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Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
Correspondence to Dr. Jan Galle, Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Joseph-Schneider-Straße, 2 D-97080, Würzburg, Germany. Phone: +49 931 201 5331; Fax: +49 931 201 3502; E-mail: j-c.galle{at}mail.uni-wuerzburg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Isolation and Oxidation of LDL
Human LDL was isolated and oxidized as described recently
(11,17).
Briefly, the native lipoprotein was prepared from pooled, fresh human plasma.
Antioxidant-free LDL (0.3 mg protein/ml) was incubated with CuSO4
(1 µM) in phosphate-buffered saline (PBS) for 30 h at 23°C. The degree
of oxidation was quantitated by the increase in relative mobility on agarose
gel, indicating an enhanced negative charge of oxidized lipoprotein.
Homogeneity of lipoproteins was tested by agarose gel electrophoresis
(REP-HDL-plus cholesterol electrophoresis; Helena Diagnostika, Hartheim,
Germany). The relative mobility of OxLDL on agarose gel electrophoresis as an
index for lipoprotein oxidation was 2.5 to 3.0 compared with native LDL.
Protein content of OxLDL and native LDL was measured using a commercially
available kit (Sigma protein kit), which is based on a modification of a
method initially described by Lowry et al.
(18). LDL concentrations are
always given as micrograms of protein per milliliter of solution. Lipoproteins
were stored at 4°C in the dark and freshly prepared every 2 weeks. During
this period, apolipoprotein B was intact and not degraded.
Culture of HUVEC and Incubation With OxLDL
HUVEC were purchased from Clonetics (Walkersville, MD) and were cultured in
endothelial basal medium supplemented with hydrocortisone (1 µg/ml), bovine
brain extract (12 µg/ml), gentamicin (50 µg/ml), amphotericin B (50
ng/ml), EGF (10 ng/ml), and 10% FCS until the fourth passage. When the fifth
passage was started, cells were cultured with a lower concentration of FCS
(2%) in the absence of EGF, if not indicated otherwise. All experiments were
done with cells of the fifth passage. The cells were incubated with OxLDL or
its buffer as control or with LPC for 22 h, before detection of proliferation
or O2- formation.
Detection of Proliferation in HUVEC
Proliferation of HUVEC was detected by quantification of the DNA-synthesis
after incubation with 3H-thymidine. For the proliferation assay, cells (1
x 105) were incubated with 3H-thymidine (0.37 MBq) for 7 h.
Afterward, cells were lysed in 300 µl of 50 mM Tris/HCl (pH 8.0), 20 mM
NaCl, 1 mM EDTA, and 1% SDS containing 500 µg/ml proteinase K and incubated
for 12 h at 56°C and centrifuged for 15 min at 15,000 x g.
The supernatant was extracted with phenol and chloroform, precipitated with
isopropanol, and resuspended in water. The amount of DNA was determined
photometrically, and the content of 3H-thymidine as indicator for
proliferation was measured in a ß-counter (Canberra-Packard GmbH,
Dreieich, Germany). Because the final activity of tritium-labeled thymidine is
strongly influenced by the time passed after the initial labeling, it is not
useful to calculate thymidine incorporation on the basis of absolute counts,
in particular when several experiments are compared. Therefore, we defined the
activity of control cells as 100%, and the activity of cells treated otherwise
was set in relation to the activity of the control cells.
Inhibition of NADPH Oxidase by Transfection With Antisense
Oligonucleotide
To inhibit the NADPH oxidase, we used in some experiments an antisense
oligonucleotide against p22phox. Specificity of inhibition was tested by
transfection of control cells with sense oligonucleotide
(19,20).
The oligonucleotides were labeled with fluorescein to determine the efficiency
of transfection. Antisense:
5'-fluo-GAT-CTG-CCC-CAT-GGT-GAG-GAC-C-phosphorothioate-3'; sense:
5'-fluo-GGT-CCT-CAC-CAT-GGG-GCA-GAT-C-phosphorothioate-3'.
For transfection, HUVEC (1 x 105) were incubated with 1 µM oligonucleotide and lipofectin (20 µl/ml medium) for 4 h. At the end of this incubation period, cells were washed with PBS and incubated either with cytochrome c and OxLDL or its respective buffer or with LPC, to measure O2- formation. To determine transfection efficiency and to detect necrotic cells, cells were stained with propidium iodide and analyzed by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA).
Detection of O2- Formation by Cytochrome C
Assay
O2- formation of HUVEC after sense and antisense
transfection was measured as described recently
(11) using the cytochrome c
assay according to the method initially described by Van Gelder and Slater
(21). Approximately 1 x
105 cells were incubated in a culture dish. The cells were kept at
37°C in a cell incubator (Heraeus-Instruments, Stuttgart, Germany) with 5%
CO2 in a humidified atmosphere. Then the cells were incubated with
OxLDL (100 µg/ml) or its respective buffer as control or with LPC (10
µM) in the presence of 20 µM cytochrome c for 4 h. Preliminary
experiments revealed that a longer incubation period with LPC was required to
obtain an increase in the signal. Therefore, cells were incubated for 18 h
with LPC before transfection with sense or antisense oligonucleotides.
