© 2000 American Heart Association, Inc.
Vascular Biology |
Lesions in Ryanodine Channels in Smooth Muscle Cells Exposed to Oxidized Low Density Lipoprotein
From the Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, and the Department of Physiology, University of Manitoba, Winnipeg, Canada.
Correspondence to Dr Grant N. Pierce, Director, Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6. E-mail gpierce{at}sbrc.umanitoba.ca
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Abstract |
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Abstract—The purpose of the present investigation was to investigate the subcellular basis responsible for the loss of vasoreactivity in atherosclerotic vessels. We have chosen to focus on the potential of oxidized low density lipoprotein (oxLDL), an important atherogenic agent, to alter sarcoplasmic reticulum (SR) structure and function. Vascular smooth muscle cells (VSMCs) were exposed for 1 to 6 days to low concentrations of minimally oxidized LDL. ATP was used to probe SR function in VSMCs. ATP can increase [Ca2+]i in control VSMCs because of a release of Ca2+ from the SR. However, after chronic exposure to oxLDL, cells lose their ability to increase [Ca2+]i in response to ATP. These cells also exhibit a depressed rise in [Ca2+]i after exposure to ryanodine. These effects were associated with a decreased immunoreactivity for the ryanodine-sensitive Ca2+-release channels in the SR of oxLDL-treated cells. Immunohistochemical analysis of aortic sections obtained from rabbits fed a cholesterol-supplemented diet revealed a significant decrease in the immunoreactivity for ryanodine channels in the plaque and in the medial layer underlying the plaque. In summary, our data identify oxLDL as a component within the atherosclerotic milieu capable of inducing a decrease in smooth muscle ryanodine channel density. This alteration is associated with a significant defect in the ability of the SR within the smooth muscle cell to regulate Ca2+. These lesions may contribute to the altered vasoreactivity exhibited by atherosclerotic vessels.
Key Words: Ca2+ • atherosclerosis • sarcoplasmic reticulum
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The vascular smooth muscle cell (VSMC) is critical for regulating vasomotor tone in the arterial system. At a cellular level, the sarcoplasmic reticulum (SR) helps to accomplish this through an efficient release and reuptake of cytoplasmic Ca2+.1 2 3 One of the mechanisms through which Ca2+ is released into the cytoplasm to support contraction occurs through ryanodine-sensitive release channels located in the SR membrane.4 5 6 Changes in the manner by which these proteins regulate intracellular Ca2+ will ultimately have significant effects on cell contraction, vascular tone, and blood flow.
Atherosclerosis is an important pathological state that appears to alter vascular reactivity and tone. Atherosclerotic vessels have been shown by a number of independent investigators to exhibit a loss of reactivity to a variety of vasoactive agents.7 8 9 10 The mechanism whereby atherosclerosis changes the cell to induce this depression in smooth muscle cell tone is unclear. Furthermore, the factor within the atherosclerotic state that is responsible for this effect on the VSMC is not clear. In view of the importance of the ryanodine-sensitive channels in the SR for regulating VSMC [Ca2+], it was reasonable to investigate whether there were any changes in the content and function of this protein in atherosclerotic vessels. We have chosen to examine oxidized LDL (oxLDL) as a candidate compound that may be responsible for effecting any change in Ca2+ regulation by SR in VSMCs. OxLDL is an important atherogenic factor that has the capacity to alter cell Ca2+.11 12
The present investigation had 2 objectives. First, we wanted to determine whether atherosclerosis could induce an alteration in the content of an important Ca2+ regulatory SR protein in vivo. Second, we wanted to isolate 1 atherogenic factor, oxLDL, under cell culture conditions and examine its potential to modify this protein and SR function in general in VSMCs.
