BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ARTICLE NO.

221, 708715 (1996)

0661

Apoptosis in Vascular Smooth Muscle Cells: Role of Cell Shrinkage

Sergei N. Orlov,1 Than-Vinh Dam, Johanne Tremblay, and Pavel Hamet2
Centre de Recherche Htel-Dieu de Montral, Universit de Montral, Montral, Qubec, Canada
Received March 7, 1996
Cell volume decrease is known to be one of the earliest steps of apoptosis in immune system cells. In this
study, we compared the kinetics of apoptosis and cell volume adjustment in cultured vascular smooth muscle
cells (VSMC) from the aorta of normotensive BrownNorway (BN.1x) as well as spontaneously hypertensive
(SHR) rats and in MardinDarby canine kidney (MDCK) cells. The transfer of VSMC to serum-deprived
medium led to a transient cell volume decrease and to increased apoptosis. Both the cell volume decrease and
apoptosis displayed faster kinetics in SHR than in BN.1x VSMC. Increased tonicity of serum-deprived medium
by the addition of 200 mM mannitol augmented apoptosis in VSMC by 2.5- to 3-fold. In contrast to VSMC,
neither apoptosis nor the cell volume of MDCK cells was affected by serum deprivation. Apoptosis in MDCK
cells was also insensitive to tonicity of serum-deprived medium. There results demonstrate an initial volume
decrease in VSMC undergoing apoptosis and suggest that this phenomenon is involved in triggering the
apoptotic process. 1996 Academic Press, Inc.

Cell volume decrease is one of the universal morphological markers of programmed cell death
(PCD) or apoptosis in immune system cells (1,2). Thus, the induction of apoptosis in rat thymocytes by g-irradiation leads to a reduction in their volume from 107 mm3 to 75 mm3 (3). This drastic
cell volume decrease results in an increase of cell density which enables the separation of intact and
apoptotic thymocytes (4) and HL-60 cells (5) by isopycnic centrifugation on Percoll gradients.
Both the mechanism of cell volume decrease in cells undergoing apoptosis and its relationship
to triggering PCD are poorly understood. Data on the shrinkage of apoptotic cells have been mainly
obtained using a Coulter Counter. It is known, however, that this technique allows precise comparison of the volume of cells with the same shape (6) whereas in apoptotic cells shrinkage is
accompanied by shape transition (1,2,7,8). The measurement of intracellular water volume with
radioactive compounds like [3H]2O or [14C]-urea largely overcomes this problem. Recently, we
reported that activation of cAMP signalling pathways in vascular smooth muscle cells (VSMC)
leads to rapid loss of intracellular water (9) followed by intensification of apoptosis (10). From
these data we speculated that cell volume decrease may be involved in triggering PCD in VSMC
(11). To verify our hypothesis, in the present study, we compared the kinetics of adjustment of
intracellular water volume and chromatin cleavage in VSMC and Mardin-Darby canine kidney
(MDCK) cells in serum-deprived and hyperosmotic media. Taking into account the augmented
level of apoptosis in cardiovascular tissue of spontaneously hypertensive rats (SHR) (10), the
kinetics of cell volume adjustment and apoptosis were also compared in VSMC from SHR and
normotensive Brown-Norway (BN.1x) rats. Our results demonstrate an initial volume decrease in
VSMC undergoing apoptosis and supports the hypothesis of the involvement of cell shrinkage in
triggering PCD.
MATERIALS AND METHODS
VSMC were obtained by explant methods from the aortas of 10- to 13-week-old males BN.1x rats (Institute of Biology,
Charles University, Prague, Czech Republic) or SHR (Institute of Physiology, Academy of Science, Czech Republic) as
1
Invited Researcher from the Laboratory of Biomembranes, Faculty of Biology, MV Lomonosov Moscow State University, Moscow, Russia.
2
To whom correspondence should be addressed at Laboratory of Molecular Pathophysiology, Centre de Recherche de
lHtel-Dieu de Montral, 3850 St. Urbain St., Montral, Qubec H2W 1T8, Canada. Fax: (514) 843-2753.

