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Am J Physiol Heart Circ Physiol 292: H1836-H1846, 2007. First published December 8, 2006; doi:10.1152/ajpheart.01079.2006
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Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through MAPK pathway

Eugene O. Apostolov,1 Alexei G. Basnakian,1,2 Xiaoyan Yin,1 Ercan Ok,1 and Sudhir V. Shah1,2

1Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences and 2Central Arkansas Veterans Healthcare System, Little Rock, Arkansas

Submitted 2 October 2006 ; accepted in final form 1 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ability of modified low-density lipoptoteins (LDLs) to induce both proliferation and death of endothelial cells is considered to be a mechanism of early atherosclerosis development. We previously showed that carbamylated LDL (cLDL) induces human coronary artery endothelial cell (HCAEC) death in vitro. This effect is similar to the atherogenic action of oxidized LDL (oxLDL) that induces the proliferation and death of endothelial cells. The present study was designed to analyze a potential proliferative effect of cLDL and whether proliferation caused by modified LDLs is related to cell death. Cultured HCAECs were exposed to different concentrations of modified LDL or native LDL for varying periods of time. Cell proliferation measured by bromodeoxyuridine incorporation and S-phase analysis was dose-dependently increased in the presence of cLDL (6.25–200 µg/ml). The proliferation induced by cLDL or oxLDL was associated with cell death and increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). Inhibition of cLDL- or oxLDL-induced proliferation by aphidicolin (1 µg/ml) was protective against both short-term cell death measured by lactate dehydrogenase release into the medium and long-term cell viability visualized by cell multiplication. Inhibition of ERK phosphorylation led to a significant decrease of DNA synthesis and cell rescue from injury by modified LDLs, while inhibition of JNK phosphorylation had an only partial rescue effect without involvement in cell proliferation. These data are the first evidence that endothelial cell death induced by cLDL or oxLDL is mediated by cell proliferation through the mitogen-activated protein kinase pathway.

carbamylation; carbamylated low-density lipoprotein; oxidized low-density lipoprotein; mitogen-activated protein kinase; atherosclerosis

CARBAMYLATION is a spontaneous, nonenzymatic reaction of protein modification by cyanate derived from urea, which is normally present in human plasma and is elevated in uremic patients (27). Chronic kidney disease in humans is associated with a several times increased risk of developing cardiovascular diseases because of accelerated atherosclerosis (12, 32, 47). The mechanism of uremia-induced atherosclerosis is not quite understood. We previously showed (2, 36, 37) that carbamylated low-density lipoprotein (cLDL) induces injury or dysfunction of endothelial cells and is present in human plasma in high quantities. Our recent observation (38) that cLDL induces cell death of human coronary artery endothelial cells (HCAECs) in vitro provided support for this hypothesis.

It is commonly accepted that modified LDLs are important for early atherosclerosis initiation and progression (39, 43). Endothelial dysfunction is often considered as one of the early predominant stages in atherosclerosis development (13). Oxidized LDL (oxLDL), the most intensively studied modified LDL, has been shown to induce endothelial cell death in vitro (31). The cytotoxic effect of oxLDL is often associated with cell proliferation (16). The combination of cell proliferation and cell death results in the high turnover of vascular endothelial cells, which is an important characteristic of atherosclerosis (25). Proliferating cells were found to be more sensitive to injury caused by oxLDL (3). As expected, both proliferating and dead endothelial cells can be observed in early apoptotic lesions (35).

The ability of cLDL to induce proliferation and hence modulate the damage of endothelial cells has not been examined previously. In the present study, we analyzed the proliferative effect of cLDL on HCAECs in vitro and a potential cause-effect relationship between modified LDL-induced proliferation and cell death. Our data strongly suggest that cLDL is capable of inducing endothelial cell proliferation and that the proliferation induced by either cLDL or oxLDL occurs through the mitogen-activated protein kinases (MAPKs) and is essential for endothelial cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Native, carbamylated, and oxidized LDLs. Human native LDL (nLDL) and all chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. cLDL was prepared as previously described (2, 38). oxLDL was prepared as described by Kume et al. (28). After modification, all LDLs were dialyzed separately against the same buffer. The dialysis buffer after the second or third dialysis had neither a cytotoxicity nor a proliferation effect on cells in control experiments.

