© 2001 American Heart Association, Inc.
Editorials |
Retinoids: New Insight Into Smooth Muscle Cell Growth Inhibition
From the Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, NY.
Correspondence to Joseph M. Miano, PhD, Center for Cardiovascular Research, Box 679, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail Joseph_Miano{at}urmc.rochester.edu
Key Words: retinoid • restenosis • smooth muscle proliferation and differentiation • growth factors/cytokines
The successful management of vascular occlusive diseases, particularly those due to procedural interventions for preexisting atherosclerosis, will likely require the targeting of numerous cellular processes rather than any 1 gene, protein, or signaling pathway. This fact is underscored by the failure of virtually all clinical trials employing pathway- or factor-specific therapeutics to limit restenosis after percutaneous vascular interventions. Retinoids are natural and synthetic derivatives of vitamin A that exert myriad effects on such cellular processes as growth, apoptosis, differentiation, and migration. As such, retinoids could be potential therapeutics to test in the context of vascular occlusive disease.1 2 3 4 Clinically, the prototypic natural retinoid, all-trans retinoic acid (atRA), effects a nearly 100% rate of remission in patients with acute promyelocytic leukemia.5 Given the similarities in the pathogenesis of neoplasia and vascular occlusive lesions (ie, increased cell growth and loss of cellular differentiated properties), it is surprising that retinoids have only recently been examined with respect to cells of the vascular wall.
Many retinoids exert their pleiotropic effects through
the binding and activation of nuclear retinoid receptors. There are 2
families of retinoid receptors, each of which comprises 3 distinct
genes. The retinoic acid receptors (RAR 
, ß, and 
) bind atRA
and its 9-cis stereoisomer
(9cRA), whereas the more weakly expressed retinoid X receptors (RXR
, ß, and ) bind
9cRA.6 Numerous retinoids
have been synthesized and tested for receptor selectivity as a means of
reducing the side effects associated with natural retinoid therapy.
Many of these synthetic retinoids have recently found clinical utility
for a number of diseases.4
Ligand-activated retinoid receptors dimerize (preferentially as
an RAR-RXR heterodimer), recognize, and bind
cis elements (called retinoic
acid–response elements, or RAREs) in the genome to activate
gene transcription. Identifying retinoid-responsive target genes is
critical in defining the molecular actions of these potent, biological
response modifiers, although other posttranscriptional processes are
likely to play a role as well (see below).
In recent years, retinoids have been examined for their influence on vascular smooth muscle cell (SMC) growth and differentiation, inasmuch as these processes are thought to be of some relevance in the pathogenesis of vascular occlusive disease.1 2 3 Thus, there is a growing body of in vitro data demonstrating that retinoids antagonize growth factor–stimulated SMC hyperplasia while in some cases promoting a more differentiated SMC phenotype.1 2 3 Because cultured SMCs and the aorta express most of the retinoid receptors and display retinoid receptor activity in vitro,7 it is hypothesized that these observed effects on SMC phenotype are related to retinoid receptor–mediated changes in the SMC transcriptome.8 Indeed, studies using retinoid receptor–selective agonists with reduced toxicity have shown inhibitory effects on SMC growth.9 10 11 Collectively, these in vitro data prompted a series of in vivo studies that examined the effects of atRA and other retinoids on the vessel wall’s response to injury. Remarkably, all in vivo studies to date have documented desirable changes in vessel wall geometry with retinoid administration after vascular injury. Such changes in vessel geometry include an attenuation in neointimal mass,10 12 13 14 15 16 an outward remodeling of the vessel wall,12 13 17 and accelerated reendothelialization.15 The decrease in neointimal mass was shown in most of the aforementioned studies to be associated with reduced SMC DNA synthesis. At this point in time, we have very little insight into the mechanisms through which retinoids exert these desirable effects on SMC growth and neointimal mass.
In this issue of
Arteriosclerosis,
Thrombosis, and Vascular Biology, Wakino et
al18 report on a series of
well-designed and executed experiments that offer important mechanistic
insights into how retinoids exert antigrowth effects in SMCs.
Consistent with mRNA data in cultured rat aortic
SMCs,7 they found that human
coronary artery SMCs (HCASMCs) express 5 of the 6 retinoid
receptors (only RXR was not detected). The authors then used a panel
of retinoids with varying selectivity for retinoid receptors to show
dose-dependent inhibition of platelet-derived growth factor
(PDGF)/insulin-stimulated HCASMC growth, a finding that is
consistent with another recent study showing 9cRA inhibition of
HCASMC growth.19 Flow
cytometry experiments were then performed that revealed a significant
decrease in the number of cells entering the S phase, which suggested
that retinoids might be interfering with the cell cycle machinery.
These results are similar to those of a previous
study,7 which suggested that
early G1 events in SMCs might be the targets of
retinoids and their activated receptors. Accordingly, Wakino et
al18 examined the effects of
retinoids on PDGF-induced phosphorylation of the
retinoblastoma (RB) protein, inasmuch as RB
phosphorylation represents a pivotal, early
cell-cycle progression
event.20 Under normal
quiescent conditions, RB is hypophosphorylated, which
promotes sequestration of the E2F family of transcription factors. On
growth stimulation, the RB protein is phosphorylated
(on some 16 serine-threonine residues) via several
cyclin/cyclin-dependent kinase (cdk) complexes, resulting in the
release of E2F. Transcriptionally active E2F stimulates expression of
growth-related genes such as
c-myc, cyclins A and E, DNA
polymerase-, and thymidine
kinase.21 Wakino et
al18 showed that each
retinoid could dose-dependently inhibit RB
phosphorylation after PDGF stimulation, although a
corresponding decrease in E2F-dependent transcription (eg, decreased
c-myc expression) was not
studied. Importantly, there was a strong correlation between each
retinoid’s ability to inhibit SMC growth and block RB
phosphorylation, suggesting that the inhibition of SMC
growth was a result of RB
hypophosphorylation.
