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(Circulation Research. 1996;79:871-880.)
© 1996 American Heart Association, Inc.

Articles

Mildly Oxidized LDL Evokes a Sustained Ca²⁺-Dependent Retraction of Vascular Smooth Muscle Cells

Nathalie Auge, Guylene Fitoussi, Jean-Loup Bascands, Marie-Therese Pieraggi, Didier Junquero, Philippe Valet, Jean-Pierre Girolami, Robert Salvayre, Anne Negre-Salvayre

the Laboratory of Biochemistry, Metabolic Disease, INSERM CJF-9206 (N.A., G.F., M.-T.P., R.S., A.N.-S.), Pharmacologie moleculaire et physiopathologie renale, INSERM U388 (J.-L.B., J.-P.G.), and Regulations adrenergiques et adaptation metabolique, INSERM U 317 (P.V.), Institut Louis Bugnard, INSERM/University Paul Sabatier, Toulouse, and Centre de Recherche Pierre Fabre, Maladies cardio-vasculaire I, Castres (D.J.), France.

Correspondence to Dr A. Negre-Salvayre or Prof R. Salvayre, Laboratoire de Biochimie "Maladies Metaboliques," CHU Rangueil, 1 Avenue J. Poulhes, 31054 Toulouse Cedex, France.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca²⁺]_i) in cultured arterial smooth muscle cells. Concomitant with the [Ca²⁺]_i rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca²⁺]_i rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12±2 hours) and sustained (14±2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (200 µg apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant -tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.

Key Words: oxidized LDL • calcium • smooth muscle cells • vascular retraction • atherosclerosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
LDL delivers cholesterol to peripheral cells and is also involved in atherogenesis.^{1 2} Ox-LDL, which has been detected in atherosclerotic lesions,^{3 4} is thought to play an important role in atherogenesis because several of its properties are potentially involved in the genesis of lesions: chemoattraction of monocytes in the subendothelial space, deviation of LDL metabolism toward macrophage and foam cell formation, cytotoxicity, induction of growth factor expression, and changes in the anticoagulant properties of the EC surface.^{5 6 7} These in vitro findings (on cultured cells used as experimental model systems) are relevant to the in vivo histopathological changes in the arterial wall that are observed during atherogenesis and that finally lead to the formation of the atherosclerotic plaque and the "triggering" of thrombotic events.^{6 8} In addition to structural changes in atherosclerotic arteries,⁹ arterial hyperresponsiveness to different vasoconstrictors has been demonstrated in hypercholesterolemic animals^{10 11 12} ; these alterations in vascular response are potentially involved in vascular spasm.^{13 14}

We recently reported that mildly Ox-LDL but not native LDL was able to elicit a delayed, intense, and sustained rise in [Ca²⁺]_i in cultured lymphoid cell lines and ECs.^{15 16} Because an increase in [Ca²⁺]_i is a key event in the contraction of smooth muscle, it was suggested that Ox-LDL might be able to induce retraction of vascular SMCs.

The data show that mildly Ox-LDL also evoked a rise in [Ca²⁺]_i in cultured arterial SMCs and subsequent intense, sustained, cellular retraction. This cell retraction was prevented by agents that block increases in [Ca²⁺]_i and by ML-9, an inhibitor of MLCK. These data suggest that Ox-LDL may be considered an additional factor involved in the alterations of vascular response in atherosclerotic arteries and that this pathogenic cell response can be prevented (at least in vitro) by protective agents that block the [Ca²⁺]_i peak evoked by Ox-LDL.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemicals
Fura 2-AM was purchased from Molecular Probes; ML-9 from Tebu; polyhema, -tocopherol, bovine serum albumin (fatty acid free), MTT, prazosin, EDTA, and EGTA from Sigma Chemical Co; ionomycin from Calbiochem; RX-821002 from Reckitt and Colman; and RPMI 1640 with L-alanyl-L-glutamine, FCS, trypsin-EDTA, penicillin, and streptomycin from GIBCO. Other reagents and chemicals were obtained from Merck or Prolabo. Nisoldipine was a generous gift from Bayer AG.

Culture of Bovine Aortic SMCs and ECs
Bovine aortic SMCs (AG 08133A) were obtained from the National Institute on Ageing cell repository (Camden, NJ) and used between passages 10 and 20. The cells were sparsely seeded in Petri dishes coated with polyhema (which decreases cell adhesion to the plastic and permits maximal cell retraction¹⁷ ) and grown in RPMI 1640 medium containing 10% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL). Twenty-four hours before LDL incorporation, the medium was removed and replaced with serum-free RPMI 1640 medium. Under the culture conditions for this study, these SMCs exhibited some classic features of the "contractile state," such as a relatively high level of -actin (compared with that of ß-actin) and desmin.^{18 19 20}

Human umbilical vein ECs (CRL-1998) were obtained from the American Type Culture Collection and routinely grown in RPMI 1640 containing 10% FCS (control conditions) or grown in RPMI 1640 without any additives for LDL oxidation experiments.