A colorimetric method using diacetyl monoxime was used to measure the degree of carbamylation in LDL preparations (50). Oxidation of LDL was quantified by thiobarbituric acid reactive substance assay (15). The electrophoretic mobility of nLDL, cLDL, and oxLDL was determined in 0.5% agarose gel-0.2% bovine serum albumin (wt/vol) as described by Nobel (34). All three LDL isoform preparations were adjusted to 1 g protein/l with PBS containing 200 µmol/l EDTA, kept at 4°C away from light, and used within 2 wk after preparation. If sediment appeared during storage it was removed by low-speed centrifugation, and only soluble fractions of the LDL modifications were used for experiments.

Cell culture and LDL treatment of cells. HCAECs were supplied by Cambrex (Walkersville, MD) in passage 3 and used between passages 4 and 6. Cells were treated with 0–200 µg/ml LDLs in serum-free EGM-2-MV medium (Cambrex) for 1–24 h. The vehicle solution in the same medium was used as a control.

Bromodeoxyuridine assay. The bromodeoxyuridine (BrdU) cell proliferation assay (Oncogene, Cambridge, MA) was used according to the manufacturer's manual to identify cells (3.5 x 103 cells/well) in the S phase of the cell cycle. Briefly, cells were seeded into a 96-well plate (3.5 x 103 cells/well) and grown overnight in EGM-2-MV medium supplemented with 5% fetal bovine serum (FBS). The cells were rinsed with serum-free medium once and then exposed to 0–200 µg/ml LDL in serum-free medium. In most of the experiments pulse labeling of synthesized DNA was used. For this, the BrdU label was added 1 h before the end of the experiment. Cells were fixed, denatured, and probed with anti-BrdU antibody. Absorbance was measured at dual wavelengths of 450 and 540 nm in a microplate reader. Proliferation was expressed as a percentage of absorbance of the treated cells to the absorbance of the nontreated control cells.

Fluorescence-activated cell sorting analysis. HCAECs were cultured in six-well plates (2 x 105 cells per well) in 5% FBS-supplemented medium overnight and then washed and treated with 6.25 or 200 µg/ml LDL for 2–24 h in serum-free medium. Cells were detached from plastic with a scraper and mixed with the cells floating in the culture medium. The combined cells were precipitated at 220 g for 5 min. The cells were washed in PBS, pH 7.4, centrifuged, and fixed in 500 µl of 70% ice-cold ethanol overnight. The cells were precipitated at 500 g for 5 min, briefly vortexed, stained with 0.05 mg/ml propidium iodide-0.01% RNase A-0.01% bovine serum albumin for 30 min at room temperature, and immediately analyzed with a Becton Dickinson FACScan flow cytometer (San Jose, CA). The percentage of cells in different cell cycle stages was calculated with the Becton Dickinson CELLQUEST software package.

Cytotoxicity assay. HCAECs were seeded in a 96-well plate (3.5x103 cells/well, 100 µl EGM-2-MV medium supplemented with 5% FBS per well) and grown overnight for adequate attachment. On the following day, the medium was changed to 80 µl of serum-free medium with LDL. After 1- to 24-h exposure in a humidified incubator, the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) was used. Briefly, 10 µl of lysis solution was added 45 min before the end of the experiments to untreated cells in order to determine maximal lactate dehydrogenase (LDH) release. The plate was centrifuged at 220 g for 5 min. The supernatant medium (50 µl) was transferred to another 96-well plate. To each well, 50 µl of the mixture of lyophilized diaphorase-lactate-NAD+ in Tris-buffered tetrazolium dye 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride and Triton X-100 was added, and the plate was incubated at room temperature for 30 min in the dark. Absorption was assayed at 490 nm as described above. Medium, volume correction, and spontaneous LDH release controls were applied. Cytotoxicity was expressed as the ratio of absorption of released LDH to that of total LDH.

