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European Heart Journal (1999) 20, 406–420
Article No. euhj.1998.1308, available online at http://www.idealibrary.com on
Review Article
Cell cycle regulatory molecules (cyclins, cyclin-dependent
kinases and cyclin-dependent kinase inhibitors) and the
cardiovascular system
Potential targets for therapy?
J.-M. Li*‡ and G. Brooks*†
*Cardiovascular Cellular and Molecular Biology, Cardiovascular Research, The Rayne Institute,
St. Thomas’ Hospital, London; †Division of Cell and Molecular Biology, School of Animal and Microbial Sciences,
University of Reading, Whiteknights, Reading, U.K.
The cell cycle as a target for the
treatment of cardiovascular diseases
Our understanding of the mechanisms that control
proliferation of vascular smooth muscle cells, endothelial cells and cardiac myocytes has increased significantly in recent years. Now certain molecules involved in
modulating the cell cycle machinery in these cell types
could serve as suitable targets for the treatment of
certain cardiovascular diseases, such as restenosis and
the repair of myocardial tissue following infarction. The
following sections will provide an overview of the cell
cycle machinery and discuss how this controls the
growth of vascular smooth muscle cells, endothelial cells
and cardiac myocytes.
The cell cycle machinery — a brief
overview
The mammalian cell cycle
Normal cellular growth can be divided into five distinct
phases (Fig. 1). Quiescent cells are found in the G0 phase
Key Words: cardiac myocyte, cyclin, cyclin-dependent
kinase, cyclin-dependent kinase inhibitor, hypertrophy,
vascular smooth muscle cell.
of the cell cycle and exist in a state where mRNA and
protein syntheses are minimal. A cell may remain in this
state for many years, but can re-enter the cycle at the
first gap (G1) phase when stimulated e.g. following
binding of a growth factor to its extracellular receptor[2].
During G1, the cell synthesizes a series of mRNAs and
proteins that are necessary for DNA synthesis (S phase)
following which the cell enters a second gap phase (G2
phase). During G2 the cell synthesizes additional
mRNAs and proteins in preparation for cell division or
mitosis (the M phase), where the cell divides into two
daughter cells[2]. A number of checkpoints exist within
the cell cycle that ensure that cell division proceeds
normally. For example, the primary cell cycle checkpoint in mammalian cells is called the restriction point
and occurs at the G1-S transition (Fig. 1). Once a cell has
passed this point it is committed to a further round of
DNA replication and cell division, except in fully differentiated cells such as adult cardiac myocytes where S
phase can occur in the absence of cell division due to
binucleation and polyploidy[2]. An additional cell cycle
checkpoint exists at the G2-M transition, which prevents
cells with incompletely replicated or damaged DNA
from entering mitosis. Thus, normal cellular proliferation is under tight regulatory controls that monitor
whether conditions are satisfactory for a particular cell
to complete a round of division.
Revision submitted 7 September 1998, and accepted 9 September
1998.
Cell cycle regulatory molecules
‡Present address: Cardiovascular Medicine, King’s College School
of Medicine and Dentistry, Bessemer Road, London, U.K.
The mammalian cell cycle is regulated by the sequential
formation, activation and inactivation of a series of cell
cycle regulatory molecules, that include a group of
regulatory subunits called the cyclins and a group of
catalytic kinase subunits known as cyclin-dependent
Correspondence: Dr Gavin Brooks, Division of Cell and Molecular
Biology, School of Animal and Microbial Sciences, University of
Reading, P.O. Box 228, Reading, Berkshire RG6 6AJ, U.K.
0195-668X/99/060406+15 $18.00/0
1999 The European Society of Cardiology
Review
407
Mitogenic signal
P
cyclin
G0
CDK
CDKI
P
Checkpoint
cyclin
P
CDK
Checkpoint
M
G1
G2
S
cyclin
CDK
P
NA
PC
P
cyclin
E2F
Rb
DP
CDK
E2F
DP
CDKI
P
cyclin
5
3
cyclins A, E and DHFR
CDK
Figure 1 Diagram to show the progression of the mammalian cell cycle and the relationship between
regulatory molecules involved in cell cycle control.
Table 1
Molecules
Cyclins
CDKs
CDKIs
Others
Cell cycle regulatory molecules and where they act in the cell cycle
Cell cycle
G0G1
G1-S
S
G2
M
C, D
4,5,6
p15, p16, p18, p19, p21, p27, p57
p53, pRb, p107, p130
A, E, H, X
CDC2, 2, 5, 7
p21, p27, p57
pRb, E2F, p107, p130
A, E, B
CDC2, 2, 5
p21, p27, p57
E2F, PCNA
B, F, A, H
CDC2, 7
p21
p53
A, B
CDC2
p21
PCNA
References
[2,4,5,8,117–119]
[2,4,5,117–120]
[2,4,5,8,9,11,13]
[12–14,18–22,117,121]
CDKs=cyclin-dependent kinases; CDKIs=cyclin-dependent kinase inhibitors; pRb=retinoblastoma protein; PCNA=proliferating cell
nuclear antigen.
kinases[2–4]. Different cyclins bind specifically to different
cyclin-dependent kinases to form distinct complexes at
specific phases of the cell cycle and thereby drive the cell
from one stage of the cycle to another (Table 1). The
cyclins are a family of proteins which, as their name
suggests, are synthesized and destroyed during each cell
cycle. To date, eight cyclins have been described, cyclins
A, B1,2,3, C, D1,2,3, E, F, G and H, which all share
an 150 amino acid region of homology called the
‘cyclin box’ that binds to the N-terminal end of specific
cyclin-dependent kinases[2,5]. Cyclins C, D and E are
short-lived proteins that function mainly during the
G1 phase and at the G1-S transition before being
destroyed via the ubiquitin pathway[3]. Cyclins A and B,
on the other hand, are mitotic cyclins that remain
stable throughout interphase, but are rapidly proteolysed during mitosis also by a ubiquitin-dependent
pathway[2]. Little information is currently available
regarding the recently described cyclins F and G,
whereas another new cyclin, cyclin H, has been shown to
form complexes specifically with CDK7 to produce an
enzyme known as cyclin-dependent activating kinase
that is involved in the activation of CDC2 and CDK2
kinases[6,7].
Eur Heart J, Vol. 20, issue 6, March 1999
408
J.-M. Li and G. Brooks
The cyclin-dependent kinases are a family of
protein kinases which bind to, and are activated by,
specific cyclins. To date, at least seven cyclin-dependent
kinases have been described viz. CDC2 (CDK1), CDK2,
CDK3, CDK4, CDK5, CDK6 and CDK7. Cyclindependent kinases 4, 5, and 6 complex mainly with the
cyclin D family and function during the G0/G1 phases of
the cycle; CDK2 can also bind the members of the cyclin
D family, but more commonly associates with cyclins A
and E and functions during the G1 phase and during the
G1-S transition. As mentioned above, CDK7 is found in
association with cyclin H and is able to phosphorylate
either CDC2, CDK2 or the C-terminal domain of the
largest subunit of RNA polymerase II, in addition to
the TATA box-binding protein or transcription factor
II E (TFIIE)[6,7]. Finally, CDC2 binds to cyclins A
and B and functions in the S, G2 and M phases of the
cell cycle.
