Uremic Toxins Induce THP-1 Monocyte Endothelial Adhesion and Migration through Specific miRNA Expression.

Atherosclerosis is initiated by the activation of endothelial cells that allows monocyte adhesion and transmigration through the vascular wall. The accumulation of uremic toxins such as indoxyl sulphate (IS) and p-cresol (PC) has been associated with atherosclerosis. Currently, miRNAs play a crucial role in the regulation of monocyte activation, adhesion, and trans-endothelial migration. The aim of the present study is to evaluate the effect of IS and PC on monocyte adhesion and migration processes in monocytes co-cultured with endothelial cells as well as to determine the underlying mechanisms. The incubation of HUVECs and THP-1 cells with both IS and PC toxins resulted in an increased migratory capacity of THP-1 cells. Furthermore, the exposure of THP-1 cells to both uremic toxins resulted in the upregulation of BMP-2 and miRNAs-126-3p, -146b-5p, and -223-3p, as well as the activation of nuclear factor kappa B (NF-κB) and a decrease in its inhibitor IĸB. Uremic toxins, such as IS and PC, enhance the migratory and adhesion capacity of THP-1 cells to the vascular endothelium. These toxins, particularly PC, contribute significantly to uremia-associated vascular disease by increasing in THP-1 cells the expression of BMP-2, NF-κB, and key miRNAs associated with the development of atherosclerotic vascular diseases.

Características clínicas de los pacientes con accidente cerebrovascular de tipo isquémico admitidos durante el periodo de ventana terapéutica en el Servicio de Urgencias del Hospital de Clínicas / Clinical characteristics of patients with ischemic stroke admitted during the therapeutic window period in the Emergency Department of the Hospital de Clinicas

Introducción: Las enfermedades cardiovasculares son la primera causa de muerte. El accidente cerebrovascular isquémico es un problema de salud pública. Objetivos: Determinar las características clínicas de los pacientes con accidente cerebrovascular de tipo isquémico admitidos durante el periodo de ventana terapéutica en el Servicio de Urgencias del Hospital de Clínicas en el periodo 2018 - 2020. Materiales y métodos: Estudio observacional, descriptivo, retrospectivo, transversal. Los sujetos fueron los pacientes de sexo masculino y femenino, mayores de 18 años admitidos en la Unidad de Ictus del Servicio de Urgencias del Hospital de Clínicas en el periodo de ventana terapéutica comprendido entre junio del año 2018 y septiembre del año 2020. Resultados: Se incluyó en el estudio 512 pacientes. La media de edad fue 65 ± 12,1 años. El sexo más frecuente fue el masculino con (58,7%) y la mayoría proceden del Departamento Central (61,3%). Los factores de riesgo más frecuentes fueron la hipertensión arterial (83,3%), el sobrepeso (34,7%) y la diabetes mellitus tipo 2 (27,3%). Presentaron infarto moderado (41,8%) y la trombólisis fue realizada en el (16%) de los pacientes. Conclusión: Los pacientes que presentaron accidente cerebrovascular de tipo isquémico admitidos en el periodo de ventana terapéutica fueron en su mayoría del sexo masculino, edad media de 65 años, los factores de riesgo cardiovasculares más frecuentes fueron la hipertensión arterial, el sobrepeso y la diabetes mellitus tipo 2, el infarto moderado fue la más frecuente y escasa cantidad recibieron trombólisis.

