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Am J Physiol Heart Circ Physiol 277: H1100-H1106, 1999;
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Vol. 277, Issue 3, H1100-H1106, September 1999

Vascular smooth muscle cell growth arrest on blockade of thrombospondin-1 requires p21Cip1/WAF1

Donghui Chen1, Kun Guo1, Jihong Yang1, William A. Frazier3, Jeffrey M. Isner1, and Vicente Andrés1,2

1 Department of Medicine (Cardiology), St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135; 2 Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, 46010 Valencia, Spain; and 3 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is thought to play an important role in the pathogenesis of atherosclerosis and restenosis. Previous studies have implicated the extracellular matrix protein thrombospondin-1 (TSP1) in mitogen-dependent proliferation of VSMCs. In this study, we investigated the molecular mechanisms involved in TSP1-mediated regulation of VSMC growth. Neutralizing A4.1 anti-TSP1 antibody inhibited the activity of the G1/S cyclin-dependent kinase 2 (cdk2) and blocked the induction of S-phase entry, which normally occurs in serum-stimulated VSMCs. This growth-inhibitory effect was associated with a marked induction of p21Cip1/WAF1 (p21) expression in A4.1-treated VSMCs. Moreover, addition of A4.1 antibody to VSMCs markedly increased the level of p21 bound to cdk2. Thus growth arrest on antibody blockade of TSP1 may be mediated by the cdk inhibitory protein p21. Consistent with this notion, anti-TSP1 antibody inhibited [3H]-thymidine incorporation in wild-type but not in p21-deficient mouse embryonic fibroblasts (MEFs). Together, these data suggest that p21 plays an important role in TSP1-mediated control of cellular proliferation.

cell cycle control; p21Cip1/WAF1; thrombospondin; extracellular matrix


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXTRACELLULAR MATRIX (ECM) plays a critical role in highly specialized cellular functions, including differentiation, migration, and proliferation (5, 21, 45, 46). The composition and structure of the ECM differs from tissue to tissue and can undergo continuous changes within the same tissue, thereby having both temporal and spatial effects on cells that come into contact with it. These changes result in part from the regulation of the synthesis and secretion of the glycoproteins that are incorporated into the ECM.

Unlike terminally differentiated myocytes, mature smooth muscle cells can reenter the cell cycle in response to physiopathological stimuli (35). Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of vascular occlusive disease, including atherosclerosis, restenosis after angioplasty, and bypass graft occlusion. Inhibition of VSMC proliferation has been shown to attenuate restenosis after balloon angioplasty in several animal models (12, 50). VSMC proliferation induced by growth factors in vitro and balloon injury in vivo is associated with changes in the expression of ECM proteins and their corresponding cellular receptors, which may play an important role as physiological regulators of cell cycle progression during atherosclerosis and restenosis (2). One of the ECM components for which dramatic regulatory changes have been observed is thrombospondin-1 (TSP1), a member of a family of related glycoproteins (TSP1 through TSP5) (3, 4, 10, 24, 25, 34, 41). TSP1 is secreted by numerous cell types, including platelets, endothelial cells, macrophages, fibroblasts, and VSMCs (19, 20, 25, 31, 33, 43). TSP1 expression is rapidly upregulated on serum or growth factor stimulation of cultured VSMCs (11, 28, 30), and TSP1 protein and mRNA are elevated with both intimal hyperplasia and hypercholesterolemia in vivo (26, 42-44, 56). Although TSP1 appears to be important for the proliferation of VSMCs (27, 29) and fibroblasts (38) it inhibits endothelial cell growth in vitro (54) and angiogenesis in vivo (13, 18). However, the molecular mechanisms by which TSP1 exerts these cell type-specific functions are not well understood.