Reduction of cytochrome c in the supernatant was detected photometrically at
550 nm. Specificity of the assay was tested by coincubation with SOD (100
U/ml), which prevented the increase in the reduction of cytochrome c. Only the
SOD-inhibitable reduction of cytochrome c was used for calculation. At the end
of the experiments, the protein content of the cells in each dish was
determined (Sigma protein kit). O2- formation was
expressed in nanomoles of O2- per milligram of
protein.
Statistical Analyses
Data are presented as means ± SEM of n experiments.
Proliferation rates are expressed as percentage of control proliferation
induced by 2% FCS. The statistical differences were determined using
Student-Newman-Keuls Test and Dunn's Test (Sigma Stat Software Program, Jandel
Scientific, San Rafael, CA). Differences were considered significant at an
error probability of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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3H-thymidine incorporation of HUVEC under basal conditions (2% FCS) and after treatment with OxLDL (1 to 20 µg/ml, 22 h of incubation) is shown in Figure 1. OxLDL significantly increased the proliferation rate of HUVEC with a maximum effect at 5 µg/ml. Therefore, the following experiments were performed with this concentration. It should be noted that the increase in thymidine incorporation as measured in our experiments exceeds thymidine incorporation as seen in the context with DNA repair, indicating that it indeed reflected proliferation.
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Prevention of Proliferation in HUVEC by SOD/Catalase and by Diphenylene Iodonium. To investigate whether O2- might be involved in the induction of proliferation in HUVEC, we incubated the cells with OxLDL in the presence of the O2--catabolizing enzymes SOD and catalase. SOD and catalase were given in combination because O2- formation in the presence of an excess of SOD in relation to catalase might result in the generation of highly toxic hydroxyl radicals (22). SOD (10 U/ml) and catalase (10 U/ml) almost completely prevented the stimulation of proliferation by OxLDL (Figure 2). Thus, oxygen radicals seem to be essential for the stimulation of proliferation by OxLDL. To determine whether the membrane-bound NADPH oxidase is the source for enhanced O2- formation, we studied the effect of the NADPH oxidase inhibitor DPI (1 µM) on OxLDL-induced proliferation of HUVEC. As shown in Figure 3A, the presence of DPI completely prevented proliferation induced by 5 µg/ml OxLDL, suggesting that indeed NADPH oxidase was involved. To exclude that DPI had an unspecific effect on cell proliferation, we studied its impact on proliferation induced by the O2- generating system xanthin/xanthine oxidase. Coincubation of HUVEC with xanthin (50 µM) and xanthine oxidase (0.0001 U/ml) for 22 h induced proliferation of the cells in a similar fashion as OxLDL. DPI did not block cell proliferation induced by exogenously produced O2-, confirming that it did not influence the cell cycle downstream of O2- formation (Figure 3B).
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Lysophosphatidylcholine Induces Proliferation of HUVEC. We next analyzed whether the effects of OxLDL can be mimicked by LPC, a compound formed during oxidation of LDL that also can stimulate O2- formation in vascular cells (23). Incubation of the cells with LPC (10 µM) significantly enhanced cell proliferation (Figure 4). In analogy to the OxLDL effects, this enhancement could be abolished by coincubation with DPI (1 µM) (Figure 4). Thus, LPC may at least in part be responsible for OxLDL-induced cell proliferation.
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O2- Formation in HUVEC
To provide additional evidence for our assumption that NADPH oxidase is the
target enzyme for OxLDL in the context of stimulation of
O2- formation and upregulation of cell proliferation, we
transfected HUVEC with sense and antisense oligonucleotides against p22phox,
the 
-subunit of cytochrome b-558 (the membrane-bound part of NADPH
oxidase), and measured O2- formation directly in
transfected cells using the cytochrome c assay. Sense transfection was
performed as a control.
Transfection of HUVEC With Sense and Antisense Oligonucleotides Against p22phox. To determine the transfection efficiency in HUVEC, we used fluorescein-labeled oligonucleotides that enabled us to quantify the transfection rate by flow cytometry. Figure 5 shows that the initial transfection rate after 4 h of incubation with the oligonucleotide and lipofectin was approximately 90%. Because incubation of the cells with OxLDL and cytochrome c for detection of O2- formation required an additional 4 h, we analyzed the stability of the transfection and quantitated the transfection rate. As depicted in Figure 5, approximately 80% of the cells were still transfected after 8 h.
