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(Circulation Research. 1995;76:412-417.)
© 1995 American Heart Association, Inc.

Articles

Rapamycin-FKBP Inhibits Cell Cycle Regulators of Proliferation in Vascular Smooth Muscle Cells

Steven O. Marx, Thottala Jayaraman, Loewe O. Go, Andrew R. Marks

From the Cardiovascular Institute, Molecular Medicine Program, Department of Medicine, and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, NY.

Correspondence to Andrew R. Marks, Box 1269, Mount Sinai School of Medicine, One Gustave L. Levy Pl, New York, NY 10029.

	Abstract

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Abstract Multiple growth factors can stimulate quiescent vascular smooth muscle cells to exit from G0 and reenter the cell cycle. The macrolide antibiotic rapamycin, bound to its cytosolic receptor FKBP, is an immunosuppressant and a potent inhibitor of cellular proliferation. In the present study, the antiproliferative effects of rapamycin on human and rat vascular smooth muscle cells were examined and compared with the effects of a related immunosuppressant, FK520. In vascular smooth muscle cells, rapamycin, at concentrations as low as 1 ng/mL, inhibited DNA synthesis and cell growth. FK520, an analogue of the immunosuppressant FK506, is structurally related to rapamycin and binds to FKBP but did not inhibit vascular smooth muscle cell growth. Molar excesses of FK520 blocked the antiproliferative effects of rapamycin, indicating that the effects of rapamycin required binding to FKBP. Rapamycin-FKBP inhibited retinoblastoma protein phosphorylation at the G1/S transition. This inhibition of retinoblastoma protein phosphorylation was associated with a decrease in p33^cdk2 kinase activity. These observations suggest that rapamycin, but not FK520, inhibits vascular smooth muscle cell proliferation by reducing cell-cycle kinase activity.

Key Words: immunophilin • accelerated arteriosclerosis • antiproliferation • transplantation • FK506

	Introduction

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Abnormal vascular smooth muscle cell (VSMC) proliferation is involved in restenosis following percutaneous transluminal angioplasty (PTCA) and accelerated arteriosclerosis after cardiac transplantation.^{1 2 3} Restenosis occurs after 30% to 40% of the procedures,^{1 4} limiting the utility of PTCA. Accelerated arteriosclerosis in coronary arteries of the donor heart is a major factor limiting long-term survival of cardiac transplant recipients.^{3 5 6} Common to both pathological processes is an injury to the vascular endothelial cell barrier resulting in activation of VSMC proliferation. Multiple signaling pathways can trigger a proliferative response in VSMC. The complexity of cell growth signaling has made it difficult to achieve adequate control of VSMC proliferation in patients.

Much attention has focused on understanding the mechanisms underlying the proliferative response in VSMC. It has been proposed that identifying the regulators of this proliferative response in VSMC may lead to therapeutic strategies aimed at blocking or inhibiting VSMC growth. After deendothelialization of arteries by mechanical injury during PTCA, or by an immune mechanism in transplant recipients, VSMC leave their quiescent state (G0/G1) and enter the cell cycle. Recent studies have shown that early response genes including c-fos and c-myc are induced after exit from G0.^{7 8} Cell-cycle kinases including p34^cdc2 and mitogen-activated protein kinase homologues appear to be involved in signaling VSMC growth, leading to induction of early response genes.⁹ On the other hand, transforming growth factor-ß₁ inhibits smooth muscle cells causing a G1 arrest^{10 11} that is associated with a decrease in p34^cdc2 kinase activity.¹² These and other similar observations have led a number of investigators to focus on cell-cycle regulators as potential therapeutic targets for inhibiting VSMC proliferation. For example, antisense oligonucleotides to c-myc, c-myb, c-fos, cyclin A, p34^cdc2 kinase, and proliferating cell nuclear antigen have been used with varying degrees of success to inhibit VSMC proliferation.^{11 13 14}

Recent studies in a rat heart transplantation model have suggested that the macrolide antibiotic FK506, currently used as an immunosuppressant after some types of organ transplant, may accelerate transplant coronary arteriosclerosis.^{15 16 17} In animal models, rapamycin, also a macrolide antibiotic, appears to retard the development of accelerated arteriosclerosis after allograft transplantation and restenosis following mechanical injury.^{15 18 19 20}

