Ana Merino

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 β-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.

On-Line Hemodiafiltration Reduces the Proinflammatory CD14+CD16+ Monocyte-Derived Dendritic Cells: A Prospective, Crossover Study

It is not known whether high convective transport may have a role in modulating the chronic inflammation of hemodialysis (HD) patients. The aim of this study was to evaluate the effect of on-line hemodiafiltration (OL-HDF) on proinflammatory peripheral monocytes: Percentage of CD14+CD16+ cells and their telomere length and spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6). In a prospective, crossover study, 31 patients who were on high-flux HD (HF-HD) were evaluated. Patients underwent the following sequence of treatments (4 mo each): HF-HD (basal), OL-HDF (period 1), HF-HD (period 2), OL-HDF (period 3), and HF-HD (period 4). The dialysis characteristics were similar in the two modalities; the only difference was a higher convective transport in the OL-HDF than in the HF-HD. All patients who were on OL-HDF periods showed a significantly lower number of CD14+CD16+ cells than on HF-HD (18.5 +/- 2.3 basal versus 13.6 +/- 2.9 period 1 and 13.9 +/- 2.3 period 3; P = 0.001). By contrast, HF-HD restored the number of CD14+CD16+ cells to the basal values (19.2 +/- 2.8 and 18.6 +/- 1.4, periods 2 and 4, respectively; NS). During OL-HDF periods, the reduction of CD14+CD16+ was paralleled by a decreased number of short telomere cells. Spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6) was increased in HF-HD as compared with OL-HDF. In conclusion, these results demonstrate that as compared with HF-HD, OL-HDF markedly reduces the number of proinflammatory CD14+CD16+ cells and the production of TNF-alpha and IL-6. Future studies are needed to assess the possible therapeutic effect of convective transport on chronic inflammation that is associated with HD.

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 β‐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 cLDLwas 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.—Carracedo, J., Merino, A., Briceño, C., Soriano, S., Buendía, P., Calleros, L., Rodriguez, M., Martín‐Malo, A., Aljama, P., Ramírez, R. Carbamy‐lated low‐density lipoprotein induces oxidative stress and accelerated senescence in human endothelial progenitor cells. FASEBJ. 25, 1314–1322 (2011). www.fasebj.org

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 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.—Buendía, P., Montes de Oca, A., Madueño, J. A., Merino, A., Martín‐Malo, A., Aljama, P., Ramírez, R., Rodríguez, M., Carracedo, J., Endothelial microparticles mediate inflammation‐induced vascular calcification. FASEB J. 29, 173–181 (2015). www.fasebj.org

Microinflammation and endothelial damage in hemodialysis.

BACKGROUND Chronic kidney disease (CKD) stage 4-5 patients have increased cardiovascular morbidity and mortality rates compared with the general population. Chronic inflammation has been proposed as a cardiovascular risk factor. We have previously demonstrated that the majority of CKD patients show a microinflammatory state with an increased percentage of CD14+/CD16+ monocytes in peripheral blood, even in patients who do not show clinical evidence of inflammatory disease. However, the role played by these microinflammatory cells on the endothelial damage that precede the development of cardiovascular disease has not been investigated. METHODS To study the effect of microinflammation on endothelial cell injury we have developed an experimental co-culture model in which isolated CD14+/CD16+ cells were seeded in 24-well tissue-culture plates. Human umbilical vein endothelial cells were placed on the top of the culture well in a insert that permitted intercellular soluble network communication. To stimulate the release of proinflammatory products, monocytes were activated with substimulating doses of bacterial DNA. Endothelial injury was characterized measuring intracellular reactive oxygen species activity and cell apoptosis. RESULTS Only CD14+/CD16+ cells released proinflammatory cytokines when they were stimulated by bacterial DNA. In the culture wells in which inflammatory cytokines were detected, endothelial cells showed an increased reactive oxygen species activity and features of apoptosis. CONCLUSIONS Our results support the hypothesis that independently of uremia, in CKD stage 4-5 patients microinflammation mediated by CD14+/CD16+ cells induces endothelial damage and thus may contribute to the increased risk of atherosclerosis and cardiovascular disease that has been reported in this population.

Effect of Different Dialysis Modalities on Microinflammatory Status and Endothelial Damage

BACKGROUND AND OBJECTIVES We studied the relationship between microinflammation and endothelial damage in chronic kidney disease (CKD) patients on different dialysis modalities. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Four groups of CKD stage 5 patients were studied: 1) 14 nondialysis CKD patients (CKD-NonD); 2) 15 hemodialysis patients (HD); 3) 12 peritoneal dialysis patients with residual renal function >1 ml/min (PD-RRF >1); and 4) 13 peritoneal dialysis patients with residual renal function <or=1 ml/min (PD-RRF <or=1). Ten healthy subjects served as controls. CD14(+)CD16(+) cells and apoptotic endothelial microparticles (EMPs) were measured by flow cytometry. Serum vascular endothelial growth factor (VEGF) was measured by ELISA. RESULTS CKD-NonD and HD patients had a higher percentage of CD14(+)CD16(+) monocytes than PD groups and controls. CD14(+)CD16(+) was similar in the PD groups, regardless of their RRF, and controls. The four uremic groups displayed a marked increase in apoptotic EMPs and VEGF compared with controls. Apoptotic EMPs and VEGF were significantly higher in HD patients than in CKD-NonD and both PD groups. However, there were no significant differences between CKD-NonD and the two PD groups. There was a correlation between CD14(+)CD16(+) and endothelial damage in CKD-NonD and HD patients, but not in PD and controls. CONCLUSIONS There was an increase in CD14(+)CD16(+) only in CKD-NonD and HD patients. In these patients, there was a relationship between increased CD14(+)CD16(+) and endothelial damage. These results strongly suggest that other factors unrelated to the microinflammatory status mediated by CD14(+)CD16(+) are promoting the endothelial damage in PD, regardless of their RRF.