LDL Isolation and Oxidation
LDL was isolated from pooled, fresh sera by sequential ultracentrifugation on a vertical rotor,²¹ dialyzed against 150 mmol/L NaCl containing 0.3 mmol/L EDTA, and sterilized by filtration (0.2-µm Millipore membrane); the purity of the LDL preparation was assured by electrophoresis on agarose gel. Native LDL (stock solution, 6 mg apoB per milliliter) was stored at 4°C under N₂ for no longer than 2 weeks. For each experiment, 2 mg apoB per milliliter LDL (ie, stock solution diluted three times in 150 mmol/L NaCl) was exposed to UVC radiation (254 nm, 0.5 m-W/cm² for 2 hours under control conditions or for various times when indicated) with or without 2 µmol/L CuSO₄ as previously described²² to obtain mildly Ox-LDL (ie, Ox-LDL with moderate TBARS levels [4 to 5 nmol TBARS per milligram apoB]). Ox-LDL was immediately incorporated (at concentrations indicated in the text) in the culture medium (because the final concentration was generally 150 µg apoB per milliliter, the final dilution factor was 36).

Alternatively (when indicated in the text), mildly Ox-LDL was obtained by incubating LDL (200 µg apoB per milliliter for 18 hours) with CRL-1998 subconfluent ECs. Cells (200 000 per well) were seeded in six-multiwell culture dishes (Nunc), grown for 16 hours in RPMI 1640 supplemented with 10% FCS, then grown in serum-free RPMI 1640 for 48 hours, and finally incubated for 18 hours with 200 µg apoB per milliliter in RPMI 1640. Under these conditions cell-derived Ox-LDL contained 3.5±0.5 nmol TBARS per milligram apoB.

Lipid peroxidation was evaluated by the TBARS assay according the fluorimetric procedures of Yagi.²³ Protein concentrations were determined with the procedure of Lowry et al.²⁴

Cytotoxicity of Ox-LDL
Cytotoxicity was determined under previously reported conditions,^{15 25} with slight modification. LDL (Ox or native) was added to the culture medium at the concentrations indicated in the text, and cell viability was determined during and after the 24-hour pulse by leakage of cellular LDH (LDH Roche assay kit, MA kit 10) or with the MTT test according to Denizot and Lang,²⁶ which measures the ability of cells to reduce a soluble, yellow, tetrazolium salt to an insoluble purple formazan precipitate.

Determination of [Ca²⁺]_i
The [Ca²⁺]_i was determined in SMC populations by using fura 2-AM, which is hydrolyzed by intracellular carboxylesterases to liberate fura 2, a trappable, intracellular, fluorescent calcium indicator. In brief, at the required time (hourly during the observation), one culture dish of SMCs (grown on glass coverslips) was used for each determination (all cultures were treated under the same conditions). Cells were incubated in RPMI medium buffered with 10 mmol/L HEPES containing 0.5% BSA and 2 µmol/L fura 2-AM for 45 minutes. After the cultures were washed, [Ca²⁺]_i determinations were performed at dual excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm and calculated by the ratio method of Grynkiewicz et al²⁷ by use of a spectrofluorometer (model JY3C, Jobin-Yvon) and a Spex Fluorilog spectrofluorometer set for the dual wavelength excitations, as previously described.^{22 25 28} Emission at 520 nm was collected by a photomultiplier and passed to a Spex system microcomputer that averaged the emitted light collected for 0.5 second at each excitation wavelength. Autofluorescence values from unloaded cells were subtracted from the fura 2–loaded cell fluorescence values at each excitation wavelength before the fluorescence ratio 340:380 was calculated.²⁸ As mentioned previously, [Ca²⁺]_i was calculated from the equation of Grynkiewicz et al²⁷ : [Ca²⁺]_i=K_dx[(R-R_min)/(R_max-R)]xS, where K_d (224 nmol/L) is the dissociation constant of the fura 2–Ca²⁺ complex and the parameters (which depend on the optical system used) were R_min=0.9, R_max=16, and S=4. Moreover, calibration and compartment studies of fura 2 in situ have been performed with 10 µmol/L ionomycin with 0.5 mmol/L calcium or 1 mmol/L MnCl₂, followed by cell permeabilization with 10 or 100 µmol/L digitonin, respectively, according to published methods.^{29 30}

Determination of Ca²⁺ Pump ATPase Activity
Cultured SMCs were harvested by careful trypsinization and immediately afterward were washed twice in RPMI 1640 and suspended in the buffer used for determination of Ca²⁺ pump ATPase activity according to the method of Wu et al.³¹ Cellular ATP concentrations were determined with an ATP kit (Calbiochem) and an LKB luminometer (model 1250 M). The results were expressed as ATP/ATP₀, where ATP and ATP₀ are the ATP concentrations determined at the indicated time and at t=0, respectively.

Cell Surface Determination
SMCs were seeded in Petri dishes coated with polyhema. Then the cells were incubated under the culture conditions described above, with or without native or Ox-LDL (150 µg apoB per milliliter under control conditions or variable concentrations, indicated in the text, for dose-response examination) or with the contractile agonists Ang II or Epi (used as a positive control). Cells were observed repeatedly at 37°C with a camera-equipped, inverted microscope (Nikon TMS microscope equipped with a thermostatted stage with a Sony CCD video camera), and the surfaces of isolated cells (ie, cross-sectional area of each cell in the plane of observation) were evaluated with an image analysis system (Biostat, AES Image Co) under conditions recently described in detail³¹ and expressed as surface units or percent of the value at t=0.

For studying the reversibility of cell retraction induced by mildly Ox-LDL, cells were pulsed with Ox-LDL for 16 hours, then washed once with RPMI 1640, and grown in complete medium (containing 20% FCS) until the end of the experiment.