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Methods |
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LDL Isolation and Oxidation
LDL was isolated from the plasma of cholesterol-fed New Zealand White rabbits as described previously.12 13 The LDL fraction was extensively dialyzed with 0.15 mol/L NaCl and 0.1 mmol/L EDTA (pH 7.4), sterile-filtered, and stored at 4°C in the dark. The protein and cholesterol (free and esterified) content of LDL was measured as described.14 15 16 17 Auto-oxidation of LDL during and before use was detected by an absence of malondialdehyde-reactive products and oxidized cholesterol.18 The EDTA concentration in native LDL was reduced before LDL oxidation. Native LDL was diluted 10-fold in 150 mmol/L NaCl (pH 7.4) solution and oxidized by incubation with a solution of 50 µmol/L FeCl3 and 0.25 mmol/L ADP for period of 3 hours at 37°C. The extent of LDL oxidation was evaluated by (1) a modest increase of thiobarbituric acid–reactive substances,12 19 (2) minimal electrophoretic mobility on agarose gels (with use of the Chiron Diagnostic Lipoprotein System), and (3) a 21% depletion of
-tocopherol content as measured by
high-performance liquid
chromatography.20
Vascular Smooth Muscle Cells
Primary cultures of VSMCs were by explant
techniques.21 The aorta from a male New Zealand White
rabbit (2.5 to 3 kg body weight) was isolated and cut into 2- to 3-mm
sections and transferred to a culture dish with growth medium. This
medium consists of 20% FBS in DMEM and 5% antibiotic-antimycotic
(GIBCO-BRL). After initial migration, the aortic sections were allowed
to proliferate for another 7 days before they were transferred to a new
culture dish. VSMCs from the second phase of migration were used in our
experiments. To induce differentiation, VSMCs were incubated (for 5 to
6 days) in a serum-free medium, as described elsewhere,22
that contained DMEM with 5 µg/mL transferrin, 200 µmol/L
ascorbate, 1 nmol/L selenium, 10 nmol/L insulin, 2.5 µmol/L
pyruvate, and 1% fungizone.
Chronic Treatment of VSMCs With OxLDL
VSMCs from the first or second passage were used in all of our
experiments. The cells were serum-deprived for 6 days before exposure
to oxLDL.22 In chronic experiments, VSMCs were exposed for
a period of up to 6 days to different concentrations of freshly
prepared oxLDL (0.001 to 0.025 mg cholesterol per
milliliter LDL). The medium containing freshly oxidized LDL was added
daily to the cultured cells. The control cells were also kept in
culture for the same period of time as the treated cells. The
cytotoxicity of different concentrations of oxLDL was assessed by 2
tests: (1) LDH released into the culture medium23 and (2)
ethidium homodimer staining of cell nuclei (LIVE/DEAD EukoLight
Viability/Cytotoxicity Assay Kit, Molecular Probes, Inc).
Immunocytochemistry
VSMC phenotype was identified by using monoclonal
antibodies against smooth muscle -actin, myosin, and caldesmon
(Sigma-Aldrich). For the identification of ryanodine receptors
within VSMCs, quiescent cells were fixed and incubated with monoclonal
anti-ryanodine antibodies (Affinity Bioreagents Inc). These cells were
then incubated with a secondary antibody conjugated to FITC. The
fluorescent images were obtained with a Bio-Rad MRC-600
confocal system connected to a Nikon Diaphot 300
epifluorescence microscope. Background reactivity was checked
in the presence of preimmune sera or in the absence of primary
antibody.
Immunohistochemistry
The aortas from both control and 0.5%
cholesterol–fed (12 to 14 weeks on diet) male New Zealand
White rabbits were cut into 5-mm sections and placed in a mold covered
with O.C.T. embedding compound (Sakura Finetek). These molds were
frozen and cut into 7-µm sections with a Leitz 1720 Cryostat. Before
use, these slides were fixed in a cold 1:1 solution of acetone and
methanol (-20°C) for 15 minutes. The sections were then blocked with
5% skim milk and incubated with monoclonal anti-ryanodine receptor
antibody (1:100) or smooth muscle–specific
monoclonal anti-myosin antibody overnight at 4°C. The aortic sections
were then incubated with anti-mouse IgG–biotinylated whole
antibody (from goat, 1:20), followed by streptavidin conjugated to
Texas Red (1:20, Amersham Life Science Inc). The fluorescent
photographs were registered on Fuji Provia 400 film with an Olympus
BH-2 epifluorescence microscope.