Several cyclin/cdk complexes actively phosphorylate RB, including cyclin D/cdk4/6, cyclin A/cdk2, and cyclin E/cdk2.21 Wakino et al18 showed that 2 pan-RAR agonists (atRA and TTNBP) decreased protein expression of cyclin D1. An RXR-selective agonist (AGN4204) inhibited cyclin D1 only at the highest concentration, whereas a pan-RAR/RXR agonist (9cRA) had no effect on cyclin D1 levels. These results contrast with those of Chen and Gardner,9 who showed that atRA and TTNBP induced cyclin D1 at both the mRNA and protein level in aortic SMCs. The reasons for these divergent data probably relate to the cell type (coronary versus aortic SMCs, which represent 2 distinct SMC lineages) as well as species differences. All retinoids tested inhibited cyclin A protein levels.18 Cyclin A is an E2F-dependent gene, so it will be informative to assess its steady-state mRNA expression profile with each retinoid. None of the retinoids tested in the study by Wakino et al18 had any effect on the expression of cyclin E or the cdk’s, which may explain why RB phosphorylation was not completely inhibited with each retinoid. Taken in aggregate, the decrease in growth factor–induced RB phosphorylation with retinoids appears to be a consequence of impaired expression, and thus activity, of cyclin D/cdk4/6, and/or cyclin A/cdk2. The findings of Wakino et al18 are in perfect agreement with numerous reports from the cancer field showing a similar inhibition of RB phosphorylation on retinoid treatment.22 23 24 25 26 What remains to be shown is whether the activities of the retinoid-targeted cyclin/cdk complexes are reduced in SMCs with a substrate such as histone H1 or RB itself.
Whereas cyclin/cdk complexes promote the
phosphorylation of RB, there is yet another family of
proteins, the cyclin-dependent kinase inhibitors (cdkI’s),
that bind to cdk’s, neutralize their activity, and thus minimize the
phosphorylation of
RB.21 Levels of cdkI’s are
high in quiescent cells; however, on mitogenic stimulation
or vascular injury, the levels of cdkI’s are rapidly downregulated,
which creates a "permissive" condition for RB
phosphorylation and cell cycle
progression.27 In HCASMCs,
Wakino et al18 found that
p27Kip1 was the only cdkI to decrease with
mitogenic stimulation. In contrast,
p15INK4b and
p16INK4a levels did not change, and
p21Cip1 actually increased with mitogens.
Growth factor–induced downregulation of the cdkI
p27Kip1 was prevented with each
retinoid.18 Interestingly,
this apparent stabilization of p27Kip1
protein levels appeared to be related to an extended half-life of the
protein, because addition of cycloheximide further increased
p27Kip1 levels in cells treated with each
retinoid. A similar posttranscriptional mechanism for retinoid-mediated
p27Kip1 stabilization was proposed in a B
lymphocyte–transformed cell
line.28 Wakino et
al18 noted
higher-molecular-weight forms of p27Kip1 in
cell lysates from retinoid-treated
HCASMCs.18 The authors
speculated that these could represent ubiquitinated forms of
p27Kip1, a finding that has recently been
observed in atRA-treated neuroblastoma cells whose growth and RB
phosphorylation are both inhibited with
retinoids.29 Thus, the
extended half-life of p27Kip1 observed in
retinoid-treated HCASMCs could be due to a reduction in
proteasome-dependent protein degradation, although this specific
mechanism awaits formal testing. It will also be important to show that
p27Kip1 actually binds its corresponding
cyclin/cdk complex, leading to diminished activity in HCASMCs treated
with retinoids. In summary, in HCASMCs,
p27Kip1 downregulation coupled with the
reduced expression of cyclins A and D appears to be sufficient for the
hypophosphorylation of RB. It should be noted that
there remains the possibility that retinoids induce or activate
a protein phosphatase (PP1?) that could dephosphorylate
RB, thereby achieving the same end of reducing cell cycle
progression.21 The figure
(Figure
)
illustrates some of the more salient effects that retinoids such as
atRA have on vascular SMCs.
|
As principal "gatekeepers" of RB activity and the cell cycle, cyclin/cdk’s and their inhibitors represent logical targets for therapy of vascular occlusive diseases. The functional importance of RB phosphorylation has already been demonstrated in rat and porcine arterial models of neointimal formation.30 The report by Wakino et al18 in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology provides the first in-depth analysis of the cell cycle and RB phosphorylation in retinoid-treated SMCs. It should be emphasized that therapies directed solely toward cell cycle regulators may not interfere with other complex aspects of human vascular occlusive disease, such as vessel (adventitial) remodeling, cellular migration, inflammation, and thrombosis. It remains to be discovered how retinoids impinge on these and other aspects of the vessel wall’s response to insult and whether retinoids may be efficacious in the treatment of human vascular disorders.
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