(Circulation. 1999;100:659-665.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Flavopiridol Inhibits Smooth Muscle Cell Proliferation In Vitro and Neointimal Formation In Vivo After Carotid Injury in the Rat
From the Division of Cardiology and Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston (J.R., A.S.M., Z.H., C.H., C.A.B., L.J.T., V.D.S., C.P.), and Hoechst Marion Roussel, Inc, Bridgewater, NJ (J.A.D.).
Correspondence to Cam Patterson, MD, University of Texas Medical Branch, Division of Cardiology, 9.138 Medical Research Bldg, 301 University Blvd, Galveston, TX 77555-1064. E-mail cpatters{at}utmb.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Smooth muscle cell (SMC) proliferation is a critical component of neointimal formation in many models of vascular injury and in human lesions as well. Cell-cycle inhibition by gene transfer techniques can block SMC proliferation and lesion formation in animal models, although these methods are not yet applicable to the treatment of human disease. Flavopiridol is a recently identified, potent, orally available cyclin-dependent kinase inhibitor.
Methods and Results—Using human aortic SMCs, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in nearly complete inhibition of basic fibroblast growth factor–induced and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone H1 phosphorylation, but had no effect on mitogen-activated protein kinase activation. Induction of the cell cycle–related proteins cyclin D1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neointimal formation in rat carotid arteries after balloon injury. Flavopiridol 5 mg/kg reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury.
Conclusions—Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.
Key Words: muscle, smooth • growth substances • angioplasty • carotid arteries
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The cellular responses to vascular injury—cell dysfunction, activation, dedifferentiation, proliferation, and migration—culminate in clinical events such as restenosis. Smooth muscle cell (SMC) proliferation is a common feature of vascular injury models, and SMCs are the major cellular component of neointimal lesions.1 Renewed interest in inhibiting SMC proliferation has accompanied the increased use of stents for the treatment of coronary disease, because in-stent restenosis is almost entirely dependent on SMC hyperplasia.2 It is estimated that as many as 100 000 patients with in-stent restenosis required treatment in 1997 alone3 ; therefore, an effective inhibitor of SMC hyperplasia would have profound clinical and economic ramifications.
Efforts to inhibit SMC proliferation in vascular injury models, either by modulating cellular mediators of the proliferative response or by interfering with the cell-cycle machinery, have provided insights into neointimal formation. Cell-cycle progression is a tightly controlled event regulated positively by cyclin-dependent kinases (Cdks) and their cyclin-regulatory subunits4 and negatively by Cdk inhibitors and tumor suppressor genes, such as retinoblastoma protein (Rb).5 Adenovirus-mediated overexpression of the endogenous Cdk inhibitors p21 and p27kip1 or of a constitutively active form of Rb blocks neointimal formation in the rat carotid injury model.6 7 8 Such studies support the general hypothesis that cell-cycle inhibition is an attractive means of intervention in vascular lesion formation.
Although genetic interventions have aided in the dissection of the mechanisms regulating neointimal formation, they suffer from the shortcoming of not being, at present, clinically suitable for the treatment of vascular disease in humans. A water-soluble, low-molecular-weight compound with specific cell cycle–regulatory effects, particularly one with oral activity, would have applicability both experimentally and, potentially, clinically. The recently identified flavone flavopiridol is a Cdk inhibitor that potently blocks the activity of Cdk2, cell division cycle 2 (Cdc2), and Cdk4.9 10 11 12 In contrast to other pharmacological inhibitors of Cdks, flavopiridol is remarkable for its kinase selectivity, its oral availability, and its potency, being effective in nanomolar concentrations.12 These features result in a favorable side effect profile that has led to testing of flavopiridol in phase 1 clinical trials for the treatment of refractory neoplasms.13 Given these properties, we have examined the ability of flavopiridol to inhibit SMC proliferation in vitro and after balloon injury to the rat carotid artery.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Flavopiridol {L86-8275, (–)-cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-benzopyran-4-one} was provided by Hoechst Marion Roussel, Inc. Basic fibroblast growth factor (bFGF) was purchased from Collaborative Biochemical and thrombin from Sigma.