In the present study, we sought to compare the effects of rapamycin and FK520 on VSMC proliferation. We found that rapamycin, but not FK520, inhibits cell growth in both human and rat VSMC. This inhibition of cell growth by rapamycin was associated with decreases in cell-cycle kinase activity at the G1/S and G2/M transitions. Phosphorylation of retinoblastoma protein (pRb), a marker for cell-cycle progression, was also reduced. Our data suggest that inhibition of cell-cycle kinases by rapamycin contributes to its potent antiproliferative effects in VSMC. Moreover, because the mechanism of the antiproliferative effect of rapamycin involves inhibition of cell-cycle kinases, rapamycin should block VSMC growth regardless of the stimulus that initiates the VSMC proliferative response. FK520, an analogue of FK506, nonsignificantly accelerated VSMC growth and increased the activity of the cell-cycle regulators. Thus, compared with FK520, rapamycin has antiproliferative properties that might make it a better choice for use in cardiac transplant recipients in whom VSMC proliferation is a potentially serious problem.

	Materials and Methods

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Reagents
Rapamycin was a gift from Wyeth-Ayerst Research Laboratory (Dr Suren Sehgal), and FK520 (an FK506 analogue) was provided by Dr John Siekierka (Merck). [³H]Thymidine was from NEN, and polyclonal anti-p34^cdc2 kinase antibody was a gift from Dr Hiroaki Kiyokawa (Memorial Sloan-Kettering Cancer Institute). Polyclonal antibodies to cyclin D (raised against the C-terminal domain of human cyclin D) and cdk2 (raised against a synthetic peptide in the C-terminal domain of human cdk2) were from Upstate Biotechnology Inc. pRb antibody was from Pharmingen.

Cell Culture
Rat aortic smooth muscle cells (RASM, isolation No. 1120)^{21 22 23} and human aortic smooth muscle cells were gifts from Dr Mark Taubman (Mount Sinai School of Medicine). RASM (passages 8 to 11) were cultured in Dulbecco's modified essential medium (DMEM) plus 20% fetal calf serum (FCS, GIBCO), 100 U/mL penicillin, and 100 µg/mL streptomycin as previously described.²¹ Medium was changed every 48 hours. When cultured in 20% FCS, RASM double approximately every 16 to 20 hours. Human aortic smooth muscle cells from ascending aorta obtained from the donor at the time of cardiac transplantation were cultured in DMEM plus 20% FCS. After plating, rapamycin (100 ng/mL) and FK520 (100 ng/mL) were added directly to DMEM. Cell proliferation analyses were performed by counting triplicate plates at the indicated times during a 7-day period by using a Coulter counter. Cell viability was determined with trypan blue stain for each experiment. Results represent the mean values from three separate experiments; error bars represent the standard error of the mean.

DNA Synthesis
For determination of DNA synthesis, [³H]thymidine incorporation was measured, and microcultures of 5000 cells were established in quadruplicates in flat-bottom 96-well microtiter plates in the presence and absence of varying concentrations of drugs. After 48 hours, each culture was pulsed with 1 µCi [³H]thymidine and harvested 16 to 20 hours later by using a Cambridge Technology PHD Harvester. [³H]Thymidine incorporation was measured in a liquid scintillation counter. The competition experiment with FK520 was performed with 2 ng/mL rapamycin and concentrations of FK520 between 2 and 500 ng/mL.

Flow Cytometric Analysis
Cells were treated with either 100 ng/mL rapamycin or FK520 for 24 hours, harvested, and washed in ice-cold phosphate-buffered saline (PBS), fixed in 70% ethanol, and stored overnight at 4°C before analysis. Cells were then washed once with ice-cold PBS treated with RNAse (1 hour at 37°C, 500 U/mL). Cellular DNA was stained with propidium iodide (50 µg/mL). Cell-cycle determination was performed by using a Coulter analyzer. Results represent a minimum of 3000 cells assayed for each determination.

Preparation of Cellular Lysates
RASM growing in log phase were plated at 30% confluence. After 24 hours in DMEM+20% FCS, plates were washed twice with PBS and transferred to DMEM+0.5% FCS for 72 hours to achieve quiescence. Plates were then stimulated with 20% FCS and treated with either no drugs (control), 100 ng/mL rapamycin, or 100 ng/mL FK520. After the indicated time period, plates were washed twice with ice-cold PBS, and cell lysates were prepared using Rb lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 120 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L NaF, 0.2 mmol/L Na₃VO₄, 10 mmol/L ß-glycerophosphate, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 10 µg/mL soybean trypsin inhibitor, and 0.5% Nonidet P-40). Cells were scraped off the bottom of the plates, and lysates were rocked for 1 hour at 4°C. Lysates were stored at -70°C. Protein concentration was measured by using the Bradford reagent (Bio-Rad), with bovine serum albumin used as a standard.

