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1
* Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA;
Department of Medicine (Cardiology), St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA; and
Laboratory of Vascular Biology, Instituto de Biomedicina de Valencia (IBV-CSIC), Spanish Council for Scientific Research, 46010 Valencia, Spain
1Correspondence: Laboratory of Vascular Biology, Instituto de Biomedicina de Valencia (IBV-CSIC), 46010 Valencia, Spain. E-mail: vandres{at}ibv.csic.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: p27 • adenovirus • gene therapy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Similarly, the requirement for a tumor to elaborate a
vascular infrastructure in order to grow and metastasize is well
documented (2
, 3)
. Therefore, a better understanding of
the molecular mechanisms that control blood vessel formation is
important for the development of novel therapies to augment or inhibit
postnatal angiogenesis.
Angiogenesis involves the remodeling of the surrounding extracellular
matrix and the migration of endothelial cells along a chemotactic
gradient. Moreover, endothelial cells must leave their normal quiescent
state and reenter the cell cycle during angiogenesis. Progression
through the mammalian cell cycle requires the activation of
cyclin-dependent kinases (CDKs) through their association with
regulatory subunits called cyclins (4
, 5)
. Different
CDK/cyclin holoenzymes are orderly activated at specific phases of the
cell cycle. Active CDK/cyclin complexes phosphorylate the
retinoblastoma gene product (pRb) and the related pocket proteins p107
and p130 from mid-G1 to mitosis, which in turn regulate the activity of
members of the E2F family of transcription factors (6
, 7)
.
CDK activity is negatively regulated by specific cell cycle regulatory
proteins, termed CDK inhibitors (CKIs), which associate with and
inhibit the activity of CDKs (4
, 8)
. CKIs of the CIP/KIP
family (p21, p27, and p57) bind to and inactivate both the cyclin
D-CDK4 and cyclin E-CDK2 complexes, whereas members of the INK4 family
(p15, p16, p18, p19) are specific for CDK4- and CDK6-containing
holoenzymes. We and others have suggested that up-regulation of p27 may
limit the growth of vascular smooth muscle cells (VSMCs) at late time
points after balloon angioplasty (9
, 10)
. Consistent with
this possibility, overexpression of p27 efficiently blocked mitogen-
and c-fos-dependent induction of cyclin A promoter activity in cultured
VSMCs (9
, 11)
, and adenovirus-mediated overexpression of
p27 attenuated neointimal thickening in balloon-injured arteries
(9
, 12)
. In contrast, recent studies using p27 knockout
mice have demonstrated that the loss of p27 results in prolonged
proliferation of cardiac myocytes (13)
and enhanced
atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice
(14)
. p27 has also been implicated in the regulation of
cardiomyocyte hypertrophy (13)
and angiotensin
II-stimulated VSMC hypertrophy (15
, 16)
.
The studies cited above identify p27 as an important regulator of the
phenotypic response of cardiac and vascular myocytes to hyperplastic
and hypertrophic stimuli in vitro and in vivo. It has also been
suggested that alterations in p27 expression regulate the proliferation
of cultured human umbilical vein endothelial cells (HUVECs) and bovine
aorta endothelial cells during the transition from contact inhibition
to the proliferative state (17)
. However, little is known
about the role of p27 on endothelial cell migration and angiogenesis.
In the present study, we describe the generation of an adenoviral
system that allows doxycycline-dependent overexpression of p27. Using
this inducible system, we investigated the effect of forced
overexpression of p27 on proliferation and migration of HUVECs in vitro
and on angiogenesis in a murine model of surgically induced hind limb
ischemia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
pACCMV-TetON was generated by subcloning into the multiple cloning site
of pACCMVpLpA (19)
an EcoRI/BamHI
restriction fragment isolated from pTetON (Clontech, Palo Alto, CA)
that contained the rtTA cDNA downstream from the CMV promoter. To
generate p
E1Sp1A-TRE-p27, a XhoI/HindIII p27
expression cassette was subcloned into pE1Sp1A (Microbix Biosystems,
Ontario, Canada) digested with XhoI and HindIII.
The 
1.5 kb p27 expression cassette contained a FLAG epitope-tagged
human p27 cDNA downstream from the TRE and CMV minimal promoter.
Recombinant adenovirus Ad-TetON and Ad-TRE-p27 were generated by
homologous recombination in human embryo kidney 293 cells
(20)
cotransfected with pJM17 (21)
and
pACCMV-TetON or pE1Sp1A-TRE-p27, respectively. Viral stock titers
were determined by plaque assay. For the hind limb ischemia studies
(see below), viruses were purified by CsCl density gradient
centrifugation.