Cell Culture
Human aortic smooth muscle cells (HASMCs) were obtained from
Clonetics and were cultured as previously described.14
Before experiments were performed, cells were growth-arrested at 80%
confluence for 48 hours with medium containing 0.2% FBS.
Cell-Proliferation ELISA
Cell proliferation was measured by ELISA (Amersham Life
Science). HASMCs were grown in 96-well plates and made quiescent. Cells
were treated with 10 ng/mL bFGF, 2 U/mL thrombin, or vehicle for 24
hours. Flavopiridol 75 nmol/L was administered 1 hour before growth
factor treatment. 5-Bromo-2'-deoxyuridine (BrdU) was added to a final
concentration of 10 µmol/L during the last 2 hours of treatment.
BrdU incorporation was measured as described.15 Results
are expressed as mean±SEM for 12 samples per condition.
Cell Counts
Growth-arrested HASMCs grown to 50% confluence in 6-well plates
were treated with or without flavopiridol 75 nmol/L or bFGF 10 ng/mL.
At intervals after treatment, cells were trypsinized and cell numbers
determined with a hemocytometer.
Western Blot Analysis
Western blot analysis was performed as previously
described.14 The primary antibodies were anti–cyclin D1
antibody (M-20, Santa Cruz), anti–proliferating cell nuclear antigen
(PCNA) antibody (PC10, Sigma), a
phosphorylation-specific p44/42 (Erk1/Erk2)
mitogen-activated protein (MAP) kinase antibody (New England
Biolabs), and anti-Rb antibody (G3-245, Pharmingen), which recognizes
the phosphorylated (pRb) and highly
phosphorylated (ppRb) Rb species.
Cdk Activity
Quiescent HASMCs were treated for 24 hours, and total cell
lysates were prepared. The kinase assay was performed with a histone H1
kinase assay kit (Upstate Biotechnology) according to the
manufacturer's instructions. Results are expressed as the mean±SEM
for 3 samples and are representative of 3 independent
experiments.
In-Gel Kinase Assay
Quiescent HASMCs were treated with growth factors for 30
minutes, and total cell lysates were prepared. In some experiments,
HASMCs were pretreated for 60 minutes with 30 µmol/L PD98059,
flavopiridol, or vehicle. Proteins were resolved on a
polyacrylamide gel that was copolymerized with myelin basic
protein. The gel was treated with [
-32P]ATP,
and autoradiography was performed as
described.15
Trypan Blue Exclusion
HASMCs were grown in 5-cm dishes and growth-arrested. Cells were
treated with flavopiridol 75 nmol/L or tumor necrosis factor-
(TNF-) 50 ng/mL for the indicated times. After the removal of the
medium, 0.4% trypan blue was added to the dishes. After 5 minutes, the
cells in the dishes were counted. Blue cells were counted as nonviable
cells.
Rat Carotid Injury Model
Injury to the rat carotid artery was performed essentially as
described.1 Adult male Sprague-Dawley rats (400 to
500 g, Zivic-Miller, Zelienople, Pa) were
anesthetized with ketamine 2 mg/kg and xylazine 4
mg/kg. The left internal carotid artery was then cannulated with a 2F
embolectomy catheter. The balloon was inflated and withdrawn across the
artery to produce a distending and denuding injury. Immediately after
surgery and for 4 days thereafter, rats were given flavopiridol 5 mg/kg
in water, or water alone, by gavage. At specified times after carotid
injury, rats were anesthetized and perfusion-fixed. Right and
left carotid arteries were removed and distended by injection of 4%
paraformaldehyde through the lumen, after which they
were dehydrated and stored at 4°C. Immunohistochemistry was performed
as previously described14 with the PCNA antibody and an
anti-Cdk2 antibody (M2-G, Santa Cruz).
Image Analysis
The extreme distal and proximal regions of each artery were
removed. Ten intermediate cross sections (8 µm each) taken
500 µm apart were analyzed from each artery. Slides were
fixed and stained with hematoxylin and eosin as previously
described.14 Under a Nikon Diaphot 300 microscope, each
cross section was captured as a digital image with a Hamamatsu video
camera. Medial and neointimal areas were determined by use
of NIH Image software. Lesion size was expressed as the
neointima/media ratio. Results for each group were
expressed as the mean±SEM.