Immunocytochemistry and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining. Immunocytochemical staining of LDL-treated cells with monoclonal proliferating cell nuclear antigen (PCNA) or Ki-67 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was performed as described by Langer et al. (30). HCAECs were treated with 200 µg/ml LDL for 24 h as described above. The cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde (pH 7.0), and probed overnight with anti-PCNA and anti-Ki-67 at 1:1,000 and 1:400 dilutions, respectively. Primary antibody was detected with 1:500 diluted anti-mouse-AlexaFluor 594 conjugate (Molecular Probes, Eugene OR). For terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, the cells were washed and probed with an in situ cell death detection kit (Roche). Cells were then washed, counterstained with 4',6-diamidino-2-phenylindole (DAPI), mounted under coverslips with an Prolong Antifade kit (Molecular Probes), and analyzed under an Axioskope 2 (mot plus) microscope (Carl Zeiss, Göttingen, Germany) with green filter set no. 31001, red filter set no. 11010v2, and blue filter set no. 31000 from Chroma Technology (Rockingham, VT). Images and acquisitions were done with an AxioCam Mrm digital camera (Carl Zeiss) and Axiovision 3.1 software (Carl Zeiss).

Western blotting. All antibodies for Western blotting were purchased from Cell Signaling Technology (Danvers, MA). The total protein was extracted from cells, run through 12% SDS-PAGE at 30 µg/lane, and transferred to nitrocellulose membranes. The membranes were stained with Ponceau S to control for equal protein load as described elsewhere (22). After incubation in blocking solution (4% nonfat milk), membranes were incubated with polyclonal antibody to p-ERK (1:1,000), p-JNK (1:500) or p-MAPK p38 (1:1,000) overnight at 4°C. Membranes were washed and incubated with a 1:1,000 dilution of secondary antibody for 1 h and tested with a chemiluminescence system (Pierce). Total MAPK proteins were detected in the same membranes after stripping and reprobing with anti-ERK (1:1,000), anti-JNK (1:500), or anti-p38 MAPK (1:1,000).

Protein measurement. Protein was measured with the bicinchoninic acid protein assay (Pierce, Rockford, IL). Bovine serum albumin was used as a standard.

Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed with ANOVA and Student's t-test. Multiple comparisons were performed by t-test with the Bonferroni adjustment. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of endothelial cell proliferation by cLDL. We tested the ability of cLDL to affect BrdU incorporation in a wide range of concentrations (1–400 µg/ml). This range included concentrations of 6.25–12.5 µg/ml and 200 µg/ml, which has been described to induce proliferation and cytotoxicity of oxLDL in vitro (16, 19). Our data showed that the BrdU incorporation induced by cLDL was higher than that by nLDL (Fig. 1). As shown in Fig. 1, at both low and high concentrations of cLDL two elevations of BrdU incorporation were observed. The first was at 2 h of incubation, and the second was at the last time point of 24 h. Incubation beyond 24 h was not used because it was associated with significant cell death. The 2-h peak of BrdU incorporation was higher than the 24-h elevation, reaching 280%, 370%, and 610% above nontreated controls at concentrations of 6.25, 12.5, and 200 µg/ml cLDL, respectively. At 24 h, BrdU incorporation reached 170%, 280%, and 430% at the same cLDL concentrations, respectively.


Figure 1
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  		Fig. 1. Time course of human coronary artery endothelial cell (HCAEC) bromodeoxyuridine (BrdU) incorporation induced by carbamylated low-density lipoprotein (cLDL; {blacklozenge}) or native LDL (nLDL; {triangleup}) at doses of 6.25 (A), 12.5 (B), and 200 (C) µg/ml. To measure the rate of proliferation induced by cLDL compared with nLDL, HCAECs were incubated with the LDLs up to 24 h, and proliferation was measured by pulse-labeling with BrdU. Cells treated with vehicle (PBS) were considered to be baseline (100%). n = 4 per point. *P < 0.05, **P < 0.01, ***P < 0.001 compared with nLDL-treated cells.

 
To determine whether cLDL affects the cell cycle, cells treated with cLDL or nLDL were subjected to cell cycle analysis by fluorescence-activated cell sorting (FACS) (Fig. 2). This experiment showed that the cell cycle of cLDL-treated cells was significantly changed by increasing the number of cells in S phase after both 2-h and 24-h impacts with low (6.25 µg/ml) or high (200 µg/ml) cLDL concentrations, confirming that increased BrdU incorporation was associated with scheduled DNA synthesis. Cells treated with 200 µg/ml of cLDL had a pre-G1 peak (G0 or so-called apoptotic peak), which resulted from DNA fragmentation and cell death.


Figure 2
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  		Fig. 2. Scheduled DNA synthesis induced by cLDL (B, D, F, H) vs. nLDL (A, C, E, G). Phases of the cell cycle were detected after 2-h (A–D, I) and 24-h (E–H, J) treatments with the vehicle or LDLs by FACS analysis with propidium iodide DNA content staining. n = 5 per point. *P < 0.05 compared with untreated control; #P < 0.05 compared with nLDL.

