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.

Protein Carbamylation: A Marker Reflecting Increased Age-Related Cell Oxidation.

Carbamylation is a post-translational modification of proteins that may partake in the oxidative stress-associated cell damage, and its increment has been recently proposed as a "hallmark of aging". The molecular mechanisms associated with aging are related to an increased release of free radicals. We have studied whether carbamylated proteins from the peripheral blood of healthy subjects are related to oxidative damage and aging, taking into account the gender and the immune profile of the subjects. The study was performed in healthy human volunteers. The detection of protein carbamylation and malondialdehyde (MDA) levels was evaluated using commercial kits. The immune profile was calculated using parameters of immune cell function. The results show that the individuals from the elderly group (60⁻79 years old) have increased carbamylated protein and MDA levels. When considered by gender, only men between 60 and 79 years old showed significantly increased carbamylated proteins and MDA levels. When those subjects were classified by their immune profile, the carbamylated protein levels were higher in those with an older immune profile. In conclusion, the carbamylation of proteins in peripheral blood is related to age-associated oxidative damage and to an aging functional immunological signature. Our results suggest that carbamylated proteins may play an important role at the cellular level in the aging process.

Markers of endothelial damage in patients with chronic kidney disease on hemodialysis.

Patients with Stage 5 chronic kidney disease who are on hemodialysis (HD) remain in a chronic inflammatory state, characterized by the accumulation of uremic toxins that induce endothelial damage and cardiovascular disease (CVD). Our aim was to examine microvesicles (MVs), monocyte subpopulations, and angiopoietins (Ang) to identify prognostic markers in HD patients with or without diabetes mellitus (DM). A total of 160 prevalent HD patients from 10 centers across Spain were obtained from the Biobank of the Nephrology Renal Network (Madrid, Spain): 80 patients with DM and 80 patients without DM who were matched for clinical and demographic criteria. MVs from plasma and several monocyte subpopulations (CD142+/CD16+, CD14+/CD162+) were analyzed by flow cytometry, and the plasma concentrations of Ang1 and Ang2 were quantified by ELISA. Data on CVD were gathered over the 5.5 yr after these samples were obtained. MV level, monocyte subpopulations (CD14+/CD162+ and CD142+/CD16+), and Ang2-to-Ang1 ratios increased in HD patients with DM compared with non-DM patients. Moreover, MV level above the median (264 MVs/µl) was associated independently with greater mortality. MVs, monocyte subpopulations, and Ang2-to-Ang1 ratio can be used as predictors for CVD. In addition, MV level has a potential predictive value in the prevention of CVD in HD patients. These parameters undergo more extensive changes in patients with DM.

Dietary magnesium supplementation prevents and reverses vascular and soft tissue calcifications in uremic rats.

Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magnesium (0.6%), improved bone homeostasis as compared to basal dietary magnesium (0.1%). Results of our study also suggested that the protective effect of magnesium on vascular calcification was not limited to its action as an intestinal phosphate binder since magnesium administered intraperitoneally also decreased vascular calcification. Oral magnesium supplementation also reduced blood pressure in uremic rats, and in vitro medium magnesium decreased BMP-2 and p65-NF-κB in TNF-α-treated human umbilical vein endothelial cells. Finally, in uremic rats with established vascular calcification, increasing dietary magnesium from 0.1% magnesium to 0.6% reduced the mortality rate from 52% to 28%, which was associated with reduced vascular calcification. Thus, increasing dietary magnesium reduced both vascular calcification and mortality in uremic rats.

Hemodiafiltration With Endogenous Reinfusion Improved Microinflammation and Endothelial Damage Compared With Online-Hemodiafiltration: A Hypothesis Generating Study.

Hemodiafiltration with endogenous reinfusion (HFR) after ultrafiltrate passage through a resin cartridge combines adsorption, convection, and diffusion. Our prospective single-center crossover study compared HFR and online-hemodiafiltration (OLHDF) effects on two uremic toxins and 13 inflammatory, endothelial status, or oxidative stress markers. After an 8-week run-in period of high-flux hemodialysis, 17 eligible stable dialysis patients (median age 65 years, 10 male) without overt clinical inflammation were scheduled for four 8-week periods in the sequence: HFR/OLHDF/HFR/OLHDF. Relative to OLHDF, HFR was associated with greater indoxyl sulfate removal and lesser abnormalities in all other study variables, namely circulating interleukin-6, tumor necrosis factor-alpha, proportions of activated proinflammatory (CD14+CD16+, CD14++CD16+) monocytes, endothelial progenitor cells, apoptotic endothelial microparticles, vascular endothelial growth factor, vascular cellular adhesion molecule, angiopoietins 2 and 1, annexin V, and superoxide dismutase. Differences were significant (P < 0.05) in median values of 13/15 variables. Study period comparisons were generally consistent with dialysis technique comparisons, as were data from the subgroup completing all study periods (n = 9). Our investigation provides hypothesis-generating results suggesting that compared with OLHDF, HFR improves protein-bound toxin removal, inflammatory and endothelial status, and oxidative stress.