Statistical Analysis
When appropriate, data from quantitative studies were expressed
as the mean±SEM. For multiple treatment groups, a factorial ANOVA
followed by Fisher's least significant difference test was applied.
Statistical significance was accepted at P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Flavopiridol Inhibits HASMC Proliferation
Because flavopiridol can inhibit proliferation in multiple tumor cell lines, we tested the hypothesis that its use would block the growth of human SMCs. Growth-arrested HASMCs were treated with bFGF 10 ng/mL for 24 hours in the presence of increasing concentrations of flavopiridol, and proliferation was measured by an ELISA-based assay. Compared with untreated cells, proliferation of bFGF-treated cells was increased 5.4-fold (Figure 1A
).
Pretreatment for 1 hour with as little as 50 nmol/L flavopiridol
significantly decreased HASMC proliferation (to 3.9-fold,
P<0.05), an effect that was nearly maximal at
concentrations of 75 nmol/L.
|
P<0.05 vs treatment with bFGF in absence of
flavopiridol. B, HASMCs were treated with bFGF, thrombin, or vehicle in
presence or absence of flavopiridol, and BrdU incorporation was
measured. *P<0.05 vs untreated cells.
**P<0.05 vs treatment with bFGF alone.
To test the generality of the effects of flavopiridol on SMC
proliferation, we examined its effect on mitogenesis elicited by
thrombin 2 U/mL. Flavopiridol 75 nmol/L significantly and potently
inhibited both bFGF- and thrombin-induced HASMC proliferation (5.4-fold
versus 1.8-fold and 2.4-fold versus 0.7-fold, respectively,
P<0.05, Figure 1B
). We performed cell counts to
confirm that the effect of flavopiridol on cell-cycle progression in
HASMCs truly reflected changes in proliferation. bFGF 10 ng/mL induced
a 3-fold increase in cell number after 3 days of treatment (Figure 2). As in the ELISA-based assays,
flavopiridol 75 nmol/L efficiently blocked bFGF-induced
proliferation.
|
), bFGF and flavopiridol (•),
or vehicle (
) for times indicated, and cell numbers after treatment
were determined. Results are expressed as cell counts per well
x103.
Flavopiridol Inhibits Cdk Activity and Cell Cycle–Related Gene
Expression in HASMCs
To assess the specific effect of flavopiridol on the cell-cycle
machinery, we measured histone H1 kinase activity in cellular lysates
from growth factor–stimulated HASMCs. Phosphorylation
of histone H1 reflects the activities of Cdc2 and Cdk2.16
Treatment of HASMCs with bFGF and thrombin resulted in 4.4-fold and
3.6-fold increases, respectively, in histone H1 kinase activity (Figure 3). These increases in cyclin-dependent
kinase activity were totally blocked by pretreatment with flavopiridol
75 nmol/L.
|
By Western blot analysis, we also addressed whether
flavopiridol influenced growth factor–induced regulation of cell
cycle–related proteins. Cyclin D1 protein levels were upregulated
6.3-fold and 3.2-fold, respectively, in response to bFGF and thrombin
treatment (Figure 4), an effect that
could be blocked by pretreatment with flavopiridol. Similarly,
increased expression of PCNA was also blocked by flavopiridol
pretreatment. As a final measure of cell cycle–related proteins, we
examined Rb phosphorylation in response to growth
factor expression using an antibody that recognizes pRb.
Phosphorylation inactivates Rb and allows
progression through the S phase to proceed. Analysis of Rb
phosphorylation is particularly relevant because Rb is
a target of Cdk2 and Cdk4 in vivo. Both thrombin and bFGF induced
hyperphosphorylation of Rb, an effect that was
inhibited by flavopiridol. Taken together, these results indicate that
flavopiridol influences the expression and activity of
G1- and S phase–related cell-cycle control
elements in HASMCs in association with its
growth-inhibitory effects.