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Reduced O2- Formation in Antisense Transfected HUVEC After Stimulation With OxLDL or LPC. O2- formation of sense or antisense transfected cells under basal, unstimulated conditions and after incubation with OxLDL is shown in Figure 6A. Under basal conditions, sense and antisense transfected cells produced equal amounts of O2-. In sense transfected cells, incubation with OxLDL (100 µg/ml for 4 h) resulted in a significant enhancement of O2- formation. In antisense transfected cells, the enhancement of O2- formation was significantly lower (Figure 6A), suggesting that NADPH oxidase contributes to O2- formation in HUVEC after stimulation with OxLDL. At lower concentrations of OxLDL, no increase in O2- formation could be detected with the cytochrome c assay.
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The impact of LPC on O2- formation of sense or antisense transfected cells is shown in Figure 6B. In analogy to cell proliferation results, 10 µM LPC mimicked the OxLDL effect. Stimulation of antisense transfected cells with LPC resulted in a significantly lower enhancement of O2- formation, providing additional evidence that LPC acts at least in part via stimulation of NADPH oxidase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Atherosclerotic vascular lesions are characterized by increased cellular turnover involving endothelial cells (3,4). In atherosclerotic plaques, cell proliferation occurs together with apoptotic and necrotic cell death (5,6). Accumulation of OxLDL is another characteristic of atherosclerotic lesions, and OxLDL is thought to play a major role in the pathogenesis of atherosclerosis (7,8). Therefore, we studied its impact on both cell death and proliferation of HUVEC. In a recently published study, we showed that OxLDL—prepared according to an identical protocol as in this article—induces apoptotic and to a minor degree necrotic cell death of HUVEC (11). On the first glimpse, these observations are in contrast to the data presented here, which indicate that OxLDL stimulates cell proliferation in the same cell type. However, the dose of OxLDL required to induce apoptotic cell death (60 to 200 µg/ml) was significantly higher than the dose with the maximum effect on cell proliferation in the present study (5 µg/ml). Thus, we propose that OxLDL has a dual effect on the cell cycle. OxLDL can induce both proliferation and cell death in HUVEC, strongly depending on its concentration.
Several lines of evidence indicate that the OxLDL effect on the cell cycle is linked to its capacity to stimulate O2- formation. Using the cytochrome c assay, we showed that OxLDL indeed stimulates HUVEC to produce O2-, confirming previous studies from our laboratory (11). SOD and catalase, enzymes that catabolize O2-, prevented OxLDL-induced cell proliferation. Cell proliferation could also be induced by O2- generated through the xanthine/xanthine oxidase reaction in the absence of OxLDL. These results are in line with previous studies suggesting that enhanced O2- formation, induced, for example, by angiotensin II, is a factor that regulates vascular or tubular cell growth (13,24,25). Because the data on cell proliferation were obtained with OxLDL at a concentration of 1 to 20 µg/ml, we attempted to measure stimulation of O2- formation within the same concentration range. However, the cytochrome c assay was not sensitive enough to detect increases of O2- formation at these low concentrations.
Our experiments suggest that the endothelial NADPH oxidase was the major
source for O2- formation. DPI, a flavoprotein inhibitor,
abolished the effect of OxLDL on cell proliferation. That DPI had no impact on
cell proliferation induced by O2- generated through the
xanthine/xanthine oxidase reaction in the absence of OxLDL argues against an
unspecific effect of DPI on cell cycle regulation downstream to
O2- formation. Additional evidence for a central role of
NADPH oxidase in the OxLDL-induced O2- formation is
provided by the experiments with antisense transfected cells. HUVEC
transfected with antisense oligonucleotides against p22phox, the
-subunit of the membrane-bound part of NADPH oxidase, cytochrome b-558,
showed a significantly reduced response to OxLDL in terms of
O2- formation. It would have been interesting to study
the impact of OxLDL on proliferation in transfected cells. However, antisense
transfected cells were not stable enough for proliferation experiments.
To examine which compound of OxLDL may be responsible for stimulation of O2- formation and of cell proliferation, we attempted to mimic the OxLDL effects with LPC. The rationale to perform these experiments is based on the fact that only oxidized LDL induces oxidative stress, whereas the native form is without effect (11); the latter observation hints at the lipid peroxidation process itself. LPC is a byproduct of cholesterol esterification, accumulates in LDL during its oxidative modification (26), and has been shown to increase O2- formation in vascular smooth muscle cells through a step involving activation of protein kinase C (23). Protein kinase C, in turn, is involved in activation of NADPH oxidase (27,28). As shown here with synthetic LPC, a similar mechanism may take place in endothelial cells. Furthermore, LPC had a stimulatory effect on cell proliferation, comparable to that of OxLDL. The NADPH oxidase inhibitor DPI abolished the LPC-induced cell proliferation. Thus, LPC may be the compound of OxLDL responsible for stimulation of O2- formation. Indeed, LPC is known to stimulate protein kinase C (23).
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Conclusion |
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Acknowledgments |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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activates a
p22phox-based NADH oxidase in vascular smooth muscle.
Biochem J 329:653
-657, 1998
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