 
Our data showed that the BrdU incorporation induced by nLDL and cLDL was dose dependent (Fig. 3, A and B). After the 2-h impact, the BrdU incorporation induced by cLDL was approximately two to three times higher than the proliferation induced by nLDL. The 24-h incubation was associated with an ~50% increase of the BrdU incorporation rate above nLDL.


Figure 3
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  		Fig. 3. Dose dependence of HCAEC proliferation and cell death induced by cLDL ({blacklozenge}) or nLDL ({triangleup}) at 2-h (A, C) and 24-h (B, D) incubation periods. BrdU incorporation in cells incubated with vehicle (PBS) was considered to be 100%. Cell death was measured by lactate dehydrogenase (LDH) release. n = 4 per point. *P < 0.01, **P < 0.001 compared with nLDL-treated cells.

 
Association of cLDL-induced HCAEC proliferation with cell death. To study whether proliferation of cLDL-treated endothelial cells is associated with cell death, the cytotoxicity of cLDL was measured with the LDH release assay. This experiment revealed that cell proliferation was accompanied by cell death with both short and long incubations with cLDL (Fig. 3, C and D). The difference between the 2-h and 24-h impact of proliferation was that at 2 h, cell death induced by cLDL was only 30% higher than that induced by nLDL, while at 24 h it was twofold higher. Thus the short incubation of the endothelial cells with cLDL was associated mainly with proliferation, whereas the prolonged exposure to cLDL was predominantly associated with cell death.

To independently assess the induction of cell death, we used the TUNEL assay. This assay showed that cLDL induced much more apoptotic cell death then the vehicle or nLDL (Fig. 4A), thus confirming the LDH release results above. There have been several reports that serum and its components protect endothelial cells from the injury caused by modified LDLs (7, 29). To determine whether cLDL may cause cell death in the presence of serum, endothelial cells were treated with cLDL in complete medium supplemented with serum (5%). Although cells seemed to be more protected compared with treatment in serum-free medium, we found that cLDL maintains its ability to cause cell death even in the presence of serum (Fig. 4B).


Figure 4
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  		Fig. 4. Induction of apoptotic DNA fragmentation in HCAECs by cLDL. HCAEC were exposed to vehicle control, 200 µg/ml nLDL, or 200 µg/ml cLDL for 24 h in serum-free (A) or complete (B) medium, fixed, and processed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method as described in MATERIALS AND METHODS. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bars, 100 µm.

 
We further analyzed whether cLDL-induced proliferation and death take place in the same endothelial cells and whether there may be a link between these two processes. For this, immunostaining for PCNA or Ki-67, the commonly used markers of cell proliferation, and TUNEL labeling, the marker of apoptosis, were applied. HCAECs were treated with 200 µg/ml cLDL or oxLDL for 24 h and double-stained for PCNA or Ki-67 and TUNEL. This approach showed that nuclear overexpression of PCNA and Ki-67 induced by cLDL was often associated with apoptosis detected by TUNEL (Fig. 5, A and B). Surprisingly, our data showed that the exposure of endothelial cells to 200 µg/ml oxLDL for 24 h resulted in the same effect as cLDL: the proliferation of the cells determined by the PCNA- and Ki-67-positive nuclei was associated with apoptosis. Although oxLDL was shown previously to induce proliferation and death of endothelial cells (16, 19), a possible link between these two processes has not been demonstrated. The TUNEL-positive reaction of vehicle control or nLDL was lower than that with the modified LDLs and did not correlate with either PCNA or Ki-67 immunostaining. A similar experiment with treatment of endothelial cells in complete medium containing 5% serum showed that nuclear colocalization of the markers of proliferation and TUNEL was present in both cLDL- and oxLDL-treated cells regardless of the presence of serum in the medium (Fig. 5, C and D).