|
Flavopiridol Has No Effect on MAP Kinase
Phosphorylation or Activity
To ensure that flavopiridol was acting specifically at the level
of the cell cycle, rather than nonspecifically on upstream kinase
pathways, we measured phosphorylation and activity of
Erk1 (p44 MAP kinase) and Erk2 (p42 MAP kinase). We chose these kinases
because they are immediately upstream of transcriptional events
occurring in response to growth stimuli and downstream of a number of
critical mitogenic signaling pathways.17 An
intact response by MAP kinases indicates that the upstream
mitogenic pathways are also intact. We measured the
phosphorylation status of Erk1 and Erk2 with an
antibody that specifically recognizes the
phosphorylated and, hence, activated forms. As
a control in these experiments, we used PD98059, a selective
inhibitor of MAP kinase activation. Increased amounts of
phosphorylated Erk1 and Erk2, compared with untreated
cells, were detected after treatment of HASMCs with thrombin and bFGF
for 30 minutes (Figure 5, top).
Phosphorylation of Erk1 and Erk2 by both thrombin and
bFGF was blocked by pretreatment with PD98059 but not with
flavopiridol. To confirm these findings, we measured Erk1 and Erk2
activity by an in-gel kinase assay (Figure 5, bottom). Again, we
found that Erk1 and Erk2 activities were increased in response to
thrombin and bFGF, an effect that was inhibitable by PD98059 but not by
flavopiridol. These experiments, in conjunction with those
presented in Figures 3 and 4, provide evidence
that the effects of flavopiridol on HASMC proliferation are due to a
specific arrest of the cell-cycle machinery by blocking Cdk activity
without affecting upstream signaling events.
|
Flavopiridol Does Not Decrease HASMC Viability
Previous reports of flavopiridol activity in other cell types have
demonstrated that, depending on the cell line, flavopiridol either may
induce growth arrest without affecting viability or may cause
apoptosis.12 18 19 20 21 We therefore assessed whether
flavopiridol decreased the viability of HASMCs. Quiescent HASMCs were
treated with flavopiridol 75 nmol/L, vehicle, or TNF- 50 ng/mL, a
cytokine known to induce apoptosis in this cell
type.22 Although TNF- potently decreased the viability
of HASMCs, flavopiridol had no such effect (Figure 6). We have noted that with higher
concentrations and longer incubations, some decreases in viability in
the presence of flavopiridol may occur (not shown). However, under the
conditions tested, flavopiridol primarily induces growth arrest,
without affecting SMC viability.
|
Flavopiridol Inhibits SMC Proliferation and Neointimal
Formation In Vivo in a Rat Carotid Injury Model of Vascular
Injury
We used the well-established rat carotid injury model to examine
whether flavopiridol induces growth arrest of SMCs in vivo, as it does
in vitro. We administered flavopiridol orally at a dose of 5 mg/kg once
daily, beginning on the day of injury and for 4 days thereafter,
because this time period covers the initial induction of Cdk2 and the
first wave of SMC proliferation in this model.23 24 Mean
intimal and medial areas were quantified 7 and 14 days after injury,
and neointimal lesion size was expressed as the ratio of
the neointimal to the medial area. The treated and
untreated groups included 12 animals each. The
neointima/media ratio at 7 days was 1.00±0.05 in arteries
of vehicle-treated rats and 0.65±0.04 in flavopiridol-treated rat
arteries, a reduction of 35.0% (Figure 7). At 14 days, the
neointima/media ratio was 1.08±0.04 in vehicle-treated
rats and 0.66±0.03 in flavopiridol-treated rats, a reduction of
38.9%. These effects were statistically significant at both time
points (P<0.05). Representative
arterial sections are shown in Figure 8.
