(Hypertension. 1995;25:511-516.)
© 1995 American Heart Association, Inc.
Articles |
Low-Density Lipoprotein Induces Vascular Adhesion Molecule Expression on Human Endothelial Cells
From the Franz Volhard Clinic and the Max-Delbrück Center for Molecular Medicine, University Hospitals Rudolf Virchow (H.H., D.S., W.Z., F.C.L.), the Department of Medicine-Nephrology, Klinikum Steglitz University Hospital (H.H., S.P., A.D.), and the Department of Biochemistry, Free University (M.K.), Berlin, Germany.
Correspondence to Hermann Haller, MD, Franz Volhard Klinik, Wiltberg Strasse 50, 13122 Berlin, Germany.
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Abstract |
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Abstract We tested the hypothesis that low-density lipoprotein (LDL) and its acetylated form influence surface expression of vascular adhesion molecules on human endothelial cells. Vascular adhesion molecule surface expression was assessed with flow cytometry on cultured endothelial cells with a modified enzyme-linked immunosorbent assay. LDL acetylation was determined by chromatography. Monocyte adhesion to endothelial cells was assessed with U937 cells by direct counting. Tumor necrosis factor-
(10 ng/mL), a positive
control, induced a time-dependent expression of vascular adhesion
molecules (P<.05), which peaked at 5 hours. Incubation of
endothelial cells with LDL (1.3 to 26.0 mmol/L) led to an increase in
expression at 2 and 5 hours (P<.05). Prolonged (24-hour)
exposure to LDL resulted in a second peak. The effect of acetylated LDL
on expression was not different from that of native LDL. Incubation
with the protein kinase C inhibitor staurosporine (5x10-8
mol/L) blocked the effects of both native and acetylated LDL completely
(P<.05). The calcium channel blocker nitrendipine
(10-7 mol/L) did not influence the expression of vascular
adhesion molecule at 2 and 5 hours but did reduce the effect of LDL on
expression at 24 hours. LDL (2.6 mmol/L) also induced a significant
increase in the surface expression of intercellular adhesion molecule-1
but did not affect the expression of endothelial adhesion molecules.
LDL (2.6 mmol/L) induced a significant increase in monocyte binding. We
conclude that LDL can induce the expression of vascular adhesion
molecules on endothelial cells. This effect is mediated by protein
kinase C and partially inhibited by calcium channel blockade. Our
results suggest that LDL may increase the recruitment of monocytes
through a protein kinase C–mediated increase in adhesion molecule
surface expression.
Key Words: cell adhesion molecules • endothelium • lipoproteins, LDL • protein kinase C • calcium channel blockers
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Introduction |
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Hypertension and hyperlipidemia both play a role in atherogenesis by interaction with endothelial cells.1 2 Both may foster monocyte recruitment within the vascular wall and promote adhesion to intimal surfaces.3 4 Monocyte adhesion may involve interaction with adhesion molecules on endothelial cells.5 The vascular cell adhesion molecule–1 (VCAM) is an inducible receptor that mediates endothelial adhesion of monocytes and lymphocytes.6 7 8 VCAM is expressed on the surface of endothelial cells overlying early "fatty streak" lesions.6 In rabbits fed cholesterol, the expression of VCAM precedes the subendothelial accumulation of monocytes,7 which suggests a role for VCAM in the early stages of vascular damage.9
The effect of a high-cholesterol diet on VCAM expression in vivo suggests that low-density lipoprotein (LDL) or LDL modified by oxidation (oLDL) may be responsible for the induction of VCAM.9 Cultured endothelial cells incubated with LDL10 or oLDL11 12 showed an increased stickiness of monocytes; however, specific adhesion molecules were not studied in these experiments. Kume et al13 showed that VCAM expression is enhanced by lysophosphatidylcholine, a component of LDL. They observed no effect of native LDL or oLDL on VCAM expression in cultured endothelial cells but suggested instead that the biological properties of modified LDL may be different in living organisms than in in vitro studies. We tested the hypothesis that LDL may influence the surface expression of VCAM on human umbilical endothelial cells. We examined both native and modified LDL. To test the results of the latter, we chose to acetylate the molecule.