Introduction: Cardiovascular diseases are the leading cause of death. Ischemic stroke is a public health problem. Objectives: To determine the clinical characteristics of patients with ischemic stroke admitted during the therapeutic window period in the Emergency Department of the Hospital de Clínicas in the period 2018 - 2020. Materials and methods: Observational, descriptive, retrospective, cross-sectional study. The subjects were male and female patients, over 18 years of age admitted to the Stroke Unit of the Emergency Service of the Hospital de Clínicas in the therapeutic window period between June 2018 and September 2020. Results: Included 512 patients in the study. The mean age was 65 ± 12,1 years. The most frequent sex was male with (58.7%), most of them come from the central department (61.3%). The most frequent risk factors were arterial hypertension (83.3%), overweight (34.7%) and type 2 diabetes mellitus (27.3%). They presented moderate infarction (41,8%). Thrombolysis was performed in (16%) of the patients. Conclusion: The patients who presented ischemic stroke admitted in the therapeutic window period were mostly male, mean age 65 years, the most frequent cardiovascular risk factors were arterial hypertension, overweight and mellitus diabetes type 2, moderate infarction was the most frequent and few received thrombolysis.

Chitosan-collagen-hydroxyapatite membranes for tissue engineering.

Tissue engineering is growing in developing new technologies focused on providing effective solutions to degenerative pathologies that affect different types of connective tissues. The search for biocompatible, bioactive, biodegradable, and multifunctional materials has grown significantly in recent years. Chitosan, calcium phosphates collagen, and their combination as composite materials fulfill the required properties and could result in biostimulation for tissue regeneration. In the present work, the chitosan/collagen/hydroxyapatite membranes were prepared with different concentrations of collagen and hydroxyapatite. Cell adhesion was evaluated by MTS assay for two in vitro models. Additionally, cytotoxicity of the different membranes employing hemolysis of erythrocytes isolated from human blood was carried out. The structure of the membranes was analyzed by X-rays diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal stability properties by thermogravimetric methods (TGA). The highest cell adhesion after 48 h was obtained for chitosan membranes with the highest hydroxyapatite and collagen content. All composite membranes showed good cell adhesion and low cytotoxicity, suggesting that these materials have a significant potential to be used as biomaterials for tissue engineering. Graphical abstract.

An Expert Perspective on Phosphate Dysregulation With a Focus on Chronic Hypophosphatemia.

Because of their rarity, diseases characterized by chronic hypophosphatemia can be underrecognized and suboptimally managed, resulting in poor clinical outcomes. Moreover, serum phosphate may not be measured routinely in primary care practice. Authors participated in several working sessions to advance the understanding of phosphate homeostasis and the causes, consequences, and clinical implications of chronic hypophosphatemia. Phosphate levels are regulated from birth to adulthood. Dysregulation of phosphate homeostasis can result in hypophosphatemia, which becomes chronic if phosphate levels cannot be normalized. Chronic hypophosphatemia may be underrecognized as serum phosphate measurement is not always part of routine analysis in the primary care setting and results might be misinterpreted, for instance, due to age-specific differences not being accounted for and circadian variations. Clinical consequences of chronic hypophosphatemia involve disordered endocrine regulation, affect multiple organ systems, and vary depending on patient age and the underlying disorder. Signs and symptoms of chronic hypophosphatemic diseases that manifest during childhood or adolescence persist into adulthood if the disease is inadequately managed, resulting in an accumulation of clinical deficits and a progressive, debilitating impact on quality of life. Early identification and diagnosis of patients with chronic hypophosphatemia is crucial, and clinical management should be started as soon as possible to maximize the likelihood of improving health outcomes. Furthermore, in the absence of a universally accepted description for "chronic hypophosphatemia," a definition is proposed here that aims to raise awareness of these diseases, facilitate diagnosis, and guide optimal phosphate management strategies by improving monitoring and assessment of patient response to treatment. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Complicaciones de la nefrolitotomía percutánea como tratamiento de la litiasis renal coraliforme