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described elsewhere (12). This study was performed on VSMC between 10 and 16 passages. MDCK cells obtained from
the American Type Culture Collection (Rockville, MD, USA) were used between 40 and 60 passages. Before experimentations VSMC and MDCK cells were plated in 24-well dishes and allowed to grow in Dulbecos modified Eagle medium
(DMEM) containing 10% calf serum for 2448 and 120168 hr, respectively.
To quantify apoptosis, chromatin cleavage was estimated by a technique originally described for thymocyte suspension
(13) and adapted here for cells growing on plastic supports. Cells in 24-well dishes were supplied with 1 ml of DMEM
containing 10% calf serum (Gibco, Burlington, Ont., Canada) and 1 mCi/ml [3H]-thymidine (Amersham, Mississauga, Ont.,
Canada). After 24 hr they were washed twice with 2 ml of DMEM and incubated in fresh DMEM containing 10% calf
serum. They were washed again 24 hr later and incubated with DMEM containing 10% calf serum (control) or 0.2% calf
serum (serum-deprived medium) with additions indicated in the figure and table legends. At time intervals indicated in the
same legends, the medium was aspirated and the cells were treated with ice-cold lysed buffer (10 mM EDTA, 10 mM
tris-HCl, 0.5% triton X100 (pH 8.0). After 15 min, the lysed buffer was transferred into tubes and the cells were harvested
from the plates with 1% SDS/4 mM EDTA mixture (fraction F1). The lysate were centrifuged (12,000g, 10 min), the
supernatant (fraction F2) was transferred into scintillation vials, the pellet was treated with 1% SDS/4 mM EDTA mixture
(fraction F3), and fractions F1 and F3 were combined (fraction F4). The relative content of intracellular chromatin fragments
was determined as (A2/(A2+A4))100%, where A2 and A4 are the radioactivity of fractions F2 and F4, respectively.
DNA fragmentation in fractions F2 and F4 was compared by 39-end labelling assay. The cells (z107 per cm2 plate) were
treated with lysate buffer as indicated above, and the cell lysate was centrifuged at 12,000g for 10 min. Then, the
supernatant (fraction F2) was transferred for DNA extraction whereas the pellets and cells in plates were treated with
SDS/EDTA mixture and combined (fraction F4). The DNA was extracted from these fractions using phenol/chloroform
methods (14). To estimate DNA fragmentation, 39-ends were labelled by terminal deoxynucleotidyl transferase (tdt) assay
with Gavrieli methods (15) as modified previously (10) and analyzed with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA, USA).
The equilibrium distribution of [14C]-urea was used to measure intracellular water space. To initiate isotope uptake, 20
ml of [14C]-urea (Amersham) in PBS was added to cells incubated in 1 ml of DMEM with 10 or 0.5% calf serum to adjust
the final concentration to z4 mCi/ml. It has been shown that the kinetics of [14C]-urea uptake by VSMC plateau in 810
min (9). In the present experiments, [14C]-urea uptake was terminated in 60 min. The cells were washed 4 times with 3 ml
of ice-cold medium containing 100 mM MgCl2 and 20 mM HEPES-tris buffer (pH 7.4) and lysed with 1 ml of 1% SDS/4
mM EDTA mixture. The volume of intracellular water (V1 - pl/cell) was calculated as Vi 4 VoAi/Aon where Ai and Ao
are the radioactivity of [14C]-urea in the cell lysate and incubation medium, respectively (cpm), n is the cell number per well,
and Vo is the volume of incubation medium (pl) used for Ao determination. Cell content in wells was measured after
harvesting with 0.5 ml of 0.05% trypsin in Ca2+- and Mg2+-free PBS.