Cell cycle progression is facilitated by the sequential activation of a family of cyclin-dependent kinases (cdks), which requires their association with specific subunits called cyclins (15, 32, 49). cdk2 activity is negatively regulated by members of the growth suppressor family of cdk inhibitors (CKIs), including p21Cip1/WAF1 (p21) and p27Kip1 (p27) (14, 16, 37). In the present study we investigated the molecular pathways through which TSP1 regulates VSMC proliferation in vitro by using neutralizing anti-TSP1 monoclonal antibody A4.1. Our results demonstrate that A4.1-mediated growth arrest in serum-stimulated VSMCs is associated with the inhibition of cdk2-dependent kinase activity. Expression of p21 and its association with cdk2 complexes were induced on addition of A4.1 antibody to serum-stimulated VSMCs. Moreover, A4.1 antibody blocked DNA synthesis in wild-type mouse embryonic fibroblasts (MEFs) but not in cells derived from p21-deficient mice. Together, these data demonstrate that antibody blockade of TSP1 inhibits cell cycle progression in a p21-dependent manner, and suggest the involvement of p21 in TSP1-mediated regulation of cellular proliferation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Cell culture. Cells were incubated at 37°C in a humidified 5% CO2-95% O2 atmosphere in medium supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 0.25 mg/ml streptomycin, and serum as indicated. Primary rat aortic VSMCs were isolated essentially as described (39) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS, growth medium). MEFs derived from wild-type and p21-deficient mice (8) were maintained in DMEM containing 10% FBS. To enrich the population of cells in G0/G1, cultures were serum-starved for 3 days in DMEM supplemented with 0.2% FBS. The C2C12 murine skeletal myoblast cell line was obtained from American Type Culture Collection. Terminal differentiation of C2C12 myoblasts maintained in 20% FBS-DMEM was induced by switching cultures to 2% heat-inactivated horse serum-DMEM (differentiation medium). Under these conditions, C2C12 cells permanently exit the cell cycle and then express differentiation markers (1).

Anti-TSP1 antibody. The mouse monoclonal anti-TSP1 antibody A4.1, which recognizes the trypsin-resistant 70-kDa core of TSP1 (40), was used in this study. Specificity of this antibody has been previously established by Western blotting against samples of whole platelets, serum, and purified proteins (40). Mouse nonspecific IgM antibody MOPC-104E (M2521) was used as a control (Sigma Chemical).

[3H]thymidine uptake analysis. Rat VSMCs and MEFs were plated in 24-well tissue culture plates in DMEM supplemented with 10% FBS. Cells were rendered quiescent by incubation for 3 days in DMEM containing 0.2% FBS and then cultures were restimulated with growth medium for 16-24 h. When indicated, cells were treated with different concentrations of anti-TSP1 antibody A4.1 (25-100 µg IgM/ml of medium). Cells were incubated in growth medium containing 3 µCi/ml of [3H]thymidine (6.7 Ci/mmol, Dupont NEN) for the last 4-6 h. Cells were washed three times with PBS and incubated with cold 10% TCA for 1 h. After we removed the TCA solution, we rinsed the cells three times with water and the precipitated material was solubilized with 0.25 N NaOH. Tritium content in the sodium hydroxide solution was determined by adding Scintiverse II (Fisher Scientific) and measured using a Beckman LS 5000TD scintillation counter. The experiments were performed in triplicate wells. Parallel cultures of VSMCs and MEFs in 24-well plates were collected by trypsinization, and the cell numbers were determined under microscopy with a hemocytometer.

FACS analysis. Rat VSMCs were plated in 100-mm petri dishes in growth medium and allowed to attach before being transferred to DMEM containing 0.2% FBS. After 72 h in low serum, cultures were restimulated by the addition of growth medium. Cells were trypsinized 24 h after addition of serum, then washed three times with PBS and fixed in 70% ethanol overnight at 4°C. DNA was stained with PBS containing 50 µg/ml each of propidium iodide and RNase A (Boehringer Mannheim). Cell cycle profile was determined at the Core Flow Cytometry Facility of the Dana Farber Cancer Institute (Boston, MA) using a Beckton Dickinson Vantage flow cytometer and Lysis II cell cycle analysis software. All experiments were performed in triplicate.