Endothelial microparticles mediate inflammation-induced vascular calcification.

Stimulation of endothelial cells (ECs) with TNF-α causes an increase in the expression of bone morphogenetic protein-2 (BMP-2) and the production of endothelial microparticles (EMPs). BMP-2 is known to produce osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that EMPs from TNF-α-stimulated endothelial cells (HUVECs) contained a significant amount of BMP-2 and were able to enhance VSMC osteogenesis and calcification. Calcium content was greater in VSMCs exposed to EMPs from TNF-α-treated HUVECs than EMPs from nontreated HUVECs (3.56 ± 0.57 vs. 1.48 ± 0.56 µg/mg protein; P < 0.05). The increase in calcification was accompanied by up-regulation of Cbfa1 (osteogenic transcription factor) and down-regulation of SM22α (VSMC lineage marker). Inhibition of BMP-2 by small interfering RNA reduced the VSMC calcification induced by EMPs from TNF-α-treated HUVECs. Similar osteogenic capability was observed in EMPs from both patients with chronic kidney disease and senescent cells, which also presented a high level of BMP-2 expression. Labeling of EMPs with CellTracker shows that EMPs are phagocytized by VSMCs under all conditions (with or without high phosphate, control, and EMPs from TNF-α-treated HUVECs). Our data suggest that EC damage results in the release of EMPs with a high content of calcium and BMP-2 that are able to induce calcification and osteogenic differentiation of VSMCs.

Effect of uraemia on endothelial cell damage is mediated by the integrin linked kinase pathway.

KEY POINTS: Patients with chronic kidney disease have a higher risk of developing cardiovascular diseases than the general population. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which have poor clearance by conventional dialysis. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity and in this study we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. For the first time, we demonstrate the implication of ILK in the protection against endothelial cell damage (inhibition of proliferation, toxicity, oxidative stress and programed cell death) induced by uraemic serum from chronic kidney disease patients and uraemic toxins. This molecular mechanism may have clinical relevance because it highlights the importance of maintaining high levels of ILK activity to help preserve endothelial integrity, at least in early stages of chronic kidney disease. ABSTRACT: Patients with chronic kidney disease (CKD) have a higher risk of developing cardiovascular diseases. Their vascular endothelium is dysfunctional, among other things, because it is permanently exposed to uraemic toxins, several of which, mostly protein-bound compounds such as indoxyl sulfate (IS) and p-cresyl sulphate, having poor clearance by conventional dialysis, induce endothelial toxicity. However, the molecular mechanism by which uraemic toxins regulate early stages of endothelial dysfunction remains unclear. Recent studies have demonstrated the important role of integrin-linked kinase (ILK) in the maintenance of endothelial integrity. In this study, we investigate the involvement of ILK in the mechanism underlying vascular endothelial damage that occurs in uraemia. First, we show that incubation of EA.hy926 cells with human uraemic serum from CKD patients upregulates ILK activity. This ILK activation also occurs when the cells are exposed to IS (25-100 µg ml(-1)), p-cresol (10-100 µg ml(-1)) or both combined, compared to human serum control. Next, we observed that high doses of both toxins together induce a slight decrease in cell proliferation and increase apoptosis and reactive oxygen species production. Interestingly, these toxic effects displayed a strong increase when the ILK protein is knocked down by small interfering RNA, even at low doses of uraemic toxins. Abrogation of AKT has demonstrated the ILK/AKT signalling pathway involved in these processes. This study has demonstrated the implication of ILK in the protection against endothelial cell damage induced by uraemic toxins, a molecular mechanism that could play a protective role in the early stages of endothelial dysfunction observed in uraemic patients.

Endothelial damage and vascular calcification in patients with chronic kidney disease.

Vascular calcification (VC) is a frequent complication of chronic kidney disease (CKD) and is a predictor of cardiovascular morbidity and mortality. In the present study, we investigated the potential involvement of endothelial microparticles (MPs) and endothelial progenitor cells (EPCs) in the generation of VC in CKD patients. The number of circulating EMPs is greater in patients with VC than without VC (307 ± 167 vs. 99 ± 75 EMPs/µl, P < 0.001). The percentage of EPCs is significantly lower in patient with VC than in patients without VC (0.14 ± 0.11% vs. 0.25 ± 0.18%, P = 0.002). The number of EPCs expressing osteocalcin (OCN) was higher in VC patients (349 ± 63 cells/100,000) than in non-VC patients (139 ± 75 cells/100,000, P < 0.01). In vitro, MPs obtained from CKD patients were able to induce OCN expression in EPCs from healthy donors; the increase in OCN expression was more accentuated if MPs were obtained from CKD patients with VC. MPs from CKD patients also induced OCN expression in vascular smooth muscle cells and fibroblasts. In CKD patients, the rise in endothelial MPs associated with a decrease in the number of EPCs, suggesting an imbalance in the processes of endothelial damage and repair in CKD patients, mainly those with VC. Our results suggest that EPCs, through OCN expression, may directly participate in the process of VC.