Tension Measurements on Rat or Rabbit Aortic Rings
Male New Zealand White rabbits (2.0 to 2.2 kg; ESD, Chatillon, France) were anesthetized with sodium pentobarbital (80 mg/kg IV), and thoracic aortas were isolated and placed in cold Krebs-Henseleit buffer ([in mmol/L] 118 NaCl, 4.7 KCl, 1.2 Na₂PO₄, 1.2 MgSO₄, 2 CaCl₂, 25 NaHCO₃, and 10 glucose, bubbled with 95% O₂–5% CO₂). Blood vessels were cleaned of fat and cut into rings (4 mm long) according to Furchgott and Zawadzki³² and as previously described.³³ When indicated, the endothelium was mechanically removed by inserting the tip of a forceps into the lumen and gently rolling the ring back and forth on a moistened filter paper. Rings were suspended horizontally between two stainless steel stirrups in organ chambers containing Krebs-Henseleit solution at 37°C and bubbled with 95% O₂–5% CO₂. One stirrup was anchored to a steel plate, whereas the other was connected to a force transducer (UC2, Gould Electronics), and isometric tension was recorded (13-G4615-30 signal amplifier coupled to a TA-11 printer, Gould Electronics). The rings were stretched in a stepwise manner to the optimal point of their length–active tension relation and then allowed to equilibrate for another 45 minutes. Removal of the endothelium was confirmed functionally by the lack of ACh (10^-6 mol/L)-induced relaxation of contracted rings. The contractile response of rings to classic agonists (60 mmol/L KCl or 100 nmol/L NE, used as positive controls) was tested before and after exposure to Ox-LDL. The effects of native or Ox-LDL on isometric tension change were examined by changing the medium every 12 hours and replacing it with fresh medium containing the same compounds. Four additional sets of experiments were performed under similar conditions with aortic rings from Wistar rats.

Electron Microscopy
SMCs were fixed with 2.5% glutaraldehyde in 0.3 mol/L cacodylate-HCl buffer, pH 7.4, dehydrated with ethanol and isoamyl acetate, coated with gold, and then examined by scanning electron microscopy in a Jeol 25 microscope as previously used.¹⁵

Data Analysis
Results are expressed as mean±SEM and statistical evaluation was performed by unpaired Student's t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rise of [Ca²⁺]_i Evoked by Ox-LDL in Cultured Arterial SMCs
The basal level of [Ca²⁺]_i was 80±20 nmol/L in aortic SMC populations grown under control conditions (ie, without lipoproteins). In cells pulsed with mildly Ox-LDL (150 µg apoB per milliliter containing 5.4±0.4 nmol TBARS per milligram apoB), the basal level of [Ca²⁺]_i was 100±20 nmol/L. At 10 hours, the [Ca²⁺]_i began to rise slowly, leading to a sustained and intense rise in [Ca²⁺]_i (Fig 1A), with the maximum (2100±280 nmol/L) occurring at 16±1.5 hours. Then the [Ca²⁺]_i slowly decreased to basal levels after 3 to 4 hours. In contrast, in cells pulsed with native LDL, we observed no significant peak at the time of the intense [Ca²⁺]_i peak (16 hours). It is noteworthy that this delayed and intense [Ca²⁺]_i peak was different from the moderate and sharp [Ca²⁺]_i peaks that appeared during the first minutes after contact with LDL, culminating at 390±40 nmol/L (data not shown) as previously reported.^{34 35 36} At no time during the [Ca²⁺]_i peak did we detect any cell loss (by MTT test) or any impairment of cell membrane permeability (as evaluated by LDH release; Fig 1B). This strongly suggests that the [Ca²⁺]_i peak evoked by Ox-LDL did not result from nonspecific alterations in plasma membrane permeability, as previously shown in and discussed about lymphoid cells and ECs.^{15 16} Moreover, to investigate the potential for compartmentalization of the probe, we performed experiments in which intracellular fura 2 fluorescent dye (in cells treated with Ox-LDL for 15 hours, ie, at the time of [Ca²⁺]_i peak) was quenched with 1 mmol/L extracellular MnCl₂ in the presence of 10 µmol/L ionomycin (under conditions described in References 29 and 30). As shown in Fig 1C, most of the calcium–fura 2 complex was quenched by Mn²⁺ and was therefore cytosolic (with the assumption that ionomycin is located mainly in the plasma membrane and that Mn²⁺ is introduced into the cytosolic compartment). Moreover, permeabilization of different intracellular compartments by increases in the concentration of digitonin²⁹ showed that most of the fura 2 probe was released by 10 µmol/L digitonin, whereas further and more extensive permeabilization (by 100 µmol/L digitonin) released only low amounts of the probe (Fig 1D). These data suggest that most of the probe is cytosolic, but we cannot rule out the possibility that part of the signal is coming from subcellular compartments.

View larger version (20K): [in this window] [in a new window]   Figure 1. Comparative time course of [Ca2+]i (A) and cytotoxicity indices (B), ie, MTT test (circles) and released LDH (squares) in SMC populations continuously pulsed with a fixed concentration of native (open symbols) or oxidized (filled symbols) LDL (150 µg apoB/mL of UV+copper–Ox-LDL containing 5.1±0.4 nmol TBARS/mg apoB). Cells were grown in RPMI 1640. At t=0, LDL (oxidized or native) was added to the medium and [Ca2+]i determined at the indicated time by using the fluorescent calcium probe fura 2-AM, as indicated in "Materials and Methods" (excitation, 340 and 380 nm; emission, 510 nm; ratio mode calculation). Mean±SEM of four experiments. C, Quenching of intracellular fura 2 fluorescent dye in cells treated with Ox-LDL for 15 hours, ie, at the time of the [Ca2+]i peak with 1 mmol/L extracellular MnCl2 and 10 µmol/L ionomycin under conditions described in References 29 and 30 (continuous monitoring with a Spex Fluorilog spectrofluorometer). D, Selective permeabilization of intracellular compartments by increasing concentrations of digitonin according to Reference 29. Fluorescence of cells treated with Ox-LDL for 15 hours, ie, at the time of [Ca2+]i peak and labeled with 2 µmol/L fura 2, was continuously monitored (Spex Fluorilog spectrofluorometer). Digitonin (10 and 100 µmol/L) was added when indicated by arrows.

