Iododeoxyuridine (IUdR) uptake in
proliferating smooth muscle cells:
An
in vitro model to assess drug effects on intimal hyperplasia
Yonghua
Xu MD1
Mandar R Jagtap, BS1
Tam Garland, DVM PhD1
Jun Ying, PhD1
Ronald C McGarry, MD2
Marc
S. Mendonca, PhD2
Gordon
McLennan, MD1*
From the Departments of
Radiology (1) and Radiation Oncology (2) at Indiana University Medical Center,
550 N. University Boulevard, Indianapolis, IN 46202
Please send all
Correspondence to:
Gordon McLennan, MD
Director, Interventional
Radiology Research Laboratory
950 West Walnut St, E124
Indianapolis, IN 46202
Phone: (317) 278-9827
Fax: (317) 278-7793
(Abstract)
Purpose:
IUdR is a halogenated pyrimidine
recognized as the thymidine substitution in DNA. When labeled with I-125 it can
be used as a carrier to incorporate the isotope into DNA and target the
dividing cells. The purpose was to assess the maximum uptake of IUdR by
proliferating smooth muscle cells in vitro to determine the optimal
concentration to be administrated in an in vivo experiment.
The long-term goal of this project is to utilize radioactive IUdR to inhibit
smooth muscle cell proliferation and restenosis of arteries after balloon
angioplasty in vivo.
Method and Materials:
Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stimulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5mM, 10mM, 20mM, 30mM, 40mM, respectively, in proliferating SMCs with control for 1, 3, 5, 7day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-stained with anti- IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentration and duration was determined by FACS. All data were repeated three times at each time point.
The doubling
times, growth curve and cell density of the proliferating SMCs were
investigated using Beckman Coulter Particle Counter and digital
microscopy.
Results:
The percentage
of proliferating SMCs uptaking IUdR increased from 1 to 5 days incubation with
all concentrations of IUdR; In day 5, the uptake rate reached the peak value,
then decreased by 7 days. IUdR uptake
on day 5 was higher with concentrations of 10mM and 20mM. The doubling times of the SMCs were prolonged with IUdR
concentration increasing, while the proliferating cell number and density
compared with control decreased obviously by day 5 (p<0.05) and by day 7
(P<0.01).
Conclusion:
The peak time to
uptake IUdR was 5 days and optimal concentration of IUdR was between10mM to 20mM for proliferating
SMCs to uptake in vitro. IUdR itself
could inhibit the SMCs’ proliferation and the inhibitory effect was related to
the concentration.
Overview:
In the United States, 926,000
angioplasties, including 500,000 percutaneous transluminal coronary angioplasty
(PTCA) procedures, were performed in 1998 (1), A major cause of angioplasty failure continues to be
arterial restenosis of the target vessel (2), manifesting as late arterial re-narrowing at the site of
intervention. As shown in several experimental and human postmortem studies (3-5), smooth muscle cell (SMC) proliferation
is the major component for the intimal hyperplasia process, which largely
causes restenosis after angioplasty. Despite considerable effort to develop a
therapeutic pharmacologic strategy to efficiently suppress SMC proliferation,
there has not been a clinically effective drug to prevent restenosis following
angioplasty (6).
Another therapeutic alternative to prevent intimal hyperplastic coronary and
peripheral restenosis is external or endoluminal radiation therapy (7-11).
The effectiveness of radiation therapy is, however, also limited by late
restenosis and “edge effects” (11), which may be related to
excessive radiation dose to normal tissue (13,14).
Targeted radionuclide therapy allows inhibition of SMC proliferation with
limited radiation exposure to surrounding tissue. To achieve this, the nuclide
must have short range of penetration and the carrier must be targeted
differential to the proliferating cells (25). One such nuclide is 5-iodo-2’-deoxyuridine (IUdR), a halogenated thymidine analog that competes with
thymidine to incorporate into the newly synthesized DNA
during the S-phase of the mitotic cells (12). This nucleotide labeled with I-125 is incorporated into the DNA of
rapidly dividing cells where the isotope will exert short-range effect to cause
double-stranded DNA breaks, thus prohibiting these cells from further division,
whereas neighboring non-dividing cells are unaffected (15). This cell cycle-specific molecular radiotherapy strategy may be
exploited to maximize efficacy for inhibiting smooth muscle cell proliferation
associated with intimal hyperplasia, and minimize toxicity for normal cells.
IUdR has long been regarded as a
potent radiosensitizer, but its clinical use is limited because of its low
incorporation ratio during conventional administration schedules, which could
not maintain high concentration in local site (16, 17). The insufficient incorporation of radioactive DNA precursor may be
avoided by locoregional delivery (15). For all therapeutic options,
local drug delivery should in theory be the preferred way of ensuring that adequate
drug is delivered to the pathological site without the risks of side effects. We have
developed a periadventitial injectable drug delivery system that can keep the delivered drug for relatively longer time and concentration at
the target site (28,29).