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.

Senescent CD14+CD16+ monocytes exhibit proinflammatory and proatherosclerotic activity.

In elderly subjects and in patients with chronic inflammatory diseases, there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype, whose origin and functional relevance has not been well characterized. In this study, we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells, and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence, intracellular cytokine expression, ability to interact with endothelial cells, and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p < 0.05) and increased expression of ß-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%, respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209, release of cytokines in response to low-intensity stimulus, and increased capacity to sustain lymphocyte proliferation. Finally, compared with CD14(++)CD16(-) cells, CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary, our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells, with increased inflammatory activity and ability to interact with endothelial cells. Therefore, accumulation of senescent monocytes may explain, in part, the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases.

Post-dilution high convective transport improves microinflammation and endothelial dysfunction independently of the technique.

BACKGROUND/AIMS: We examined the effects of different online hemodiafiltration techniques on microinflammation and endothelial damage/repair. METHODS: The study was designed as a prospective crossover study. Flow cytometry was used to measure CD14(+)CD16(+) monocytes, apoptotic endothelial microparticles (EMPs), and endothelial progenitor cells (EPCs). RESULTS: Patients treated with high-flux hemodialysis showed a marked chronic inflammatory state (HF-HD 11 ± 2) versus healthy subjects (HS 3.9 ± 2.3; p < 0.05). High convective transport, independent of the technique used, improves microinflammatory parameters (OL-HDF 7.3 ± 2.1 or MID 6.5 ± 3.4; p < 0.05) and the endothelial damage/repair balance compared to HF-HD (EPCs HF-HD 0.3 ± 0.2), with no differences found between the two modalities (EPCs OL-HDF 0.6 ± 0.1, MID 0.6 ± 0.2; p < 0.05). CONCLUSION: An increase in convective transport improves the microinflammatory state and the endothelial damage/repair of these patients independently of the technique used.

Sex differences in markers of oxidation and inflammation. Implications for ageing.

Sexual dimorphism is a key factor to consider in the ageing process given the impact that it has on life expectancy. The oxidative-inflammatory theory of ageing states that the ageing process is the result of the establishment of oxidative stress which, due to the interplay of the immune system, translates into inflammatory stress, and that both processes are responsible for the damage and loss of function of an organism. We show that there are relevant gender differences in a number of oxidative and inflammatory markers and propose that they may account for the differential lifespan between sexes, given that males display, in general, higher oxidation and basal inflammation. In addition, we explain the significant role of circulating cell-free DNA as a marker of oxidative damage and an inductor of inflammation, connecting both processes and having the potential to become a useful ageing marker. Finally, we discuss how oxidative and inflammatory changes take place differentially with ageing in each sex, which could also have an impact on the sex-differential lifespan. Further research including sex as an essential variable is needed to understand the grounds of sex differences in ageing and to better comprehend ageing itself.

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.

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.

Carbamylated low-density lipoprotein induces oxidative stress and accelerated senescence in human endothelial progenitor cells.

Carbamylated low-density lipoprotein (cLDL) plays a role in atherosclerosis. In this study we evaluate the effect of uremia on LDL carbamylation and the effect of cLDL and oxidized LDL (oxLDL; 200 µg/ml) on number, function, and genomic stability of endothelial progenitor cells (EPCs) obtained from healthy volunteers. cLDL was generated after incubation of native LDL (nLDL) with uremic serum from patients with chronic kidney disease (CKD) stages 2-4. Oxidative stress was measured by flow cytometry and fluorescent microscopy, mitochondrial depolarization by flow cytometry, senescence by ß-galactosidase activity and telomere length, and DNA damage by phosphorylated histone H2AX (γH2AX). The percentage of cLDL by uremic serum was related to the severity of CKD. Compared with nLDL, cLDL induced an increase in oxidative stress (62±5 vs. 8±3%, P<0.001) and cells with mitochondrial depolarization (73±7 vs. 9±5%, P<0.001), and a decrease in EPC proliferation and angiogenesis. cLDL also induced accelerated senescence (73±16 vs. 12±9%, P<0.001), which was associated with a decrease in the expression of γH2AX (62±9 vs. 5±3%, P<0.001). The degree of injury induced by cLDL was comparable to that observed with oxLDL. This study supports the hypothesis that cLDL triggers genomic damage in EPCs, resulting in premature senescence. We can, therefore, hypothesize that EPCs injury by cLDL contributes to an increase in atherosclerotic disease in CKD.