In vitro assays with adenovirus
HUVECs were isolated from human umbilical cords as described
previously (22)
. Cells were grown onto 1.5%
gelatin-coated plates (Sigma, St. Louis, MO) in medium M199
supplemented with 20% fetal bovine serum (FBS) (both from
BioWhittaker, Walkersville, MD), 0.4% bovine brain extract (Clonetics,
San Diego, CA), 0.5% penicillin/streptomycin, and 0.5% Fungizone.
HUVECs were used between passages 3 and 8. Cells were maintained at
37°C in a humidified atmosphere of air with 5%
CO2.
To assess the level of p27 overexpression using the adenoviral system,
HUVECs were seeded in 60 mm culture dishes, grown to 80%
confluency, and incubated for 24 h with Ad-TetON and Ad-TRE-p27
with or without doxycycline (1 µg/ml, Sigma) in the culture medium.
Preparation of whole cell lysates and Western blot analysis was as
described previously (9)
. Rabbit polyclonal anti-p27
antibodies (1/250, Ab-1, Oncogen Research Products, Cambridge, MA) were
used to detect adenovirus-encoded p27, which displayed slower
electrophoretic mobility than endogenous p27 due to the presence of an
amino-terminal FLAG tag. Recombinant p27 expression could not be
detected using monoclonal anti-FLAG antibody.
HUVECs for flow cytometry were seeded in 6-well dishes and
serum-starved in medium containing 2% FBS (without bovine brain
extract). After 2 days of serum starvation, 6 x
107 pfu of each Ad-TetON and Ad-TRE-p27 were
added for an additional 16 h with or without doxycycline. Infected
cells were washed twice with PBS, fed fresh medium containing 20% FBS
with or without doxycycline, and harvested 12 h later for flow
cytometry as described previously (11)
.
To assess the effect of doxycycline on cell proliferation, asynchronously growing HUVECs exposed to different concentrations of doxycycline were incubated for 5 h with 3H-thymidine (1 µCi/ml, Amersham, United Kingdom), after which cells were washed with PBS and incubated for 1 h with ice-cold 5% trichloroacetic acid. Labeled DNA was extracted with 0.5N NaOH and counted in a scintillation counter (Wallac, Turku, Finland).
Migration assays were performed using a 48-well chemotaxis chamber (Neuroprobe, Bethesda, MD). After exposing HUVECs to Ad-TRE-p27 and Ad-TetON (multiplicity of infection of 30 and 15, respectively) for 16 h, cells were trypsinized and resuspended at 3 x 105/ml in chemotaxis medium (M199 supplemented with 0.5% bovine serum albumin) before being placed in the upper wells of a chemotaxis chamber. The lower wells contained either control medium or PDGF-BB (10 ng/ml) (Sigma). Upper and lower wells were separated by a 8 µm pore polyvinylpyrrolidone-free polycarbonate filter (Poretics, Livermore, CA) precoated with fibronectin (20 µg/ml, Sigma). Chemotaxis chambers were incubated for 4 h at 37°C in a humidified atmosphere of air with 5% CO2. Thereafter, the filters were fixed and stained with Diff Qick (DADE, Aguada, Puerto Rico). Cells that had not migrated were removed from the upper surface. After air drying, the coverslips were mounted onto glass slides and migration was quantified by counting the migrated cells in 20 randomly selected high power fields (200x).
To assess the effect of doxycycline on cell migration, HUVECs labeled with the fluorescent dye Calcein-AM were placed in FALCON HTS FluoroBlock inserts (8.0 µm pore size, 2x104 cells/insert) (Becton Dickinson, Bedford, MA) in serum-free medium. To induce migration, the lower chamber contained complete medium. When indicated, doxycycline (1 µg/ml) was added to both chambers. Chemotaxis was assessed by detecting the fluorescence of cells migrating to the lower chamber using a Victor 4120 multilabel counter (Wallac).
To examine the formation of tubelike structures, HUVECs were seeded on 4-well chamber slides (5x104 cells/well) coated with Matrigel (Collaborative Biomedical Products, Bedford, MA) and incubated for 24 h in medium containing 5% FBS. Control cells were not infected with adenovirus. When indicated, cells were coinfected with Ad-TetON (7.5x105 pfu) and Ad-TRE-p27 (1.5x106 pfu) with (1 µg/ml) or without doxycycline in the medium.