Determination of Cyclin-Dependent Kinase Activities
Activities of p34^cdc2 and p33^cdk2 kinases were analyzed essentially as described previously,²⁴ with some modifications. Protein extracts (100 µg) were diluted to 500 µL with RIPA buffer (50 mmol/L Tris-HCl, pH 7.4, 250 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na₃VO₄, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]). RASM lysates were immunoprecipitated with either anti-p34^cdc2 kinase C-terminus–specific antiserum²⁵ or a human polyclonal anti-p33^cdk2 kinase antibody. Protein A–Sepharose beads (20 µL) were added and gently rocked for 1 hour at 4°C. Samples were centrifuged and washed twice with RIPA buffer, twice with RIPA without NaCl, and twice with kinase assay buffer (mmol/L: Tris-HCl 50, pH 7.4, MgCl₂ 10, and dithiothreitol 1). Phosphorylation was carried out in 25 µL of kinase buffer with the addition of 0.1 mg/mL of histone H1 (Boehringer Mannheim) and 50 µCi [-³²P]ATP for 15 minutes at 28°C. The reaction was terminated with the addition of 6 µL of 6x Laemmli's sample loading buffer and boiled for 5 minutes. Samples (15 µL) were analyzed on a 12% SDS-polyacrylamide gel. Gels were dried for 2 hours and analyzed by using [-³²P]ATP standards and a phosphorimager.

Measurement of Retinoblastoma Protein Levels
Protein extracts (50 µg) were size-fractionated on 7.5% gels and transferred to nitrocellulose overnight at 60 V. Filters were blocked in PBS containing 0.1% Tween-20 (PBS-T) and 5% dry milk for 1 hour at 30°C, followed by incubation overnight with anti-pRb antibody (1/1000 dilution) at 4°C. The filters were washed with PBS-T, then incubated with the secondary antibody conjugated to peroxidase (goat anti-mouse IgG) for 1 hour at 4°C, and washed; signals were detected by using the chemiluminescence detection system (ECL) followed by exposure to Kodak XAR film. Autoradiographic signals were quantified by scanning the gels by use of a Macintosh computer with ADOBE PHOTOSHOP and IMAGE 1.44 software. The ratio of hyperphosphorylated to hypophosphorylated pRb was calculated for each time point and plotted. Results are shown for a representative experiment. This experiment was repeated three times, and similar results were obtained each time.

Measurement of Cyclin-Dependent Kinase and Cyclin Protein Levels
Protein extracts (50 µg) were electrophoresed on separate 12% SDS-polyacrylamide gels and transferred to nitrocellulose overnight at 45 V. Filters were blocked in PBS containing 0.1% Tween 20 and 5% dry milk for 1 hour at room temperature, followed by incubation overnight with anti-p34^cdc2 antibody (1/1000 dilution), anti-p33^cdk2 antibody (2.5 µg/mL), or anti–cyclin D antibody (2.5 mg/mL). The filters were then washed, incubated with goat anti-rabbit IgG conjugated to peroxidase for 1 hour at 4°C, and washed again; signals were detected by using the chemiluminescence detection system (Bio-Rad) and exposed to Kodak XAR films. Results are shown for representative experiments. These experiments were repeated three times, and similar results were obtained each time.