In addition to a series of positive regulators
of the cell cycle, i.e. the cyclins and cyclin-dependent
kinases, a family of negative regulators, called cyclindependent kinase inhibitors, also exist and exert effects
during the cell cycle[5,8–10]. In mammalian cells, the
cyclin-dependent kinase inhibitors can be divided
structurally into two distinct families: the INK4 family
and the KIP/CIP family (reviewed in[9]). The INK4
family includes p14, p15 (INK4B), p16 (INK4A), p18
(INK4C) and p19 (INK4D), which inhibit specifically
cyclin D/CDK4 and cyclin D/CDK6 complexes and are
involved in G1 phase control. The KIP/CIP family
includes p21 (CIP1/WAF1/SDI1), p27 (KIP1) and p57
(KIP2), which are 38–44% identical in the first 70 amino
acid region of their amino termini, a region that is
involved in cyclin binding and kinase inhibitor
function[9–11]. The CIP/KIP family displays a broader
specificity than the INK4 family, since members interact
with, and inhibit the kinase activities of, cyclin E/CDK2,
cyclin D/CDK4, cyclin D/CDK6, cyclin A/CDK2 and
cyclin B/CDC2 complexes, and function throughout
the cell cycle (reviewed in[9]). The tumour suppressor
protein, p53, also plays an important role in cell cycle
arrest at the G1 and G2 checkpoints subsequent to
inducing apoptosis[12–14]. The p53 protein has a central
sequence-specific DNA binding domain and a transcriptional activation domain at its amino-terminus
and, in response to DNA damage, it can induce the
transcription of the cyclin-dependent kinase inhibitor,
p21, which inhibits the activation of various G1 cyclin/
cyclin-dependent kinase complexes[11,14].
A variety of other gene products, e.g. the proliferating cell nuclear antigen, the retinoblastoma protein
and members of the E2F family of transcription factors,
can also interact with, and modulate the activities of,
cyclin/cyclin-dependent kinase complexes (Fig. 1 and
Table 1). Proliferating cell nuclear antigen is a cofactor
of DNA polymerase ä, and is required during DNA
replication and repair. In mammalian cells, proliferating
cell nuclear antigen is found associated with the cyclin
B/CDC2 complex and p21[11,15]. p21 can bind directly to,
and inhibit the function of, proliferating cell nuclear
Eur Heart J, Vol. 20, issue 6, March 1999
antigen[15–17]. Retinoblastoma protein is a negative regulator of cell growth and is expressed throughout the cell
cycle (reviewed in[18,19]). The ability of retinoblastoma
protein to suppress cellular proliferation is controlled
by its cell cycle dependent phosphorylation, such that
the protein is hypophosphorylated in the G0/G1 phase
(active form) of the cell cycle and becomes highly
phosphorylated (inactive form) following the activation
of CDC2 and cyclin-dependent kinases 2, 4, 5 and 6
containing complexes beginning in late G1 and continuing through until M phase. Only the hypophosphorylated form of retinoblastoma protein can interact with
a variety of cellular proteins, such as the transcription
factor E2F, to inhibit cellular proliferation e.g. by
inhibiting E2F transcriptional activity[18,19]. Two other
proteins, p107 and p130, share structural homology and
functional activity with retinoblastoma protein, and so
are called retinoblastoma protein-related proteins[20,21].
Different members of the retinoblastoma protein family
apparently regulate specific E2F family members such
that retinoblastoma protein shows higher binding affinity to E2F-1, 2 and 3, whereas p107 and p130 display
greater affinities towards E2F-4 and 5[21,22]. The E2F
transcription factor family comprises two distinctly related subfamilies, E2F and DP[20,23,24]. One subunit of
E2F combines with one subunit of DP to form an active
heterodimer which is essential for high-affinity DNA
binding, transcriptional activity, and binding to retinoblastoma protein-related proteins[20,25,26]. Following
activation of the cell cycle activated G1 cyclin/cyclindependent kinase complexes e.g. cyclin D/CDK4 phosphorylate the retinoblastoma protein component of the
E2F-DP-retinoblastoma protein complex, resulting in
dissociation of E2F-DP from retinoblastoma protein.
The free E2F-DP heterodimer functions as a transcription factor and controls the expression of various genes
that are essential for the G1 to S phase transition and for
DNA replication e.g. cyclin A, dihydrofolate reductase
and cyclin E[26,27]. Furthermore, it has been suggested
that different E2F members, together with their primary
retinoblastoma protein protein regulators, control different sets of genes at different stages of the cell cycle[22]
and recently it has been shown that E2F family members
can also interact directly with cyclin/cyclin-dependent
kinase complexes[25].
Recent studies have demonstrated the importance of E2F transcription factors and other cell cycle
regulatory molecules in controlling the growth of vascular smooth muscle cells both in vitro and in vivo. The
following sections will review our current understanding
of cell cycle control in vascular disease and discuss
recent studies that have targeted certain cell cycle
regulatory molecules in cardiovascular cells.
Cell cycle control in vascular disease
The rapid and recent advances in our understanding
of cell cycle control in various mammalian systems has
Review
Table 2
mRNA
Protein
Activity
409
Expression of cell cycle regulatory molecules in vascular smooth muscle cells
Species
Quiescent
Mitogen stimulated
Reference
human
bovine
rat
human
human
CDC2
cyclin D1; CDKs 2 and 4
CDC2
cyclin D1; CDKs 2 and 4
cyclin A
cyclin E; CDK12; p21 and p27
cyclins D1, D2, D3, C, G, A, E, B;
CDKs 2, 4, 5 and CDC2; pRb
CDC2
cyclins A and E; CDK2; p21
[38]
human
human
human
rat
rat
human
rat
rat
cyclin E; CDK2; p21
cyclins D2, D3, C and G; CDKs 2, 3, 4 and 5; pRb
CDC2
cyclin E; CDK2; p21 and p27
p21
pRb
CDC2
CDKs 2 and 4
CDC2
led to the feasibility of targeting certain cell cycle
regulatory molecules in vascular smooth muscle cells for
the treatment of diseases such as atherosclerosis and
restenosis. Novel approaches for the treatment of such
diseases are of paramount important to reduce the
mortality and morbidity associated with such disorders.