Furthermore, the basal level of cellular ATP (Fig 2A) and the activity of the ATP-dependent calcium pumps were not significantly altered up to the end of the [Ca²⁺]_i peak (Fig 2B). It is noteworthy that ATP consumption was only slightly altered (decreased by 12%) by adding to the cell preparation 100 nmol/L thapsigargin, a cell-permeable, specific inhibitor of endoplasmic reticular calcium pumps.³⁷ This suggests that the method used in the present study mainly evaluates the activity of the plasma membrane ATP-dependent calcium pump. These data strongly suggest that the [Ca²⁺]_i rise is not due to a defect in the calcium pumps and may explain the decay portion of the [Ca²⁺]_i peak (which probably results from translocation of calcium from the cytosolic compartment to outside the cell or intracellular stores).

View larger version (16K): [in this window] [in a new window]   Figure 2. ATP levels (A) and calcium pump ATPase activities (B) in SMCs pulsed with 150 µg apoB/mL of native (open symbols) or oxidized (containing 4.7±0.3 nmol TBARS/mg apoB of UV+copper–Ox-LDL; filled symbols). A, At the indicated times cells were washed and harvested and cellular ATP level was immediately determined (cellular ATP level at t=0 was 2.1±0.2 nmol ATP/106 cells). B, Calcium pump ATPase activities were determined with (circles) or without (squares) extracellular calcium (0.1 mmol/L) at the indicated times (0, 9, 16, and 22 hours). The value at t=22 hours was evaluated with (100 nmol/L; thaps, dotted line) or without (ox 22) thapsigargin. Mean±SEM of three experiments.

Reduction in Size (Cell Retraction) of Vascular SMCs Evoked by Ox-LDL
SMCs treated by Ox-LDL exhibited reductions in size (ie, in cross-sectional areas of the cell in the plane of observation and referred to as cell retraction), as shown by phase-contrast microscopy (Fig 3) and scanning electron microscopy (Fig 4). The cell retraction induced by Ox-LDL was time dependent, as shown by time course experiments (Fig 5) in which changes in the shape of individual cells were monitored continuously. LDL oxidized by UVC alone (with 3.3±0.2 nmol TBARS per milligram apoB) induced less cell retraction than did LDL oxidized by UVC and copper (LDL with 4.6±0.2 nmol TBARS per milligram apoB; Fig 5A). In contrast, cells grown with native LDL did not exhibit any significant reduction in cross-sectional area (Fig 5A and 5B). Cell retraction elicited by Ox-LDL (150 µg apoB per milliliter) began simultaneously with the rise in [Ca²⁺]_i (evaluated by the fura 2 ratiometric method on the whole cell population), reached maximal retraction (60±10%) after 16 hours, and was sustained for several hours thereafter (Fig 5B). Cell retraction that began at 12 hours was maximal 4 hours later under the conditions used in the legend to Fig 5B. Follow-up of each cell showed variability in their response to Ox-LDL: most cells exhibited an intense retraction, but some were relatively less responsive (Fig 5A and 6).

View larger version (184K): [in this window] [in a new window]   Figure 3. Phase-contrast photomicrographs of SMCs grown on polyhema. A and B, Control cells; C-F, cells pulsed for 14 (C and D) and 24 (E and F) hours with Ox-LDL (150 µg apoB/mL LDL, oxidized by UV+copper and containing 4.6±0.2 nmol TBARS/mg). Bar=50 µm.

View larger version (45K): [in this window] [in a new window]   Figure 4. Scanning electron microscopy of SMCs grown in RPMI 1640 supplemented or not with 150 µg Ox-LDL (same batch as in Fig 3, ie, LDL oxidized by UV+copper and containing 4.6±0.2 nmol TBARS/mg). A, Controls; B-D, cells cultured continuously with 150 µg apoB/mL Ox-LDL (for 14, 18, and 24 hours, respectively). Bars=10 µm.

View larger version (46K): [in this window] [in a new window]   Figure 5. Time course of retraction in individual cells evoked by Ox-LDL. Cells were grown on Petri dishes coated with polyhema in RPMI 1640 medium containing 150 µg apoB/mL of native LDL (open circles) or LDL oxidized by UV+copper (same batch as in Fig 3, ie, Ox-LDL containing 4.9±0.3 nmol TBARS/mg; filled circles) or LDL oxidized by UV only (Ox-LDL containing 3.3±0.2 nmol TBARS/mg apoB; open squares). The surface of each cell was continuously monitored between 10 and 20 hours by using a microscope equipped with a thermostatted stage as indicated in "Materials and Methods." A, Time course of cross-sectional area of individual cells pulsed with native LDL (open symbols) or LDL oxidized by UV (open squares) or by UV+copper (filled circles). Mean cell surface at t=0 (100%) was 1180±120. B, Relative time course of mean±SEM of cross-sectional areas (circles) determined on 20 cells (mean cross-sectional surface at t=0, ie, 100%, was 1220±110) and of [Ca2+]i (determined on the cell population with fura 2-AM; triangles, broken line) of cells pulsed with 150 µg apoB/mL native LDL (open circles) or UV+copper–Ox-LDL (filled circles) (same batch as in Fig 5A).