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Methods |
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Culture Procedures
Human umbilical vein endothelial cells were isolated from umbilical cords by chymotrypsin treatment. The cords were cleaned with isotonic NaCl buffer at room temperature and were incubated for 25 minutes at 37°C with 1% chymotrypsin in phosphate-buffered saline (PBS, Seromed). Endothelial cells were then removed by centrifugation (400g for 10 minutes). The pellet was resuspended in M-199 (Seromed) with 20% fetal calf serum, 1% L-glutamine, 1% nonessential amino acids (Seromed), 1% HEPES (GIBCO), 1% Na-pyruvate, and 1% Schutz medium (Seromed), as well as with streptomycin and penicillin. Primarily cultured cells were grown for 3 to 4 days and were subcultured. Subcultures 1 and 2 were used for the experiments.
Preparation of LDL and Acetylated LDL
LDL was isolated from citrated normolipemic human plasma by
sequential ultracentrifugation. Acetylation of the LDL fraction was
carried out by the incubation LDL with a saturated solution of
Na-acetanhydride for 1 hour at 4°C. Acetylated LDL (acLDL) was then
dialyzed against a 0.9% NaCl solution containing 10 mmol/L sodium
phosphate (pH 7.4) and 1 mmol/L EDTA for 12 hours and then
filter-sterilized. The purity of the fractions was assessed by gel
electrophoresis; all preparations were assayed for protein content and
total cholesterol and are expressed as LDL cholesterol
concentrations.
Flow Cytometry
Surface expression of VCAM, intercellular adhesion
molecule–1 (ICAM-1), and endothelial leukocyte adhesion molecule
(ELAM) was measured by flow cytometry.14 Endothelial cell
cultures were stimulated with LDL or tumor necrosis factor-
(TNF-) as indicated, washed, and removed from tissue culture by
treatment with 0.02% EDTA. Cells were stained with monoclonal
antibodies–specific antibody for VCAM, ICAM-1, and ELAM (all
antibodies from Biermann) and fluorescein isothiocyanate and were
analyzed on an automated fluorescence-associated cell separation system
(FACScan, Becton Dickinson Immunocytometry Systems).
Enzyme-Linked Immunosorbent Assay for Expression of VCAM-1, ICAM-1,
and ELAM
Experiments were carried out on 96-microtiter plates. The
endothelial cells were seeded in the medium as described above and
incubated at 37°C for 2 to 3 days. Then the medium was changed, and
the stimulants were added. LDL or acLDL was dissolved in ethanol (stock
solution, 1 mol/L) and added to the medium at the final concentration
as described. Ethanol was added to the control cells in similar
concentrations in all experiments. The medium was removed after 2, 5,
12, and 24 hours, and the plates were incubated with the specific
antibody for VCAM, ICAM-1, and ELAM (all antibodies from Biermann) at a
concentration of 1:1000 in PBS Dulbecco (Seromed) with 0.2% Tween-20
(Serva) for 30 minutes at room temperature. The cells were then washed
twice with PBS. Thereafter, a peroxidase-conjugate IgG antibody (goat,
anti-mouse) was added (1:20 000, 30 minutes, room temperature),
followed by two washes with PBS. The color reaction was begun by adding
the substrate tetramethylbenzidine dihydrochloride (Sigma Chemical Co).
Chromophore development was determined by measurement of optical
density at 450 nm with a microtiter plate reader (Dynatech) and stopped
by the addition of 2 mol/L sulfuric acid. Wells were read against blank
controls containing cells incubated without the primary antibody. The
reported data are derived from optical density readings well within the
linear portion of the development curve (10 minutes). Values are
expressed as optical density (OD). The relation between enzyme-linked
immunosorbent assay (ELISA) and flow cytometry was assessed for TNF-
(100 ng/mL) and tissue plasminogen activator (10, 50, and 100 nmol/L)
at different time points (6, 12, and 24 hours). We observed similar
changes and a close correlation in surface expression of VCAM-1 and
ICAM-1 with the two different methods.