RESUMEN Introducción: La nefrolitotomía percutánea es la primera opción terapéutica para la litiasis renal coraliforme. Objetivo: Caracterizar a los pacientes con complicaciones de la nefrolitotomía percutánea para el tratamiento de la litiasis renal coraliforme. Método: Se estudió una serie de 191 pacientes, operados mediante nefrolitotomía percutánea. Variables estudiadas: tipo de litiasis coraliforme, posición para la técnica, condición de libre de litiasis después de la operación, presencia de complicaciones, momento, tipo y grado según clasificación de Clavien-Dindo. Se hallaron frecuencias absolutas, relativas y se utilizó el test de ji cuadrado para determinar asociación entre variables. Resultados: El 86,9 % tenía menos de 60 años, 67,0 % eran masculinos, 61,7 % presentaba comorbilidades. La litiasis coraliforme era parcial o total (30,3 % y 46,5 %, respectivamente). En 60,2 % afectaba el riñón izquierdo; 58,1 % se operaron en supino y 70,2 % quedaron libre de litiasis con la nefrolitotomía percutánea monoterapéutica. Ocurrieron complicaciones en 19,9 %; 16,2 % fueron postoperatorias, 14,1 % infecciosas, 7,8 % Clavien-Dindo I y 5,2 % IIIb. El tipo de litiasis y la posición de la nefrolitotomía percutánea no se asociaron con las complicaciones (p> 0,05). El grado de la complicación no se relacionó con el tipo de litiasis (p> 0,05). Conclusiones: Las complicaciones postoperatorias más frecuentes son las relacionadas con la infección y el sangrado; predominan ligeramente en los pacientes con litiasis coraliformes parcial, total y en los operados en supino; el grado Clavien-Dindo de las complicaciones, es mayor en las litiasis coraliformes más complejas.

ABSTRACT Introduction: Percutaneous nephrolithotomy is the first therapeutic option for staghorn kidney stones. Objective: To characterize patients with complications of percutaneous nephrolithotomy for the treatment of staghorn renal lithiasis. Method: A series of 191 patients, operated by percutaneous nephrolithotomy, was studied. Variables studied: type of staghorn lithiasis, position for the technique, stone-free condition after the operation, presence of complications, time, type and grade according to the Clavien-Dindo classification. Absolute and relative frequencies were found and the chi-square test was used to determine the association between variables. Results: 86.9 % were less than 60 years old, 67,0 % were male, 61,7 % had comorbidities. The staghorn lithiasis was partial or total (30,3 % and 46,5 %, respectively). In 60,2 % it affected the left kidney; 58.1 % underwent supine surgery and 70,2 % were stone free with monotherapeutic percutaneous nephrolithotomy. Complications occurred in 19,9 %; 16,2 % were postoperative, 14,1 % infectious, 7,8 % Clavien-Dindo I, and 5,2 % IIIb. The type of lithiasis and the position of the percutaneous nephrolithotomy were not associated with complications (p> 0,05). The degree of complication was not related to the type of lithiasis (p> 0,05). Conclusions: The most frequent postoperative complications are those related to infection and bleeding; they slightly predominate in patients with partial and total staghorn stones and in those operated on in the supine position; the Clavien-Dindo grade of complications is higher in the more complex staghorn stones.

Hemodiafiltration with ultrafiltrate regeneration reduces free light chains without albumin loss in multiple myeloma patients.

BACKGROUND: Acute kidney injury (AKI) occurs in 12-20% of multiple myeloma (MM) patients. Several studies have shown a reduction of free light chains (FLC) using hemodialysis with High-Cut-Off membranes. However, this technique entails albumin loss. Hemodiafiltration with ultrafiltrate regeneration is a technique that includes a process of adsorption. The aim of this study was to evaluate the effectiveness of hemodiafiltration with ultrafiltrate regeneration in reducing FLC levels without causing albumin loss. METHODS: This is an observational study (2012 to 2018) including nine patients with MM (5 kappa, 4 lambda) and AKI. All patients were treated with chemotherapy and hemodiafiltration with ultrafiltrate regeneration. Blood Samples (pre and post-dialysis) and ultrafiltrate were collected pre and post-resin at 5 min after initiation of the session and 5 min before the end of the procedure. RESULTS: The serum levels of kappa and lambda were reduced by a 57.6 ± 10% and 33.5 ± 25% respectively. Serum albumin concentration remained unchanged after the procedure. In the ultrafiltrate, the mean FLC reduction ratio shortly after initiation of the dialysis procedure was: 99.2 and 97.06% for kappa and lambda respectively, and only 0.7% for albumin; and at the end of the session the percent reduction was: 63.7 and 33.62% for kappa and lambda respectively, and 0.015% for albumin. Patients clinical outcome was: 33.3% recovered renal function, 22.2% died during the first year and 44.4% required maintenance dialysis. CONCLUSIONS: Hemodiafiltration with ultrafiltrate regeneration reduces FLC levels without producing a significant loss of albumin; and, FLC removal is maintained throughout the session. Therefore, hemodiafiltration with ultrafiltrate regeneration may be considered an effective adjunctive therapy in patients with MM.