View larger version (20K): [in this window] [in a new window]   Figure 6. Dose dependence of cell retraction. SMCs were pulsed with increasing concentrations of UV+copper–Ox-LDL (containing 4.3±0.3 nmol TBARS/mg, oxidized under standard conditions), and cross-sectional area (each point represent the area of one cell) was determined after a 16-hour pulse. Horizontal bars indicate mean values.

The cell retraction induced by Ox-LDL was also dependent on their concentration in the medium (Fig 6). Under conditions described in the legend to Fig 6, the maximal cell retraction observed at 16 hours was obtained with the higher dose of Ox-LDL (ie, 300 µg apoB per milliliter). Cell surface area was reduced by 39±8% with 100 µg apoB per milliliter Ox-LDL (P<.01) and by 63±7% with 200 µg apoB per milliliter Ox-LDL (P<.001).

We observed that cells were responsive to classic contractile agonists, but with different time courses. Ang II (100 nmol/L) and Epi (100 nmol/L) evoked a rapid and sharp [Ca²⁺]_i peak, with a maximum at 950±70 and 650±60 nmol/L, respectively, as determined by the fura 2 ratiometric method. The shortening of SMCs induced by Ang II or Epi was concomitant with the [Ca²⁺]_i rise (determined with fura 2-AM) and reversible at the end of the [Ca²⁺]_i peak (data not shown).

Reversibility of cell retraction was examined under various culture conditions (Fig 7). Retraction of cells that were pulsed continuously with high concentrations (>200 µg apoB per milliliter) of Ox-LDL was not reversible, and the cells became progressively detached and died as shown by the release of LDH into the medium (Fig 7). With lower doses (50 or 100 µg apoB per milliliter Ox-LDL), cell retraction was reversible when Ox-LDL was removed from the culture medium after 16 hours (ie, at the beginning of cell retraction) and in cells grown for the following "chase" period in complete, fresh culture medium containing 20% FCS. As shown in Fig 7, retraction was persistent after the decline in the [Ca²⁺]_i peak, and complete cell relaxation occurred 20±3 hours after cell retraction began, ie, 10 hours after the end of the [Ca²⁺]_i peak. When cells were pulsed with intermediate doses (150 µg apoB per milliliter) of Ox-LDL for 16 hours and chased in complete, fresh culture medium, retraction of most of the cells was reversible, but a significant number remained retracted and later became detached (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 7. Test of reversibility of SMC retraction. SMCs were pulsed for 16 hours with 100 or 200 µg apoB/mL (circles and squares, respectively) of UV+copper Ox-LDL (containing 4.9±0.2 nmol TBARS/mg) and the medium was then discarded and replaced with fresh, complete (20% FCS) culture medium. Mean±SEM of cross-sectional areas of 20 cells was monitored during the experiment and expressed as a percentage of the cell surface at t=0 (100%=1250±150). With 200 µg apoB/mL, some contracted cells became progressively detached and died (at the time indicated by the arrow). Concomitant time course of [Ca2+]i (determined on the cell population with fura 2-AM) of cells pulsed with UV+copper–Ox-LDL (broken line). Inset reports the activities of LDH released into the medium at t=16 (black bars) and 36 (striped bars) hours, expressed as a percent of the level in controls at the same time.

All the cells used in these experiments were responsive to Epi (100 nmol/L) before exposure to Ox-LDL. In contrast, after exposure to Ox-LDL, the contractile response to the same agonist was only effective under the reversible conditions shown in Fig 7 (ie, with 50 or 100 µg apoB per milliliter Ox-LDL in pulse-chase experiments). Under toxic conditions (continuous pulse with 200 µg apoB per milliliter Ox-LDL), we observed neither reversibility of cell retraction nor a response to agonists after exposure to Ox-LDL. These data suggest that the sustained cell retraction elicited by Ox-LDL is reversible under nontoxic conditions (relatively low concentration of Ox-LDL) but irreversible with higher doses, which lead to cell detachment and death.

To investigate the specificity of the cell retraction induced by Ox-LDL in vascular SMCs, we examined the morphology of two types of nonmuscle cell (fibroblasts and ECs) treated with Ox-LDL. With relatively high concentrations (200 µg apoB per milliliter), both cell types exhibited a sustained and irreversible retraction, followed by cell detachment and death. At low, nontoxic doses, we observed no significant cell retraction during the 24-hour observation period.

Relation Between LDL Oxidation Level and SMC Retraction
A large range of mildly Ox-LDL containing increasing levels of TBARS was obtained by increasing the exposure time to UV. As shown in Fig 8, cell retraction was highly correlated with irradiation times of 1 hour (P<.01) or longer (P<.001). Cell retraction was well correlated with LDL oxidation levels (TBARS levels), as shown in the inset to Fig 8 (r=.93). In the same way, when LDL oxidation was inhibited by antioxidants (oxidation of 2 mg apoB/mL with 100 µmol/L -tocopherol or 20 µmol/L probucol), the ability of the LDL to induce retraction was inhibited by a similar proportion to oxidation (Fig 9). The effect of antioxidants did not result from a direct inhibitory effect on SMCs, since LDL preparations containing antioxidants were diluted in the culture medium by a factor of 13. At this final concentration (7.5 µmol/L -tocopherol or 1.5 µmol/L probucol), the tested antioxidants did not exhibit any inhibition of retraction induced by Ox-LDL, therefore excluding a direct effect at the cellular level.

