1 Servicios de Hemoterapia-Hemostasia and 2 Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain
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Abstract |
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Methods. ECs were cultured with growth media supplemented with pooled sera from healthy donors. Semiconfluent ECs were incubated for 24 h with media supplemented with pools of control or uraemic sera. Cell proliferation was assessed through morphometric analysis and by flow cytometry evaluation of cell cycle. To investigate if uraemic medium induces apoptosis in ECs, we used a combination of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay and activation of caspase-3 using flow cytometry. Changes in the phosphorylation levels of MAPK were evaluated in cell lysates by western blotting.
Results. Exposure to uraemic media caused an alteration in the morphology of ECs, showing irregular shape and size. The number of ECs at S+G2M phase in the cell cycle was found to be increased when exposed to uraemic media for 24 h (28.4±2.9 vs 20.2±2.6% in control ECs). There was a transient increase in levels of phosphorylation of MAPK in both cells, although these levels were significantly higher in ECs exposed to uraemic media, especially after 5 min. In contrast, no signs of apoptosis were observed in ECs incubated with uraemic medium at the conditions applied.
Conclusions. Under our experimental conditions, uraemic medium accelerates proliferation of ECs, but it does not seem to induce apoptosis. The increased proliferation observed could be related to a higher MAPK activity in these cells. Although the enhanced atherosclerosis cannot be explained on the basis of an apoptotic process, the proliferative status could contribute to intimal proliferation, which is considered to be an earlier step in the development of atherosclerosis.
Keywords: apoptosis; atherothrombosis; cell cycle; endothelial cells; MAPKs; uraemia
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Introduction |
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The vascular endothelium has been recognized as a complex endocrine organ that regulates many physiological functions such as vascular tone, vascular smooth muscle cell growth and migration, vascular permeability to solutes and blood cells, and regulation of haemostasis, among others [3]. In this regard, there is in vivo [4] and in vitro evidence of endothelial dysfunction in uraemia [57]. We have demonstrated that exposure of endothelial cells in culture (ECs) to growth media containing uraemic serum results in quantitative and qualitative changes in the vascular subendothelium, characterized by a less intricate network of fibrils [5] and an increased thrombogenicity of the extracellular matrix generated by ECs with an enhanced expression of tissue factor [6]. In addition, under the experimental conditions assayed, we observed an augmented expression of adhesion receptors on the cell surface, shedding of adhesion molecules and increased levels of markers of endothelial dysfunction [7]. Endothelial dysfunction has been considered to be the initial event in the development of atherosclerosis [8].
The development of atherosclerosis has been related to abnormal proliferation and signs of apoptosis of endothelial and smooth muscle cells [9], with presence of apoptotic macrophages and T-lymphocytes in atherosclerotic plaques [10]. The changes observed previously in endothelial cells exposed to uraemic media [5,6] suggested the presence of an enhanced apoptosis. In this regard, studies performed in patients with chronic renal failure have reported the occurrence of increased apoptosis in circulating cells [11].
In the present study we have investigated the potential effect of uraemic media on proliferation and apoptosis of ECs, two key events in the progression of atherosclerosis. For this purpose, ECs were exposed to growth media containing pooled samples of serum obtained from patients with end-stage renal disease undergoing haemodialysis. We evaluated changes in cell morphology and cycle using light microscopy and flow cytometry techniques. Apoptosis was assessed in these cells by two different methods, using flow cytometry analysis. An analysis of the phosphorylation kinetics of MAPK p42/44 and p38 was also carried out.
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Subjects and methods |
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Human endothelial cell culture
ECs were isolated from human umbilical cord veins [12]. Cells were grown with culture medium (MEM 199; Gibco BRL, Life Technologies, Scotland, UK) supplemented with 1 mM glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin and 20% pooled human serum. In those experiments performed to assess the effect of uraemic media, sera were obtained from a group of patients with chronic renal failure undergoing haemodialysis. Cells were identified as endothelial cells both morphologically and by the presence of von Willebrand Factor, as detected by immunofluorescence. ECs were grown at 37°C in a 5% CO2 humidified incubator. The culture medium was changed every 48 h. After the second passage, ECs were subcultured on 1% gelatin-coated glass coverslips and on 80 cm2 Roux flasks.