Western blot analysis, immunoprecipitation/Western blotting, and cdk2-dependent kinase assay. Subconfluent, starvation-synchronized rat VSMCs (in 100-mm plates) were switched to growth medium with or without the addition of the indicated amounts of either control IgM or A4.1 anti-TSP1 antibodies for 16 h. Cells were washed three times with cold PBS, resuspended in 500 µl of lysis buffer (50 mM Tris · Cl pH7.4, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, 2 µg/ml aproptinin, 2 µg/ml leupeptin, 1 µM phenylmethylsulfonyl fluoride) and passed through a 26-gauge, 0.5-in. needle several times. Insoluble material was cleared by centrifugation at 13,000 rpm for 10 min at 4°C. Protein concentration of lysates was determined using the Bradford reagent (BioRad). Protein extract (50 µg) was subjected to electrophoresis on 12% SDS-PAGE and transferred to Immobilon-P (Amersham). Membranes were blocked overnight at 4°C with buffer A (0.2% Tween-20 in PBS) containing 5% nonfat milk and then were incubated at room temperature for 3 h with the indicated primary antibodies diluted in buffer A containing 2% nonfat milk. The following antibodies were used in this study: anti-p21 (sc-397, 1:200), anti-p27 (sc-528, 1:250), anti-p53 (sc-99, 1:250), anti-cdk2 (sc-163, 1:500), anti-cyclin A (sc-751, 1:200), and anti-cyclin E (sc-481, 1:250) (Santa Cruz Biotechnology). After several washes with buffer A, immunocomplexes were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Life Science) according to the recommendations of the manufacturer. Autoradiographs of Western blots were scanned and band intensity was determined after background subtraction using a densitometric program (Sigma Gel, Jandel Scientific).

For immunoprecipitation/Western blot-coupled assays, 200 µg of cell extract was precleared with 20 µl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) for 30 min at 4°C, after which samples were incubated with 2 µg of anti-cdk2 antibodies for 3 h at 4°C. Immunocomplexes were precipitated with 20 µl of Protein A/G PLUS-Agarose beads at 4°C for 1 h. Pellets were washed three times with lysis buffer and subjected to Western blotting with anti-cdk2 antibodies as described above.

Cdk2-dependent kinase assays in cell lysates were performed using histone H1 (Boehringer Mannheim) and [gamma -32P]ATP (DuPont NEN) substrates as previously described (7). The reaction mixtures were separated on 12% SDS-PAGE. Gels were stained with Coomassie blue (Sigma Chemical), dried, and autoradiographed.

Statistics. All results are means ± SE. Statistical significance was evaluated using ANOVA followed by Scheffé's procedure for more than two means. P < 0.05 was interpreted to indicate statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neutralizing A4.1 anti-TSP1 antibody blocks serum-inducible DNA synthesis in VSMCs. We first investigated the effects of neutralizing A4.1 monoclonal anti-TSP1 antibody on [3H]thymidine incorporation on serum restimulation of starvation-synchronized rat VSMCs. To this end, cells were incubated for 72 h in 0.2% FBS-DMEM and then stimulated with growth medium (10% FBS-DMEM) for 24 h, with or without the addition of A4.1 antibody. As shown in Fig. 1A, serum restimulation of VSMCs treated with control IgM led to an approximately sixfold increase in [3H]thymidine incorporation. However, increasingly higher concentrations of anti-TSP1 antibody reduced serum-inducible [3H]thymidine uptake, with the highest amount of A4.1 antibody tested completely blocking [3H]thymidine incorporation in serum-restimulated VSMCs.


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  		Fig. 1.   Antibody blockade of thrombospondin-1 (TSP1) inhibits serum-inducible vascular smooth muscle cell (VSMC) proliferation. A: serum-starved VSMCs (solid bar) were stimulated with DMEM supplemented with 10% fetal bovine serum (FBS) and control mouse IgM (100 µg/ml, hatched bar) or with the indicated concentrations of A4.1 anti-TSP1 antibody. After 18 h, [3H]thymidine was added to the medium (3 µCi/ml) and cells were incubated for an additional 6 h. [3H]thymidine uptake was determined in TCA precipitates in counts/min (cpm). Each bar represents mean ± SE of 3 independent experiments. * Statistically significant differences in A4.1-treated cells vs. cells treated with control mouse IgM (P < 0.01). B: A4.1 antibody blocked S-phase entry in serum-stimulated VSMCs. Starvation-synchronized VSMCs were switched to growth medium (10% FBS) supplemented with either control IgM or A4.1 antibody (100 µg/ml). After 24 h, cells were washed with PBS and harvested by trypsinization. Cells were then fixed in 70% ethanol overnight at 4°C and stained with propidium iodide. DNA content was analyzed by flow cytometry to determine cell cycle distribution. Each bar represents mean ± SE of 3 independent experiments. * Statistically significant differences in A4.1-treated cells vs. cells treated with control mouse IgM (P < 0.01).