View larger version (26K): [in this window] [in a new window]   Figure 8. Influence of the level of LDL oxidation by UV+copper (A) or ECs (B) on contraction of vascular SMCs. A, Contraction induced by increasingly Ox-LDL, obtained by exposure of LDL to UV+copper under conditions indicated in "Materials and Methods" for various times (0-5 hours). SMCs were pulsed with a fixed concentration of Ox-LDL (150 µg apoB/mL) for 16 hours, and the cross-sectional area of cells (40 for each concentration) was determined. Horizontal bars indicate mean values. Inset shows the relation between mean levels of LDL oxidation (modulated by increasing UV irradiation time and expressed as nmol TBARS/mg apoB) and means of related cell surface areas. B, Effect of native LDL (0) and LDL oxidized for 18 hours (18) by ECs (containing 3.5±0.2 nmol TBARS/mg apoB).

View larger version (38K): [in this window] [in a new window]   Figure 9. Inhibition of LDL oxidation by antioxidants and of the subsequent cell contraction (indirect inhibitory effect). LDL was supplemented or not with antioxidants -tocopherol (T, 100 µmol/L) or probucol (P, 20 µmol/L), subjected to the oxidizing effect of UV+copper under standard conditions, and added to the medium for testing their contractile effect. Cell contraction (A) was determined on 40 individual cells (after a 16-hour pulse with 150 µg apoB/mL), and LDL oxidation was evaluated by TBARS levels (B). Mean±SEM of three experiments. **P<.05, ***P<.01.

Because LDL oxidation can be mediated by cultured vascular cells,^{6 7 8} we tested the effect of LDL oxidized by human ECs. As shown in Fig 8B, EC–Ox-LDL evoked retraction in SMCs that was similar to that of LDL oxidized by UV and copper (relative to the TBARS content). These data suggest that cell retraction is independent of the mechanism of LDL oxidation, the same phenomenon being observed with UV-, UV and copper–, and cell-mediated oxidation and is rather related to the level of LDL oxidation.

Effect of Ox-LDL on Tension in Vascular Rings
To examine the potential pathophysiological relevance of retraction evoked by Ox-LDL in cultured vascular SMCs, we examined the effect of Ox-LDL on vascular tone. Testing of ring function by contractile agonists (NE and KCl) showed that the rings could be used (contractile response) for as long as 30 hours, after which time the contractile response was no longer reliable. When rings with intact endothelium were used (responsive to NE-induced contraction and ACh-induced relaxation), we observed an early and slight increase in ring tension similar to that observed by Sachinidis et al,³⁴ but no later tension change was observed with either 150 µg apoB/mL of oxidized or native LDL. When deendothelialized rings (responsive to NE-induced contraction [Fig 10A] but unresponsive to ACh-induced relaxation) were used, Ox-LDL (150 µg apoB per milliliter) evoked a delayed (lag period of 12 hours) and sustained increase in vascular tone that was spontaneously reversible 15±2 hours later (ie, spontaneous relaxation occurred at 27±2 hours; Fig 10B and 10C). The contractile response was dose dependent above a threshold of 50 µg apoB per milliliter of mildly Ox-LDL and lower concentrations induced no significant rise in tension, whereas 100 µg apoB per milliliter and higher doses induced progressively increasing responses. Maximal response (3 g) was obtained with 150 µg apoB per milliliter. Under these conditions the contractile response induced by mildly Ox-LDL was reversible, and the contractile response of rings induced by 60 mmol/L KCl or 100 nmol/L NE was preserved after exposure to Ox-LDL. With higher doses of Ox-LDL (300 µg apoB per milliliter), we observed no reversibility in the contractile response evoked by Ox-LDL. The sustained contractile response induced by 150 µg apoB per milliliter Ox-LDL was not inhibited by a combination of -adrenergic blockers (10 µmol/L of the ₁-antagonist prazosin and 10 µmol/L of the ₂-antagonist RX821002, Fig 10D), thus suggesting that the increase in tension caused by Ox-LDL was not mediated via -adrenergic stimulation (consistent with the data in cultured SMCs). Under similar conditions native LDL did not induce any sustained rise in ring tension (Fig 10E). Similar results were obtained with rat aortic rings. All these data suggest that Ox-LDL can evoke a sustained contractile response in aortic rings similar to that observed in isolated, cultured, vascular SMCs.

View larger version (18K): [in this window] [in a new window]   Figure 10. Representative tracing of isometric tension of deendothelialized rabbit aortic rings without LDL (A), with UV+copper–Ox-LDL containing 4.5±0.3 nmol TBARS/mg) (B-D), or with 150 µg apoB/mL native LDL (E). The delayed, sustained, and reversible increase in isometric tension contraction of the rings induced by Ox-LDL was not inhibited by a combination of two -adrenergic antagonists (10 µmol/L of the 1-antagonist prazosin and 10 µmol/L of the 2-antagonist RX821002; D). NE indicates contraction induced by 100 nmol/L NE (deendothelialization of rings was tested by ACh, which induces relaxation in rings with intact endothelium). Each experiment was done at least in triplicate and gave similar results (typical curves from each experiment). Number of experiments: controls (no LDL), n=8; native LDL, n=4; Ox-LDL, n=8; Ox-LDL with -blockers, n=3.