Cell Adhesion Assay
Experiments were carried out on 96-microtiter plates as
described previously.15 The endothelial cells were seeded
in the medium and incubated at 37°C for 2 to 3 days. The medium was
then changed and LDL added. LDL was dissolved in ethanol (stock
solution, 10 mmol/L) and added to the medium at the final concentration
as described. Ethanol was added to the control cells in similar
concentrations in all experiments. The medium was removed after 24
hours. U937 cells were then added at a concentration of 105
cells per well, and the titer plates were incubated for 30 minutes at
37°C. The plates were then washed three times with medium, and the
adherent U937 monocytes were stained with neutral red diluted in medium
(1:2 vol%). After the plates were washed, the adherent monocytes were
counted under the microscope. For each measurement, seven to eight
wells were analyzed. The intra-assay variability for these measurements
was 15%.
Statistics
Statistical analysis was carried out on a Macintosh II
computer (Apple Inc) with a commercial program (STATVIEW,
Cricket Software Inc). The results are expressed as mean±SEM. The
nonparametric Wilcoxon test and a one-way ANOVA were used. Differences
were considered to be significant when the probability value was
.05.
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Results |
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Fig 1 shows the VCAM surface expression on endothelial cells as assessed by flow cytometry. The ordinate represents the absolute cell number. The abscissa shows fluorescence intensity expressed in a logarithmic fashion. TNF-
(positive control) caused a dramatic shift of the fluorescence curve to
the right, indicating increased surface expression of VCAM compared
with controls. LDL also shifted the fluorescence curve to the right,
albeit to a lesser degree. Fig 2 shows the mean number
of cells with positive VCAM surface expression for controls, LDL
treatment, and TNF- as determined by flow cytometry. The effect of
LDL was different (n=12, P<.05) from controls; the effect
of TNF- was different from both LDL and controls (n=14,
P<.05).
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We used a second technique to identify VCAM expression—namely, a modified ELISA. Fig 3 shows these results. Incubation of cultured human umbilical vein endothelial cells with LDL resulted in a dose-dependent increase in VCAM expression. The fiducial limits given in this and subsequent experiments represent the mean±SEM of at least 12 separate experiments. Concentrations of LDL at 1.3 mmol/L induced a significant increase in VCAM expression above baseline (P<.05). The elevated values stabilized after 2.6 mmol/L. LDL concentrations at 26 mmol/L and those above (data not shown) actually decreased VCAM expression, probably because of damage to the cells.
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Fig 4 shows the effects of exposure to LDL and acLDL 2.6
mmol/L over time. A biphasic response was seen with an initial prompt
increase (n=6, P<.05), followed by a decrease over 12
hours, with a subsequent increase (n=6, P<.05) again at 24
hours. These significant changes were small compared with the
stimulatory effects of TNF- shown in Fig 3. They represent
approximately 15% of the TNF- effect.
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We next investigated possible mechanisms of LDL-induced VCAM
expression. These results are shown in Fig 5. When
endothelial cells were incubated with LDL (2.6 mmol/L) and the protein
kinase C inhibitor staurosporine (10-8 mol/L) together for
5 hours, the stimulatory effect of LDL on VCAM expression was abolished
completely (Fig 5, top and bottom). In preliminary experiments, we
examined the effects of staurosporine at this dose
(5x10-8 mol/L) on growth and cell behavior and found no
toxic effects (data not shown). At the concentrations used in our
experiments, there was also no significant effect of staurosporine on
VCAM expression. However, higher concentrations of staurosporine
(>10-7 mol/L) induced an increase in VCAM expression.
While staurosporine completely blocked the LDL effect on VCAM
expression, the effect of TNF- on VCAM surface expression was only
partially inhibited (data not shown). Incubation of the cells with LDL
and the calcium antagonist nitrendipine (10-7 mol/L) also
reduced the VCAM surface expression significantly but only after 24
hours. In contrast, nitrendipine showed no significant effect on VCAM
surface expression at 5 hours (Fig 5, top). Nitrendipine had no
significant effect on basal VCAM expression even at higher
concentrations. LDL and acLDL responses and their inhibition were
similar in all experiments.
