Paula Buendía

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.

Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome

Summary.  Background: One of the described mechanisms leading to thrombosis in antiphospholipid syndrome (APS) is overexpression of tissue factor (TF) in the monocytes and endothelial cells of patients with antiphospholipid antibodies (aPL). Vascular endothelial growth factor (VEGF) may stimulate monocyte TF expression through its receptor, the tyrosine kinase Flt‐1. Objectives: This study aimed to analyze the following in monocytes of 55 primary APS patients: VEGF and Flt‐1 expression levels, their potential regulation by aPL, and the association of VEGF and Flt‐1 expression with the increased TF expression found in APS patients. Results: Purified monocytes from APS patients showed higher levels of VEGF and Flt‐1 than healthy donors, which further correlated with immunoglobulin G (IgG) anticardiolipin titers and TF expression rank. Moreover, monocyte VEGF and Flt‐1 levels were significantly higher in patients with than in patients without previous thrombosis. In vitro, IgG from APS patients increased monocyte VEGF and Flt‐1 expression in a dose‐dependent manner. VEGF and Flt‐1 expression was significantly inhibited by the p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580; this suggests the involvement of this kinase in the aPL‐induced VEGF and Flt‐1 upregulation. Conclusions: Our data show, for the first time in vivo, that monocytes from primary APS patients have an increased expression of VEGF and Flt‐1. Furthermore, in vitro results indicated that this cytokine is produced by monocytes when treated with aPL, and that the p38 MAPK signaling pathway plays an important role. Thus, VEGF might act as a regulatory factor in aPL‐mediated monocyte activation and TF expression, thereby contributing to the proinflammatory–prothrombotic phenotype of APS patients.

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

Global effects of fluvastatin on the prothrombotic status of patients with antiphospholipid syndrome

Objective Numerous mechanisms have been proposed to explain the thrombotic/proinflammatory tendency of antiphospholipid syndrome (APS) patients. Prothrombotic monocyte activation by antiphospholipid antibodies involves numerous proteins and intracellular pathways. The anti-inflammatory, anticoagulant and immunoregulatory effects of statins have been aimed as a therapeutic tool in APS patients. This study delineates the global effects of fluvastatin on the prothrombotic tendency of monocytes from APS patients. Methods Forty-two APS patients with thrombosis and 35 healthy donors were included in the study. APS patients received 20 mg/day fluvastatin for 1 month. Blood samples were obtained before the start, at the end and 2 months after the end of treatment. Results After 1 month of treatment, monocytes showed a significant inhibition of tissue factor, protein activator receptors 1 and 2, vascular endothelial growth factor and Flt1 expression that was related to the inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B/Rel DNA-binding activity. Proteomic analysis showed proteins involved in thrombotic development (annexin II, RhoA and protein disulphide isomerase) with altered expression after fluvastatin administration. In-vitro studies indicated that the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by fluvastatin might inhibit protein prenylation and MAPK activation. Conclusion The data from this study support the belief that fluvastatin has multiple profound effects in monocyte activity, which might contribute to thrombosis prevention in APS patients.

Proteomic analysis in monocytes of antiphospholipid syndrome patients: Deregulation of proteins related to the development of thrombosis

OBJECTIVE Antiphospholipid antibodies (aPL) are closely related to the development of thrombosis, but the exact mechanism(s) leading to thrombotic events remains unknown. In this study, using proteomic techniques, we evaluated changes in protein expression of monocytes from patients with antiphospholipid syndrome (APS) related to the pathophysiology of the syndrome. METHODS Fifty-one APS patients were included. They were divided into 2 groups: patients with previous thrombosis, and patients with recurrent spontaneous abortion. As controls, we studied patients with thrombosis but without aPL, and age- and sex-matched healthy subjects. RESULTS The proteins that were more significantly altered among monocytes from APS patients with thrombosis (annexin I, annexin II, protein disulfide isomerase, Nedd8, RhoA proteins, and Hsp60) were functionally related to the induction of a procoagulant state as well as to autoimmune-related responses. Proteins reported to be connected to recurrent spontaneous abortion (e.g., fibrinogen and hemoglobin) were also determined to be significantly deregulated in APS patients without thrombosis. In vitro treatment with IgG fractions purified from the plasma of APS patients with thrombosis changed the pattern of protein expression of normal monocytes in the same way that was observed in vivo for monocytes from APS patients with thrombosis. CONCLUSION For the first time, proteomic analysis has identified novel proteins that may be involved in the pathogenic mechanisms of APS, thus providing potential new targets for pathogenesis-based therapies for the disease.

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.

Differential expression of protease‐activated receptors in monocytes from patients with primary antiphospholipid syndrome

OBJECTIVE To investigate the expression of protease-activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS). METHODS Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1-4 genes was measured by quantitative real-time reverse transcription-polymerase chain reaction. Cell surface TF and PARs 1-4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti-PAR-1 (ATAP2) or anti-PAR-2 (SAM11) to test the effect of blocking the active site of PAR-1 or PAR-2. RESULTS Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR-2 as compared with the control groups. PAR-1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR-3 expression was not significantly altered. PAR-4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR-2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR-2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR-1 and PAR-2. Inhibition studies suggested that there was direct cross-talk between TF and PAR-2, such that inhibition of PAR-2 prevented the aCL-induced expression of TF. CONCLUSION These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS.

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.