Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease.

AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.

Plasma glycocalyx pattern: a mirror of endothelial damage in chronic kidney disease.

Background: Endothelial damage and cardiovascular disease complicate chronic kidney disease (CKD). The increased atherogenicity observed in patients with CKD can be linked to microinflammation and endothelial damage. Circulating endothelial glycocalyx degradation products, such as perlecan and decorin, tend to be elevated in CKD. We aimed to explore the association between the plasma perlecan and decorin levels and this pro-inflammatory and atherogenic state by studying monocyte subpopulations and intracellular adhesion molecule (ICAM)-1 expression in patients with CKD. Methods: We studied 17 healthy controls, 23 patients with advanced CKD, 25 patients on haemodialysis, 23 patients on peritoneal dialysis and 20 patients who underwent kidney transplantation. Perlecan and decorin levels were evaluated using enzyme-linked immunosorbent assays, and the monocyte phenotype was analysed using direct immunofluorescence and flow cytometry. Results: The plasma perlecan levels were higher in patients with CKD than in the healthy controls. These levels were associated with a higher prevalence of ICAM-1+ monocytes. Conversely, patients with advanced CKD (pre-dialysis) had higher plasma decorin levels, which were associated with a reduced ICAM-1 expression per monocyte. Conclusions: Elevated perlecan levels in CKD may be associated with a higher prevalence of ICAM-1+ monocytes and a pro-inflammatory phenotype. Elevated decorin levels may act as a negative regulator of ICAM-1 expression in monocytes. Therefore, perlecan and decorin may be related to inflammation and monocyte activation in CKD and may act as potential markers of endothelial damage.

New mechanisms involved in the development of cardiovascular disease in chronic kidney disease.

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs' role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients.

Nuevos mecanismos implicados en el desarrollo de la enfermedad cardiovascular en la enfermedad renal crónica / New mechanisms involved in the development of cardiovascular disease in chronic kidney disease

La enfermedad renal crónica (ERC) tiene una alta incidencia mundial y una tendencia ascendente que afecta principalmente a personas de edad avanzada. Cuando la ERC está muy avanzada se requiere el uso de terapias renales sustitutivas para prolongar la vida (diálisis o trasplante renal) y, pese a que la diálisis mejora muchas complicaciones de la ERC, la enfermedad no revierte de manera completa. Estos pacientes presentan un aumento del estrés oxidativo, inflamación crónica y aumento de la liberación de vesículas extracelulares (VE), que provocan daño endotelial y el desarrollo de distintas enfermedades cardiovasculares (ECV). De hecho, los pacientes con ERC desarrollan de forma prematura enfermedades asociadas a una edad avanzada, como es el caso de las ECV. Las VE desempeñan un papel muy importante en el desarrollo de ECV en pacientes con ERC, ya que su número aumenta en el plasma y su contenido se modifica. Las VE de pacientes con ERC generan disfunción endotelial, senescencia y calcificación vascular. Además, los miRNA libres o transportados en las VE junto a otros componentes vehiculados en estas VE promueven disfunción endotelial, eventos trombóticos y calcificación vascular en los pacientes con ERC, entre otros efectos. En esta revisión se describen los factores clásicos y el papel de nuevos mecanismos que intervienen en el desarrollo de la ECV asociada a la ERC, con especial hincapié en el papel de las VE en el desarrollo de enfermedades cardiovasculares en un contexto de ERC. Además, se expone el papel de las VE como herramienta diagnóstica y como diana terapéutica, actuando sobre su liberación o contenido para intentar evitar el desarrollo de ECV en enfermos renales crónicos. (AU)

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs’ role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients. (AU)

Endothelial Senescence and the Chronic Vascular Diseases: Challenges and Therapeutic Opportunities in Atherosclerosis.

Atherosclerosis is probably one of the paradigms of disease linked to aging. Underlying the physiopathology of atherosclerosis are cellular senescence, oxidative stress, and inflammation. These factors are increased in the elderly and from chronic disease patients. Elevated levels of oxidative stress affect cellular function and metabolism, inducing senescence. This senescence modifies the cell phenotype into a senescent secretory phenotype. This phenotype activates immune cells, leading to chronic systemic inflammation. Moreover, due to their secretory phenotype, senescence cells present an increased release of highlighted extracellular vesicles that will change nearby/neighborhood cells and paracrine signaling. For this reason, searching for specific senescent cell biomarkers and therapies against the development/killing of senescent cells has become relevant. Recently, senomorphic and senolityc drugs have become relevant in slowing down or eliminating senescence cells. However, even though they have shown promising results in experimental studies, their clinical use is still yet to be determined.