Indeed, atherosclerosis is the principal cause of heart
attack, stroke and gangrene of the extremities, and is
responsible for about 50% of all mortality in the U.S.A.,
Europe and Japan[28,29]. Both endothelial cells and vascular smooth muscle cells are thought to play a role in
this disease such that endothelial dysfunction is believed
to play a critical role in the early stage of atheroma in
native arteries, and is associated with the development
of severe neointimal lesions in human vein grafts[30],
whereas rapid pathological vascular smooth muscle cell
proliferation is the prominent feature of neointimal
formation[31,32]. Conventional treatments for atherosclerosis principally involve surgical approaches, such as
bypass grafting, endarterectomy, and percutaneous
transluminal coronary angioplasty (PTCA). Although
PTCA has proved to be a very successful approach for
the treatment of ischaemic injury during the past decade,
enthusiasm for this intervention has decreased recently
due to subsequent and significant symptomatic
restenosis which occurs in about one third of cases and
is resistant to most available pharmacological interventions[33]. The failure of conventional therapy in
restenosis has led to investigators turning their attentions to alternative approaches, such as targeting
expression and activities of specific cell cycle regulatory
molecules, in an effort to inhibit vascular smooth
muscle cell proliferation and to improve endothelial cell
function after mechanical injury.
The following sections will outline the expression
of cell cycle regulatory molecules in vascular smooth
muscle cells and endothelial cells and describe some of
the in vitro and in vivo studies that have targeted specific
molecules to inhibit cellular proliferation.
pRb
CDC2
CDKs 2 and 4
CDC2
CDC2
[42]
[39]
[43]
[44]
[38]
[43]
[122]
[34]
[35]
[44]
[34]
[35]
Expression of cell cycle regulatory molecules
in vascular smooth muscle cells
The expression of cell cycle regulatory molecules in
vascular smooth muscle cells has been examined
extensively at the mRNA level using cells isolated from
different species and sources such as aortic tissue
from rat[34–41], bovine[38,42], and human sources and
from human umbilical arteries[38,39,43,44]. Table 2
summarizes the expression of various cell cycle regulatory molecules in quiescent vascular smooth muscle cells,
which express significant mRNA levels of cyclin G,
cyclin C, CDK4 and CDK5. Expression of cyclin D1,
cyclin D2, cyclin D3, CDK3 and CDK2 mRNAs are low
in these cells and no mRNA levels are detectable for
cyclins A, E, and B, and CDC2[39,42,44]. Quiescent vascular smooth muscle cells have also been shown to
express high levels of hypophosphorylated retinoblastoma protein consistent with the fact that these cells
are not in cycle. Serum stimulation of human vascular
smooth muscle cells increases significantly the mRNA
expression of cyclins D1, D3, A, E, and B, CDK2 and
CDC2[38,39,44], although no effects on the mRNA expressions of cyclins D2, C and G, and CDK4 were observed.
Interestingly, serum stimulation of human vascular
smooth muscle cells suppresses the mRNA expression of
CDK3 and CDK5, suggesting that these cyclindependent kinases may not be involved in the proliferative response of vascular smooth muscle cells after serum
stimulation. Alternatively, cyclin-dependent kinases 3
and 5 may function as growth suppressors in vascular
smooth muscle cells and maintain these cells in a differentiated and quiescent state. Indeed, CDK5 and its
co-activator, Xp35.1, have recently been demonstrated
to play a significant role in regulating early embryonic
muscle formation in Xenopus by suppressing myogenic
gene expression, including MyoD and MRF4[45].
Homocysteine, which stimulates DNA synthesis
and cellular proliferation in vascular smooth muscle
Eur Heart J, Vol. 20, issue 6, March 1999
410
J.-M. Li and G. Brooks
cells, is elevated in patients at increased risk of thrombotic and atherosclerotic vascular disease and represents
an independent risk factor for these diseases[46]. Interestingly, homocysteine has been shown to induce significantly cyclin A expression and to up-regulate the kinase
activity of CDC2[34,35], whereas TGFâ1 and TNP-470 (a
fumagillin analogue, which has been reported to suppress vascular smooth muscle cell proliferation) inhibits
CDK2 and CDC2 kinase activities, respectively, resulting in an inhibition of DNA synthesis and cellular
proliferation[38,42]. Similar results on cell growth have
been reported in other cell systems exposed to TGFâ,
since it is known that this cytokine induces expression
of the cyclin-dependent kinase inhibitor molecule,
p21[38,47]. In addition, treatment of lung epithelial cells
with TGFâ has been shown to induce p15 (INK4B)
expression that binds to and inhibits cyclin D-dependent
kinases[48]. This induction of p15 prevents p27 (that
is predominantly bound to cyclin D/CDK4 without
inhibiting this kinase in dividing cells) from binding to
these CDK complexes and promotes p27 binding and
inhibition of cyclin E/CDK2[48]. Thus, TGFâ-mediated
cell cycle arrest is a complex and multifaceted process.
The results reported above were obtained from
in vitro studies using cultured vascular smooth muscle
cells. Recently, an in vivo study has investigated the
effects of PTCA on the expression and activities of
certain cell cycle regulatory molecules in human coronary and rat carotid arteries[49]. Expression of CDK2
protein and CDK2 kinase activity were very low in
uninjured rat carotid artery wall, whereas cyclin A,
cyclin E, and proliferating cell nuclear antigen protein
expression were not detectable in this tissue by immunoblotting. However, a marked induction of cyclins A and
E, CDK2 and proliferating cell nuclear antigen protein
expression was observed in rat carotid arteries, concomitant with an increase in the level of CDK2 kinase
activity, within the first 2 days following balloon angioplasty, with these high levels of protein expression being
sustained for up to 10 days following injury consistent
with the period when neointimal growth is most
rapid[31,32]. Subsequently, the expression of these molecules returned to control levels in surrounding areas,
although levels remained upregulated in the intimal
lesion for up to 14 days after injury[49]. These findings
were substantiated when tissues from human atherosclerotic lesions obtained by directional atherectomy
were analysed. In accordance with the data obtained
from animal studies, abundant expression of CDK2,
cyclins A, E and proliferating cell nuclear antigen were
observed in regions of human restenotic lesions which
were marked by vascular smooth muscle cell proliferation[49] thereby implicating these molecules in the increased growth of these cells following injury. Sylvester
and colleagues[50] have recently extended these studies
and investigated the mechanisms that regulate transcription of the cyclin A gene following angioplasty. These
authors showed that a Ras-dependent mitogenic signalling pathway is essential for normal stimulation of cyclin
A promoter activity and DNA synthesis in rat vascular
Eur Heart J, Vol. 20, issue 6, March 1999
smooth muscle cells. In addition, E2F was shown to be
essential for both serum- and c-fos-dependent induction
of cyclin A expression[50]. Evidence for differences in the
proliferative, senescent and apoptotic responses of vascular smooth muscle cells derived from normal vessels
and those from human atherosclerotic plaques being due
to alterations in cell cycle proteins has been reported
recently by Bennett and colleagues[51]. These authors
demonstrated that human plaque vascular smooth
muscle cells have slower rates of cellular proliferation
and senesce earlier than cells from normal vessels due
to a defect in the phosphorylation of retinoblastoma
protein. Furthermore, disruption of the retinoblastoma
protein-E2F complex and inhibition of p53 were shown
to be required for plaque vascular smooth muscle cells
to proliferate without apoptosis. This latter study highlights the fact that cells present in diseased vascular
tissue differ significantly in terms of their cell cycle
machinery from cells found in normal vessels. Thus, it
may be possible to specifically target those cell cycle
molecules that are altered in atherosclerotic/restenotic
cells as an alternative approach to therapy.