Therefore, it can allow IUdR to incorporate into the proliferating cells
sufficiently.
Our long-term purpose is to use radioisotope labeled IUdR
to inhibit SMC proliferation and restenosis after angioplasty. Prior to
using it, this in vitro study was performed to determine
the optimum concentration of IUdR for proliferating SMCs to uptake, and investigate the
effect of nonradioactive IUdR on vascular SMCs proliferation. Such data would be used as a guide for further experiments.
Materials and Methods:
Tissue
harvest and cell isolation: Porcine thoracic aorta was
harvested and placed in sterile Dulbecco’s Modified Eagles (DMEM) medium
with 10% FBS and antibiotics. The endothelium was scraped off and the vessels
were cut into 1mm2 pieces & placed in endothelial side down into
6 well plates (Fisher scientific, Hampton NH). One mL of culture media was
added to each well and it was ensured that the tissue was lying on the bottom
of the well. The tissue was placed in an incubator at 370 C in 5%CO2.
Media was replenished every 4-5 days. After 2 weeks the cells were
treated with 1% trypsin (Sigma-Aldrich, St. Louis, MS) for 1-3 minutes with gentle
agitation. The cells were pipetted to create a single cell suspension and
transferred to a T-25 flask with 10% FBS-DMEM, 100ug/mL of streptomycin and
100U/mL of penicillin (Sigma-Aldrich, St. Louis, MS). Media were changed twice
a week until the cells were confluence.
Cell culture and IUdR Treatment:: Standard techniques for cell culture were used for
propagation. SMCs between passage 2 and 5 were used for all experiments. The
SMCs were plated in 6 well plates at densities of 5 x 104 per well
(for growth assay) or in T-25 flasks (Fisher scientific, Hampton NH) at densities of 2 x 105 (for flow
cytometry) in 5% FBS-DMEM medium to keep cells quiescent. After 24h, the cells
attached to the bottom of the plates were stimulated by 10% FBS medium and
insulin to proliferate (18). IUdR (Sigma-Aldrich, St. Louis, MS) was added at 0mM(control), 5mM, 10mM, 20mM, 30mM, 40mM, respectively, in proliferating SMCs
for 1, 3, 5, 7day incubation.
Cell growth
assays: After the end of the incubation period, the cells were released from the plates by
trypsinization. The total cell number (established by direct counting using Beckman
Coulter Particle Counter) and cell density (established with digital
camera imaging through an inverted microscopy) were used to determine the doubling times and growth curves of the
proliferating SMCs.
Flow cytometry assay: The cells incubated with or without different IUdR concentration
were harvested by trypsinization, washed with cold phosphate-buffered saline
(PBS), PH 7.0, and incubated with 300u/mL DNAse in HBSS at 37oC for
60 minutes, After pelleting, the IUdR incorporated cells were labeled with
20uL/ml of FITC-conjugated anti-IUdR antibody (Becton Dickinson, San Jose, CA).
Additionally the total cells were stained with propidium iodide (10ug/mL).
Two-color flow cytometry analysis was performed with the use of a
fluorescence-activated cell scanner (FAC Sort; Beckton Dickinson), The ratio of IUdR-labeled cells to total cell population for each IUdR
concentration and duration was analyzed using Cell Quest and WinMDI2.8
software. All data were repeated three times at each time point.
Statistics Analysis The SMC proliferation data were analyzed with a two-way ANOVA
model (IUdR concentration and incubation period). On day 1, 3, 5, and 7 after
incubation with IUdR, the means of cell number were compared using Bonferroni
multiple comparison test for each concentration. The doubling time data was
analyzed with a one-way ANOVA model (concentration) and Bonferroni multiple
comparison tests were applied to compare the mean of doubling time for each
concentration. Finally, a piecewise
regression model was used to investigate the IUdR uptake data. For each
concentration, the slopes of uptake were compared before and after day 5
following treatment. The Bonferroni multiple comparison test was used to
compare predicted means of uptake of each concentration at each day 1, 3, 5 and
7 after treatment. All statistical analyses were done using SAS 8.0 (SAS
Institute Inc., Cary, NC, USA.)
and Splus 2000 (MathSoft Inc., USA.).
Results
Effect of IUdR on morphology of proliferating SMCs: There is a clear
morphological reduction of cell numbers after IUdR exposure, which is
concentration-dependent. As shown in Fig.1, SMC density significantly decreased
with the IUdR concentration increasing after 5 days incubation.
Control 5 m M 10 m M
20 m M 30 m M 40 m M
Fig.1: IUdR inhibits the SMCs proliferation in concentration-dependent manner.