Direct regulation of fibroblast growth factor 23 by energy intake through mTOR.

To test the hypothesis that fibroblast growth factor 23 (FGF23) is directly regulated by energy intake, in vivo and in vitro experiments were conducted. Three groups of rats were fed diets with high (HC), normal (NC) and low (LC) caloric content that resulted in different energy intake. In vitro, UMR106 cells were incubated in high (HG, 4.5 g/l) or low glucose (LG, 1 g/l) medium. Additional treatments included phosphorus (P), mannitol, rapamycin and everolimus. Intestinal absorption of P and plasma P concentrations were similar in the three groups of rats. As compared with NC, plasma FGF23 concentrations were increased in HC and decreased in the LC group. A significant correlation between energy intake and plasma FGF23 concentrations was observed. In vitro, mRNA FGF23 was significantly higher in UMR106 cells cultured in HG than in LG. When exposed to high P, mRNA FGF23 increased but only when cells were cultured in HG. Cells incubated with HG and mechanistic target of rapamycin (mTOR) inhibitors expressed low mRNA FGF23, similar to the values obtained in LG. In conclusion, this study shows a direct regulation of FGF23 production by energy availability and demonstrates that the mTOR signaling pathway plays a central role in this regulatory system.

Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration.

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Parathyroidectomy in dialysis patients: Indications, methods, and consequences.

Secondary hyperparathyroidism, characterized by increased PTH synthesis and secretion, is often seen in advanced stages of chronic kidney disease. Excessive proliferation of parathyroid cells leads to the development of diffuse hyperplasia that subsequently progresses to nodular histology. Refractory hyperparathyroidism occurs when parathyroid glands fail to respond to medical therapy. Parathyroidectomy (PTX), surgical resection of parathyroid glands, is usually performed in cases of persistent serum levels of PTH above 1000 pg/mL associated with hypercalcemia or when hyperparathyroidism is refractory to conservative therapy. Parathyroidectomy can be carried out using different procedures: subtotal PTX or total PTX with or without parathyroid autotransplantation. Parathyroid surgery may have undesirable consequences due to PTH oversuppression, such as the development of adynamic bone disease; hungry bone syndrome is quite common after this surgery. However, PTX improves survival and parameters of mineral metabolism. Parathyroidectomy needs to be considered in those patients with severe hyperparathyroidism with a poor response to pharmacological treatment and with distinct undesirable effects of PTH on bone and mineral metabolism parameters.

Higher Proportion of Non-1-84 PTH Fragments in Peritoneal Dialysis Patients Compared to Hemodialysis Patients Using Solutions Containing 1.75 mmol/l Calcium.