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To determine whether the effect of LDL on VCAM expression was specific
for this surface adhesion molecule, we also measured the effect of LDL
on ICAM-1 and ELAM. LDL (2.6 mmol/L) induced a slight but significant
increase in endothelial surface expression of ICAM after 2 and 6 hours,
while the surface expression of ELAM was not affected by exposure of
endothelial cells to LDL. The results (n=16 experiments for ICAM-1 and
n=8 experiments for ELAM at all time points) for ICAM were 88±23 to
125±21 at 2 hours (P=NS) and 186±31 at 6 hours
(P<.05). Those for ELAM were 56±20 to 74±19 at 2 hours
and 82±31 at 6 hours (P=NS). Similar results were obtained
when fluorescence-associated cell separation system analysis was
used to evaluate ICAM-1 and ELAM expression. In the last set of
experiments, we investigated the functional significance of the
LDL-induced upregulation of endothelial cell adhesion molecules.
Endothelial cells were exposed to LDL (2.6 mmol/L) for 6 and 24 hours
and incubated with monocytes for 30 minutes, and the adherent cells
were counted under the microscope. As Fig 6 shows, LDL
after 24 hours induced a significant increase in monocyte adhesion to
endothelial cells (n=18, P<.01). A stimulatory effect of
LDL on monocyte adhesion was also observed after 6 hours of incubation
(4.7± 0.7 cells per well, n=18, P<.01). The LDL effect was
approximately 20% that of the positive control TNF-–induced
monocyte binding. We then tried to specifically block the
VCAM-dependent adhesion using specific antibodies. However, our
antibodies did not block VCAM-dependent adhesion. Instead, the
antibodies augmented the LDL-induced effect on monocytes by 30%
(P<.05), indicating that the antibodies do not block the
specific binding site.
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Discussion |
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Activated endothelial cells may be important in the development of arteriosclerotic vascular disease.1 2 For instance, they may exhibit altered permeability, express binding sites for monocytes, and perhaps secrete monocyte activators. Activated endothelial cells may also secrete oxygen free radicals that can modify LDL, act as procoagulant surfaces, or synthesize and secrete mitogens and chemoattractants. The mechanisms that activate endothelial cells are imperfectly defined. Hemodynamic factors resulting from hypertension and cholesterol or its oxidized derivatives, cigarette smoking, or indirect mediators (eg, TNF-
generated in the vessel wall in
response to one of the above stimuli) are being investigated. Monocyte-derived macrophages participate in atherogenesis and may play a role in hypertension-induced vascular injury and organ hypertrophy. We recently identified monocyte participation in hypertension-induced renal damage16 and found that the proto-oncogene for the receptor of monocyte colony stimulating factor is expressed within perivascular monocytic infiltrates found in hypertension-induced cardiac hypertrophy.17 A primary step in monocyte recruitment is the attachment of this blood-borne cell onto a surface—in the case of the vessel wall, the endothelial surface. Little is known of the actual sequence of events; however, the surface expression of VCAM has been shown to be important to this process.18 The link between hyperlipidemia and monocyte attachment to endothelium is established.19 Pritchard et al20 demonstrated that when added to endothelial cells, native LDL increased monocyte binding to the cell surface. Other investigators demonstrated the importance of VCAM surface expression in the monocyte recruitment process.6 7 8 9
We demonstrated that LDL leads to a significant increase in VCAM
surface expression on human endothelial cells by means of two separate
and different techniques. The effect of LDL was small compared with the
TNF-–mediated response. We could have attempted to pursue the issue
with Northern or Western blotting. However, it is unlikely that these
semiquantitative techniques would elucidate the LDL effect further.
A similar response of ICAM to stimulation with LDL was also
observed; however, no effect on ELAM could be demonstrated. This
observation suggests that LDL exerts a specific stimulatory effect on
adhesion molecules of the integrin family but not on the selectins. We
admit that the effects are modest; however, chronic vascular disease
takes years to develop. Any clinical implications of these findings
should be drawn with caution.