In vitro assays with retrovirus
Recombinant retrovirus were generated using the retroviral
vectors pBabePuro-p27wt and
pBabePuro-p27ck- (gift of B. Amati), which
encode for wild-type p27 and a mutated p27 protein that is unable to
interact with CDKs and cyclins (23)
, respectively.
Infection of asynchronously growing HUVECs was performed as suggested
by the supplier of the PT67 packaging cells (Clontech). Uninfected
HUVECs were used for control. Cells were trypsinized, counted with an
hemocytometer, and plated on 12-well culture dishes at a density of
3 x 103 cells/well. Quantification of
3H-thymidine incorporation into DNA and p27
Western blot analysis were performed as indicated above for the studies
with adenoviral vectors.
Hind limb ischemia studies
Unilateral hind limb ischemia in male C57/BL6J mice was produced
by occlusion of the femoral artery as described previously
(24)
. One week before surgery, mice were randomly divided
into - doxycycline and + doxycycline group (at 1 mg/ml, in
drinking water containing 2.5% sucrose). Immediately after surgery,
animals received in the left hind limb an i.m. injection consisting of
60 µl of each Ad-TetON (1x108 pfu/ml) and
Ad-TRE-p27 (1x109 pfu/ml). Western blot analysis
was performed to assess the expression of endogenous and adenovirally
encoded p27 in cell extracts prepared from ischemic muscle specimens
pooled from three mice retrieved 3 and 28 days after surgery. Serial
assessment of hind limb blood flow was performed using a PIM 2.0 laser
Doppler perfusion imager (Lisca, Mahwah, NJ) as described (24
, 25)
. Calculated perfusion was expressed as a ratio of left
(ischemic) to right (control) limb.
To determine capillary density, whole nonischemic and ischemic limbs of two mice from each group were immediately fixed in methanol overnight. Histological sections (5 µm thick) were prepared from paraffin-embedded tissue samples. Endothelial cells were identified by histochemical staining with biotinylated Griffin Simpliciforia lectin I isolectin B4 (Vector, Burlingame, CA), followed by peroxidase-conjugated ultrastreptavidin (Signet, Dedham, MA) and DAB substrate (Vector). Sections were counterstained with Gill’s hematoxylin solution (Sigma). Six microscopic fields containing cross sections of muscle fibers were randomly selected for each animal (at a magnification of 66x). Areas of 0.06 mm2 were photographed and printed on high quality photographic paper. Capillaries were identified as structures with positive staining for isolectin B4 and appropriate morphology. Neither vessel lumen nor red blood cells were used to define a microvessel. The number of capillaries per muscle fiber was counted in each photograph, and the counts of the six chosen areas were averaged for each mouse. To eliminate interobserver variations, a single investigator, who was blinded to the treatment regimens, performed all counts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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schematizes the adenoviral system generated to achieve
inducible p27 expression. Ad-TetON directs expression of the rtTA
regulatory protein from the CMV promoter. rtTA is a fusion of amino
acids 1–207 of the tet repressor (rTetR) and the carboxyl-terminal
activation domain (130 amino acids) of the VP16 protein of herpes
simplex virus. A second adenoviral vector, Ad-TRE-p27, directs
expression of human p27 from the tet response element (TRE) and a
minimal CMV promoter (PminCMV). Under basal
conditions, transcription from Ad-TRE-p27 is minimal because rtTA is in
its inactive conformation. Inducible transcription of the p27 cDNA is
achieved via activation of rTetR-VP16 by exposure to tetracycline or
its derivative doxycycline. We tested this system by Western blot
analysis of control and infected HUVECs (Fig. 1B
). Whereas
endogenous p27 was detected at similar levels in all conditions tested,
adenovirally encoded p27 was detected only in cells coinfected with
Ad-TetON and Ad-TRE-p27 when doxycycline was added to the culture
medium. Moreover, the level of ectopically expressed p27 increased
gradually as the amount of Ad-TRE-p27 augmented from 1.5 x
105 to 4.5 x 106 pfu.
|
Serum-inducible proliferation of HUVECs is inhibited by
overexpression of p27
To test this system functionally, we examined the cell cycle
kinetics of HUVECs by flow cytometry (Fig. 2A
). In the absence of doxycycline, cultures coinfected with
Ad-TetON and Ad-TRE-p27 entered the cell cycle after serum
restimulation when compared with starvation-synchronized HUVECs, as
indicated by a significant reduction in the proportion of cells in
G0/G1 and increased S-phase entry. In marked contrast, infected cells
exposed to doxycycline at 1 µg/ml failed to progress through the cell
cycle despite serum refeeding. Control experiments showed that the
addition of doxycycline up to 100 µg/ml fails to inhibit de novo DNA
synthesis in serum-stimulated cultures of HUVECs (Fig. 2B
).