Effect of EGTA, Nisoldipine, and ML-9 on [Ca²⁺]_i Rise and Cell Retraction Induced by Mildly Ox-LDL
To better understand the mechanism of cell retraction evoked by Ox-LDL and to attempt to inhibit its potential pathogenic effect, we investigated the effects of various potential inhibitors. As expected, ML-9, a relatively selective inhibitor of MLCK,³⁸ effectively inhibited SMC retraction induced by Ox-LDL (P<.01) despite a lack of inhibition of the [Ca²⁺]_i peak (Fig 11). Because the SMC response is calcium dependent³⁹ and the sustained [Ca²⁺]_i rise elicited by Ox-LDL is largely dependent on calcium influx,¹⁶ we tried to prevent cell retraction by inhibiting the rise in [Ca²⁺]_i. The calcium chelator EGTA (0.5 mmol/L added at t=8 hours)^{16 22} and the dihydropyridine calcium channel blocker nisoldipine (10 µmol/L)²⁵ inhibited the sustained and intense [Ca²⁺]_i peak (P<.01), presumably by blocking [Ca²⁺]_i influx, and subsequently inhibited cell retraction induced by Ox-LDL (P<.01). Because -tocopherol exhibits a "direct" cytoprotective effect by blocking the sustained [Ca²⁺]_i rise elicited by Ox-LDL,⁴⁰ we tested its possible inhibitory effect on SMC retraction induced by Ox-LDL. As shown in Fig 11, 100 µmol/L -tocopherol also blocked the [Ca²⁺]_i peak elicited by Ox-LDL in SMCs and the subsequent cell retraction (P<.01).

View larger version (32K): [in this window] [in a new window]   Figure 11. Test of inhibition of cell retraction (direct inhibition at the cellular level) and of the [Ca2+]i rise by the calcium chelator EGTA (E, 0.5 mmol/L), the dihydropyridine calcium channel blocker nisoldipine (N, 10 µmol/L), the MLCK inhibitor ML-9 (M, 10 µmol/L), and the antioxidant -tocopherol (T, 100 µmol/L). The tested agents and 150 µg apoB/mL of UV+copper–Ox-LDL (containing 4.7±0.2 nmol TBARS/mg) were added simultaneously to the SMC medium at t=0. At t=18 hours the [Ca2+]i was determined on the cell population by using fura 2-AM, and cell retraction was evaluated as previously described. Mean±SEM of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
LDL oxidation can be induced by cultured cells from the arterial wall (see reviews in References 5 through 8), and Ox-LDL is present in vivo in atherosclerotic lesions,^{3 4} where it is thought to play a major role in atherogenesis. In addition to the wide variety of biologic properties of Ox-LDL,⁸ we report a novel property of mildly Ox-LDL, its apparent ability to evoke a sustained and intense contraction of cultured aortic SMCs and arterial rings, which is potentially involved in the pathophysiology of atherosclerotic arteries.

The [Ca²⁺]_i peak evoked by mildly Ox-LDL in cultured aortic SMCs is consistent with that observed in lymphoid cells and ECs.^{15 16} Such a [Ca²⁺]_i peak is distinctive by its time course and intensity and is different from the usually rapid response to Ca²⁺-mobilizing agonists⁴¹ and from the early, relatively moderate, and transient [Ca²⁺]_i rise induced by LDL.^{34 35 36} In ECs a similar [Ca²⁺]_i peak was also observed with nontoxic doses of mildly Ox-LDL¹⁵ but not with native LDL. The mechanism whereby the [Ca²⁺]_i peak is induced by mildly Ox-LDL is largely unknown. It is not due to a loss of plasma membrane integrity (as shown by the lack of LDH release or ⁵¹Cr leakage¹⁵ ). It does not result from cellular oxidative stress, because at the time of the [Ca²⁺]_i peak, we detected neither ATP depletion nor any defect in calcium pump activity. This contrasts with the effect of redox cycling chemicals, which induce depletion of cellular glutathione and ATP and a subsequent inhibition of the calcium pumps.⁴² The [Ca²⁺]_i rise seems to result from as-yet-unknown nonclassic pathogenic intracellular signaling that is triggered by lipid peroxidation derivatives contained in mildly Ox-LDL.⁴³ The hypothetical steps, which may help to explain the very long lag period of the [Ca²⁺]_i peak, can be summarized as follows: Mildly Ox-LDL must be taken up by cells, internalized, and degraded in the lysosomal compartment and thereby liberate lipid peroxidation derivatives, which attack their cytoplasmic targets (these "metabolic" steps seem to explain the first 3 to 4 hours of the lag period) and trigger a cascade of pathogenic intracellular signals (detected during the 6 hours preceding the sustained calcium influx). The influx of extracellular Ca²⁺ was suggested by inhibition of the [Ca²⁺]_i peak by EGTA, which depletes extracellular free Ca²⁺, and by dihydropyridine calcium channel blockers, which block Ca²⁺ influx (Reference 25 and this article). This has recently been confirmed by determining the ⁴⁵Ca uptake.⁴⁴ Experiments with manganese/ionomycin and digitonin argue that much of the Ca²⁺ is not compartmentalized, but we cannot exclude the possibility that some of the signal is coming from subcellular compartments.