RESULTS AND DISCUSSION

DNA degredation is viewed as one of the biochemical markers of apoptosis in the majority of
cells studied so far (16). To estimate DNA degradation in cells with low level apoptosis, particularly in VSMC, a method for DNA 39-end labelling with tdt was developed (15,17). Using this
method, it was shown that serum-deprivation increases the content of low molecular weight DNA
fragments (<1,500 bp) in cultured VSMC (10). However, the method is hardly applicable for
quantitative analysis of a large number of samples obtained in different experiments and run on
different gels. To overcome this problem, we used the chromatin cleavage assay which was
originally applied for the study of apoptosis in thymocyte suspension (13). As seen in Fig. 1, intact
DNA and high molecular weight DNA fragments (z20,000 bp) were localized in the F4 fraction
only, whereas the F2 fraction was dominated by low molecular weight DNA fragments. The data
presented in Table 1 show that serum-deprivation induced approximately the same increment of
apoptosis in VSMC as measured by 39-end labelling of low molecular weight DNA fragments or
by chromatin cleavage. These results were also corroborated by data on the increment of apoptotic
cells in serum-deprived medium, recognized by their condensed shape under phase-contrast microscopy (Fig. 2). Comparing the 39-end labelling and chromatin cleavage in samples from SHR
and BN.1x VSMC under basal conditions as well as in serum-deprived medium and after forskolin
stimulation (data not shown), a highly significant level of correlation (r 4 0.97; n 4 8) was also
observed. These results validated the further usage of chromatin cleavage as a marker of apoptosis
in the rest of our investigation.
As seen in Fig. 3a, in accordance with previously-reported data (10,18), chromatin cleavage was
very low in the presence of 10% calf serum (0.20.4%). In the next 48 hr, it increased linearly to
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FIG. 1. Blot showing typical DNA laddering of pellet (fraction F4) and lysate (fraction F2) of vascular smooth muscle
cells from BN.1x rats incubated for 24 hr in serum-derived medium (DMEM containing 0.2% calf serum). 1 mg of DNA
samples with molecular weight standard or with DNA extracted from F4 or F2 fractions was labeled by tdt assay, as
described in Materials and Methods.

1.40 0.26 and 1.93 0.30% without any significant alterations of cell volume (Fig. 3b). After 24
hr of serum-deprivation, chromatin cleavage was augmented by 3.91 0.45-fold in BN.1x and 9.31
0.74-fold in SHR VSMC (p<0.01), similar to recently-reported data on enhanced apoptosis in
target organ of hypertension (10). Data from the present study demonstrate the faster kinetics of
apoptosis induced by serum deprivation in SHR VSMC compared to BN.1x rats. Thus, maximal
apoptosis in SHR VSMC was observed 6 hr after serum-deprivation whereas in BN.1x it continued
to increase for 48 hr. The transfer of VSMC to serum-deprived medium was accompanied by cell
shrinkage (Fig. 4b). The kinetics of cell shrinkage were faster in SHR than in BN.1x VSMC. Thus,
in 3 hr, VSMC volume was decreased by 13 3 and 33 4% in BN.1x and SHR, respectively. In
contrast to VSMC, neither chromatic cleavage nor cell volume was affected by serum-deprivation
TABLE 1
Effect of Serum Deprivation on 39-End Labelling of Low
Molecular Weight DNA Fragments and Chromatin Cleavage
in Cultured Vascular Smooth Muscle Cells
Calf serum
(%)
10%
0.2%

39-end labelling
(arbitrary units)

Chromatin cleavage
(%)

980
3423

0.91 0.11
3.12 0.28

To determine 39-end labelling of DNA fragments and of

chromatin cleavage, VSMC from BN.1x rats were incubated for
24 hr in DMEM containing 10 or 0.2% calf serum. 39-end
labelling of low molecular weight DNA fragments (<1500 bp)
and chromatin cleavage assay were performed as described in
the Materials and Methods section. The values of 39-end labelling obtained from estimates of [32P]-dCTP incorporation with
the PhosphorImager system and the means S.E. (n44) of
chromatin cleavage obtained with the same cells are given.
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FIG. 2. Phase-contrast microscopy of vascular smooth muscle cells from BN.1x rats grown in DMEM with 10% calf
serum (A) or for the last 24 hr in serum-derived medium (0.2% calf serum) (B). Arrows indicate apoptotic cells. The cells
were photographed with vericolor III film under a phase contrast microscope at an original magnification of 100.

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FIG. 3. Kinetics of chromatin cleavage (a) and cell volume (b) in vascular smooth muscle cells (VSMC) from BN.1x
rats (1) and SHR (2) incubated in the presence of 10% calf serum. VSMC were resuspended in DMEM containing 10% calf
serum and seeded in 24-well plates 48 hr before measurement. Means S.E. obtained in experiments performed in
quadruplicate are given.