|
|
To demonstrate directly that flavopiridol inhibited SMC proliferation,
we stained sections for PCNA expression in
representative fields from each artery and determined
the percentage of PCNA-positive nuclei in the neointima. At
7 days, 31.1±7.2% of nuclei in injured arteries from untreated rats
were PCNA-positive, whereas only 11.8±1.5% of injured arteries in
flavopiridol-treated rats were PCNA-positive (Figure 7;
P<0.05). At 14 days, PCNA-positive nuclei were present
in 10.4±2.0% of neointimal cells from untreated but in
only 4.2±0.5% of neointimal cells from treated injured
rat arteries (P<0.05). Similarly, Cdk2-positive cells were
much less common in the neointima of flavopiridol-treated
rats (Figure 9, A and C) than in arteries
from untreated rats (B and D) at both 7 and 14 days after injury.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We have examined whether the novel Cdk inhibitor flavopiridol is a suitable candidate for inhibiting SMC proliferation in vivo, particularly in the setting of vascular injury. Previous attempts to target the cell-cycle machinery therapeutically for the treatment of vascular disease have provided a rationale for the present studies6 7 ; however, methods used to this end have relied on gene transfer technologies to inhibit cell-cycle progression. At present, formidable obstacles prevent the clinical application of these techniques.25 During the course of our studies, it was reported that CVT-313, a recently identified compound that also has Cdk-inhibitory properties but at micromolar concentrations, can also inhibit neointimal formation; however, it was required that CVT-313 be instilled into the carotid artery at the time of injury to produce this effect.26 In contrast, we show that flavopiridol, when administered orally, can potently inhibit neointimal formation, to a degree comparable to other clinically relevant agents.8 27 28 The oral activity of flavopiridol makes it virtually unique among agents shown to be active in animal models of vascular injury. Its selectivity, potency, and ease of administration make flavopiridol an excellent candidate for examining the therapeutic benefits of cell-cycle inhibition in vivo in human vascular lesions.
We chose to administer flavopiridol orally in a concentration half that which inhibits tumor growth in a nude mouse xenograft model.19 It is notable that flavopiridol concentrations of 75 nmol/L result in nearly complete inhibition of SMC proliferation in our studies, whereas median serum concentrations of 425 nmol/L were attained at doses below the toxic threshold in phase 1 human studies of refractory carcinoma.13 Our results suggest that much lower doses of cell-cycle inhibitors than those used for neoplasia may be effective in the setting of vascular diseases, such as restenosis, with the concomitant benefit of increased tolerability.
Although we have demonstrated that flavopiridol induces growth arrest without affecting the viability of HASMCs in culture and have shown decreased neointimal formation after flavopiridol treatment in vivo, we cannot be sure that cell-cycle arrest is the only factor reducing neointimal formation in carotid lesions. Flavopiridol can induce growth arrest with or without inducing apoptosis, depending on the cell type observed.21 Interestingly, flavopiridol inhibits apoptosis in PC12 cells that have been terminally differentiated, yet it induces apoptosis in undifferentiated PC12 cells that are proliferating.20 Although our in vitro experiments were performed under conditions that would mimic the phenotype of SMCs before injury, it is possible that SMCs may respond differently to flavopiridol after injury and may even undergo apoptosis. Although the role of apoptosis in vascular lesions is unclear, expression of the Fas ligand in SMCs induces apoptosis and blocks neointimal formation in rabbits after balloon injury,29 suggesting that if flavopiridol does indeed induce apoptosis of SMCs in vivo, as it does in proliferating PC12 cells, this may be a salutary phenomenon in the context of neointimal formation.
Our results indicate that flavopiridol can inhibit SMC proliferation and, hence, neointimal formation in a well-accepted small animal model of vascular disease. It must be pointed out that the relevance of inhibition of SMC proliferation is controversial in human vascular lesions and may differ depending on the nature of the lesion and the time at which observations of proliferation are made. The proliferative index of SMCs in human atherectomy specimens is remarkably low,30 although these specimens may not reflect proliferative changes at earlier, more critical stages in lesion development. In addition, arterial remodeling independent of neointimal growth may account for a significant proportion of luminal obstruction after angioplasty in humans.31 In contrast, indices of mitotic activity in SMCs are much higher (25% PCNA-positive) in atherectomy specimens from human lesions with in-stent restenosis, consistent with the established role of SMC hyperplasia, but not remodeling, in this process.2 As stent placement and the clinical problem of in-stent restenosis increases, so will the need for an effective means to arrest SMC hyperplasia and neointimal formation. Because flavopiridol is a potent, orally available drug with specific Cdk-inhibitory activity and because safe doses of flavopiridol are known in humans, flavopiridol can be considered a pharmacological candidate for prevention of in-stent restenosis in humans.
|
Acknowledgments |
|---|
This work was supported by NIH grants HL-03658 and AG-15234 to Cam Patterson. The authors thank Joann Aaron for editing and Dr Rick Stouffer for critical reading of the manuscript.
|
Footnotes |
|---|
Dr Ruef and A.S. Meshel contributed equally to this work.