The pool of sera from uraemic patients was obtained from 10 patients with end-stage renal disease on maintenance haemodialysis, five men and five women, mean age 57±4.7 years, mean time on haemodialysis 80±20.9 months (mean±SEM). The cause of renal failure in these patients was: polycystic kidney disease (two), chronic glomerulonephritis (three), chronic pyelonefhritis (two), nephrosclerosis (one), haemolitic uraemic syndrome (one) and bilateral nephrectomy (one). The mean values for the biochemical parameters measured were 79.4±3.1 mg/dl of glucose, 64.2±5.9 mg/dl BUN, 9.2±0.8 mg/dl creatinine, 139.7±0.9 mEq/l Na+, 5.48±0.3m Eq/l K+, 9.7±0.2 mg/dl Ca2+. Blood samples were always obtained immediately before initiating the haemodialysis session. Sera samples were filtered through 0.22 µM filters (MillexTM, Millipore Corp., Bedford, MA) before supplementing the growth media. These samples proved to be endotoxin free. All patients were dialysed for 4 h three times a week with cellulose acetate membranes and bicarbonate containing dialysate. The pool of control sera was obtained from healthy subjects. Informed consent was obtained from patients for serum utilization.
Coverslips processing and morphometric analysis
After reaching semiconfluency, ECs grown on coverslips were incubated, for 24 h, with growth media containing 20% pooled sera from healthy donors or from uraemic patients. ECs monolayers were fixed with 4% paraformaldehyde in 0.15 M phosphate-buffered saline (PBS), pH 7.4 (4°C, 10 min). Following extensive washing with PBS, fixed coverslips were stained with toluidine blue (0.02% in distilled water) for 10 min at 22°C and mounted in aqueous media. Representative images from ECs were captured by a digital camera (Leica DC300F) connected to a light microscope (Polyvar, Reichert-Jung, Wien, Austria). The effect of control or uraemic sera on proliferation was assessed through morphometric analysis of ECs in six different experiments (n=6). Number of cells in division stages (metaphase) were determined in 10 random microscope fields in each experiment, using always the same magnification (x320).
Flow cytometry evaluation of cell cycle
ECs grown on 80 cm2 Roux flasks were exposed to growth media containing 20% pooled sera from healthy donors or from uraemic patients. After 24 h of incubation, cells were detached with a solution of 0.25% trypsin and 0.02% EDTA. Cells were then resuspended with Hanks' balanced salt solution (Gibco BRL, Life Technologies) and adjusted to 1x106 cells/ml. Cells were washed twice with PBS and then fixed with methanol. For fixation, 50 µl of PBS were added to the pellet followed by 950 µl of methanol at -20°C, for 10 min on ice. Following fixation, all samples were washed twice in 1 ml PBS to remove methanol. After the final wash, cells were resuspended in 500 µl of PBS containing 0.1 µg/ml RNase A (Sigma) and 5 µg/ml propidium iodide (PI, Sigma), and incubated overnight at 4°C, always protected from light. Flow cytometry data were collected on a FACScan (BDIS, San Jose, CA). Single laser analysis was carried out using an air-cooled 15 mW argon laser, operating at 488 nm. Forward scatter, side scatter and FL3 (PI fluorescence) were collected on a linear scale. Forward and side scatter gains were adjusted so those cells occupied between one- and two-thirds of the scatterplot. Doublets and cell aggregates were removed from a dot plot of FL3-A (PI area) vs FL3-W (PI width), using a gate around the singlet population. Analysis was carried out using CellQuest (BDIS) and Modfit (Verity) Software. The fluorescence of at least 20 000 events was measured at a low flow rate to ensure low coefficient of variation.
Evaluation of tyrosine phosphorylation of MAPK in cell lysates
To evaluate the effect of uraemic sera on MAPK p42/44 and p38 activity in EC, confluent monolayers, grown in six-well dishes, were serum starved during 18 h before experiments were performed by replacing their growth media with media containing 0.5% pooled human serum. This experimental procedure ensures basal levels of kinase activity. Cells were then incubated with growth media containing 20% pooled sera from healthy donors or from uraemic patients for 0, 30s, 1, 5, 15, 30 and 60 min, at 37°C. Then samples were lysated with Laemmli's buffer (125 mM TrisHCl, 2% SDS, 5% glycerol and 0.003% bromophenol blue) containing 2 mM sodium orthovanadate and 0.625 mg/ml N-ethylmaleimide, as phosphatase and protease inhibitors. After 15 min at 4°C, lysated EC were removed from the coverslip by scrapping, collected in an Eppendorf, sonicated during 15 s and heated at 90°C for 5 min. Samples were kept at -20°C until electrophoretic analysis was performed.
Samples corresponding to ECs lysates were resolved by 8% SDSpolyacrylamide gel electrophoresis. Proteins present in the gels were transferred to nitrocellulose membranes (Bio-Rad, CA). After blocking for non-specific binding with Tris-buffered saline (2.4 g Tris base, 8 g NaCl; adjust pH to 7.6 with HCl) containing 0.1% Tween-20 and 5% non-fat dry milk, western blots were probed with a rabbit polyclonal IgG anti-MAPK p42/44 or with a sheep polyclonal IgG anti-Phospho-MAPK p42/44 (Upstate Biotechnology, Barcelona, Spain) and a rabbit polyclonal IgG anti-MAPK p38 or with a rabbit polyclonal IgG anti-Phospho-MAPK p38 (Cell Signaling Technology, Inc., New England Biolabs, UK). The excess of antibody was removed by extensive washing and blots were developed by the enhanced chemiluminiscence method (ECL) (Amersham Pharmacia Biotek, Essex, UK). Protein bands were densitometrically analysed (Kodak Digital Science 1D, Eastman Kodak Company, Rochester, NY).