of MDCK cells (Fig. 5). These results indicate a relationship between cell shrinkage and development of apoptosis in serum-deprived VSMC.
A trivial explanation of the cell volume decrease might be that the new modal density adopted
by apoptotic cells is secondary to reorganization of the nucleus and intracellular organelles, reflecting their nuclear density triggered by chromatin condensation. However, this explanation is
probably true for terminal steps of apoptosis and is unlikely for the initial cell volume decrease
observed in our study. Indeed, as seen in Fig. 4, maximal shrinkage of VSMC from BN.1x and SHR
after their transfer to serum-deprived medium was noted before the intracellular content of chromatin fragments reached maximum values. This statement is also supported by data obtained with
other in vitro models of PCD. Thus, thymocytes exposed to g-irradiation shrank in 2 distinct stages:
initial volume decreased from 99 mm3 to 76 mm3 followed by a gradual decline to 57 mm3 in the
next few hours. Probably, the latter stage reflected nuclear density and cell shape alteration whereas
the first stage preceded chromatin condensation (19).
To further verify the role of cell volume decrease in triggering of PCD, we studied the effect of
hyperosmotic shrinkage. We demonstrated previously that the addition of 200 mM sucrose to
isosmotic medium decreases VSMC volume by 35 6%. Hyperosmotically-shrunken VSMC do
not undergo a regulatory volume increase in that their volume is stable, at least for the next 2 hr
(9). Much the same effect has been observed with mannitol in MDCK cells (unpublished data).
Table 2 shows that hyperosmolality did not affect chromatin cleavage in VSMC under control
conditions but augmented apoptosis induced by serum-deprivation by 2-to 3-fold. These results
suggest that the initial cell volume decrease is not sufficient for apoptosis induction when VSMC
are protected by serum-derived factors but is involved in triggering or developing of PCD under
serum-deprivation.
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FIG. 4. Kinetics of chromatin cleavage (a) and cell volume (b) in vascular smooth muscle cells (VSMC) from BN.1x
rats (1) and SHR (2) incubated in serum-deprived (0.2%) medium. Before the experiment, the cells were grown for 48 hr
in the presence of 10% calf serum. The average initial volume of VSMC from BN.1x and SHR in 10% calf serum (2.16
0.04 and 2.24 0.03 pl/cell, respectively, see Fig. 3b) was taken as 100%. Means S.E. obtained in experiments
performed in quadruplicate are given.

Neither serum-deprivation (Fig. 5) nor osmolality (Table 2) affected apoptosis in MDCK cells.
These results have potential physiological implications. Indeed, in contrast to myocytes functioning
in medium with a stable serum protein concentration and osmolality, kidney epithelial cells are well
adapted for survival in serum-depleted and essentially unisotonic media. These results are also in
TABLE 2
Effect of Hyperosmotic Shrinkage on Apoptosis in Vascular Smooth
Muscle Cells from BN.1x Rats and MDCK Cells
Chromatin cleavage
(%)

Calf serum,

Mannitol,

mM

VSMC

MDCK

0
200
0
200

0.89 0.07
0.90 0.20
2.75 0.28
6.16 0.64*

2.01 0.18
2.38 0.12
2.44 0.43
2.61 0.30

10
10
0.2
0.2

Chromatin cleavage was measured after 24 hr of incubation in DMEM

containing 10% or 0.2% calf serum with or without 200 mM mannitol. For
more details see Materials and Methods and Fig. 35 legends. Data are the
means S.E. from 3 (VSMC) and 2 (MDCK) experiments performed in
quadruplicate. * p<0.001, compared with mannitol-free serum-deprived
medium.
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FIG. 5. Kinetics of chromatin cleavage (a) and cell volume (b) in MDCK cells during incubation in DMEM containing
10% (1) or 0.2% (2) calf serum. Before the experiment, MDCK cells were grown for 120 hr in the presence of 10% calf
serum. Means S.E. obtained in experiments performed in quadruplicate are given.

accordance with low level apoptosis in the renal medulla as compared with the cortex and cardiovascular tissue of mice subjected to g-irradiation (11).
Thus, our study demonstrates that the initial volume decrease in VSMC undergoing apoptosis in
serum-deprived medium is caused by loss of intracellular water and indicates that it is involved in
triggering or promoting the apoptotic program. Transport systems mediating the movement of
intracellular osmolytes and osmotically-obliged water as well as volume-sensitive elements of the
apoptotic process should be further investigated.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical Research Council of Canada (MT-10802, MT-10803) and the Heart
and Stroke Foundation of Canada. Johanne Tremblay is the recipient of a senior scholarship from Fonds de la Recherche
en Sant du Qubec and of a grant from the Medical Research Council of Canada (MT-11463). Sergei Orlov is the recipient
of a visiting professorship from the International Society of Hypertension, Pfizer Pharmaceutical Company. The technical
assistance of Monique Poirier, the secretarial skills of Jose Bdard-Baker and the editorial help of Ovid Da Silva are
appreciated.

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