The sustained and intense (tonic) retraction of SMCs is clearly calcium dependent, because it was inhibited by agents that block the [Ca²⁺]_i peak. This retraction is probably mediated through activation of the calcium/calmodulin-dependent enzyme MLCK,^{39 45} since cell retraction was inhibited by ML-9, a relatively specific inhibitor of MLCK³⁸ that does not inhibit the [Ca²⁺]_i peak. Because cell retraction persisted largely after the decline in the [Ca²⁺]_i peak, it is not inconceivable that an additional and calcium-independent mechanism could be involved in the maintenance of cell retraction (reviewed in Reference 46). Retraction induced by nontoxic doses of mildly Ox-LDL was reversible, and vascular response to the contractile agonists NE and KCl was preserved after exposure to Ox-LDL, whereas retraction induced by higher doses was irreversible and led to cell detachment and death. As previously discussed, cytotoxicity did not result directly from cell retraction, since effective concentrations of ML-9 blocked the cell retraction evoked by Ox-LDL (Fig 11) but did not prevent cell death (data not shown). In contrast, cytotoxicity seemed to be directly linked to the [Ca²⁺]_i peak,^{16 22} since EGTA and the calcium channel blocker nisoldipine blocked the [Ca²⁺]_i rise, cell retraction, and the cytotoxic effect of Ox-LDL. Cell retraction induced by Ox-LDL was also inhibited by an effective concentration of -tocopherol (100 µmol/L), which blocked the pathogenic intracellular signaling leading to the rise in [Ca²⁺]_i as previously observed in ECs.^{40 47} The molecular mechanism of this inhibitory effect is still unknown.

Our results (an intense and sustained [Ca²⁺]_i rise) in cultured cells may be compared with the data of Strickberger et al,⁴⁸ who reported a fivefold increase in ⁴⁵Ca²⁺ influx and [Ca²⁺]_i in atherosclerotic blood vessels of cholesterol-fed rabbits. The steady state increase in [Ca²⁺]_i observed by Strickberger et al⁴⁸ may be a consequence of the sustained [Ca²⁺]_i peak observed in SMC cultures that are relatively synchronized by 24-hour incubation in serum-free medium just before the addition of LDL and subsequent intracellular storage of calcium, clearly observed in dying cells.⁴⁹

Ox-LDL has been shown to impair vascular relaxation and to potentiate vasoconstriction to various agonists.^{11 12 50} To our knowledge the present article is the first to report that mildly Ox-LDL can induce sustained contraction of arterial SMCs and vascular rings. The delayed and sustained response of vascular rings evoked by mildly Ox-LDL reported in this article is different from the immediate response mediated by lysophosphatidylcholine⁵¹ due to inhibition of NO production by ECs,^{12 50 51} because contraction was delayed and observed only in deendothelialized rings in our study. This may be explained by at least two possible mechanisms: (1) Intact endothelium may reduce the diffusion of LDL in the vascular wall and reduce the concentration of Ox-LDL in contact with SMCs. Because the contractile effect is concentration dependent, the contractile effect of Ox-LDL may likewise be inhibited. Note that in the atherosclerotic vascular wall, Ox-LDL is present in the lesion and in direct contact with SMCs. (2) Intact endothelium may secrete compounds able to maintain vasodilation tone and to inhibit the contractile response (eg, NO). The endothelium-independent, delayed, and sustained contractile effect elicited by Ox-LDL in vascular SMCs reported here may constitute an additional mechanism leading to increases in vascular tone. This effect is probably independent of catecholamine release in the rings treated with Ox-LDL, as suggested by the lack of inhibition of a combination of the -adrenergic antagonists prazosin and RX-821002.⁵²

This contractile effect of Ox-LDL may occur in vivo, since Ox-LDL is present in the subendothelium of the atherosclerotic arterial wall and in direct contact with SMCs in atherosclerotic fibrous plaques.^{3 4} Local contraction of SMCs could lead to a sustained reduction (or spasm) of lumen size in atherosclerotic arterial segments. Moreover, it cannot be excluded that irreversible contraction of isolated cells and vascular rings induced ex vivo by high concentrations of Ox-LDL, followed by cell death, could also occur in the atherosclerotic arterial wall and lead to local necrosis at the center of the atheromatous plaque.

Another interesting feature of the present study was the demonstration that SMC contraction evoked by mildly Ox-LDL can be effectively blocked in two ways: (1) by preventing LDL oxidation with antioxidants, such as -tocopherol or probucol, and (2) by blocking the pathogenic intracellular signaling elicited by mildly Ox-LDL by various cytoprotective agents, such as -tocopherol or dihydropyridine calcium channel blockers, which efficiently protect cultured cells against Ox-LDL in vitro.^{25 40}

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine EC = endothelial cell Epi = epinephrine LDH = lactate dehydrogenase ML-9 = 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine MLCK = myosin light chain kinase MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NE = norepinephrine Ox-LDL = oxidized LDL polyhema = poly(2-hydroxyethyl methacrylate) SMC(s) = smooth muscle cell(s) TBARS = thiobarbituric acid–reactive substances

	Acknowledgments

 
This work was supported by grants from INSERM, Region Midi-Pyrenees (9308181), Ministere de l'Enseignement Superieur et de la Recherche (JE-174), Fondation pour la Recherche Medicale, and the European Communities (PL 931790). Dr Auge was the recipient of a fellowship from Ministere de l'En-seignement Superieur et de la Recherche. The authors thank J.C. Thiers for the photomicrographs and C. Mora and M. Troly for technical assistance.

Received August 14, 1995; accepted June 18, 1996.

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