Carbamylated darbepoetin derivative prevents endothelial progenitor cell damage with no effect on angiogenesis

Erythropoietin (EPO) prevents cell apoptosis induced by oxidative stress. Carbamylated EPO maintains the tissue-protective activities of the unmodified EPO but does not stimulate erythropoiesis. This study evaluates whether carbamylated erythropoietin is as effective as recombinant human erythropoietin in protecting endothelial progenitor cells (EPCs) from apoptosis without stimulating erythropoiesis. Experiments were performed in an erythroid cell line (UT-7) and in human EPCs. Cell signals regulating proliferation and apoptosis (Jak-2, Akt, Erk1/2, NFkappaB and Stat-5) were measured by Western blotting. In human EPCs, cell senescence, apoptosis and proliferation were assessed by acidic beta-gal and measurement of telomere length, TUNEL and PCNA labeling, respectively. Angiogenesis was evaluated using the endothelial tube formation assay. In UT-7, carbamylated erythropoietin (C-darbe) induced phosphorylation of the anti-apoptotic Jak-2/Akt signal and, as opposed to recombinant human erythropoietin (darbe), did not produce a significant activation of cell proliferating signals. Darbe increased the percent of proliferating EPCs and promoted angiogenesis. By contrast, C-darbe failed to stimulate proliferation of EPCs. Both C-darbe and darbe equally reduced apoptosis and senescence. Thus, C-darbe protects EPCs from apoptosis and does not increase erythropoiesis.

CD14+CD16+ monocytes from chronic kidney disease patients exhibit increased adhesion ability to endothelial cells.

Chronic kidney disease (CKD) patients present an inflammatory process that induces endothelial damage and therefore plays a role in the high rates of cardiovascular morbidity and mortality reported in these patients. Although new therapies have reduced the elevated serum levels of inflammatory mediators such as cytokines and CRP in CKD patients, the rise in the level of activated immunocompetent cells is maintained in peripheral blood, which appears to play a prominent role in the endothelial damage suffered by these patients. CD14+CD16+ monocytes are a subset of activated monocytes that are found in greater numbers in the peripheral blood of CKD patients. The increased presence of these cells is related to the endothelial damage suffered by these patients. However, the mechanism through which these cells damage the vascular endothelium is still unclear. One of the characteristics that differentiate CD14+CD16+ monocytes is their powerful ability to produce inflammatory cytokines, which may be responsible for causing damage to endothelial cells. However, it is difficult to imagine that the cytokines produced by a relatively small proportion of these cells are capable of damaging the endothelium. For this reason, we have suggested that these cells do not release their cytokines into the bloodstream, but that they possess cellular mechanisms that lead them to produce and release cytokines after adhering to the layer of endothelial cells. This hypothesis is based on the fact that unlike the CD14++CD16- monocytes found in healthy subjects, CD14+CD16+ monocytes in CKD patients show a high level of expression of chemokines that favors their migration to the vascular wall, and a low level of chemokines such as CCR2 that would prevent such migration. Furthermore, these CD14+CD16+ monocytes express a large number of adhesion molecules, which helps them attach to endothelial cells. In view of this scenario, it is easy to suggest that a moderate number of CD14+CD16+ monocytes might well be capable of producing endothelial damage; therefore, the rise in the number of these cells in CKD patients may play an important role in the development of vascular disease.

Microinflammation in hemodialysis is related to a preactivated subset of monocytes

Increased percentage of monocytes with low CD14 expression and that co‐express CD16 (CD14+/CD16+) have been reported in hemodialysis (HD) patients. We sought to determine whether CD14+/CD16+ monocytes in HD therapy are sensibilized cells to a proinflammatory activity. Cells from 32 HD patients, and from 9 Systemic Lupus Erythematosus (SLE), 9 individuals with human immunodeficiency virus (HIV)‐1‐ and 15 healthy controls were studied. Cells were analyzed by means of flow cytometry for CD14/CD16 expression and immune function (cytokine, chemokines, and sialoadhesin expression), and phagocytosis. Increased percentage of CD14+/CD16+ monocytes was observed in HD patients. Compared with CD14++ monocytes, the CD14+/CD16+ monocytes exhibited increased expression of proinflammatory cytokines and markers of differentiated cells. In addition, these monocytes showed an increased phagocytic activity. Similarly, CD14+/CD16+ monocytes from SLE and HIV patients showed increased inflammatory activity as compared with CD14++ cells. These results support that CD14+/CD16+ monocytes from HD patients evidence characteristics of primed prestimulated proinflammatory cells, similar to data observed in SLE and HIV.