Recently, Tanner and colleagues reported the
expression profiles of certain cyclin-dependent kinase
inhibitor molecules in normal and diseased vascular
tissues[52]. Thus, immunoblotting and fluorescenceactivated cell sorting analysis showed that p27, but not
p16, protein levels were increased during serum deprivation of primary porcine aortic vascular smooth muscle
cells leading to a G1 arrest. This was proposed to be due
to binding of p27 to cyclin E/CDK2 complexes in these
cells[52]. In normal porcine arteries, p27, but not p16,
protein was expressed constitutively at low but detectable levels. However, immediately following balloon
injury, vascular smooth muscle cell proliferation increased with a concomitant decrease in p27 levels that
remained low until approximately 3 weeks after injury.
In contrast, p16 levels increased transiently following
injury such that maximal levels of this cyclin-dependent
kinase inhibitor were reached 7 days after intervention.
Three weeks following injury, when vascular smooth
muscle cell proliferation had decreased to <27%, p27
levels increased again suggesting that p27, and possibly
p16, functions as an inhibitor of cell proliferation
in injured vessels. These authors also investigated the
expression of p16, p21 and p27 in human coronary
arteries, obtained from transplant patients, that varied
in their atherosclerotic status from normal atherosclerosis through to advanced disease[52]. Interestingly,
p27 levels were abundant in non-proliferative vascular
smooth muscle cells and macrophages from all stages
of disease, whereas p21 levels were only detected in
advanced atherosclerosis; p16 was not detected at any
stage of disease. These results suggest that p27 may
function to maintain normal vascular smooth muscle
cells in G0/G1 and to inhibit cellular proliferation during
the latter phases of arterial repair. p21, on the other
hand, may function as a co-factor that is induced during
the latter phases of remodelling to inactivate cyclin/
cyclin-dependent kinase complexes and cause G1 arrest.
Review
Table 3
mRNA
Protein
Activity
411
Expression of cell cycle regulatory molecules in endothelial cells
Species
Quiescent
Mitogen stimulated
Reference
human
human
human
human
human
bovine
human
human
human
human
human
CDK2
cyclin D1; CDKs 4 and 2
cyclins D1, E; CDKs 4, 2; E2F1
cyclins A and B
cyclin D1
CDK2, CDC2
cyclins D1, A and E; CDKs 4, 2 and CDC2
cyclins D1, A, E; CDKs 4, 2 and CDC2; E2F1
cyclins A and B
cyclin D1; CDK2
cyclin A
p53, p21
pRb
cyclin B; CDC2
CDK2, CDC2
CDK2, CDC2
[55]
p53, p21
CDK2, CDC2, pRb
cyclin B
CDK2
CDK2, CDC2
Expression of cell cycle regulatory molecules
in endothelial cells
Most data relating to the expression of cell cycle regulatory molecules in endothelial cells have been obtained
from studies on human umbilical vein endothelial cells
and are summarized in Table 3. At the mRNA level,
quiescent endothelial cells express relatively high levels
of CDK4, low levels of cyclin D1 and CDK2, and barely
detectable levels of cyclin A, cyclin E, CDC2 and
E2F1[53–56]. Kinase activities of CDC2 and CDK2, as
determined by histone H1 phosphorylation assay after
immunoprecipitation of CDC2 and CDK2, were detectable at basal levels in the lysates of quiescent endothelial
cells[53,55] whereas stimulation of these cells with growth
factors resulted in a significant increase in kinase
activities of CDK2 and CDC2 with a time course that
correlated closely with the period when cells started to
enter S phase[54–56]. In addition, the mRNA expressions
of cyclin D1, cyclin A, cyclin E, CDK2, CDC2 and
E2F1 all increased significantly within 12 h of stimulation of quiescent cells with growth factors. The
fumagillin analogue, AGM-1470, a potent inhibitor of
endothelial cell proliferation and angiogenesis, was
shown to suppress significantly the growth factorinduced increase in mRNA expression of cyclin A and
cyclin E and also inhibited CDC2 and CDK2 kinase
activities, although there was no effect on CDK4 mRNA
expression or kinase activity[53]. Furthermore, exposure
to this agent inhibited DNA synthesis and cellular
proliferation[53]. The cyclin B/CDC2 complex was undetectable at the protein level in quiescent endothelial
cells but was inducible with serum and growth factors.
Furthermore, phorbol 12-myristate 13-acetate significantly suppressed the growth factor-induced protein
expression of cyclin B and cyclin A and inhibited
endothelial cell proliferation, thereby implicating a role
for protein kinase C in modulating endothelial cell
growth[57]. Similar results were obtained with bovine
aortic endothelial cells such that cyclin A mRNA was
detectable in growing cells but was undetectable in
confluent cell cultures suggesting that endothelial cell
[53]
[56]
[57]
[54]
[58]
[123]
[55]
[57]
[55]
[53]
proliferation, was contact inhibitible and was associated
with the suppression of cyclin A gene expression[58].
Therapeutic manipulation of cell cycle
regulatory molecules in vascular
smooth muscle cells and endothelial
cells
Recent studies have illustrated the feasibility of targeting specific cell cycle molecules in cardiovascular cells
as a novel approach for drug therapy. Three major
approaches have been used viz. oligodeoxynucleotide
therapy, adenoviral and adeno-associated viral vectors
and haemagglutinating virus of Japan (HVJ)-liposomemediated transfer as described below.
Oligodeoxynucleotide therapy — antisense
and decoy strategies
The most common approach for manipulating the
expression of cell cycle molecules in cardiovascular cells
has been to use synthetic antisense oligodeoxynucleotides that are complimentary to the coding RNA
(sense) strand either to block the function of a specific
gene by interfering with its expression, or to produce unreadable double-stranded areas in the mRNA
molecule and to mark the mRNA for destruction by
ribonuclease H[59,60]. An alternative approach to
traditional antisense therapy is to design oligodeoxynucleotides that are capable of either binding to specific
DNA elements to affect DNA unwinding, or binding to
transcription factors and thereby blocking their function. This commonly is referred to as a decoy approach
(for reviews see references[59] and [60]).