Received January 12, 1999; revision received March 23, 1999; accepted April 9, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
2.
Kearney M, Pieczek A, Haley L, Losordo DW, Andres V,
Schainfeld R, Rosenfield K, Isner JM. Histopathology of in-stent
restenosis in patients with peripheral artery
disease. Circulation. 1997;95:1998–2002.
3. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am J Cardiol. 1998;81:7E–13E.[Medline] [Order article via Infotrieve]
4. Sherr CJ. Mammalian G1 cyclins. Cell. 1993;73:1059–1065.[Medline] [Order article via Infotrieve]
5. Hunter T. Braking the cycle. Cell. 1993;75:839–841.[Medline] [Order article via Infotrieve]
6. Chen D, Krasinski D, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27KIP1, an inhibitor of neointima formation in the rat carotid artery. J Clin Invest. 1997;99:2334–2341.[Medline] [Order article via Infotrieve]
7. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96:2260–2268.
8.
Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel EG,
Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular
proliferative disorders with a constitutively active form of the
retinoblastoma gene product. Science. 1995;267:518–522.
9. Losiewitz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of Cdc2 kinase activity by the flavanoid, L86–8275. Biochem Biophys Res Commun. 1994;201:589–595.[Medline] [Order article via Infotrieve]
10.
Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland
PJ. Flavopiridol induces G1 arrest with
inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast
carcinoma cells. Cancer Res. 1996;56:2973–2978.
11.
de Azevedo WF, Mueller-Dieckmann H-J, Schuze-Gahmen U,
Worland PJ, Sausville E, Kim S-H. Structural basis for specificity and
potency of a flavanoid inhibitor of human Cdk2, a cell
cycle kinase. Proc Natl Acad Sci U S A. 1996;93:2735–2740.
12.
Kaur G, Stetler-Stevenson M, Sebers S, Worland P,
Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition
with reversible cell cycle arrest of carcinoma cells by flavone
L86–8275. J Natl Cancer Inst. 1992;84:1736–1740.
13. Senderowicz AM, Headlee D, Stinson S, Lush RM, Figg WD, Pluda J, Sausville EA. A phase I trial of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasm. Proc Am Soc Clin Oncol. 1997;16:226a.
14.
Ruef J, Hu ZY, Yin L-Y, Wu Y, Hanson SR, Kelly AB,
Harker LA, Rao GN, Runge MS, Patterson C. Induction of vascular
endothelial growth factor in balloon-injured baboon
arteries. Circ Res. 1997;81:24–33.
15.
Ruef J, Rao GN, Li F, Bode C, Patterson C, Bhatnagar A,
Runge MS. Induction of rat aortic smooth muscle cell growth by the
lipid peroxidation product 4-hydroxy-2-nonenal.
Circulation. 1998;97:1071–1078.
16. Swank RA, Th'ng JP, Guo XW, Valdez J, Bradbury EM, Gurley LR. Four distinct cyclin-dependent kinases phosphorylate histone H1 at all of its growth-related phosphorylation sites. Biochemistry. 1997;36:13761–13768.[Medline] [Order article via Infotrieve]
17. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995;80:225–236.[Medline] [Order article via Infotrieve]
18.
Konig A, Schwartz GK, Mohammad RM, Al-Katib A,
Gabrilove JL. The novel cyclin-dependent kinase inhibitor
flavopiridol downregulates Bcl-2 and induces growth arrest and
apoptosis in chronic B-cell leukemia lines. Blood. 1997;90:4307–4312.
19. Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Malspeis L, Grever M, Sausville EA, Fieberg HH. Flavopiridol (L86–8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin Cancer Res. 1997;3:273–279.[Abstract]
20.
Park DS, Rarinelli SE, Greene LA.