These results demonstrate that doxycycline-dependent overexpression of
p27 can block cell cycle progression of serum-stimulated HUVECs.
|
Additional control experiments were performed to assess whether the
inhibitory effect of ectopically expressed p27 might be due to
nonspecific interference with basic cellular functions resulting from
massive overexpression. Cultures of asynchronously growing HUVECs were
infected with retroviral vectors encoding for wild-type p27
(p27wt) or a double mutant that had lost the
ability to form stable complexes with cyclins and CDKs
(p27ck-) and fails to inhibit CDK2 activity
(23)
. Consistent with our data from adenoviral vectors
(Fig. 2A
), retrovirus-mediated overexpression of
p27wt greatly inhibited DNA synthesis compared
with uninfected cells (Fig. 3A
). In marked contrast, retrovirally encoded
p27ck- slightly enhanced DNA synthesis. These
differences occurred despite the similar level of overexpression of
p27wt and p27ck- (Fig. 3B
).
|
Migration of HUVECs and tubulogenesis is inhibited by
overexpression of p27
We next used two well-established assays to assess the potential
regulatory role of p27 on cell motility. First, the effect of p27
overexpression on cell migration was tested in a modified Boyden
chamber (Fig. 4A
). Addition of PDGF-BB to HUVECs coinfected with Ad-TetON
and Ad-TRE-p27 and maintained in the absence of doxycycline
significantly increased the number of migrated cells over basal
migration in unstimulated cells. However, addition of doxycycline at 1
µg/ml significantly reduced both basal and PDGF-BB-induced migration
of infected cells. Control experiments demonstrated that doxycycline by
itself, at the concentration used in these studies, does not inhibit
migration of HUVECs (Fig. 4B
).
|
Endothelial cells plated on a Matrigel substrate attach and migrate to
form tubelike structures (26
, 27)
. Therefore, we also
investigated the effect of p27 on HUVEC motility in this in vitro model
system. Cultures of HUVECs were uninfected or coinfected with Ad-TetON
and Ad-TRE-p27 and seeded on Matrigel-coated culture dishes. As shown
in Fig. 5
, addition of doxycycline to infected HUVECs reduced tubulogenesis
relative to both uninfected cultures and infected cells in the absence
of doxycycline.
|
Overexpression of p27 inhibited blood flow recovery and reduced
capillary density in ischemic limbs
The in vitro results presented thus far demonstrate that ectopic
overexpression of p27 can inhibit endothelial cell functions essential
for angiogenesis (i.e., cell proliferation and migration). To examine
whether inducible overexpression of p27 can indeed impair de novo blood
vessel formation, we performed gene transfer experiments in a murine
hind limb model of acute ischemia. Immediately after surgery, all mice
received Ad-TetON and Ad-TRE-p27 via an i.m. injection in the injured
leg. Western blot analysis demonstrated doxycycline-dependent
overexpression of adenovirally encoded p27 in ischemic muscle specimens
retrieved 3 and 28 days after surgery (Fig. 6A
). Angiogenesis was quantified by laser Doppler perfusion
imaging and measurement of capillary density. As revealed by Doppler
analysis of Fig. 6B
, hind limb blood flow in the -
doxycycline group of adenovirally infected mice was precipitously
reduced after surgery (day 0), increased steadily between postsurgery
days 7, 14, and 21, and remained elevated by day 28. A similar
precipitous reduction in hind limb blood flow occurred in mice
receiving adenovirus and doxycycline; however, in contrast to -
doxycycline group, flow recovery was negligible up to postoperative day
28 (Fig. 6B
). This corresponded to a significant reduction
in capillary density in the ischemic hind limb at day 35 after surgery
in mice receiving adenovirus and doxycycline (Fig. 6C
).
Control experiments with uninfected animals demonstrated that
doxycycline by itself, at the concentration used in these studies, does
not inhibit blood flow recovery (Fig. 7
). Collectively, these results demonstrate that doxycycline-dependent
overexpression of p27 can impair blood flow recovery and reduce
capillary density in a murine model of hind limb ischemia.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 29)
. Together, these studies suggest that members of
the CKI family of growth suppressors are important regulators of cell
migration.