We next performed flow cytometry analysis to further characterize the effect of A4.1 antibody on VSMC proliferation. In serum stimulated cultures, ~60 and 30% of the cells were in G1 and S phase, respectively (Fig. 1B). Whereas treatment of VSMCs with control IgM did not significantly affect this cell cycle profile, addition of A4.1 antibody decreased the cell population in S phase to ~10% and augmented the population in G1 to ~81%. Thus, consistent with previous studies (29), these data demonstrate that neutralization of TSP1 function in serum-stimulated VSMCs leads to inhibition of DNA synthesis and accumulation of cells in G1.

Neutralizing A4.1 anti-TSP1 antibody blocks serum-inducible cdk2-dependent kinase activity in VSMCs. Cdk2 function is required for progression through G1 and S phase (15, 32, 49). When assayed in vitro using anti-cdk2 antibodies and histone H1 as substrate, cdk2-dependent kinase activity was downregulated in both starvation-synchronized skeletal muscle cells (SKMCs) and VSMCs compared with asynchronously proliferating cells (Fig. 2A, lane Q vs. P). Consistent with the irreversibility of cell cycle exit in striated myocytes, serum-restimulated SKMCs disclosed impaired induction of cdk2-dependent kinase activity (lane Q + FBS). In marked contrast, serum-restimulated VSMCs upregulated cdk2-dependent kinase activity to a level similar to that seen in asynchronously growing cultures, demonstrating that VSMCs can reversibly regulate cdk2 function in response to mitogens. Because conditions that promote cell growth arrest were associated with inhibition of cdk2 function, we sought next to investigate the effect of A4.1 antibody on cdk2-dependent kinase activity in VSMCs. As shown in Fig. 2B, VSMCs treated with control IgM efficiently upregulated cdk2-dependent kinase activity on serum refeeding. In contrast, addition of A4.1 antibody to the medium blocked the normal serum-dependent induction of cdk2 activity. Thus inhibition of cdk2 activity may contribute to the cell cycle inhibitory activity of anti-TSP1 antibodies in VSMCs.


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  		Fig. 2.   A4.1 antibody blocks serum-inducible cyclin-dependent kinase 2 (cdk2)-dependent kinase activity. Cell extracts were immunoprecipitated with anti-cdk2 antibodies, and the kinase activity in the immunopellets was assayed by using histone H1 and [gamma -32P]ATP as substrates. Reaction mixtures were separated onto 12% SDS-PAGE, and the gels were dried and autoradiographed. Arrows point to phosphorylated histone H1. A: C2C12 skeletal muscle cells (SKMC) and VSMCs were maintained in growth medium (lane P, proliferating) or were serum starved for 3 days (lane Q). Parallel dishes were also serum-restimulated for 18 h after starvation (lane Q + FBS). B: cell extracts were prepared from starvation-synchronized VSMCs (0.2% FBS, lane 1) and from cells that had been serum-restimulated for 16 h with growth medium after serum starvation (10% FBS, lanes 2-4). Serum-stimulated cells were treated with control mouse IgM (lane 3) or A4.1 anti-TSP1 antibody (lane 4) (100 µg/ml).