Inhibitors of cyclin-dependent kinases promote survival of
post-mitotic neuronally differentiated PC12 cells and sympathetic
neurons. J Biol Chem. 1996;271:8161–8169.
21.
Parker BW, Kaur G, Nieves-Niera W, Taimi M, Kohlhagen
G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM.
Early induction of apoptosis in hematopoietic cell lines after
exposure to flavopiridol. Blood. 1998;91:458–465.
22.
Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P.
Apoptosis of vascular smooth muscle cells induced by in vitro
stimulation with interferon-, tumor necrosis factor-, and
interleukin-1ß. Arterioscler Thromb Vasc Biol. 1996;16:19–27.
23.
Schwartz SM, deBlois D, O'Brien ERM. The intima: soil
for atherosclerosis and restenosis. Circ
Res. 1995;77:445–465.
24. Wei GL, Krasinski K, Kearney M, Isner JM, Walsh K, Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ Res. 1997;80:418–426.
25.
de Young MB, Dichek DA. Gene therapy for
restenosis. Circ Res. 1997;82:306–313.
26.
Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman
RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz PG, Wang X, Wick MM,
Shiffman D. CVT-313, a specific and potent inhibitor of
CDK2 that prevents neointimal proliferation. J
Biol Chem. 1997;272:29207–29211.
27.
Sirois MG, Simons M, Edelman ER. Antisense
oligonucleotide inhibition of PDGFR-ß receptor
subunit expression directs suppression of intimal thickening.
Circulation. 1997;95:669–676.
28. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med. 1996;2:293–298.[Medline] [Order article via Infotrieve]
29.
Sata M, Perlman H, Muruve DA, Silver M, Ikebe M,
Libermann TA, Oettgen P, Walsh K. Fas ligand gene transfer to the
vessel wall inhibits neointima formation and overrides the
adenovirus-mediated T cell response. Proc Natl Acad Sci
U S A. 1998;95:1213–1217.
30.
O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N,
Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM.
Proliferation in primary and restenotic coronary
atherectomy tissue: implications for anti-proliferative therapy.
Circ Res. 1993;73:223–231.
31.
Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LR,
Wong SC, Hong MD, Kovach JA, Leon MB. Arterial remodeling
after coronary angioplasty: a serial intravascular ultrasound
study. Circulation. 1996;94:35–43.
This article has been cited by other articles:
 |
|
 |
|
  E. J.W. Wallitt, M. Jevon, and P. I. Hornick Therapeutics of Vein Graft Intimal Hyperplasia: 100 Years On Ann. Thorac. Surg., July 1, 2007; 84(1): 317 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Wang, Z. Liu, Q. Li, M. Karpurapu, V. Kundumani-Sridharan, H. Cao, N. Dronadula, F. Rizvi, A. K. Bajpai, C. Zhang, et al. An Essential Role for gp130 in Neointima Formation Following Arterial Injury Circ. Res., March 30, 2007; 100(6): 807 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Andres Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential Cardiovasc Res, July 1, 2004; 63(1): 11 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Taylor and C. A. McNamara Regulation of Vascular Smooth Muscle Cell Growth: Targeting the Final Common Pathway Arterioscler Thromb Vasc Biol, October 1, 2003; 23(10): 1717 - 1720. [Full Text] [PDF]
|
|
|
|
 |
|
 V. Sriram and C. Patterson Cell Cycle in Vasculoproliferative Diseases : Potential Interventions and Routes of Delivery Circulation, May 15, 2001; 103(19): 2414 - 2419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamasaki, K. Miyoshi, N. Oda, M. Watanabe, H. Miyake, J. Chan, X. Wang, L. Sun, C. Tang, G. McMahon, et al. Weekly Dosing With the Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor SU9518 Significantly Inhibits Arterial Stenosis Circ. Res., March 30, 2001; 88(6): 630 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ruef, S.-Q. Liu, C. Bode, M. Tocchi, S. Srivastava, M. S. Runge, and A. Bhatnagar Involvement of Aldose Reductase in Vascular Smooth Muscle Cell Growth and Lesion Formation After Arterial Injury Arterioscler Thromb Vasc Biol, July 1, 2000; 20(7): 1745 - 1752. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||