Background: The prevalence of low- turnover bone disease (LTBD) in peritoneal dialysis (PD) patients is higher than in hemodialysis (HD) patients. LTBD patients may be at risk for vascular calcification, and cardiovascular disease. Current therapy for chronic kidney disease metabolic bone disorders (CKD-MBD) is guided by biochemical parameters, as bone biopsy is not used in routine clinical care. Methods: We assessed intact PTH (iPTH: 1-84PTH plus non-1-84PTH), 1-84PTH, and the 1-84PTH/non-1-84PTH ratio in 129 hemodialysis and 73 PD prevalent patients dialyzed with solutions containing 1.75 mmol/L calcium. Results: Hemodialysis and PD patients presented similar iPTH and tCa values and prevalence of putative LTBD as defined according to KDOQI iPTH cut-off levels or 1-84 PTH levels. However, iCa accounted for a higher percentage of tCa in PD (53%) than in hemodialysis (39%) p < 0.001, and the 1-84PTH/non-1-84PTH ratio was lower in PD than in hemodialysis patients (0.44 ± 0.12) vs. (0.60 ± 0.10), p < 0.001. The prevalence of putative LTBD when using the coexistence of 1-84PTH/non-1-84PTH ratio < 1.0 and iPTH < 420 pg/m, was higher in PD than in hemodialysis patients (73 vs. 16% respectively, p < 0.001). In a multivariate logistic regression analysis, dialysis modality was the main determinant of the 1-84PTH/non-1-84PTH ratio. Conclusion: Solutions containing 1.75 mmol/L calciums are associated to a higher proportion of non-1-84PTH fragments in PD than in HD patients. Different analytical criteria result in widely different estimates of LTBD prevalence, thus impairing the ability of clinicians to optimize therapy for CKD-MBD.

Novel Urinary Biomarkers For Improved Prediction Of Progressive Egfr Loss In Early Chronic Kidney Disease Stages And In High Risk Individuals Without Chronic Kidney Disease.

Chronic kidney disease is associated with increased risk of CKD progression and death. Therapeutic approaches to limit progression are limited. Developing tools for the early identification of those individuals most likely to progress will allow enriching clinical trials in high risk early CKD patients. The CKD273 classifier is a panel of 273 urinary peptides that enables early detection of CKD and prognosis of progression. We have generated urine capillary electrophoresis-mass spectrometry-based peptidomics CKD273 subclassifiers specific for CKD stages to allow the early identification of patients at high risk of CKD progression. In the validation cohort, the CKD273 subclassifiers outperformed albuminuria and CKD273 classifier for predicting rapid loss of eGFR in individuals with baseline eGFR > 60 ml/min/1.73 m2. In individuals with eGFR > 60 ml/min/1.73 m2 and albuminuria <30 mg/day, the CKD273 subclassifiers predicted rapid eGFR loss with AUC ranging from 0.797 (0.743-0.844) to 0.736 (0.689-0.780). The association between CKD273 subclassifiers and rapid progression remained significant after adjustment for age, sex, albuminuria, DM, baseline eGFR, and systolic blood pressure. Urinary peptidomics CKD273 subclassifiers outperformed albuminuria and CKD273 classifier for predicting the risk of rapid CKD progression in individuals with eGFR > 60 ml/min/1.73 m2. These CKD273 subclassifiers represented the earliest evidence of rapidly progressive CKD in non-albuminuric individuals with preserved renal function.

AGES: Automated Gas Environment System for in situ neutron powder diffraction.

High fluxes available at modern neutron and synchrotron sources have opened up a wide variety of in situ and operando studies of real processes using scattering techniques. This has allowed the user community to follow chemistry in the beam, which often requires high temperatures, gas flow, etc. In this paper, we describe an integrated gas handling system for the general-purpose powder diffraction beamline Powgen at the Spallation Neutron Source. The Automated Gas Environment System (AGES) allows control of both gas flow and temperature (room temperature to 850 °C), while measuring the partial pressure of oxygen and following the effluent gas by mass spectrometry, concurrent with neutron powder diffraction, in order to follow the structural evolution of materials under these conditions. The versatility of AGES is illustrated by two examples of experiments conducted with the system. In solid oxide fuel cell electrode materials, oxygen transport pathways in double perovskites PrBaCo2O5+δ and NdBaCo2O5+δ were elucidated by neutron diffraction measurements under atmosphere with oxygen partial pressures (pO2) of 10-1 to 10-4 (achieved using mixtures of nitrogen and oxygen) and temperatures from 575 to 850 °C. In another example, the potential oxygen storage material La1-xSrxFeO3 was measured under alternating flows of 15% CH4 in N2 and air (20% O2 in N2) at temperatures from 135 to 835 °C. From the oxygen stoichiometry, the optimal composition for oxygen storage was determined.

Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.

A high precision gas flow cell for performing in situ neutron studies of local atomic structure in catalytic materials.

Gas-solid interfaces enable a multitude of industrial processes, including heterogeneous catalysis; however, there are few methods available for studying the structure of this interface under operating conditions. Here, we present a new sample environment for interrogating materials under gas-flow conditions using time-of-flight neutron scattering under both constant and pulse probe gas flow. Outlined are descriptions of the gas flow cell and a commissioning example using the adsorption of N2 by Ca-exchanged zeolite-X (Na78-2xCaxAl78Si144O384,x ≈ 38). We demonstrate sensitivities to lattice contraction and N2 adsorption sites in the structure, with both static gas loading and gas flow. A steady-state isotope transient kinetic analysis of N2 adsorption measured simultaneously with mass spectrometry is also demonstrated. In the experiment, the gas flow through a plugged-flow gas-solid contactor is switched between N215 and N214 isotopes at a temperature of 300 K and a constant pressure of 1 atm; the gas flow and mass spectrum are correlated with the structure factor determined from event-based neutron total scattering. Available flow conditions, sample considerations, and future applications are discussed.

High phosphate induces a pro-inflammatory response by vascular smooth muscle cells and modulation by vitamin D derivatives.

In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an in vitro model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1ß, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10-8 M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10-10 and 10-12 M attenuated but 10-8 M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.

Procaine Inhibits Osteo/Odontogenesis through Wnt/ß-Catenin Inactivation.

INTRODUCTION: Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. METHODS: In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. RESULTS: Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/ß-catenin pathway, observing a loss of nuclear ß-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3ß and an increase of phosphorylated ß-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3ß, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3ß and ß-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. CONCLUSIONS: Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/ß-catenin pathway through the increase of Gsk3ß expression and ß-catenin phosphorylation.

Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs.

Angiotensin II prevents calcification in vascular smooth muscle cells by enhancing magnesium influx.

BACKGROUND: Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). Low magnesium levels are associated with VC, and recent in vitro studies confirm a protective role of magnesium, which is mediated by its entry into the VSMCs through the Transient Receptor Potential Melastatin 7 (TRPM7) channel. The role of Angiotensin II (Ang II) on VC is still unclear. As Ang II is able to stimulate TRPM7 activity, we hypothesize that it might prevent VC. Thus, the aim of this study was to dissect the direct effect of Ang II on VC. MATERIALS AND METHODS: We worked with a model of high phosphate (HP)-induced calcification in human aortic smooth muscle cells, which resembles the CKD-related VC. RESULTS: Addition of Ang II to cells growing in HP decreased calcification, which was associated with the upregulation of the osteogenic factors BMP2, Runx2/Cbfa1, Osterix and ALP. A reduction of magnesium entry into the HP-calcifying cells was found. The treatment with Ang II avoided this reduction, which was reversed by the cotreatment with the TRPM7-inhibitor 2-APB. The protective effect of Ang II was related to AT1R-induced ERK1/2 MAPKinase activation. HP-induced calcification was also associated with the upregulation of the canonical Wnt/beta-catenin pathway, while its downregulation was related to attenuation of calcification by Ang II. CONCLUSION: As hypothesized, Ang II prevented phosphate-induced calcification in VSMCs, which appears mediated by the increase of magnesium influx and by the activation of the ERK1/2 and the inhibition of the canonical Wnt/beta-catenin signalling pathways.