	Results

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Rapamycin as low as 1 ng/mL, but not FK520 at any dose tested, inhibited RASM proliferation (P<.05, Fig 1). Rapamycin also decreased [³H]thymidine incorporation in a dose-dependent manner (P<.01, Fig 2A). The inhibition of proliferation produced by rapamycin persisted at least through 7 days (168 hours) of cell culture. Similarly, rapamycin inhibited human aortic smooth muscle cell proliferation by 50% after 72 hours (P<.05; Fig 1, inset). In contrast, FK520 increased cell growth compared with control, but the differences were not significant. Cell viability, as assessed by trypan blue staining, was >99% in control, rapamycin-treated, and FK520-treated cells. The effect of rapamycin in terms of inhibiting DNA synthesis was competed by a molar excess of FK520 (Fig 2B). This result indicates that the reduction in [³H]thymidine incorporation was probably mediated by rapamycin binding to the immunophilin FKBP, since both rapamycin and FK520 share this same cytosolic receptor. FK520 at low concentrations (eg, 2.5 ng/mL) caused a small but significant (P<.05) decrease in DNA synthesis (Fig 2A), and there was a small additive effect of FK520 (only at low concentrations, eg, 8 and 16 ng/mL) combined with rapamycin in terms of decreasing DNA synthesis (Fig 2B).

View larger version (28K): [in this window] [in a new window]   Figure 1. Bar graph showing the time course for rapamycin-induced inhibition of cultured rat aortic smooth muscle cell (RASM) proliferation. The inset shows similar data for human aortic smooth muscle cells at 72 hours. Cells were treated with either no drug, 100 ng/mL rapamycin, or 100 ng/mL FK520. The results are expressed in mean cell numbers of triplicate plates; error bars represent standard deviation of the mean. The results are representative of three similar experiments. P<.05 for the comparison between control and rapamycin for both RASM and human aortic smooth muscle cells at each time point after 48 hours.

View larger version (26K): [in this window] [in a new window]   Figure 2. A, Bar graph showing the effect of immunosuppressive drugs rapamycin and FK520 on the incorporation of [3H]thymidine in cultured rat aortic smooth muscle cells. B, Bar graph showing that FK520 competes with rapamycin for binding to FKBP12 and inhibits the effects of rapamycin on [3H]thymidine uptake in cultured rat aortic smooth muscle cells. The results are a mean of quadruplicate wells, and the error bars represent standard deviation of the mean. P<.05 for the comparison between rapamycin-treated and control cells at each concentration above 0.0025 ng/mL. The results are representative of three similar experiments.

Rapamycin, but not FK520, delayed progression from G1 to S as assessed by cell-cycle analysis using propidium iodide staining. After stimulation with 20% FCS, 30% of cells progressed from G1/S and G2/M. The effect of rapamycin was to reduce progression from G1/S and G2/M to 10% of cells. pRb phosphorylation is believed to be a marker for progression from G1 to S. Hypophosphorylated pRb suppresses the progression from G1 to S,²⁶ and hyperphosphorylation generally occurs 1 to 2 hours before the G1/S transition.^{27 28} Cells in early G1 contain exclusively hypophosphorylated pRb. At an undefined point in late G1, pRb is hyperphosphorylated and remains hyperphosphorylated until M. In quiescent RASM (maintained in 0.5% FCS for 72 hours), pRb phosphorylation occurred 6 to 8 hours after stimulation with 20% FCS. Culturing cells with rapamycin (100 ng/mL) delayed the onset of pRb hyperphosphorylation in RASM by 6 hours to 12 hours after G0 and reduced the levels of phosphorylation at each time point sampled (Fig 3). In contrast, FK520 nonsignificantly accelerated the time course of pRb phosphorylation (Fig 3).

View larger version (43K): [in this window] [in a new window]   Figure 3. Bar graph showing the effects of rapamycin and FK520 on phosphorylation of retinoblastoma protein in cultured rat aortic smooth muscle cells. Cells were treated with either no drug, 100 ng/mL rapamycin, or 100 ng/mL FK520. The indicated times are in hours after G0. The positions indicating hyperphosphorylation and underphosphorylation (ppRb and pRb, respectively) are indicated at the left of each gel panel (inset above graph). The results shown are from a representative experiment. Similar results were obtained in three experiments.

Progression through the cell cycle is dependent on the activity of specific cell-cycle kinases, several of which, including cdk2 and cdk4, are thought to phosphorylate pRb. We examined the effects of rapamycin and FK520 on the activity of several cell-cycle kinases in VSMC. In RASM compared with control cells, p34^cdc2 kinase activity was decreased 16 to 20 hours after G0 by rapamycin but not by FK520 (Fig 4A). Protein levels of p34^cdc2 kinase were unchanged throughout the cell cycle (Fig 4A, insets). The decrease in p34^cdc2 kinase activity at 16 to 20 hours corresponds to the G2/M transition in RASM. At earlier time points (during the G1/S transition), p34^cdc2 kinase activity was low despite steady levels of p34^cdc2 protein, suggesting that it may have little effect at this point in the cell cycle in RASM (Fig 4A).