Antisense oligodeoxynucleotide therapy
Cell cycle regulatory molecules that are involved in the
late G1 phase and G1 to S phase transition e.g. cyclins,
cyclin-dependent kinases and proliferating cell nuclear
Eur Heart J, Vol. 20, issue 6, March 1999
412
J.-M. Li and G. Brooks
Table 4
Therapeutic manipulation of cell cycle regulatory molecules using oligonucleotides (ODNs) in animal models
Gene
ODN
Species
Blood vessel
Transport
Duration
(weeks)
Neointimal
inhibition
In vitro
inhibition
CDC2+PCNA
CDC2+PCNA
PCNA
CDK2
CDC2
CDK2+CDC2
CDK2
CDC2
cyclin B1+CDC2
CDK2
E2F
AS
AS
AS
AS
AS
AS
AS
AS
AS
AS
decoy
rabbit
rat
rat
rat
rat
rat
rat
rat
rat
mouse
rat
jugular vein graft
carotid artery
carotid artery
carotid artery
carotid artery
carotid artery
carotid artery
carotid artery
carotid artery
coronary artery
carotid artery
HVJ-liposome
HVJ-liposome
pluronic gel
HVJ-liposome
HVJ-liposome
HVJ-liposome
pluronic gel
pluronic gel
HVJ-liposome
HVJ-liposome
HVJ-liposome
2
2
2
2
2
2
4
4
2
8
2
>90%
50–6-%
80%
60%
40%
85%
55%
47%
78%
54%
74%
not done
40% VSMC
70% VSMC
not done
not done
not done
not done
not done
not done
not done
25% VSMC
Reference
[62]
[63]
[124]
[64]
[64]
[64]
[65]
[65]
[66]
[68]
[70]
PCNA=proliferating cell nuclear antigen; AS=antisense; HVJ-liposome=haemagglutinating virus of Japan-liposome-mediated transfer;
VSMC=vascular smooth muscle cells.
antigen are suitable targets for antisense oligodeoxynucleotide therapy (for summary see Table 4). By
chemically modifying the oligodeoxynucleotides, or by
packing them into liposomes with viral coat proteins,
the efficiency of delivery of such oligodeoxynucleotides
into injured vessel walls has been much improved, and
recently oligodeoxynucleotides have been delivered
intraluminally via a catheter[61]. The principal animal
model used for gene therapy studies is the ballooninjured rat carotid artery model. Several groups have
demonstrated that a single dose of antisense oligodeoxynucleotides directed against CDK2 and/or CDC2,
and proliferating cell nuclear antigen can effectively
inhibit vascular smooth muscle cell proliferation for an
extended period of several weeks following balloon
injury[62–66]. Thus, Morishita and colleagues have demonstrated that intraluminal delivery of a single bolus
dose of antisense phosphorothioate oligodeoxynucleotides directed against CDK2 or CDC2, using
HVJ-liposome-mediated transfer, significantly inhibited
neointimal formation by 60% for CDK2 and 40% for
CDC2 compared with injured vessels treated with sense
oligodeoxynucleotides or left untreated[64]. Furthermore,
the combination of two antisense oligodeoxynucleotides
(CDK2 plus CDC2) abolished neointimal formation
completely. Using FITC-labelled antisense oligodeoxynucleotides, these authors were also able to demonstrate
an immediate localization of these molecules to the
medial layer of the rat carotid artery, which persisted for
up to 2 weeks after transfection[64]. Antisense oligodeoxynucleotide therapy has also been used to prevent
atherosclerosis in rabbit jugular vein grafts reverse
interposited to the carotid artery[62,67]. Similarly, local
application of antisense oligodeoxynucleotides against
proliferating cell nuclear antigen and CDC2 resulted
in a significant preservation of normal endothelial
phenotype and function[67], accompanied by a significant
inhibition of neointimal formation[62]. Most recently,
antisense oligodeoxynucleotides have been used to prevent coronary graft arteriosclerosis following cardiac
Eur Heart J, Vol. 20, issue 6, March 1999
transplantation into mice[68]. Thus, a single intraluminal
delivery of an antisense oligodeoxynucleotide directed
against the CDK2 gene into donor hearts resulted in
oligodeoxynucleotide stability within the coronary vessel
wall and prevented arterial neointimal formation that
was shown to persist for up to 30 days following cardiac
transplantation[68].
Decoy oligodeoxynucleotide therapy
An alternative approach to antisense therapy is the use
of decoy oligodeoxynucleotides to block the binding
site(s) of certain transcription factors, such as nuclear
factor-êB[69] and E2F[70]. This method has been used
successfully to block E2F-mediated transcription using
synthetic double-stranded DNA containing a sequence
with high specificity and affinity to bind E2F, thereby
preventing the transactivation of cell cycle regulatory
genes by this transcription factor[70]. Indeed, it has been
shown that transfection of vascular smooth muscle cells
with an E2F decoy sequence inhibited significantly
serum stimulated cell proliferation in vitro. Furthermore, it was shown that local administration of the E2F
decoy oligodeoxynucleotide to balloon-injured vessels
prevented significantly neointimal formation in the rat
carotid artery wall after angioplasty[70].
Adenoviral and adeno-associated viral
vectors
Significant advances in cardiovascular gene therapy
have been made recently with the use of modified viruses
designed to carry the target gene directly into the local
arterial wall. Adenoviral vectors are one of the most
commonly used vectors for introducing genes into the
cardiovascular system since they accept relatively large
recombinant genes (up to 7·5 kilobases) and can be
propagated easily in mammalian cell culture. In
addition, infection with an adenoviral vector does not
Review
Table 5
models
413
Therapeutic manipulation of cell cycle regulatory molecules using adenoviral vectors in experimental animal
Gene
Species
Blood vessel
Delivery
method
Duration
days
Neointima
inhibition
Proliferation
inhibition
p21
p27
p21
pRb
pRb
porcine
rat
rat
rat
porcine
iliofemoral artery
carotid artery
carotid artery
carotid artery
femoral artery
catheter
catheter
catheter
catheter
catheter
28
14
20
20
21
37%
49%
46%
50%
47%
90% on VSMCs
not done
60% on VSMCs
90% on VSMCs
not done
require a replicating cell, making it advantageous for
gene delivery into quiescent vascular smooth muscle
cells, endothelial cells and/or cardiac myocytes (see
later). Because the adenovirus used for gene therapy is
replication defective, its expression is limited to only a
few critical days or weeks following angioplasty. This is
a particularly useful property for limiting neointimal
hyperplasia following PTCA since it is known that
vascular smooth muscle cell outgrowth is most rapid
during the first 3 weeks following angioplasty[31,32].