Premature Aging in Chronic Kidney Disease: The Outcome of Persistent Inflammation beyond the Bounds.

Over the last hundred years, life expectancy in developed countries has increased because of healthier living habits and the treatment of chronic pathologies causing premature aging. Aging is an inexorable, time-dependent, multifactorial process characterized by a series of progressive and irreversible physiological changes associated with loss of functional, psychological, and social capabilities. Numerous factors, such as oxidative stress, inflammation, and cellular senescence, and an irreversible geriatric syndrome known as frailty, contribute to human body deterioration in aging. The speed of aging may differ between individuals depending on the presence or absence of multiple factors (genetic and/or environment) and the subsequent misbalance of homeostasis, together with the increase of frailty, which also plays a key role in developing chronic diseases. In addition, pathological circumstances have been reported to precipitate or accelerate the aging process. This review investigated the mechanisms involved in the developing pathologies, particularly chronic kidney disease, associated with aging.

A high magnesium concentration in citrate dialysate prevents oxidative stress and damage in human monocytes in vitro.

BACKGROUND: The use of dialysis fluids (DFs) during haemodialysis has been associated with increased oxidative stress and reduced serum magnesium (Mg) levels, contributing to chronic inflammation. Since the role of Mg in modulating immune function and reducing oxidative stress has been demonstrated, the aim of this study was to characterize in vitro whether increasing the Mg concentration in DFs could protect immune cells from oxidative stress and damage. METHODS: The effect of citrate [citrate dialysis fluid (CDF), 1 mM] or acetate [acetate dialysis fluid (ADF), 3 mM] dialysates with low (0.5 mM; routinely used) or high (1 mM, 1.25 mM and 2 mM) Mg concentrations was assessed in THP-1 human monocytes. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and oxidized/reduced (GSSG/GSH) glutathione were quantified under basal and inflammatory conditions (stimulation with lipopolysaccharide, LPS). RESULTS: The increase of Mg in CDF resulted in a significant reduction of ROS production under basal and inflammatory conditions (extremely marked in 2 mM Mg; P < 0.001). These effects were not observed in ADF. Interestingly, in a dose-dependent manner, high Mg doses in CDF reduced oxidative stress in monocytes under both basal and inflammatory conditions. In fact, 2 mM Mg significantly decreased the levels of GSH, GSSG and MDA and the GSSG/GSH ratio in relation to 0.5 mM Mg. CONCLUSIONS: CDF produces lower oxidative stress than ADF. The increase of Mg content in DFs, especially in CDF, could have a positive and protective effect in reducing oxidative stress and damage in immune cells, especially under inflammatory conditions.

Microvesicles from indoxyl sulfate-treated endothelial cells induce vascular calcification in vitro.

Vascular calcification (VC), an unpredictable pathophysiological process and critical event in patients with cardiovascular diseases (CVDs), is the leading cause of morbi-mortality and disability in chronic kidney disease (CKD) patients worldwide. Currently, no diagnostic method is available for identifying patients at risk of VC development; the pathology is detected when the process is irreversible. Extracellular vesicles (EVs) from endothelial cells might promote VC. Therefore, their evaluation and characterization could be useful for designing new diagnostic tools. The aim of the present study is to investigate whether microvesicles (MVs) from endothelial cells damaged by uremic toxin and indoxyl sulfate (IS) could induce calcification in human vascular smooth muscle cells (VMSCs). Besides, we have also analyzed the molecular mechanisms by which these endothelial MVs can promote VC development. Endothelial damage has been evaluated according to the percentage of senescence in endothelial cells, differential microRNAs in endothelial cells, and the amount of MVs released per cell. To identify the role of MVs in VC, VSMCs were treated with MVs from IS-treated endothelial cells. Calcium, inflammatory gene expression, and procalcification mediator levels in VSMCs were determined. IS-treated endothelial cells underwent senescence and exhibited modulated microRNA expression and an increase in the release of MVs. VSMCs exposed to these MVs modulated the expression of pro-inflammatory genes and some mediators involved in calcification progression. MVs produced by IS-treated endothelial cells promoted calcification in VSMCs.

Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line.

Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs). Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used.

Mechanisms of Cardiovascular Disorders in Patients With Chronic Kidney Disease: A Process Related to Accelerated Senescence.

Cardiovascular diseases (CVDs), especially those involving a systemic inflammatory process such as atherosclerosis, remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD is a systemic condition affecting approximately 10% of the general population. The prevalence of CKD has increased over the past decades because of the aging of the population worldwide. Indeed, CVDs in patients with CKD constitute a premature form of CVD observed in the general population. Multiple studies indicate that patients with renal disease undergo accelerated aging, which precipitates the appearance of pathologies, including CVDs, usually associated with advanced age. In this review, we discuss several aspects that characterize CKD-associated CVDs, such as etiopathogenic elements that CKD patients share with the general population, changes in the cellular balance of reactive oxygen species (ROS), and the associated process of cellular senescence. Uremia-associated aging is linked with numerous changes at the cellular and molecular level. These changes are similar to those observed in the normal process of physiologic aging. We also discuss new perspectives in the study of CKD-associated CVDs and epigenetic alterations in intercellular signaling, mediated by microRNAs and/or extracellular vesicles (EVs), which promote vascular damage and subsequent development of CVD. Understanding the processes and factors involved in accelerated senescence and other abnormal intercellular signaling will identify new therapeutic targets and lead to improved methods of diagnosis and monitoring for patients with CKD-associated CVDs.

Hypoxia-Inducible Factor-1α: The Master Regulator of Endothelial Cell Senescence in Vascular Aging.

Aging is one of the hottest topics in biomedical research. Advances in research and medicine have helped to preserve human health, leading to an extension of life expectancy. However, the extension of life is an irreversible process that is accompanied by the development of aging-related conditions such as weakness, slower metabolism, and stiffness of vessels. It also debated that aging can be considered an actual disease with aging-derived comorbidities, including cancer or cardiovascular disease. Currently, cardiovascular disorders, including atherosclerosis, are considered as premature aging and represent the first causes of death in developed countries, accounting for 31% of annual deaths globally. Emerging evidence has identified hypoxia-inducible factor-1α as a critical transcription factor with an essential role in aging-related pathology, in particular, regulating cellular senescence associated with cardiovascular aging. In this review, we will focus on the regulation of senescence mediated by hypoxia-inducible factor-1α in age-related pathologies, with particular emphasis on the crosstalk between endothelial and vascular cells in age-associated atherosclerotic lesions. More specifically, we will focus on the characteristics and mechanisms by which cells within the vascular wall, including endothelial and vascular cells, achieve a senescent phenotype.

MicroRNA-126 regulates Hypoxia-Inducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence.

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1α in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1α, and the microvesicles released by these cells failed to carry HIF-1α. Of note, HIF-1α protein levels were restored in HIF-1α stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1α, transfection with a microRNA-126 inhibitor downregulated HIF-1α expression in early passage endothelial cells. Moreover, while HIF-1α inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1α is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease.

Endothelial Extracellular Vesicles Produced by Senescent Cells: Pathophysiological Role in the Cardiovascular Disease Associated with all Types of Diabetes Mellitus.

Endothelial senescence-associated with aging or induced prematurely in pathological situations, such as diabetes, is a first step in the development of Cardiovascular Disease (CVDs) and particularly inflammatory cardiovascular diseases. The main mechanism that links endothelial senescence and the progression of CVDs is the production of altered Extracellular Vesicles (EVs) by senescent endothelial cells among them, Microvesicles (MVs). MVs are recognized as intercellular signaling elements that play a key role in regulating tissue homeostasis. However, MVs produced by damage cell conveyed epigenetic signals, mainly involving microRNAs, which induce many of the injured responses in other vascular cells leading to the development of CVDs. Many studies strongly support that the quantification and characterization of the MVs released by senescent endothelial cells may be useful diagnostic tools in patients with CVDs, as well as a future therapeutic target for these diseases. In this review, we summarize the current knowledge linking senescence-associated MVs to the development of CVDs and discuss the roles of these MVs, in particular, in diabetic-associated increases the risk of CVDs.