Kume et al13 recently showed that the surface expression of VCAM and ICAM was induced by lysophosphatidylcholine. In contrast to our results, they did not find a stimulatory effect of native LDL on these adhesion molecules in cultured endothelial cells. This discrepancy is not easily explained. Our results showed that LDL leads to only a small rise in VCAM expression. Perhaps the magnitude of the response, coupled with differences in methodology, serves to explain the discrepancy in our findings. However, we are confident in our results because flow cytometry and specific antibodies both showed the effect of LDL on VCAM surface expression. Another possibility is that the biological properties of the LDL preparations used in the two studies were different because of different preparations. Furthermore, in our experiments only subcultured endothelial cells from passages 1 and 2 were used. It is possible that endothelial cells in higher passages are less responsive to LDL. Thus, the small effect observed in our study would have been masked.
Interestingly, we could not find a difference in the response of VCAM surface expression between native LDL and acLDL. Although acLDL is not abundant in humans, acetylation alters the biological properties of the lipoprotein similar to oxidation.21 Both oLDL and acLDL bind to a so-called "scavenger" receptor, which differs from the native LDL receptor.22 23 24 Numerous studies demonstrated that oLDL leads to a more pronounced cellular stimulation compared with native LDL.5 25 There seems to be no difference between the two forms of LDL with respect to the effects of LDL on surface expression of VCAM and ICAM. It is also possible that the observed effects of LDL in our study are not due to receptor-ligand interactions but instead reflect a nonspecific interaction of LDL with plasma membranes. Tremoli et al26 also observed no difference between the effect of LDL and acLDL on the synthesis of plasminogen activator inhibitor–1 in cultured human endothelial cells.
The inhibitory effect of the protein kinase C inhibitor staurosporine
suggests that the induction of VCAM by LDL is mediated by protein
kinase C. This enzyme complex was also shown to be involved in the
induction of adhesion molecules by other stimuli such as TNF- and
lipopolysaccharides.27 28 Although staurosporine also
inhibits other protein kinases, the inhibition of protein kinase C is
predominant at the concentrations used in our experiments. Palkama et
al29 reached similar conclusions. They also demonstrated
the activation of protein kinase C by LDL, which is in agreement with
several studies showing that LDL leads to an intracellular mobilization
of calcium through generation of inositol
trisphosphate.30 31
The calcium antagonist nitrendipine did not influence the LDL-induced surface expression of VCAM after 5 hours but instead reduced VCAM surface expression after 24 hours. This observation suggests that the early and late induction of VCAM by LDL is controlled by two different mechanisms. Because endothelial cells have no voltage-sensitive (L-type) calcium channels,32 our findings suggest that calcium antagonists exert effects on endothelial cells that are not dependent on their inhibitory effect on L-type calcium channels. We do not know for certain which intracellular mechanisms were responsible for the effect of nitrendipine. However, our findings are consistent with those of McDonagh and Rauzzino,33 who recently observed a decrease in leukocyte adhesion in the coronary microcirculation after exposure to a calcium antagonist. We believe that our calcium antagonist data may have relevance for another reason. We recently found that LDL results in a calcium signal within endothelial cells that is diminished by thapsigargin and completely blocked by the G-protein inhibitor pertussis toxin.34 In those experiments, thrombin, another activator of endothelial function, was used as the positive control for the calcium signal. Thrombin is also known to stimulate monocyte adhesion to endothelial cells.35 Although we did not examine calcium signals in the current study, we believe that the effects of nitrendipine on VCAM expression are consistent, even in the absence of L-type receptors on endothelial cells.
In summary, we observed a stimulant effect of LDL and acLDL on the
surface expression of VCAM in cultured human endothelial cells. The
effect was blocked completely by the concomitant incubation with
staurosporine, which suggests that the LDL response is mediated by
protein kinase C. The calcium antagonist nitrendipine also reduced VCAM
expression. Our results suggest that hypercholesterolemia may increase
the recruitment of leukocytes through a protein kinase C–mediated
increase in adhesion molecule surface expression. Although the effect
is small compared with agonists such as TNF-, atherogenesis is an
interactive process that takes years to develop. We believe that under
in vivo circumstances, which may include the influence of hypertension,
cigarette smoking, and other concomitant noxious influences, small
effects may be clinically significant.
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Acknowledgments |
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This work was supported by a grant in aid from the Deutsche Forschungsgemeinschaft to Dr Haller (Ha 1388-2/3).
Received January 28, 1994; first decision April 6, 1994; accepted November 28, 1994.
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