Ca2+ Measurement
Measurement of
[Ca2+]i in a single cell
was carried out by use of a Spex spectrofluorometer connected to a
Nikon epifluorescence microscope, as described in detail
previously.12 Cultured VSMCs were loaded with 2
µmol/L fura 2 for 20 minutes at 22°C in a Krebs-Henseleit buffer
(mmol/L: NaCl 120, NaHCO3 25, KCl 4.8,
KH2PO4 1.2,
MgSO4 1.25, CaCl2 1.8, and
dextrose 8.6). The cells were excited at wavelengths of 340 and 380 nm
with a xenon lamp, and emission was recorded at 505 nm. The maximum
and minimum fluorescence signals were obtained by adding
10 µmol/L 4-bromo-A23187 and 5 mmol/L EGTA, respectively,
at the end of the experiment to calibrate the signal with
[Ca2+]i.24
Statistical Analysis
Data were expressed as mean±SE. The statistical comparisons
were made by 1-way ANOVA, followed by the Student-Newman-Keuls test for
multiple comparison. Differences between means were considered
significant at P<0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Oxidative modification of LDL will result in the formation of multiple bioactive molecules that have been shown to alter cell integrity and viability in vivo and in vitro.19 25 In our experimental protocol, we used an Fe-ADP free radical–generating system to oxidize LDL. The concentration of the free radical–generating system and the time of oxidation were carefully chosen to obtain a minimally modified LDL. It is possible that oxLDL may be cytotoxic.26 27 28 The integrity of smooth muscle cells was assessed by measuring LDH release from cells chronically treated with oxLDL as function of time. Compared with control untreated cells, VSMCs incubated with 0.025 mg/mL oxLDL over a 6-day period did not release a significant amount of LDH into the media (data not shown). Subsequent experiments were undertaken at oxLDL concentrations of
0.025 mg/mL.
VSMCs chronically treated with oxLDL were stimulated with ATP. ATP
binds to purinergic receptors; this binding stimulates phospholipase C
to form inositol 1,4,5-tris-phosphate (IP3),
which stimulates Ca2+ release from the SR via the
IP3 pathway.29 30 Control smooth
muscle cells responded to ATP with an immediate sharp increase in
intracellular Ca2+ levels. However, not all the
cells treated chronically with oxLDL (0.001 to 0.025 mg
cholesterol per milliliter) responded to ATP with an
increase in intracellular Ca2+. The number of
cells that responded with an increase in intracellular
Ca2+ as function of ATP was reduced as the
concentration of oxLDL was raised. As shown in Figure 1
, VSMCs that had been incubated with
0.001 to 0.025 mg cholesterol per milliliter for 6 days
exhibited a depressed capacity to respond to ATP. The majority of cells
incubated with oxLDL at concentrations >0.01 mg/mL were no longer
responsive to ATP. All the control VSMCs (n=12) and cells treated with
0.001 mg/mL for 6 days (n=3) responded to ATP by an increase in
[Ca2+]i. Fifteen of 17
cells responded to ATP when the cells were treated with 0.005 mg/mL
oxLDL. This number was reduced to 9 of 20 cells and 3 of 14 cells when
VSMCs were treated with 0.01 and 0.025 mg/mL oxLDL for 6 days.
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Some cells did respond to ATP with a Ca2+
transient; however, the peak Ca2+ was depressed
(Figure 2). This effect was dependent on
the dose of oxLDL to which the cells were exposed. The results from a
number of cells was tabulated (Table). A
concentration-dependent dampening of the Ca2+
transient induced by ATP was observed for oxLDL-treated cells. With
respect to peak Ca2+ transients, the control
VSMCs showed 
170% increase as the result of stimulation with ATP,
whereas the VSMCs chronically treated with 0.025 mg/mL oxLDL for 6 days
demonstrated a significant attenuation in Ca2+
peak value to 112%. This alteration in peak
Ca2+ level corresponds to
Ca2+ release from SR through ryanodine
receptors.
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To test whether the attenuation in Ca2+ response
was due to factors in addition to ATP, we used endothelin-1 with the
chronically treated cells. Endothelin-1 is a
vasoconstrictive agent that binds to the endothelin-1
receptor on the smooth muscle cells, leading to activation of
phospholipase C, formation of IP3, and release of
intracellular Ca2+. Control smooth muscle cells
demonstrated a rapid increase in
[Ca2+]i as a result of
exposure to endothelin-1 (Figure 3).