The SMCs were incubated with IUdR at 0mM(control),
5mM, 10mM, 20mM,
30mM and 40mM for 5days. The densities of cells decreased
with increasing concentration of IUdR
Effect of IUdR on cell
growth of proliferating SMCs: A significant
concentration-dependent growth inhibition was observed with IUdR treatment.
After the addition of 5mM, 10mM, 20mM, 30mM and 40mM of IUdR concentration, 12%, 24%, 30%, 43% and 47% cell
growth inhibition was observed on day 5 (P<0.05), and 21%, 47%, 56%, 59% and
63% on day 7 (P<0.01), respectively (Fig.2).
Fig 2: Effects of IUdR on SMC numbers. 0mM(control), 5mM, 10mM,
20mM, 30mM or 40mM
IUdR was added to the proliferating SMC culture media. On 1, 3, 5 and 7 d, the
cells were suspended by treatment with 0.05% trypsin, and were counted to draw
cell growth curves. The data are expressed as the mean (SD) of cell counts.
Doubling time of SMC for each concentration is shown in Fig. 3. The ANOVA test results showed
significant overall differences among concentrations (P=0.02).
Bonferroni multiple comparison tests revealed that the mean doubling times at 20mM,
30mM
and 40mM of
IUdR were
significantly higher than that at control (P<0.05).
Fig 3: The doubling time was prolonged with IUdR concentration increasing. The value
shown is mean (SD)
* P<0.05 vs control.
Detection of IUdR uptake in proliferating
SMCs: IUdR uptake in
proliferating SMCs was detected by flow cytometry using FITC-conjugated
anti-IUdR antibody (Fig. 4). Means and standard deviations of uptake for
each concentration and control on day(s)1, 3, 5 and 7 after treatment are
presented in Table 1 with Bonforroni multiple tests. There was no
significant difference for uptake rates at all concentrations on day one. The uptake rates for 10mM IUdR on days 5, 20mM on days 3 and 7 were significantly
higher than the 5mM rate (P<0.05). They were, however, not significantly
different from that uptake rates at higher IUdR concentrations (P>0.05),
except that the uptake rate (44.85%) at 10mM was significantly higher than ones at
30mM and 40mM on day 5 (P<0.05). This indicated that the
concentration between 10uM and 20uM was optimal for incorporation of IUdR and
higher concentrations such as 30mM and 40mM didn’t result in higher incorporation
at peak time. This is shown in Fig. 4, where piecewise regression lines for
each concentration data are shown. All concentrations of IUdR reached peak
incorporation on day 5. The slopes were positive (P<0.05) before day 5,
indicating an increasing uptake over time, and IUdR uptake was higher with
concentrations of 10mM, and 20mM. The uptake rates
at 5mM, 10mM, and 20mM decreased after they reached their peaks on day 5
(p<0.05). Meanwhile, the slopes of 30mM and 40mM showed no significant difference after
day 5.
Fig. 4: Exemplary FACS analysis of the 5-day time point to demonstrate anti-IUdR antibody FITC intensity for quantifying the percentages of IUdR incorporated cells using histogram. The M1 region represents positive IUdR labeled cells, which stained positive for FITC conjugated anti-IUdR antibody.(percentage above M1 lines).
Table 1: IUdR uptake rate by proliferating SMCs (% IUdR labeled cells)
|
|
Control |
5mM |
10mM |
20mM |
30mM |
40mM |
|
1 day |
1.23 (0.75)a |
5.53 (2.03)a |
4.31 (0.95)a |
4.71 (1.81)a |
5.05 (1.67)a |
5.28 (4.26)a |
|
3 days |
1.25 (1.83)b |
10.46 (7.09)c |
23.87 (17.07)cd |
24.30 (8.63)d |
16.89 (12.80)d |
23.55 (6.67)d |
|
5 days |
1.00 (1.57)e |
31.12 (2.88)f |
44.85 (11.14)g |
39.86 (11.23)fg |
31.87 (5.80)f |
31.01 (12.27)f |
|
7 days |
0.25 (0.24)h |
11.92(7.36)hk |
19.97 (4.23)km |
30.38 (2.92)m |
25.78 (8.75)m |
30.82 (3.83)m |
All uptake values were mean (SD). Means followed by the same letter in a row were not significantly different by Bonferroni’s multiple tests at a 5% probability level.
Fig. 5: Percentages of IUdR labeled cells over time were analyzed
with the piecewise linear regression. The uptake rates reached peak of IUdR
incorporation for all IUdR concentrations on day 5, and 10mM and 20mM of
IUdR concentrations had higher uptake rates on day 3 and day 5. After day 5, the uptake rates decreased.