Thus, if the affected tissue could be protected during this
period, the degree of restenosis potentially could be
minimized. In addition, adenoviral vectors do not
integrate into the genome thereby reducing potential
risks of insertional mutagenesis[33]. Most often, adenoviral vectors are introduced directly through a catheter
into a balloon-injured artery immediately following
angioplasty. Cell cycle inhibitory molecules, such as
cyclin-dependent kinase inhibitors and retinoblastoma
protein, are suitable candidates for gene therapy to
prevent proliferative disorders such as restenosis (Table
5) and two independent groups of investigators have
studied the effects of overexpression of the cyclindependent kinase inhibitor molecule, p21 in vitro and
in vivo using either porcine femoral artery[71] or rat
carotid artery[72] following balloon injury. Thus, forced
expression of p21 in cultured vascular smooth muscle
cells inhibited significantly cell proliferation as compared with cells transfected with vector alone[71,72] and
direct intraluminal delivery of the p21 gene into the
injured arterial wall resulted in a significant reduction in
neointimal formation to the extent of 37% in porcine
femoral arteries[71] and 46% in rat carotid arteries[72].
Similar results were obtained using the cyclin-dependent
kinase inhibitor molecule, p27, transfected by adenovirus into the rat carotid arterial wall following balloon
injury[37]. Thus, increased expression of p27 suppressed
significantly CDK2 and cyclin A promoter activities
concomitant with the inhibition of vascular smooth
muscle cell proliferation, and produced a 49% reduction
in neointimal formation[37]. The tumour suppressor gene
product, retinoblastoma protein, has also been used as a
candidate to inhibit neointimal formation following
balloon angioplasty[72]. Thus, transfection of cultured
rat vascular smooth muscle cells with an adenoviral
vector expressing a constitutively active form of retinoblastoma protein inhibited cellular proliferation significantly[73]. Local transfection of activated retinoblastoma
Reference
[71]
[37]
[72]
[73]
[73]
protein into balloon-injured rat carotid and porcine
femoral arterial walls inhibited neointimal formation by
up to 45–50% as compared with arterial walls transfected with vector alone for both animal models[73].
One of the major problems associated with currently available adenoviral vectors is the significant
immune responses and inflammation that occur in the
infected tissues[74]. Such immune responses often result
in short-lived gene expression because of loss of transduced cells and also limit the efficacy of repeat delivery.
Vector systems based on adeno-associated viruses may
overcome certain of these limitations and are currently
being investigated for vascular gene delivery. Lynch and
colleagues[74] recently reported the successful transduction of primary cultures of rat, rabbit, monkey and
human vascular smooth muscle cells and monkey and
human endothelial cells with recombinant adenoassociated virus vectors expressing the alkaline phosphatase reporter gene. These authors also demonstrated
successful recombinant adeno-associated virus delivery
in vivo to arteries of atherosclerotic cynomolgus
monkeys[74].
One advantage of using either adenovirus or
adeno-associated virus delivery is the bystander effect
observed in surrounding non-transduced cells that
demonstrate similar effects to those cells that have been
transduced by the virus. Thus, a pronounced bystander
effect was observed in the non-transduced neighbouring
cells of rat aortic vascular smooth muscle cells transduced with an antisense cyclin G1 retroviral vector[75]. A
similar effect was observed in a recent study by Harrell
and colleagues[76] who showed that vascular smooth
muscle cells infected with an adenovirus encoding cytosine deaminase exhibited a profound bystander effect on
the growth of neighbouring cells, which did not require
cell-to-cell contact.
HVJ-liposome-mediated transfer
The human p21 gene has recently been transfected into
human aortic vascular smooth muscle cells using HVJliposome-mediated transfer[77]. The results of this study
showed a significant increase in p21 protein expression
compared with cells transfected with vector alone and
this increase in p21 was associated with a decrease in
vascular smooth muscle cell proliferation. Furthermore,
overexpression of p21 led to apoptosis in transfected
Eur Heart J, Vol. 20, issue 6, March 1999
414
J.-M. Li and G. Brooks
vascular smooth muscle cells as demonstrated by cell
shrinkage, membrane blebbing, cell rounding and DNA
laddering[77].
In a separate study, Yonemitsu et al. showed that
HVJ-liposome-mediated transfer of human wild-type
p53 cDNA into bovine aortic vascular smooth muscle
cells in vitro led to a decrease in thymidine incorporation
and S phase concomitant with a transient increase in
G2/M 2 days after gene transfection, although almost all
cells were shown to have arrested in G1 by 5 days after
transfection[78]. Interestingly, overexpression of p53 did
not lead to apoptosis in these cells despite the fact that
p53 is known to inactivate G1 cyclin-associated cyclindependent kinase activities through a direct activation of
p21 levels[11,14]. These authors showed also that wildtype p53 transfected into balloon-injured rabbit carotid
arteries suppressed neointimal formation significantly[78]. Thus, p53 overexpression in the vessel wall
may offer an approach for the treatment of restenosis
occurring after vascular intervention.
Taken together, the experiments described above
demonstrate clearly that cell cycle regulatory molecules
are suitable targets for developing alternative strategies
against restenosis after angioplasty.
Expression of cell cycle regulatory
molecules in cardiac myocytes
Mammalian cardiac myocytes proliferate actively during
fetal and early neonatal development and grow both by
hyperplasia and hypertrophy. However, the ability of
cardiac myocytes to divide ceases completely shortly
after birth with all subsequent growth of cardiac muscle
occurring by an increase in myocyte size (for review
see[5] and [10]). The adult myocyte does, however, retain
the ability to undergo DNA synthesis following e.g.
haemodynamic overload, suggesting that these cells are
capable of re-entering the cell cycle although there is no
evidence that demonstrates that such cells undergo
mitosis. However, Anversa and Kajstura[79] have recently suggested that such cells can divide, although
Soonpaa and Field[80] have questioned this proposal.
The inability of mature cardiac myocytes to undergo cell
division leads to major problems following severe injury
such as myocardial infarction, since the heart is unable
to regenerate new myocytes to replace necrotic or
damaged tissue. An understanding of the molecules
involved in controlling the growth potential of cardiac
myocytes during development may enable us to develop
alternative strategies aimed at re-initiating DNA synthesis and cell division in a controlled manner to repair
damaged myocardium.
Expression of cell cycle regulatory molecules
during cardiac development
Recently, our laboratory and others have characterized
in detail the expression of a range of cell cycle regulatory
Eur Heart J, Vol. 20, issue 6, March 1999
molecules during cardiac development in the rat[81–91].