Figure 5
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  		Fig. 5. Proliferating cell nuclear antigen (PCNA; A) and Ki-67 (B) immunostaining (red color) and TUNEL (green color) in HCAECs treated for 24 h with nLDL, cLDL, or oxidized LDL (oxLDL) (200 µg/ml each) in serum-free and complete medium (C and D, respectively). Proliferation and cell death markers (cyclin/PCNA or Ki-67 and TUNEL, respectively) have been found to coincide in some cell nuclei after cLDL or oxLDL impact (white arrowheads), while nonproliferating cells (PCNA-negative nuclei) do not manifest cell death signs (empty arrowheads). PCNA- and Ki-67-positive cells after nLDL impact are not positive for TUNEL. Counterstaining with DAPI. Bars, 25 µm.

 
Cause-effect relationship between cLDL- or ox-LDL-induced endothelial cell proliferation and cell death. Since the early peak of BrdU incorporation preceded cell death, we hypothesized that conditions of DNA synthesis may sensitize HCAECs to further cytotoxic cLDL or oxLDL impact, and thus the first 2-h peak of proliferation may contribute to the 24-h cell death. Such a phenomenon, called "mitotic cell death," has been described in several other in vitro models, including human endothelial cells (14, 18). To study the cause-effect relationships between cLDL/oxLDL-induced proliferation and cell death, proliferation was inhibited by aphidicolin and cell death was measured by LDH release. Aphidicolin, a DNA polymerase-{alpha} inhibitor, was chosen because of its ability to specifically inhibit DNA synthesis and arrest cells in G1/S or early S phase (11, 24).

High concentrations of aphidicolin have been described to produce a cytotoxic effect (40). Therefore, before the experiment the application of aphidicolin to HCAECs was tested at several concentrations between 0 and 20 µg/ml to choose the concentration that would inhibit proliferation without any cytotoxic effect. The data presented in Fig. 6 showed that at 1 µg/ml aphidicolin inhibited the proliferation of HCAECs (not treated with LDLs) to 8% of control. Since 1 µg/ml aphidicolin did not have significant cytotoxicity as measured by LDH release compared with control, this concentration was chosen for further experiments.


Figure 6
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  		Fig. 6. HCAEC proliferation and cell death induced by 2-h incubation with different concentrations of aphidicolin. BrdU incorporation (open bars) and LDH release (filled bars) were measured as described in MATERIALS AND METHODS. n = 4 per point. *P < 0.001 compared with untreated cells.

 
We observed that the presence of aphidicolin was cytoprotective against cLDL injury (Fig. 7). oxLDL-induced cell death was also prevented with aphidicolin. Therefore, this approach showed that the inhibition of proliferation and arrest of cells at G1/S phase caused significant decrease of endothelial cell death induced by cLDL.


Figure 7
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  		Fig. 7. Cell death of HCAECs induced by 200 µg/ml of cLDL or oxLDL in the absence (open bars) or presence (filled bars) of aphidicolin measured by LDH release assays. n = 4 per group. *P < 0.01 vs. cells treated without aphidicolin.

 
We next examined whether the arrest of the cells at G1/S by aphidicolin at the moment of cLDL impact would affect overall cell survival and growth by protecting proliferating cells from injury in a long-term experiment. Our data suggested that despite the short-term impact and quick change of conditions to normal, cell death occurred in the samples treated with cLDL, but not with vehicle- or nLDL-treated cells (Fig. 8). Again, treatment with oxLDL had a very similar effect on endothelial cell multiplication. Therefore, treatment with modified LDLs in the presence of aphidicolin resulted in the recovery and growth of the cells at close to the control level (Fig. 8). Thus proliferation and cell death appear to be interrelated effects of cLDL and oxLDL on endothelial cells. The inhibition of proliferation provided protection of endothelial cells.


Figure 8
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  		Fig. 8. Endothelial cell survival after a short time course (2 h) of vehicle (A, B), nLDL (C, D), cLDL (E, F), and oxLDL (G, H) impact at a dose of 200 µg/ml without (A, C, E, G; filled bars) and with (B, D, F, H; open bars) prevention of S phase with aphidicolin followed by 72-h period of growth in complete LDL-free medium. The cell number was determined by manual "blind" count. Images are representative of the group. Dashed line shows cell density at the beginning of experiments. Numbers are collected from 4 separate experiments; n = 5 per point *P < 0.01, **P < 0.001 compared with untreated control; #P < 0.01, ##P < 0.001 compared with cell treated with same LDL without aphidicolin.