Consistent with previous in vitro studies (30)
, incubation
with doxycycline up to 100 µM did not significantly inhibit the
growth of HUVECs. Similarly, we have shown that doxycycline at 1 µM
did not inhibit the migration of HUVECs nor did concentrations up to
10.4 µM impair the formation of tubelike structures
(30)
. It is also noteworthy that tetracycline analogs,
including doxycycline, have been shown to inhibit the activity of
several metalloproteinases in vitro and in vivo (31
32
33
34
35
36)
.
This inhibitory effect of doxycycline could impair matrix degradation
and therefore limit angiogenesis in the murine hind limb model of acute
ischemia. However, at the concentrations used in our studies, treatment
with doxycycline did not affect blood flow recovery.
Cellular migration and proliferation are important events during
angiogenesis (1
, 2)
. Thus, it is reasonable to presume
that the ability of p27 to inhibit cellular growth and migration
contributes to the anti-angiogenic effect of p27 overexpression
reported here. Of further interest is the wealth of evidence indicating
that angiogenesis is required for tumor growth and metastasis (2
, 3)
. Indeed, several anti-angiogenic therapies have proved
efficient at inhibiting tumor progression in animal models of cancer
(3
, 37
38
39)
. Moreover, it is appropriate to consider
previous studies showing that adenovirus-mediated overexpression of the
growth suppressors p53 (40)
and p16 (41)
inhibited angiogenesis induced in vivo by human cancer cells. Schreiber
et al. (42)
compared the efficacy of different CKIs at
inhibiting tumor formation in a polyomavirus middle T antigen model of
murine breast carcinoma. These authors demonstrated that
adenovirus-mediated overexpression of p16, p18, p21, and p27 results in
a significant delay in tumor progression, varying from 5 wk for p21 to
more than 25 wk for p27. Similarly, in vitro experiments with human
breast cancer cells MDA-MB-231 and MCF-7 infected with recombinant
adenovirus expressing p27 and p21 showed a markedly more (up to
56-fold) potent growth inhibitory effect of p27 (43)
.
Thus, p27 may be a better candidate than other CKIs for gene therapy of
tumors. p27-deficient mice are predisposed to tumorigenesis in multiple
tissues when challenged with 
irradiation or a chemical carcinogen;
p27 heterozygous mice were also more susceptible than wild-type
animals, but at intermediate rates (44)
. The sensitivity
to p27 dosage suggests that reduced p27 expression might be a common
event during tumor growth. Indeed, low expression of p27 has frequently
been found in several human cancers, and this is highly predictive of
tumor progression and patient mortality (45
46
47
48
49
50
51
52)
.
Conversely, acquired expression of p27 might be a favorable independent
prognostic indicator in patients with hepatocellular carcinoma
(53)
. It is also noteworthy that p27 expression appears to
correlate inversely with vascular cell proliferation in human
atherosclerotic tissue (10
, 54)
and that p27 inactivation
accelerates the development of atherosclerotic lesions in
hypercholesterolemic apolipoprotein E-deficient mice (14)
.
Collectively, these studies suggest that restoration of p27 function
might be effective for the treatment of cancer and atherosclerosis.
Although available scientific evidence supports the feasibility of
human gene therapy, major benefit is expected from the perfection of
current vectors and/or the development of new gene delivery strategies
(55)
. As indicated by numerous animal studies, a major
limitation of current adenoviral vectors is the substantial drop in
gene expression normally seen shortly after delivery (typically 1–2
wk). Our study shows sustained doxycycline-dependent overexpression of
adenovirally encoded p27 up to 28 days postinjection into the hind
limb. Similarly, Harding and co-workers reported persistent
doxycycline-regulated adenovirus-mediated gene expression up to 3
months after local transfer into neuronal cells in vivo
(56)
. Thus, the long-term expression achieved with
doxycycline-regulatable adenoviral system may be considered for future
experimental and clinical applications. This inducible system may
provide additional advantages over conventional gene therapy
strategies. For example, an inescapable concern when overexpressing a
therapeutic gene is the possibility of undesirable systemic effects,
especially when a potentially harmful gene is being delivered. Thus,
using an inducible strategy to maintain gene expression for a defined
length of time seems sure to mitigate inherent potential risks,
particularly in diseases for which a relatively short period of
treatment might be effective (i.e., postangioplasty restenosis). The
combined use of constitutive and inducible expression systems may be
desirable in cases where the susceptibility of target cells to a given
therapeutic agent depends on exposure to a ‘priming’ agent. Last,
future anti-cancer therapies that rely on the combination of gene
therapy and conventional strategies (i.e., chemotherapy, radiotherapy,
immunotherapy) may also benefit from inducible gene expression.
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ACKNOWLEDGMENTS |
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Received for publication February 14, 2001.
Revision received June 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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