View larger version (39K): [in this window] [in a new window]   Figure 4. A, Bar graph showing that rapamycin, but not FK520, decreases p34cdc2 kinase activity at the G2/M transition in cultured rat aortic smooth muscle cells. The insets are immunoblots showing p34cdc2 protein levels. Cells were treated with either no drug, 100 ng/mL rapamycin, or 100 ng/mL FK520. B, Bar graph showing that rapamycin, but not FK520, decreases p33cdk2 kinase activity at the G1/S transition. The insets are immunoblots showing p33cdk2 protein levels. With the exception of the earliest two time points in the control samples, the level of p33cdk2 protein remained constant throughout the cell cycle. The results shown are from representative experiments. Similar results were obtained in three experiments.

Compared with control cells, rapamycin (100 ng/mL) decreased p33^cdk2 kinase activity beginning at 10 hours through 16 hours after G0 (Fig 4B). The period from 10 to 16 hours after G0 corresponds to the time during which pRb phosphorylation is decreased by rapamycin-FKBP (Fig 3). Protein levels for p33^cdk2 kinase were unchanged throughout the cell cycle compared with control cells (Fig 4B, insets), indicating that the decrease in p33^cdk2 kinase activity was not due to a decrease in p33^cdk2 synthesis. These data suggest that the inhibition of pRb phosphorylation could at least in part be due to a decrease in p33^cdk2 kinase activity.

A regulatory role for cyclin D1 has been proposed with regard to pRb phosphorylation.^{29 30} Interactions between cyclin D1 and a variety of cyclin-dependent kinases have been reported, and the expression of D-type cyclins is regulated by growth factors.³¹ We sought to determine, on the basis of these observations, whether the antiproliferative effects of rapamycin in RASM were associated with regulation of cyclin D1. Cyclin D1 levels were elevated in control RASM at 10 hours after G0, corresponding to the onset of pRb phosphorylation. Rapamycin delayed the onset of this rise in cyclin D1 levels by 4 to 6 hours (data not shown). The reduction in cyclin D1 levels by rapamycin occurred at the point when pRb phosphorylation was reduced.

	Discussion

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Our data show that the antiproliferative effects of rapamycin in VSMC are associated with an inhibition of cell-cycle kinases, cyclins, and pRb phosphorylation. These data imply that phosphorylation of pRb plays an important role in signaling during smooth muscle proliferation. In contrast, FK520, another potentially useful drug for immunosuppression following cardiac transplantation, induces a nonsignificant increase in VSMC proliferation associated with an acceleration of the time course and extent of pRb phosphorylation (Figs 1 and 3). The antiproliferative effects of rapamycin appear to be mediated by binding to the cytosolic receptor FKBP because they are competed by FK520, a drug that shares the same receptor. However, these studies do not exclude the possibility that these drugs also interact with other binding sites in RASM.

We observed small but significant effects only at low concentrations of FK520 (2 to 20 ng/mL) in terms of inhibiting DNA synthesis. However, the physiological importance of these effects was questionable because we never observed any inhibition of RASM proliferation when using either FK520 or FK506 at any concentration. Indeed, to the contrary, we have consistently observed a small nonsignificant increase in proliferation when using either FK520 or FK506. Moreover, FK520 increases pRb phosphorylation and cell-cycle kinase activity (Figs 3 and 4), suggesting that it accelerates cell-cycle progression.

Cyclin-dependent kinases, including cdk2, have been implicated as regulators of pRb function.^{32 33 34} Although the data suggest that a cyclin-dependent kinase phosphorylates pRb in vitro, there remains some controversy as to which kinase and how many actually carry out this function in vivo. We found that rapamycin but not FK520 decreased the activity of p33^cdk2 kinase³⁵ in VSMC. This finding does not indicate that pRb phosphorylation is dependent on p33^cdk2 kinase but suggests that this kinase could be involved in regulating pRb function in VSMC. We did not examine the activities of other kinases that similarly could be playing a role in phosphorylating pRb. Indeed, determining the specific kinase(s) that phosphorylates pRb would be interesting but is not required to support the main point of the present study, which is that the antiproliferative effects of rapamycin in VSMC are associated with inhibition of regulators of cell-cycle progression. Similarly, the finding that rapamycin decreased the activity of cell-cycle kinases and the levels of a cyclin (D1) does not rule out the possibility that rapamycin could have effects on other regulators of cell growth as well.