Senescent Microvesicles: A Novel Advance in Molecular Mechanisms of Atherosclerotic Calcification.

Atherosclerosis, a chronic inflammatory disease that causes the most heart attacks and strokes in humans, is the leading cause of death in the developing world; its principal clinical manifestation is coronary artery disease. The development of atherosclerosis is attributed to the aging process itself (biological aging) and is also associated with the development of chronic diseases (premature aging). Both aging processes produce an increase in risk factors such as oxidative stress, endothelial dysfunction and proinflammatory cytokines (oxi-inflamm-aging) that might generate endothelial senescence associated with damage in the vascular system. Cellular senescence increases microvesicle release as carriers of molecular information, which contributes to the development and calcification of atherosclerotic plaque, as a final step in advanced atherosclerotic plaque formation. Consequently, this review aims to summarize the information gleaned to date from studies investigating how the senescent extracellular vesicles, by delivering biological signalling, contribute to atherosclerotic calcification.

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery.

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

Reduced TH expression and α-synuclein accumulation contribute towards nigrostriatal dysfunction in experimental hepatic encephalopathy.

PURPOSE: The present work examines α-synuclein expression in the nigrostriatal system of a rat chronic hepatic encephalopathy model induced by portacaval anastomosis (PCA). There is evidence that dopaminergic dysfunction in disease conditions is strongly associated with such expression. Possible relationships among dopaminergic neurons, astroglial cells and α-synuclein expression were sought. METHODS: Brain tissue samples from rats at 1 and 6 months post-PCA, and controls, were analysed immunohistochemically using antibodies against tyrosine hydroxylase (TH), α-synuclein, glial fibrillary acidic protein (GFAP) and ubiquitin (Ub). RESULTS: In the control rats, TH immunoreactivity was detected in the neuronal cell bodies and processes in the substantia nigra pars compacta (SNc). A dense TH-positive network of neurons was also seen in the striatum. In the PCA-exposed rats, however, a reduction in TH-positive neurons was seen at both 1 and 6 months in the SNc, as well as a reduction in TH-positive fibres in the striatum. This was coincident with the appearance of α-synuclein-immunoreactive neurons in the SNc; some of the TH-positive neurons also showed α-synuclein immunoreactivity. In addition, α-synuclein accumulation was seen in the SNc and striatum at both 1 and 6 months post-PCA, whereas α-synuclein was only mildly expressed in the nigrostriatal pathway of the controls. Astrogliosis was also seen following PCA, as revealed by increased GFAP expression from 1 month to 6 months post-PCA in both the SN and striatum. The astroglial activation level in the SN paralleled the reduced neuronal expression of TH throughout PCA exposure. CONCLUSION: α-synuclein accumulation following PCA may induce dopaminergic dysfunction via the downregulation of TH, as well as astroglial activation.

The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles.

We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.

Microvesicles from the plasma of elderly subjects and from senescent endothelial cells promote vascular calcification.

Vascular calcification is commonly seen in elderly people, though it can also appear in middle-aged subjects affected by premature vascular aging. The aim of this work is to test the involvement of microvesicles (MVs) produced by senescent endothelial cells (EC) and from plasma of elderly people in vascular calcification. The present work shows that MVs produced by senescent cultured ECs, plus those found in the plasma of elderly subjects, promote calcification in vascular smooth muscle cells. Only MVs from senescent ECs, and from elderly subjects' plasma, induced calcification. This ability correlated with these types of MVs' carriage of: a) increased quantities of annexins (which might act as nucleation sites for calcification), b) increased quantities of bone-morphogenic protein, and c) larger Ca contents. The MVs of senescent, cultured ECs, and those present in the plasma of elderly subjects, promote vascular calcification. The present results provide mechanistic insights into the observed increase in vascular calcification-related diseases in the elderly, and in younger patients with premature vascular aging, paving the way towards novel therapeutic strategies.

Ammonia Affects Astroglial Proliferation in Culture.

Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.

Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes.

Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.