durante la CVM en el modelo de nefrectomía (NX) de 5/6 en rata, intentando esclarecer así mismo el orden de los eventos que aparecen de forma temprana. Por otro lado se ha llevado a cabo un análisis de los sucesos que tienen lugar en el método de calcificación de células de la musculatura lisa vascular (VSMC) in vitro con elevada concentración de fosfato en el medio, intentando esclarecer su correlación con los sucesos in vivo y formulando un protocolo de trabajo lo más parecido posible a los procesos fisiopatológicos que ocurren in vivo. Finalmente, debido a la importancia de la hiperfosfatemia en el avance de la CVM, se ha llevado a cabo una exhaustiva caracterización exhaustiva el transporte de fosfato en el principal tipo celular presente en la túnica media de las arterias (y que además participa activamente en la CVM), las VSMC. Entre los resultados más trascendentales de esta tesis se encuentra la localización de cambios genéticos y proteicos en las aortas, así como plasmáticos anteriores al aumento del calcio en las mismas, aportando también un orden de sucesos en los momentos más iniciales de la CVM in vivo. Así mismo se ha demostrado que los protocolos de calcificación de VSMC in vitro se basan en precipitaciones homogéneas sin relación con los procesos in vivo, y hemos propuesto las condiciones mínimas que deben utilizarse. Finalmente, se ha avanzado en la descripción del transporte Na-independiente de Pi en VSMC, se han aportado datos nuevos sobre los transportadores Na-dependientes (NaPi III), y se ha caracterizado por primera vez el transporte de salida cloro-dependiente de fosfato en VSMC

Síndrome hiperosmolar hiperglicémico en un gato diabético : análisis de los disturbios ácido-base asociados mediante un modelo cuantitativo simplificado

La incidencia del síndrome hiperosmolar hiperglucémico en la especie felina es baja; sin embargo, la morbilidad y mortalidad asociadas a esta complicación diabética son elevadas. La evaluación y monitorización de la natremia, glucemia y osmolalidad son fundamentales a la hora de abordar el manejo de este tipo de urgencia endocrina en pacientes con graves desequilibrios hidroelectrolíticos y, frecuentemente, con enfermedades crónicas asociadas. En conocimiento de los autores, este es el primer caso donde se describen las anomalías ácido-base, asociadas a este tipo de desequilibrios, mediante la ecuación de Fencl-Stewart, un modelo cuantitativo simplificado especialmente útil para el análisis del estatus ácido-base en pacientes críticos

Cell Phenotype Transitions in Cardiovascular Calcification

Cardiovascular calcification was originally considered a passive, degenerative process, however with the advance of cellular and molecular biology techniques it is now appreciated that ectopic calcification is an active biological process. Vascular calcification is the most common form of ectopic calcification, and aging as well as specific disease states such as atherosclerosis, diabetes, and genetic mutations, exhibit this pathology. In the vessels and valves, endothelial cells, smooth muscle cells, and fibroblast-like cells contribute to the formation of extracellular calcified nodules. Research suggests that these vascular cells undergo a phenotypic switch whereby they acquire osteoblast-like characteristics, however the mechanisms driving the early aspects of these cell transitions are not fully understood. Osteoblasts are true bone-forming cells and differentiate from their pluripotent precursor, the mesenchymal stem cell (MSC); vascular cells that acquire the ability to calcify share aspects of the transcriptional programs exhibited by MSCs differentiating into osteoblasts. What is unknown is whether a fully-differentiated vascular cell directly acquires the ability to calcify by the upregulation of osteogenic genes or, whether these vascular cells first de-differentiate into an MSC-like state before obtaining a “second hit” that induces them to re-differentiate down an osteogenic lineage. Addressing these questions will enable progress in preventative and regenerative medicine strategies to combat vascular calcification pathologies. In this review, we will summarize what is known about the phenotypic switching of vascular endothelial, smooth muscle, and valvular cells

Síndrome hiperosmolar hiperglicémico en un gato diabético : análisis de los disturbios ácido-base asociados mediante un modelo cuantitativo simplificado

La incidencia del síndrome hiperosmolar hiperglucémico en la especie felina es baja; sin embargo, la morbilidad y mortalidad asociadas a esta complicación diabética son elevadas. La evaluación y monitorización de la natremia, glucemia y osmolalidad son fundamentales a la hora de abordar el manejo de este tipo de urgencia endocrina en pacientes con graves desequilibrios hidroelectrolíticos y, frecuentemente, con enfermedades crónicas asociadas. En conocimiento de los autores, este es el primer caso donde se describen las anomalías ácido-base, asociadas a este tipo de desequilibrios, mediante la ecuación de Fencl-Stewart, un modelo cuantitativo simplificado especialmente útil para el análisis del estatus ácido-base en pacientes críticos

Critical Parameters of the <i>In Vitro</i> Method of Vascular Smooth Muscle Cell Calcification

<div><p>Background</p><p>Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification <i>in vitro</i> and to determine the relevance to calcification <i>in vivo</i>.</p><p>Experimental Procedures and Results</p><p>Rat VSMC calcification <i>in vitro</i> was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca²⁺ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO₂ vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO₄^3--containing ACP.</p><p>Conclusions</p><p>The precipitation of ACP <i>in vitro</i> is unrelated to VC <i>in vivo</i>. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition <i>in vivo</i>.</p></div

Identifying early pathogenic events during vascular calcification in uremic rats

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.Sources of support: Grants SAF2012-33898 and SAF2015-66705-P to V.S. and MAT2014-54975-R to AM, all three from the Spanish MINECO/FEDER.Peer reviewe