Discussion:
Currently, Restenosis remains the major limitation of angioplasty,
since 30-40% of patients undergoing angioplasty would develop restenosis within
6 months (21). Vascular injury due to angioplasty triggers a cascade
of events that include endothelial denudation or dysfunction, inflammation and
SMC activation and proliferation (27). Among them, SMC proliferation
was found to be the key component, and an early event in the process of
restenosis from studies involving animal models of arterial injury. With the
use of a single balloon injury in the rat carotid artery, maximal SMC
proliferation was found in the media and intima at 4 and 7days, respectively (23,
24). Hanke(3) reported
that a peak in proliferation activity occurred in the intima 3 days after
balloon dilatation; and Banai(4) found that a peak in proliferating
activity occurred in the tunica media on day 5, with a subsequent peak in the
neointima on 7 day in the rabbit ear artery injury model. Therefore, this
experiment only focused on the first week of proliferation of the vascular
SMCs. Insulin-like growth factor-I is strongly mitogenic for SMCs and may play
one of critical roles in in-vivo proliferation of SMCs (22). In this
experiment we used 10%FBS medium with insulin to stimulate the SMCs to
proliferate from relatively quiescent status. We observed the numbers of SMCs
in this in vitro experiment exponentially increased within 7 days after initial
treatment, and rapidly increased especially on day 5 and maintained on day 7.
Many antiproliferative drugs
(such as heparin, terbinafine, trapidil, Angiopeptin, etoposide or doxorubicin
etc.) have been tested as a means to prevent vascular proliferating diseases.
However, clinical trials have generally failed to recapitulate the efficacy
documented in animal studies (6). It seems improbable that a
multifactorial disease can be treated successfully by targeting a single
mitogenic factor. So, antiproliferative therapeutics has focused on targeting
specific parts of the cell cycle as a “final common pathway” (27). In addition to pharmacological agents or
gene therapy, irradiation can be a means to achieve cell-cycle inhibition.
Local delivery of radiation, inducing DNA damage of vascular cells, has emerged
as a promising therapy to reduce intimal tissue responses following
angioplasty. Its drawbacks include nonspecific and suboptimal radiation dose
with the further consequence of potential aneurysm formation from weakening of
the vessel wall, and “edge effect” causing later restenosis (11).
IUdR is incorporated into DNA during S-phase of cell cycle, but clinical
use is limited because of its low cytotoxicity (19). To increase the killing efficiency of IUdR, it can be labeled with
I-125 and served as a carrier to incorporate the isotope into DNA of dividing
cells. Therefore, the radiolabeled IUdR is used as a cell-cycle inhibitor (26). This cell cycle-specific molecular radiotherapy strategy can be
especially attractive in antiproliferative treatment. IUdR competes with
thymidine for DNA incorporation. The
size of the extracellular and intracellular thymidine pools, therefore, is
critical to the percentage of IUdR incorporated into DNA (20). For higher incorporation rate, it is pivotal
to determine optimal concentration of IUdR, which will be used as a guide to
the selection of an appropriate radioactive IUdR dosing range.
In the present study, the data indicated that 10mM to 20mM IUdR was the optimal concentration
range for IUdR incorporation into proliferating SMCs, since it could achieve
higher uptake rate with possibly less amount of IUdR during a peak of SMC
proliferation. Though there was greater SMCs proliferation at 5mM of IUdR concentration, the IUdR uptake
rate was lower at this concentration. This may be due to decreased competition
with thymidine at the lower concentration. For IUdR concentrations of 10mM and 20mM, the uptake rates reached peak on day 5
and then decreased on day 7. The reason could be that the amount of IUdR was
exhausted in the medium as more and more proliferating SMCs had uptaken it
after day 5, thus the actual concentration decreased. It is therefore possible
that if a concentration between 10 and 20mM IUdR can be maintained in medium, for
example by prolonged release from a polymer, this situation may be resolved.
We investigated the
effect of IUdR on the proliferation of SMCs as well as the incorporation rate
of IUdR in this experiment. Our data
showed IUdR inhibited the proliferation of porcine SMCs stimulated by 10% FBS
and insulin in vitro in a concentration dependent manner on day5 and day 7.
Cell proliferation can be inhibited with cytotoxic or cytostatic mechanisms.
(….) The inhibitory effects of IUdR were likely due to cytotoxic effects, even
though it exhibits low cytotoxicity in vivo. However, we could not exclude the
effect of prolonged cell cycle arrest, which induced delayed DNA synthesis.
Our
results showed that IUdR could act as a growth inhibitor and an isotope carrier
capable of being incorporated into the dividing cells. These indicated that
this compound is a suitable candidate therapeutic agent for prevention of
restenosis following angioplasty. The inhibitory effect was concentration
dependent. The optimum concentration of IUdR for incorporation into
proliferating SMCs in vitro was between 10mM to 20mM, and the peak time for
uptake was on day 5. Further studies involving sustained drug release and
periadventitial administration of IUdR in a porcine model will be performed.
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