Thus, fetal rat cardiac myocytes express high levels of
cyclins D1, D2, D3, C, A, E and B at both mRNA and
protein levels[81,84,88,89] and additionally express high
levels of CDC2, cyclin-dependent kinases 2, 4, 5, 6 and
proliferating cell nuclear antigen proteins and high
kinase activities of CDC2 and cyclin-dependent kinases
2, 4, 5 and 6, which correlate closely with the proliferative capacity of these cells[81]. Shortly after birth, the
protein expression profile of cyclins D1, D2, D3, A, E,
CDC2 and cyclin-dependent kinases 2, 4, 5 and 6 in
cardiac myocytes becomes progressively and significantly down-regulated such that levels of each of these
cyclin and cyclin-dependent kinase molecules were
down-regulated in 2-day-old rat myocytes compared
with levels expressed in fetal myocytes obtained from the
hearts of fetuses at 18 days gestation. Furthermore,
the protein levels of cyclins A, B, D1, E and CDC2
were undetectable in adult cardiac myocytes by
immunoblotting[81–88]. Sadoshima and colleagues have
recently compared how the expressions of certain cell
cycle regulatory molecules change in cultured rat neonatal myocytes stimulated with angiotensin II (a
hypertrophic stimulus) and serum (a mitogenic stimulus)[85]. Interestingly, these authors demonstrated that
the mRNA and protein expressions of cyclins A, D1 and
D3 and the activity of CDK2 were down-regulated in
cultured neonatal cardiac myocytes exposed to angiotensin II, but were up-regulated when exposed to a
mitogenic agent such as serum. Serum increased the
expression of G1 to S phase cyclins and stimulated the
kinase activities of CDK2, CDK4 and CDC2, but failed
to stimulate significantly DNA synthesis in cultured
neonatal cardiac myocytes[85]. Recently, Flink and colleagues have reported that specific changes in E2F
complexes occur in rat cardiac myocytes during the fetal
to neonatal transition[89]. Thus, E2F is complexed with
p107 in proliferating fetal myocytes, whereas myocytes
obtained from 2-day-old animals contained E2F that
was principally associated with p130 and a lower level of
retinoblastoma protein[89]. These results strongly implicate the existence of a potent cell cycle inhibitory system
in terminally differentiated cardiac myocytes.
Recently, observations from our laboratory and
others have confirmed that, in addition to the downregulation in the expression of cyclins and cyclindependent kinases, which may contribute to the
permanent withdrawal of adult myocytes from the cell
cycle as discussed above, there is a concomitant and
specific up-regulation of the cyclin-dependent kinase
inhibitor molecules, p21 and p27, during normal development of rat cardiac myocytes[10,83,87,92]. Furthermore,
we have demonstrated, by immunocytochemistry, that
p21 and p27 are expressed in the nuclei of cardiomyocytes[82], and levels are upregulated at both mRNA and
protein levels during the fetal to adult developmental
period[83]. Despite numerous attempts, we have been
unable to demonstrate the expression of any INK4
family members at the protein level in freshly isolated
rat cardiac myocytes prepared from fetal (18 days of
Review
Expression of cell cycle regulatory molecules
during pressure overload-induced cardiac
hypertrophy
During the development of pressure overload on the
heart, the cardiac myocyte responds with an adaptive
hypertrophic growth response[94–96]. However, if the
increase in haemodynamic load persists, as occurs in
arterial hypertension or following valvular disease, longstanding cardiac hypertrophy leads to heart failure
and/or sudden cardiac death[94,96]. From a clinical point
of view, an identification of the molecular switches
which control cardiac myocyte proliferation and hypertrophy may enable us to develop strategies aimed at
regenerating new adult ventricular myocytes from
healthy cells that surround infarcted areas[97]. Accordingly, we have monitored the expression of certain cell
cycle regulatory molecules in adult myocytes during the
development of pressure overload-induced left ventricular hypertrophy in rats[82–98]. Interestingly, we observed
a transient, but significant, down-regulation of p21 and
p27 mRNA and protein levels[82,98], accompanied by a
concomitant up-regulation in the expression and activities of cyclin D2, cyclin D3, CDK4 and CDK6 complexes in left ventricular tissue during the first 2 weeks
following aortic constriction compared with hearts
obtained from sham-operated and normal (without
operation) control animals (Fig. 2)[82,98,99]. There was
also a reproducible up-regulation of CDK2 protein
expression and activity from day 7 to 14 in myocytes
after the imposition of aortic constriction, although this
did not reach statistical significance[99]. Thus, changes in
the expression and activities of certain cell cycle regulatory molecules are associated with the development of
cardiac myocyte hypertrophy, and it would appear that
the cell cycle machinery in adult cardiac myocytes
reverts to the fetal–neonatal programme of expression
during hypertrophic growth following pressure overload, in a similar manner to changes reported for other
genes/proteins e.g. contractile proteins[94,100] and TGFâ
isoforms[101,102]. In accordance with our results, Chen
2·0
Densitometry index
Aortic concentration
1·5
1·0
0·5
0
7
14
21
28
35
42
2·0
Sham operation
Densitometry index
gestation), neonatal (2 days after birth) or adult hearts
(Poolman and Brooks, unpublished observations).
Horky and colleagues have recently monitored day to
day changes of p21 positive myocytes in the rat during
the first week following birth using immunocytochemistry[87]. These investigators observed a measurable
increase in the number of p21 positive myocytes from
day 3 to day 6 (from 20% to 87%) after birth, which
correlated closely with the time when rat ventricular
myocytes switch from hyperplastic to hypertrophic
growth[93]. Therefore, it is possible that certain cyclindependent kinase inhibitors play a pivotal role in the
transition of myocyte growth from a hyperplastic to
hypertrophic phenotype by acting as a ‘brake’ to produce cardiac myocyte cell cycle arrest during early
neonatal development.
415
1·5
1·0
0·5
0
7
14
21
28
Days post-operation
35
42
Figure 2 Reciprocal expression of cyclin-dependent
kinase inhibitor (p21) and CDK4 during the development
of pressure overload-induced left ventricular hypertrophy.
Left ventricular tissues obtained from adult rats that
underwent either aortic constriction or sham-operation
were examined by immunoblotting for the protein expression of p21 ( ) and CDK4 ( ) at days 1, 3, 7, 14, 21 and
42 post-operation. Gels were scanned densitometrically
and expressed as a ratio to normal control (without
operation) values included in every experiment. Results
show meansSEM obtained from 6 rats per treatment
group at each time point after operation.
et al.[103] have used the cultured rat embryonic cell line,
H9C2, to demonstrate that insulin growth factor, which
induces myocyte differentiation, activates the p21 promoter, thereby suggesting a possible role for p21 in the
modulation of growth stimulatory pathways in cardiac
myocytes. However, it should be noted that H9C2 cells
do not mimic closely the cardiac myocyte phenotype and
results obtained with this cell line should be viewed
accordingly.