Neutralizing A4.1 anti-TSP1 antibody abrogates serum-inducible cyclin A and cyclin E expression and induces p21Cip1/WAF1. Having demonstrated that cell cycle arrest in A4.1-treated VSMCs is associated with impaired cdk2 function, we sought to elucidate the molecular mechanisms underlying this inhibitory effect. As shown by the Western blot analysis of Fig. 3, addition of A4.1 antibody to serum-restimulated VSMCs had no effect on cdk2 protein levels. Because cdk2 activity during G1 and S phase is induced in part through its association with cyclin E and cyclin A, respectively, the effect of A4.1 antibody on the expression of these regulatory subunits was also studied. Treatment of serum-restimulated VSMCs with A4.1 antibody blocked serum-inducible expression of cyclin E and cyclin A (Fig. 3). Thus inhibition of cdk2 activity by A4.1 is associated with diminished expression of its cyclin regulatory subunits.


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  		Fig. 3.   Antibody blockade of TSP1 inhibits serum-inducible cyclin A and cyclin E expression and induces p21 protein levels in VSMCs. Cell extracts (50 µg protein), prepared from VSMCs treated as indicated in Fig. 2B, were subjected to Western blot analysis using anti-cdk2, anti-cyclin E, anti-cyclin A, anti-p27, and anti-p21 antibodies. Densitometric analysis was performed to quantify the relative amount of protein in each band. Results are shown below each lane and are expressed relative to the amount in serum-starved cells (lane 1 = 100%). To determine the amount of p21 bound to cdk2, 200 µg of cell extract was immunoprecipitated with anti-cdk2 antibodies. Immunopellets were resuspended in sample loading buffer and separated by electrophoresis on 12% SDS-PAGE. After transfer, blot was subjected to Western blot analysis using anti-p21 antibodies.

Because cdk2 activity can be negatively regulated through its association with the inhibitory proteins p21 and p27, we also analyzed the effect of A4.1 antibody on these growth suppressor molecules. Whereas A4.1 antibody did not affect p27 expression, its addition to serum-restimulated VSMCs markedly upregulated p21 protein levels (Fig. 3). To test whether the induction of p21 in A4.1-treated VSMCs resulted in an increased association of p21 with cdk2-containing complexes, cell lysates were immunoprecipitated with anti-cdk2 antibodies and then the immunopellets were subjected to Western blot analysis using anti-p21 antibodies. As shown in Fig. 3, the abundance of p21 associated with cdk2 was increased by the treatment of cells with A4.1 antibody. The lack of cdk2-bound p21 in serum restimulated cells that were not treated with A4.1 antibody (Fig. 3, lanes 2 and 3) may be caused by association of p21 with cdk4-cyclin D1 holoenzymes (23, 36). These results suggest that upregulation of p21 and its increased association with cdk2, together with diminished cyclin A and cyclin E expression, may contribute to growth arrest in VSMCs treated with neutralizing anti-TSP1 antibody. In these studies, control mouse IgM had little or no effect on the expression of these cell cycle regulatory proteins, demonstrating the specificity of the effects elicited by the A4.1 antibody.

p21 is essential for growth suppression on antibody blockade of TSP1. The above results suggest that p21 plays an important role on cell cycle arrest in cells treated with A4.1 antibody. To further investigate the role of p21 on growth arrest induced on neutralization of TSP1 function, the effect of A4.1 antibody on [3H]thymidine incorporation was tested in MEFs isolated from wild-type and p21-deficient mice. As shown in Fig. 4, addition of A4.1 antibody inhibited in a dose-dependent manner [3H]thymidine incorporation in serum-stimulated wild-type MEFs. In marked contrast, addition of A4.1 to the culture medium had little or no effect on [3H]thymidine uptake in serum-stimulated p21-deficient MEFs. These findings demonstrate that p21 is essential for the cell cycle inhibitory activity of anti-TSP1 antibody.


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  		Fig. 4.   p21 is essential for A4.1-dependent cell cycle arrest in mouse embryonic fibroblasts. MEFS were isolated from wild-type (A) and p21-deficient (B) mice. Cells were serum-starved by incubation in 0.2% FBS for 72 h. Cultures were switched to growth medium (10% FBS) supplemented with either control IgM (100 µg/ml, solid bar) or with the indicated amount of A4.1 antibody. [3H]thymidine was added to the culture medium 12 h postserum stimulation, cells were incubated for an additional 4 h, and incorporated label was determined by TCA precipitation. Values are means ± SE of 3 experiments.