Apoptosis assays
ECs grown on 80 cm2 Roux flasks were exposed to media containing 20% pooled sera from healthy donors or from uraemic patients. After 24 h of incubation, cells were detached with a solution of 0.25% trypsin and 0.02% EDTA. Cells were resuspended with Hanks' balanced salt solution (Gibco BRL) and adjusted to 0.51x106 cells/ml.
For the TUNEL staining, cells were washed twice with PBS containing 0.2% BSA and then were fixed with 4% paraformaldehyde in 0.1 M NaH2PO4, pH 7.4 at 4°C for 30 min. A positive control was used: 1 mM Camptothecin, a topoisomerase I inhibitor, which induces apoptosis. Following fixation, all samples were washed twice in 1 ml PBS containing 0.2% BSA. For permeabilization, cells were suspended in 200 ml of 70% ethanol and incubated for 30 min at -20°C. In TUNEL method, 3'-OH DNA ends generated by DNA fragmentation is nick-end labelled with fluorescein-dUTP (FITC-dUTP), mediated by terminal deoxynucleotidyl transferase (TdT) [13]. After washing twice the cell pellet with PBS containing 0.2% BSA, 30 ml of TdT reaction reagent (TdT buffer, TdT and FITC-dUTP, mixed at a proportion of 18:1:1) was added to the cell pellet and incubated for 1 h at 37°C. Finally, cells were washed twice with PBS containing 0.2% BSA, and the cell pellets were suspended with 400 ml of PBS containing 0.2% BSA. Flow cytometry data were collected on a FACScan (Beckton and Dickinson, San Jose, CA), as described above.
For the caspase-3 method [14], cells were washed twice with PBS containing 0.2% BSA and then cells were incubated with caspase-3. The intracellular activity of this protein was detected using a fluorogenic substrate kit (PhiPhiluxTM G1D2, Calbiochem) accordingly with manufacturer instructions. Flow cytometry data were collected on a FACScan (Beckton and Dickinson), as described above.
Statistical analysis
Results of the experiments were expressed as mean±SEM. Statistical differences were analysed using Student's t-test for paired data (Primer of Biostatistics; McGraw-Hill). A P<0.05 was considered statistically significant.
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Results |
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Analysis of cell cycle was performed by flow cytometry using PI staining (see histograms in Figure 2). For cell-cycle calculations, DNA content histograms with coefficients of variation <7% were used. Control endothelial cells exhibited a characteristic pattern, with 20.2±2.6% of cells in S+G2M phase. Incubation of ECs with media containing uraemic serum increased significantly the number of cells in S+G2M phase to 28.4±2.9%, P<0.05.
Apoptosis in endothelial cells
The potential effect of uraemic serum on apoptosis was analysed by two different methods, one detecting DNA fragmentation (TUNEL assay) and a second measuring activation of caspase-3.
A positive control was obtained by incubating ECs with camptothecin, a topoisomerase I inhibitor, which resulted in 4.7% of dUTP-FITC positive cells. Exposure of ECs to growth media containing serum from healthy donors resulted in 0.1±0.0% dUTP-FITC positive cells. Presence of serum from uraemic patients did not modify the presence of positive cells observed in control experiments (0.1±0.0% dUTP-FITC).
Expression of activated caspase-3, measured by flow cytometry, was absent in ECs incubated with growth media either in the presence of control serum or pooled serum from uraemic patients.
Analysis of changes in the phosphorylation levels of the mitogen-activated protein kinase p42/44 and p38
The effect of exposing ECs to growth media containing uraemic sera on the phosphorylation state of MAPK p42/44 and p38 was evaluated. After reaching confluence, endothelial cells were incubated with either control or uraemic medium at different periods of time, in four different experiments. Immunoblots to confirm equal presence of protein p42/44 and p38 were performed.
Results showed that MAPK p42/44 in endothelial cells became rapidly phosphorylated after 1 min of exposure to control media. Levels of phosphorylation increased slightly after 5 min of exposure and were maintained until 30 min of incubation with control media. When cells were exposed to uraemic media, kinetics of phosphorylation for MAPK p42/44 were similar, although levels of phosphorylation were significantly higher, being maximum after 5 min of exposure. In the case of MAPK p38, endothelial cells exposed to growth media supplemented with control sera became phosphorylated after 1 min. Levels of phosphorylation increased slightly at 5 min and the phosphorylation was maintained until 15 min. When cells were exposed to growth media supplemented with uraemic sera we observed a longer-lasting (maintained until 30 min) and more intense phosphorylation than in control cells (Figure 3).