However, smooth muscle cells treated chronically with 0.025 mg/mL oxLDL
for 6 days demonstrated no rapid elevation in
[Ca2+]i. Chronically
treated cells responded in a delayed fashion (75 seconds after
treatment) with a very small increase in
[Ca2+]i (Figure 3).
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Because ATP mobilizes Ca2+ from the SR, the
depressed Ca2+ transient in cells chronically
treated with oxLDL may be due to a defect in the capacity of the SR to
regulate intracellular Ca2+. To test the
functional integrity of the SR Ca2+-release
channels, ryanodine was used on control VSMCs and on cells chronically
treated with oxLDL. Exposure of control VSMCs to ryanodine resulted in
a significant rise in
[Ca2+]i (Figure 4). Chronic exposure of VSMCs to 0.025
mg/mL oxLDL for 6 days significantly attenuated the peak
Ca2+ level in response to ryanodine (Figure 4). This resulted in 6-fold decrease in
Ca2+ release from ryanodine receptors as the
result of stimulation with 1 µmol/L ryanodine (Figure 4).
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This defect in SR regulation of intracellular
Ca2+ may be associated with a change in ryanodine
channel density. Ryanodine channels were detected via
immunocytochemical staining. With the use of confocal microscopy,
ryanodine receptors could be visualized in a striking tendril fashion
throughout the cytoplasm in control cells (Figure 5). The majority of cells examined
exhibited this tendrillike staining pattern if the cells were carefully
sectioned in a visual manner with the confocal microscope. This was
achieved because of the narrow plane of focus within the cell.
Conventional fluorescent immunocytochemical analysis
could generate only diffuse staining throughout the VSMCs. After
chronic exposure to 0.025 mg/mL oxLDL, smooth muscle cells showed a
gradual loss in ryanodine receptors by 3 days, until there was no
detectable staining for ryanodine receptors by day 6 (Figure 6).
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Therefore, our data have demonstrated a loss of ryanodine receptors as
a result of chronic treatment with oxLDL in culture. However, there is
no evidence in the literature regarding an alteration in ryanodine
receptor density in the atherosclerotic vessel wall in vivo. To examine
whether changes in ryanodine receptor density were present in
atherosclerotic tissue, aortas were examined from New Zealand White
rabbits fed a 0.5% cholesterol–supplemented diet for 12
to 14 weeks. Sections from control and cholesterol-fed
rabbits were examined with immunohistochemistry for ryanodine receptors
(Figure 7a and 7b). There was a
striking decrease in immunoreactivity for the ryanodine receptor in the
medial region under the plaque compared with control aorta (Figure 7a and 7b). This change in ryanodine receptor staining
intensity was also observed in aortic sections from the area with a
plaque and the area of the vessel that did not contain a plaque. As
shown in Figure 7b, ryanodine receptor density was more intense
in the medial region of these vessels without a plaque, whereas the
same medial layer under the plaque showed a decrease in
fluorescence intensity. This reduction in fluorescence
intensity was not due to a decrease in cell density in the medial
region of these aortic sections. When an adjacent aortic section was
stained for smooth muscle–specific myosin antibody, there was an
intense and uniform staining in medial regions of control and
atherosclerotic vessels (Figure 7c and 7d).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Atherosclerotic vessels are unresponsive to a variety of vasoactive agents.7 8 9 10 Because these agents used many different ion transport and signaling pathways to alter the contractile state of the smooth muscle, this would suggest that a general component critical to the excitation-contraction process is altered by the atherogenic milieu. Several possibilities exist. Our investigation focused on the hypothesis that the SR may become defective during atherosclerosis. Ryanodine-sensitive Ca2+-release channels are critical proteins involved in the regulation of [Ca2+]i in smooth muscle. Alterations in the function or content of these proteins have been suggested to contribute to contractile dysfunction in muscle cells during many different disease processes.31 32 Our data demonstrate that the ryanodine-sensitive channels are depressed in vivo in aortic sections of atherosclerotic plaque in cholesterol-fed rabbits. This depression is particularly evident within and under the plaque area, suggesting that a component associated with the atherogenic environment induces this lesion. OxLDL has been identified as an important atherogenic agent.25 33 Our data in a controlled cell culture environment demonstrate that oxLDL has the ability to mimic the effects that were observed in vivo on ryanodine-sensitive channels. As determined by immunohistochemical analysis, oxLDL reduced the content of ryanodine receptors in cells treated chronically with oxLDL. This conclusion is limited to immunohistochemical staining. We were unable to find an antibody that would allow us to successfully quantify protein content in rabbit VSMCs by Western blot analysis. However, the dramatic decrease in immunoreactivity demonstrated in Figure 5