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

TSP1 expression is rapidly upregulated on serum or growth factor stimulation of quiescent VSMCs (11, 28, 30). Moreover, TSP1 protein and mRNA are detected in VSMCs within atherosclerotic lesions (26, 43, 44, 56) and during the proliferative response of VSMCs to vascular injury (42). Consistent with the role of TSP1 as a positive regulator of VSMC proliferation, addition of neutralizing anti-TSP1 antibodies blocked serum-inducible VSMC proliferation (29). However, the mechanisms by which TSP1 regulates VSMC growth are not completely understood. The present study demonstrates that addition of neutralizing A4.1 antibody to cultured VSMCs blocked in a dose-dependent manner the serum-inducible activity of cdk2, a cell cycle regulator that is required for G1- and S-phase progression. Although cdk2 expression was not affected by the addition of A4.1 to VSMC cultures, p21 protein level was induced markedly in A4.1-treated cells. Importantly, exposure to A4.1 antibody also increased the interaction of p21 with cdk2 complexes, suggesting that upregulation of p21 contributes to the repression of cdk2 activity and cell-cycle arrest on neutralization of TSP1 function. Further evidence implicating p21 in growth arrest induced by anti-TSP1 antibody was provided using MEFs derived from either wild-type or p21-null mice. Indeed, A4.1 significantly blocked DNA synthesis in wild-type, but not in p21-null MEFs. These data together demonstrate that p21 is essential for A4.1-induced growth arrest, therefore implicating p21 as a downstream effector of TSP1.

Our results show that A4.1 antibody blocks the normal induction of cyclin A and cyclin E protein expression normally seen in serum-stimulated VSMCs. Together with the requirement of these regulatory subunits for cell cycle progression (14, 16, 17), these data suggest that inhibition of cyclin A and cyclin E expression may contribute to growth arrest in VSMCs exposed to A4.1 antibody. It is noteworthy that serum-dependent induction of cyclin A promoter activity in VSMCs and fibroblasts requires a functional E2F binding site (47, 52). Moreover, the CKIs p16, p21, and p27 can repress transcription of E2F target genes, including cyclin A and cdc2 (7, 9, 48, 52, 59), suggesting that blockade of cyclin A expression in VSMCs treated with A4.1 antibody may result in part from p21-dependent transcriptional repression.

The effect of CKIs, ECM components and their cellular receptors on VSMC proliferation has been the subject of recent studies. Expression of p21 and p27 in VSMCs is markedly upregulated after angioplasty at time points that coincide with the reestablishment of the quiescent phenotype (7, 53, 58). Moreover, adenovirus-mediated overexpression of p21 (6, 55, 58) and p27 (7) attenuates VSMC hyperplasia after vascular injury in vivo. Regarding the regulation of CKI expression by specific components of the ECM, Koyama et al. (22) reported that polymerized type I collagen inhibits mitogen-inducible VSMC proliferation in vitro by upregulating p27 and p21 levels, whereas monomer collagen supported cell cycle activity (22). Interestingly, treatment with neutralizing antibodies against alpha 2-integrins induced p27 and p21 expression and caused cell cycle arrest in VSMCs grown on monomer collagen (22). These findings suggest that alpha 2-integrins can sense changes in collagen structure that modulate VSMC proliferation through the regulation of CKI expression. Of note, it has been shown that the TSP receptor CD47 (IAP) can associate with alpha 2beta 1-integrin and modulate its function in VSMCs (57). Moreover, TSP-induced VSMC proliferation in vitro is regulated by beta 3-integrins, which are upregulated during injury-induced VSMC hyperplasia in vivo (51). Thus regulation of CKI expression through specific ECM components (i.e., TSP1) appears to be an important regulator of VSMC growth.

In summary, the present study demonstrates that VSMC growth arrest on antibody blockade of TSP1 is associated with the induction of p21 and repression of cyclin A and cyclin E expression. Neutralizing A4.1 antibody failed to inhibit cell proliferation in embryonic fibroblasts derived from p21-deficient mice. These results suggest that cell cycle arrest in cells treated with neutralizing A4.1 antibody results, at least in part, from p21-dependent inhibition of cdk2 function. Taken together, these data implicate p21 in TSP1-dependent regulation of cellular growth. Future studies should elucidate the molecular mechanisms underlying A4.1-dependent regulation of p21 expression.