p34^cdc2 kinase is thought to play an important regulatory role in the G2/M transition.^{36 37} The time course for inhibition of p34^cdc2 kinase activity by rapamycin suggests that this kinase may play an important role in the G2/M transition in VSMC. In another myogenic cell line, BC3H1 cells, we had previously shown that rapamycin inhibited proliferation and induced differentiation and that these effects were also associated with a reduction in p34^cdc2 kinase activity.²⁴ However, in BC3H1 cells, the decrease in p34^cdc2 kinase activity occurs at the G1/S transition. The p34^cdc2 kinase may have multiple roles in the cell cycle, depending on which cell type is examined.

The growth-inhibitory effects of rapamycin (Fig 1) in VSMC are long lasting. In contrast, inhibition of pRb phosphorylation (Fig 3) and cell-cycle kinase activity (Fig 4) appears to be more of a transient delay rather than a complete block. Moreover, examination of the cell growth data in Fig 1 shows that although growth is significantly suppressed by rapamycin, there is some slow growth in the rapamycin-treated cells. Indeed, taken together, these data suggest that rapamycin significantly lengthens the cell cycle by introducing delays at the G1/S and G2/M transition points. These delays appear to result in a marked prolongation of the doubling time for the VSMC exposed to rapamycin (Fig 1). Some of the cell-cycle kinase activity and phosphorylation of pRb observed later in the cell cycle (eg, at 16 to 20 hours) in the rapamycin-treated cells may reflect the fact that the cell cultures were not synchronized. Thus, a subset of cells was past the G1/S transition at the start of the experiment, despite culturing in low-serum medium for 72 hours to induce quiescence.

It is believed that immunologic events linked to HLA incompatibility between the donor and host may result in vascular injury, leading to VSMC proliferation. Moreover, accelerated arteriosclerosis is not limited to cardiac transplant patients, as other organ allografts are subject to similar processes.³⁸ Cyclosporin A, one of the most widely used immunosuppressants, appears to have a neutral effect on accelerated arteriosclerosis.^{6 15 38 39} The mechanisms underlying post–cardiac transplant–accelerated arteriosclerosis remain poorly understood.^{5 15 38 40} Nevertheless, VSMC proliferation is the fundamental pathology. Accelerated arteriosclerosis after cardiac transplantation occurs with similar frequency despite the use of newer immunosuppressant agents, including cyclosporin A and FK506. FK506 is currently being used as a therapeutic agent for the prevention of post–cardiac transplant rejection in humans. Our findings predict that FK506 would either be neutral in terms of VSMC proliferation or could have an adverse effect by accelerating the time course and the extent of posttransplant arteriosclerosis. Conversely, since rapamycin both immunosuppresses and blocks VSMC proliferation, it could be the preferred therapeutic agent to reduce accelerated arteriosclerosis following cardiac transplantation and might even prolong survival in cardiac transplant recipients.

Many studies have attempted to identify the factor or factors contributing to VSMC proliferation following vascular injury, particularly after PTCA.^{2 9 12 21 22 41 42 43 44 45 46} The fact that rapamycin inhibits cell-cycle dependent kinases and phosphorylation of pRb suggests that its effects on VSMC proliferation would not depend on which of the many agents capable of triggering VSMC proliferation after injury are causative. As such, rapamycin might also be a useful agent for reducing or blocking the component of post-PTCA restenosis that is due to VSMC proliferation.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (NS-29814), the New York Heart Association, the Sarah Chait and the Louis B. Mayer Foundations (Dr Marks), and a Sable grant (Dr Marx). Dr Marks is a Bristol Meyers-Squibb Established Investigator of the American Heart Association. Drs Marx and Go are ACC/Merck Fellows. We thank Drs Valentin Fuster and Mark B. Taubman for helpful discussions, Dr Taubman for providing rat and human aortic smooth muscle cells, Dr Suren Sehgal of Wyeth-Ayerst for providing rapamycin, and Dr Hiroaki Kiyokawa (Memorial Sloan-Kettering Cancer Institute) for providing anti-p34^cdc2 kinase antibody.

Received September 7, 1994; accepted December 2, 1994.

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