Julia Carracedo

NK phenotypic markers and IL2 response in NK cells from elderly people

Immunosenescence is a process that primarily affects the T cell compartment of the immune system, although age-associated immunological alterations have also been demonstrated in the NK cell phenotype and function. A significant expansion in the number of NK cells is found in aging. The NK cytotoxic capacity of total peripheral blood lymphocytes is also well preserved, not only in healthy elderly people but also in centenarians. However, NK cell killing of K562 is impaired when considered in a per-cell basis, and this defect is associated with defective signal transduction after activation more than a diminished conjugate formation or killing capacity. We have studied the phenotype of NK cells in elderly donors fulfilling the Senieur criteria. We have also studied the capacity of these cells to be activated by IL2 when different NK cell functions, other than cytotoxicity, are considered. Our results confirm the increased percentage of NK cells in the elderly due to the expansion of the CD56dim subset that also show an altered pattern of activation markers, whereas no differences were found in the CD56bright subset. The response of NK cells to IL2 was found to be impaired when proliferation, expression of CD69, and Ca2+ mobilization were considered, whereas TNF-alpha production was not significantly affected. These results suggest that human NK cells do not escape the aging process, although senescence have a differential effect on distinct NK cell biological functions, ranging from severe to negligible impairment, depending on the parameters considered.

Mediterranean diet reduces endothelial damage and improves the regenerative capacity of endothelium

BACKGROUND Endothelial dysfunction is a fundamental step in the atherosclerotic disease process. Activation or injury of the endothelium leads to a variety of inflammatory disorders, including the release of microparticles. Endothelial progenitor cells may contribute to the maintenance of the endothelium by replacing injured mature endothelial cells. OBJECTIVE We studied the influence of dietary fat on the release of endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) in elderly subjects. DESIGN Twenty healthy, elderly subjects (10 men and 10 women) consumed 3 diets following a randomized crossover design, each for 4 wk: a saturated fatty acid diet; a low-fat, high-carbohydrate diet; and a Mediterranean diet (MedDiet) enriched in monounsaturated fatty acids. We investigated total microparticles, EMPs from activated endothelial cells (activated EMPs), EMPs from apoptotic endothelial cells (apoptotic EMPs), EPCs, oxidative stress variables, and ischemic reactive hyperemia (IRH). RESULTS The MedDiet led to lower total microparticle, activated EMP, and apoptotic EMP concentrations and higher EPC numbers than did the other diets (P < 0.001). We detected lower superoxide dismutase activity (P < 0.001), a higher plasma β-carotene concentration (P < 0.001), and lower urinary isoprostane and plasma nitrotyrosine concentrations after consumption of the MedDiet than after consumption of the other 2 diets (P < 0.05). Furthermore, the occurrence of IRH was higher after consumption of the MedDiet than after consumption of the other 2 diets (P < 0.05). CONCLUSION Consumption of the MedDiet induces a reduction in endothelial damage and dysfunction, which is associated with an improvement in the regenerative capacity of the endothelium, in comparison with 2 other diets.

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

Klotho modulates the stress response in human senescent endothelial cells

Lack of Klotho expression in mice leads to premature aging and age-related diseases, including vascular diseases. The aim of this study was to determine how endothelial cell line senescence affects Klotho expression and whether intra- or extracellular Klotho has any effect on the response of senescent cells to oxidative stress. The study was performed using human endothelial cells (HUVEC); cell aging was obtained by prolongation of cell division to 42 population doublings (PD). Senescence was also obtained by exposure to TNFα, which causes cell changes resembling cellular senescence. The decline in Klotho preceded the manifestations of cell ageing: telomere shortening and β-galactosidase expression. Klotho was also reduced in cells exposed to the proinflammatory cytokine TNFα. The addition of exogenous Klotho to aging cells did not modify the proportion of cells with short telomeres or any other feature of cell aging; however, exogenous Klotho prevented the changes resembling premature cellular senescence associated with TNFα, such as the decrease in telomere length and the increase in β-galactosidase-positive cells. Likewise exogenous Klotho prevented the increases in reactive oxygen species (ROS) activity, mitochondrial potential and cell apoptosis induced by TNFα.

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.

Klotho Prevents NFκB Translocation and Protects Endothelial Cell From Senescence Induced by Uremia

In patients with renal disease, uremia raises oxidative stress and senescence in endothelial cells, which can lead to endothelial dysfunction and cardiovascular disease. Klotho protein is a β-glucuronidase capable of hydrolyzing steroid β-glucuronides. This protein is recognized as an antiaging gene, that modulate both stress-induced senescence and functional response. The aim of the study was to investigate how senescence and oxidative stress induced by uremia in endothelial cells affects Klotho expression and whether intra or extracellular Klotho has effects on the response of these cells. Senescence and oxidative stress was obtained by exposure to uremic serum. Telomere length, the enzyme β-galactosidase, and oxidative stress were studied by flow cytometry. Nuclear factor kappa B activity was determined by electrophoretic mobility shift assay. The expression of Klotho decreased with the uremia and preceded the manifestations of cell aging. Levels of intracellular Klotho decreases associated to endothelial senescence, and exogenous Klotho prevents cellular senescence by inhibiting the increase in oxidative stress induced by uremia and diminished the nuclear factor kappa B-DNA binding ability.