 
MAPKs are involved in endothelial cell proliferation and death induced by cLDL. Because several protein kinases and in particular MAPK are involved in many events during endothelial cell proliferation (33, 42, 46), we hypothesized that the MAPK pathway may be involved in the cause-effect relationship between proliferation and cell death induced by cLDL or oxLDL. In our experiments, cLDL induced significant activation of ERK1/2 at 2, 6, and 12 h points; however, no activation was found at the 24 h point (Fig. 9A). Similar to cLDL, oxLDL also caused significant activation of ERK1/2; however, phosphorylation of ERK1/2 was observed at all time points. Both cLDL- and oxLDL-induced phosphorylation of JNK/SAPK occurred only at the late time points (Fig. 9B). MAPK p38 was not activated during the entire treatment course, and the total level of all mentioned proteins was not changed (data not shown). To determine the role of the described changes in cell proliferation and death, U-0126, a MEK1/2 inhibitor, and SP-600125, an inhibitor of JNK, were applied. The doses of these compounds were elaborated in separate experiments (Fig. 10). Similar to aphidicolin, the inhibition of ERK1/2 phosphorylation led to the significant twofold depression of BrdU incorporation (Fig. 11A) and protected cells from death induced by both cLDL and oxLDL (Fig. 11B). At the same time, inhibition of JNK did not affect the proliferation and had an insignificant rescue effect.


Figure 9
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  		Fig. 9. cLDL and oxLDL induce proliferation-mediated cell death through MAPK pathway: phosphorylation (p) of ERK1/2 (A) and JNK/SAPK kinases (B). Endothelial cells were treated with cLDL or oxLDL (200 µg/ml each) for varied times. Western blotting was performed as described in MATERIALS AND METHODS. Experiments were repeated 3 times, and representative images are presented.

 

Figure 10
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  		Fig. 10. Optimization of efficiency and toxicity of chemical inhibitors U-0126 (A) and SP-600125 (B) in HCAECs by cell ELISA and LDH release, respectively. n = 4. *P < 0.05 compared with untreated control.

 

Figure 11
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  		Fig. 11. Induced by cLDL and oxLDL (200 µg/ml, 24 h), BrdU incorporation (A) was significantly inhibited by MEK inhibitor U-0126 (10 µmol/l) while JNK inhibitor SP-600125 (1 µmol/l) did not affect it. U-0126 rescued endothelial cells from modified LDL injury, while SP-600125 had little effect (B). Numbers are from 3 separate experiments; n = 4. *P < 0.05, **P < 0.01 compared with cell treated with the same LDL with vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Both cLDL and oxLDL were shown to be elevated in blood of patients with chronic kidney disease (2, 23) and have proatherogenic properties including cytotoxicity toward endothelial cells (38, 48). oxLDL has been reported to cause endothelial proliferation (16, 19). By using three approaches, which included BrdU incorporation, FACS (S phase) analysis, and cell multiplication, we have demonstrated for the first time that cLDL induces dose-dependent proliferation of endothelial cells in vitro. We have identified two elevations of cell proliferation induced by cLDL to higher levels than nLDL. Interestingly, similar elevations were dose dependent and were observed at high (200 µg/ml) and quite low (6.25 µg/ml) concentrations of cLDL. Recently, we showed (2) that the cLDL concentration in human sera varies from 0.4 to >900 µg/ml, which makes the cLDL concentrations used in the present study relevant to physiological processes in humans. Because of cell death, it was impossible to detect the activation of proliferation and multiplication of the endothelial cells after long exposure to cLDL. A similar observation was made in another study that used oxLDL (8).

The proliferation after both short- and long-term incubation with cLDL was associated with cell death detected by LDH release and TUNEL. The oxidatively modified LDL has a dual effect on endothelial cells in vitro, similar to that observed by us with cLDL (16, 19). The fact that proliferation and cell death may relate to each other and occur in the same cell has not been previously described. PCNA and Ki-67 were chosen because they are established markers of cell proliferation and have low levels of degradation (21, 45). Thus we expected both of them to remain in the nuclei while proliferation-mediated cell death occurred. These experiments showed that proliferation and subsequent death may occur in the same cell and not just be present in different subpopulations of cells. Because oxLDL showed a pattern of staining similar to that of cLDL, it is suggested that both cLDL and oxLDL may at least partially utilize a universal pathway. Such a similarity between the pathways of several other modified LDLs was reported previously (4, 41).