Recently, we have shown that during the development of pressure overload-induced left ventricular
hypertrophy, the number of myocyte nuclei arresting in
the G2 phase of the cell cycle prepared from hypertrophied hearts increased significantly by about 57%
compared with sham-operated and non-operated controls analysed by FACS[99], thereby providing evidence
for DNA synthesis and cell cycle progression of myocyte
nuclei from the G0/G1 into the G2/M phase of the
cell cycle during the development of left ventricular
Eur Heart J, Vol. 20, issue 6, March 1999
416
J.-M. Li and G. Brooks
Table 6
mRNA
Cyclins
CDKs
CDKIs
Protein
Cyclins
CDKs
CDKIs
Others
Expression of cell cycle molecules in unstimulated rat cardiac myocytes
Fetal
Neonatal
Adult
Reference
A, B, C, D1, D2, D3, E
Not reported
p57
A, B, D1, D2, D3, E
CDC2
p21, p27
C, D1, D2, D3, E
Not reported
p21, p27
[84,85,88]
A, B, C, D1, D2, D3 , E
2, 4, 5, 6 and CDC2
p57
PCNA
A, C, D1, D2, D3, E
2, 4, 5, 6 and CDC2
p21, p27
PCNA, pRb
D2, D3
2, 4, CDC2
p21, p27
Not reported
[81,84,85,88]
[85]
[82,83,86]
[81,85,88]
[82,83,86]
[85,88]
CDKs=cyclin-dependent kinases; CDKIs=cyclin-dependent kinase inhibitors.
hypertrophy. The precise mechanism for this striking
and somewhat unexpected G2/M phase arrest of adult
cardiac myocytes during left ventricular hypertrophy is
currently the subject of further investigation in our
laboratory. Experiments designed to induce forced
expression of these molecules should provide more
information about cell cycle control in cardiac myocytes
and may offer possibilities for re-initiating cell division
in adult cardiac myocytes.
Approaches taken to improve cardiac
myocyte growth by manipulation of
myocyte cell cycle control
To date, two strategies have been used in an attempt
to increase the number of myocytes, and therefore
improve cardiac function, in the heart following infarction. The first approach has utilized the transplantation of myocytes that have proliferative capacity into
infarcted myocardium. For example, direct implantation
of skeletal muscle cells into mouse ventricular myocardium[104], transplantation of fetal myocardial tissue into
the infarcted myocardium of adult rats[105], and transplantation of fetal cardiac myocytes into infarcted rat
myocardium[106] have all demonstrated that grafted
myocytes, albeit from skeletal or fetal sources, maintain
their original morphology and survive in infarcted myocardium. The results from these initial experiments are
promising and suggest that cell transplantation has the
potential to be used as an alternative therapy for
regenerating damaged myocardial tissue. The second
approach used to increase the number of viable cardiac
myocytes has been to manipulate the expression of cell
cycle regulatory molecules in these cells by gene transfer.
Rapid progress in the development of methods for gene
delivery and improvements in adenoviral vectors has
made the introduction of genes which control the expression of cell cycle dependent molecules into cardiac
myocytes feasible. Genetically engineered mice also
provide us with models with which to investigate the
physiological consequences of overexpressing or deleting
specific gene products[107]. Recently, pioneering work
of restarting the cell cycle in ventricular myocytes by
Eur Heart J, Vol. 20, issue 6, March 1999
manipulating E2F-1 and adenovirus E1A expression
has been elegantly demonstrated by the groups of
Schneider[97,108–110], Kitsis[111,112] and Bishopric[113].
Thus, Kirshenbaum et al. have shown that forced expression of the gene for human E2F-1 can mimic the S
phase reentry property of the viral protein E1A, thereby
reactivating DNA synthesis and proliferating cell
nuclear antigen expression in ventricular myocytes both
in vitro and in vivo[108,109]. Importantly, these transfected cells did not traverse the cell cycle fully and
accumulated in G2/M in an analogous fashion to that
reported for myocytes undergoing cardiac hypertrophy[99]. Kirshenbaum and Schneider have also
shown that adenovirus E1A reactivates DNA synthesis
in ventricular myocytes by repressing cardiac gene transcription via alternative pocket protein- and p300binding domains[110]. In a separate series of studies, Liu
and Kitsis showed that expression of wild-type E1A in
fetal rat myocytes stimulated DNA synthesis in up to
94% of transduced cells[111,112]. Furthermore, these
authors showed that the ability of E1A to bind to
retinoblastoma protein and related pocket proteins had
little effect on stimulation of DNA synthesis, whereas
binding of E1A to the co-activator molecule, p300, was
important for this response. Bishopric and colleagues
have extended these studies and shown that the mechanism by which E1A inhibits cardiac myocyte-specific
gene expression is dependent upon the amino-terminus
of E1A, although E1A binding to p300 was not required
for repression of cardiac myocyte-specific promoter
activity[113]. However, co-expression of p300 with E1A
did partially reverse the E1A-mediated transcriptional
repression, suggesting that the E1A–p300 interacting
domain may play a role in this process[113].
Soonpaa et al.[114] have demonstrated that overexpression of cyclin D1 in the hearts of transgenic mice
resulted in a concomitant increase in the expression of
CDK4, CDK2 and proliferating cell nuclear antigen in
cardiac myocytes with an abnormal pattern of multinucleation[114]. Adult cardiac myocytes obtained from
these transgenic mice underwent active DNA synthesis
and increased significantly the number of myocyte nuclei
as compared with cardiac myocytes obtained from
normal mice[114]. Similarly, p27 knock-out mice showed
multi-organ enlargement including the heart[115] and
Review
we have recently observed, by immunocytochemical
analyses, a significant increase in the number of myocyte
nuclei in left ventricular sections obtained from these
p27 knockout mice compared with cardiac sections
obtained from normal or heterozygous mice[116].
Although approaches for manipulating the
expression of cell cycle regulatory molecules in cardiac
myocytes are still in their preliminary stages of development, the results obtained to date are encouraging and
suggest the feasibility of restarting the cell cycle in
ventricular myocytes by this method. The precise mechanism(s) by which cell cycle regulatory molecules cause a
progressive withdrawal of cardiac myocytes from the cell
cycle remains to be determined; however, by carefully
dissecting the possible candidate molecules involved, it
should be possible to develop strategies which will
enable us to overcome the block in cell cycle progression
and to reinitiate myocyte division in a controlled
manner.
Summary and conclusions
In the preceding sections we have described the potential
for using cell cycle regulatory molecules as targets for
drug development within the cardiovascular system.
Opportunities for affecting the expression and activities
of selected cell cycle regulatory molecules exist in interventional cardiological procedures such as PTCA to
limit specifically the intimal hyperplasia of vascular
smooth muscle cells that occurs following angioplasty.
In addition, the potential for targeting the cardiac
myocyte cell cycle to re-initiate cell division in a controlled manner would provide a suitable approach for
repairing damaged areas of myocardial tissue following
an infarct. Although this approach has not been demonstrated to date in vivo, data from transgenic mouse
models and in vitro studies have implicated the cell
cycle as a suitable target for manipulation. The next few
years will enable the feasibility of this approach to be
demonstrated.
The authors would like to thank the Wellcome Trust and the
British Heart Foundation for financial support.
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