was so strong that it argues persuasively that the
expression of this protein was decreased as a result of exposure to
oxLDL. The changes in SR channel density have functional implications. The response of the cells to ATP, a known activator of SR Ca2+ release,29 30 was depressed in cells after chronic exposure to oxLDL. This is in striking contrast to the acute effects of ATP in control cells, whereby [Ca2+]i rises immediately.34 35 More direct evidence of the effects of oxLDL on SR Ca2+ release was observed with the use of ryanodine. Ryanodine can induce an increase in [Ca2+]i by opening Ca2+-release channels.36 Cells incubated chronically with oxLDL and then stimulated with ryanodine exhibited an attenuated Ca2+ transient. This is consistent with a lesion in ryanodine channel function and density. This does not exclude the possibility that oxLDL may also affect Ca2+ entry through the L-type Ca2+ channels or Na+-Ca2+ exchange. Such effects have been suggested by studies in other cell types.12 13 16
OxLDL induces changes in contractile protein content and distribution (data not shown). However, the changes in cell regulation of Ca2+ appear to occur at earlier time points after exposure to oxLDL and at lower oxLDL concentrations. Most important, the cells examined in the present study were purposefully selected to exhibit no changes in morphology as detected via light microscope. Therefore, these changes in ryanodine channel expression and function may represent an early structural change that occurs before obvious transformation from a contractile to a synthetic phenotype.
The relevance of our culture conditions to an in vivo atherosclerotic state deserves discussion. Previous studies examining the cellular effects of oxLDL have used short exposure times (seconds to hours) of cells to oxLDL.11 The present cell culture conditions used a relatively long incubation time with lower concentrations of minimally modified oxLDL. This was done to more closely approximate the in vivo situation. Atherosclerosis is a gradual process that occurs over relatively long time periods. If oxLDL is involved in atherosclerosis, it is difficult to argue that its contribution is restricted to only a few seconds or minutes or hours. It is more reasonable to propose that oxLDL is generated in small quantities over relatively long time periods. We have restricted the present study to 6 days, but our results clearly demonstrate that the duration of exposure to oxLDL is an important factor to consider in evaluating its effects. Furthermore, as discussed in Methods, the oxLDL preparation used has biochemical characterizations consistent with limited oxidation. The change in Ca2+ regulation was clearly due to an oxidized component within the LDL, because native LDL was incapable of inducing changes (data not shown). This lack of action by native LDL also demonstrates that the effects on VSMC Ca2+ were not due to an oxidation of the LDL through a release of oxygen-derived free radicals from the cells.
In summary, our data identify important changes in the expression of ryanodine-sensitive Ca2+ channels in the SR during in vivo atherosclerotic conditions. We have identified oxLDL as an important factor in inducing similar changes in vivo. These alterations are associated with important effects on the capacity of the SR to regulate [Ca2+]i in smooth muscle cells. This may explain the loss of vasoreactivity exhibited by atherosclerotic vessels. However, the depressed vasoreactivity may also be partly associated with a change in smooth muscle phenotype. During atherosclerosis, VSMCs change from a cell type abundant in contractile proteins to one of a synthetic state that has lost much of its contractile machinery.37 38 Because we have selected cells that did yet exhibit any such alterations, our data would suggest that these lesions in SR structure and function may represent an early oxLDL-induced modification that has implications for the regulation of blood flow in atherosclerotic vessels.
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Acknowledgments |
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We are grateful to the Medical Research Council of Canada for supporting this study. H. Massaeli was supported by a Studentship from the Heart and Stroke Foundation of Canada. Dr Pierce is a Senior Scientist of the Medical Research Council of Canada.
Received February 5, 1999; accepted August 30, 1999.
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