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Discussion |
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ECs covering the inner vascular layer are considered an interface with a critical role in the maintenance of balanced haemostasis [15]. Alterations of the vascular endothelium represent a critical event for the initiation of atherosclerosis [8]. Several studies have shown that patients with chronic renal failure present an impaired endothelium dependent vasodilation as a sign of endothelial cell dysfunction [4]. On the other hand, our group reported that uraemic medium induces ECs alterations characterized by a decreased cell resistance to blood flow, and an enhanced thrombogenicity of the underlying extracellular matrix, the equivalent of the vascular subendothelium [5,6]. Our present results reinforce the previous observations of endothelial damage caused in vitro by a uraemic environment.
Some of the pathophysiological factors in atherogenesis leading to endothelial dysfunction, including an increase in cytokine levels, bacterial products, advanced glycosylation-end products (AGEs), hyperhomocysteinaemia, hypercholesterolaemia and oxidized lipoproteins and their components [16]. Most of these factors have been described to be present in patients with chronic renal failure [2,17]. In haemodialysed patients there is an intense activation of blood cell elements [18]. Cell activation has been related to cytokine release. In dialysed patients, increased levels of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6 and TNF-gamma and C-reactive protein [19] have been reported. Such cytokines could probably activate endothelial cells by themselves, and they may act as mitogenic signals and increase the susceptibility of cells to further dysfunction [20].
In the present study, we report an increase in the proliferation of endothelial cells when exposed to uraemic media. Cell proliferation may be related to mitogenic extracellular signals. The mitogenic signal of many stimuli occurs through MAPKs in many cell types. The extracellular regulated kinases p44ERK1 and p42ERK2 are included in the family of MAPKs, which are associated with cellular growth and proliferation processes. Protein p38 is another MAPK associated with inflammation and growth differentiation. Activation of ERKs by extracellular stimuli implies protein phosphorylation of tyrosine and threonine residues, and translocation from the cytosol to the nucleus. The nuclear translocation is determinant for cell response as pERKs activate certain transcriptional factors.
In our study, the exposure of endothelial cells to growth media induced the activation, measured as increases in the levels of phosphorylation, of p44ERK1 and p42ERK2. This effect was significantly increased when cells were exposed to growth media containing uraemic sera. These observations paralleled the accelerated proliferation observed in these cells with other methods applied in our investigation. Moreover, according to our previous study [6], the exposure of endothelial cells to uraemic media resulted in changes in the composition of the underlying extracellular matrix, with a higher presence of tissue factor. From our studies, this effect could be related to the activation of transcriptional factors, as derived from an increased expression of mRNA for this protein [6]. Moreover, the increased levels of phosphorylation of MAPK p38 would be related with the increased expression of adhesion molecules found in a previous study performed by our group [7], indicating that uraemic media could contribute to the presence of a pro-inflammatory state.
Cell apoptosis has emerged as a key element in the complex pathophysiology underlying the initiation, progression and complications of atherosclerosis [4]. There is evidence for in situ apoptotic cell death in animal and human atherosclerotic plaques [21]. Furthermore, several studies confirm apoptotic events in lymphocytes, monocytes and polymorphonuclear leukocytes in patients with chronic renal failure [6]. However, in our experimental setting, we were unable to detect neither nick-end labelling in endothelial cells exposed to uraemic media using the TUNEL assay nor activity of caspase-3. Therefore, although apoptosis does not seem to be the underlying cause of endothelial cell dysfunction in uraemia, the increased proliferation reported in the present study, with a decreased cell resistance to flow, and changes in the distribution of adhesion receptors reported previously [6,7], could contribute to the development of atherosclerotic processes. Expression of adhesion receptors in endothelial cells may promote the recruitment of leukocytes from the bloodstream to the site of subendothelium, an event that may lead to inflammatory state and atherosclerosis development. The inflammatory state observed in uraemic patients may contribute to the risk of cardiovascular events described in these patients.
The results of our in vitro study support the concept of endothelial dysfunction in uraemia. Uraemic media accelerated cell proliferation, as observed by different methods, with increased levels of phosphorylation of MAPK p42/44 and p38 and may contribute to changes in the phenotype of these cells. In contrast, uraemic medium was not able to induce apoptosis in ECs. Results from the present work reinforce previous results by other authors and our group, suggesting a potential role of endothelial cell dysfunction in the pathogenesis of atherosclerosis in uraemic patients.
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Acknowledgments |
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Conflict of interest statement. None declared.
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Notes |
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References |
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