We observed that endothelial cell proliferation was associated with death while S-phase arrest by aphidicolin led to cell rescue from modified LDL-induced injury. Furthermore, a short course of endothelial cell treatment with cLDL or oxLDL followed by a change of medium back to normal caused profound cell death at 72 h after impact. However, the pretreatment of endothelial cells with aphidicolin was protective from cLDL- or oxLDL-induced cell death. To the best of our knowledge, this phenomenon has not been described with any of the modified LDLs. These data may suggest that cLDL and oxLDL induce fast and irreversible activation of the mechanisms leading to simultaneous cell proliferation and cell death.

We can speculate that cLDL and oxLDL act by stimulating proliferation and inducing a mitogenic effect, which results in mitotic cell death. The latter has been reported to occur because of inappropriate stimuli for cells to proliferate (1, 10). A recent study by Zettler et al. (51) suggested that small doses of oxLDL may induce either cell proliferation or cell death in serum-free and complete media, respectively. These data are in agreement with our results, suggesting greater cytotoxicity of modified LDL in the proliferating cells due to the proliferative effect of serum components. Although programmed cell death is found in some tissues and developing organs in association with proliferation, the induction of both by the same compound is not commonly observed. Vascular cells have been shown to be more susceptible to oxLDL toxicity during proliferation (8, 20, 44). In fact, cells become most sensitive to oxLDL injury during the DNA synthesis phase of the cell cycle (26), which is a result of cell cycle entry rather than DNA repair (5).

Several mechanisms were offered to explain the proliferative properties of native and modified LDLs in endothelial cells (8, 53). Some studies report the involvement of MAPKs, which were shown to participate in cell proliferation, growth, arrest, and death (6, 17, 42). Our data support studies from other groups that described that modified LDLs may cause phosphorylation of ERK1/2 in vascular endothelial and smooth muscle cells and lead to their proliferation (6, 9, 42). The pathogenic role of induced p-ERK1/2 in cLDL- and oxLDL-mediated cell death was determined by cell rescue with MEK inhibitor, which suppressed the phosphorylation of ERK1/2. Phosphorylated ERK1/2 is known to be responsible for synthesis of several cell cycle regulators moving the cell from G1 to S phase (11, 42). Therefore the protective role of MEK inhibitor in modified LDL-induced cell death can be explained by slowing down of the cell cycle and a low proliferation rate at the time of treatment with LDLs.

Multiple studies have shown the participation of JNK in endothelial cell death (33, 49, 52). p-JNK kinase expression was slightly induced by both modified LDLs. In contrast to p-ERK1/2, JNK was phosphorylated at very late time points and its inhibition caused negligible rescue of the cells, while DNA synthesis was not affected at all. We may speculate that JNK participates in some late or downstream mechanisms of modified LDL-induced cell death.

According to our data, DNA synthesis and proliferation primarily regulate cell death by making cells more susceptible to injury. It is not known whether proliferation and death are induced by the same lipoprotein component/receptor or result from the simultaneous impact of several of them with different or the same mechanisms. Therefore we conclude that 1) cLDL and oxLDL induce similar injuries in endothelial cells; 2) proliferation and DNA synthesis play a primary and initiative role in endothelial cell injury after modified LDLs impact them through a mechanism similar to mitotic cell death; and 3) MAPK mechanisms play a significant role in the endothelial cell injury induced by modified LDLs. Further studies will be necessary to investigate the role of these cLDL- and oxLDL-caused effects in vivo in the process of atherogenesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by a grant from Satellite Healthcare (A. G. Basnakian, S. V. Shah), an Arkansas Tobacco settlement award (E. O. Apostolov), Department of Veterans Affairs Merit Review Grants (A .G. Basnakian, S. V. Shah), and fellowships from the Turkish Nephrology Association and the International Society of Nephrology (E. Ok).


   ACKNOWLEDGMENTS
 
We thank Dr. Ray Biondo for editorial assistance.

The use of the facilities in the University of Arkansas for Medical Sciences Digital and Confocal Microscopy Laboratory supported by National Institutes of Health Grant 2 P20 RR-16460 (principal investigator: Larry Cornett, IDeA Network of Biomedical Research Excellence, Partnerships for Biomedical Research in Arkansas) is acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. O. Apostolov, Div. of Nephrology, Dept. of Internal Medicine, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 501, Little Rock, AR 72205 (e-mail: apostolovyevgeniyo{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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