Losartan prevents the development of the pro-inflammatory monocytes CD14+CD16+ in haemodialysis patients.

BACKGROUND: The principal cause of mortality in haemodialysis (HD) patients is cardiovascular disease, which is linked to chronic inflammation. Recent studies have demonstrated that angiotensin II receptor AT1 antagonists have anti-inflammatory properties. In this study, we evaluated the effect of losartan on CD14+CD16+ monocytes in HD patients. In addition, we developed an in vitro model to study the mechanisms by which losartan modulates these cells. METHODS: We divided 18 HD patients into two groups, based on anti-hypertensive treatment: 9 patients were treated with losartan (losartan group) and 9 received other anti-hypertensive drugs that did not affect the renin-angiotensin axis (no-losartan group). Losartan was withdrawn in five patients from the losartan group for 2 months. Ten healthy subjects were included as controls. Invitro, we studied the differentiation of monocytes from healthy donors on stimulation with interleukin (IL)-10, IL-4 and granulocyte monocytes colony-stimulating factor with or without losartan in the culture medium. RESULTS: In patients who were taking losartan, the percentage of monocytes that expressed CD14+CD16+ was lower compared with patients in the no-losartan group. The percentage of CD14+CD16+ was similar in the losartan group and healthy subjects. When losartan was withdrawn from five patients in the losartan group, the percentage of CD14+CD16+ monocytes increased compared with before withdrawal. In vitro, when we added losartan to the culture medium, CD14++CD16- monocytes failed to differentiate into CD14+CD16+ cells. CONCLUSION: Losartan acts as an immunomodulator that prevents the development of CD14+CD16+ pro-inflammatory monocytes in HD patients.

Effects of intravenous iron on mononuclear cells during the haemodialysis session.

BACKGROUND: This study analysed, in vivo and in vitro, the effects of four different intravenous iron preparations (iron gluconate, iron sucrose, iron dextran and ferric carboxymaltose) on activation and damage of mononuclear cells. METHODS: A randomized prospective study was conducted in 10 haemodialysis (HD) patients. Blood samples were collected at baseline (T0); 1 h after starting HD, just before the iron or saline administration (T1); 30 min after the iron or saline infusion (T2) and at the end of HD (T3). In addition, peripheral blood mononuclear cells from 10 healthy individuals and 9 chronic kidney disease Stage-5 (CKD-5) without HD treatment were cultured with the 4 iron preparations. RESULTS: Iron infusion during the HD session increased the percentage of mononuclear cells with reactive oxygen species (ROS) production, Inter-Cellular Adhesion Molecule-1 (ICAM-1) and apoptosis. There were no significant differences between the four iron preparations. Culture of mononuclear cells from healthy individuals and CKD-5 patients with the different iron preparations resulted in a significant increase in ROS, ICAM-1 and apoptosis as compared with control. In an additional study, the effect of original iron sucrose formulation on mononuclear cells was compared with that of one generic formulation. The generic formulation produced a greater increase in ROS, ICAM-1 and apoptosis than the original iron sucrose. CONCLUSIONS: Our results suggest that intravenous iron has deleterious effects on mononuclear cells. The four iron compounds evaluated produced similar effects on oxidative stress, cell activation and apoptosis. However, the effects of iron compounds with the same formulation were different, thus further investigation may be required to establish the safety of iron preparations that theoretically have the same composition.

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.

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.

Oxidative Stress in Patients with Advanced CKD and Renal Replacement Therapy: The Key Role of Peripheral Blood Leukocytes.

Oxidative stress plays a key role in the pathophysiology of chronic kidney disease (CKD). Most studies have investigated peripheral redox state focus on plasma, but not in different immune cells. Our study analyzed several redox state markers in plasma and isolated peripheral polymorphonuclear (PMNs) and mononuclear (MNs) leukocytes from advanced-CKD patients, also evaluating differences of hemodialysis (HD) and peritoneal dialysis (PD) procedures. Antioxidant (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH)) and oxidant parameters (xanthine oxidase (XO), oxidized glutathione (GSSG), malondialdehyde (MDA)) were assessed in plasma, PMNs and MNs from non-dialysis-dependent-CKD (NDD-CKD), HD and PD patients and healthy controls. Increased oxidative stress and damage were observed in plasma, PMNs and MNs from NDD-CKD, HD and PD patients (increased XO, GSSG and MDA; decreased SOD, CAT, GPX and GSH; altered GSSG/GSH balance). Several oxidative alterations were more exacerbated in PMNs, whereas others were only observed in MNs. Dialysis procedures had a positive effect on preserving the GSSG/GSH balance in PMNs. Interestingly, PD patients showed greater oxidative stress than HD patients, especially in MNs. The assessment of redox state parameters in PMNs and MNs could have potential use as biomarkers of the CKD progression.