    ACKNOWLEDGEMENTS

We are grateful to Dr. P. Leder for the gift of wild-type and p21-deficient MEFS and to Dr. P. Bornstein for a critical reading of the paper.


    FOOTNOTES

This paper was supported in part by National Institutes of Health Grants HL-57519 and AG-15227 (to V. Andrés); HL-40518, HL-53354, and HL-57516 (to J. M. Isner); and CA-65872 (to W. A. Frazier).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Dr. V. Andrés, Instituto de Biomedicina, CSIC, Jaime Roig 11, 46010 Valencia, Spain (E-mail: vandres{at}ibv.csic.es).

Received 4 November 1998; accepted in final form 23 March 1999.


    REFERENCES
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DISCUSSION
REFERENCES

1.   Andrés, V., and K. Walsh. Myogenin expression, cell cycle withdrawal and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J. Cell Biol. 132: 657-666, 1996[Abstract/Free Full Text].

2.   Assoian, R. K., and E. E. Marcantonio. The extracellular matrix as a cell cycle control element in atherosclerosis and restenosis. J. Clin. Invest. 98: 2436-2439, 1996[Medline].

3.   Bornstein, P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130: 503-506, 1995[Free Full Text].

4.   Bornstein, P., K. O'Rourke, K. Wikstrom, F. W. Wolf, R. Katz, P. Li, and V. M. Dixit. A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome. J. Biol. Chem. 266: 12821-12824, 1991[Abstract/Free Full Text].

5.   Carey, D. J. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Annu. Rev. Physiol. 53: 161-177, 1991[Medline].

6.   Chang, M. W., E. Barr, M. M. Lu, K. Barton, and J. M. Leiden. 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. 96: 2260-2268, 1995.

7.   Chen, D., K. Krasinski, D. Chen, A. Sylvester, J. Chen, P. D. Nisen, and V. Andrés. 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. 99: 2334-2341, 1997[Medline].

8.   Deng, C., P. Zhang, J. W. Harper, S. J. Elledge, and P. Leder. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82: 675-684, 1995[Medline].

9.   Dimri, G. P., M. Nakanishi, P.-Y. Desprez, J. R. Smith, and J. Campisi. Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein. Mol. Cell. Biol. 16: 2987-2997, 1996[Abstract].

10.   Dixit, V. M., S. W. Hennessy, G. A. Grant, P. Rotwein, and W. A. Frazier. Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin. Proc. Natl. Acad. Sci. USA 83: 5449-5453, 1986[Abstract/Free Full Text].

11.   Framson, P., and P. Bornstein. A serum response element and binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene. J. Biol. Chem. 268: 4989-4996, 1993[Abstract/Free Full Text].

12.   Gibbons, G. H., and V. J. Dzau. Molecular therapies for vascular diseases. Science 272: 689-693, 1996[Abstract].

13.   Good, D. J., P. J. Polverini, F. Rastinejad, M. M. Le Beau, R. S. Lemons, W. A. Frazier, and N. P. Bouck. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA 87: 6624-6628, 1990[Abstract/Free Full Text].

14.   Graña, X., and E. P. Reddy. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11: 211-219, 1995[Medline].

15.   Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, and S. J. Elledge. The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75: 805-816, 1993[Medline].

16.   Harper, J. W., and S. J. Elledge. Cdk inhibitors in development and cancer. Curr. Opin. Genet. Dev. 6: 56-64, 1996[Medline].

17.   Heichman, K. A., and J. M. Roberts. Rules to replicate by. Cell 79: 557-562, 1994[Medline].

18.   Iruela-Arispe, M. L., P. Bornstein, and H. Sage. Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro. Proc. Natl. Acad. Sci. USA 88: 5026-5030, 1991[Abstract/Free Full Text].

19.   Jaffe, E. A., J. T. Ruggiero, and D. J. Falcone. Monocytes and macrophages synthesize and secrete thrombospondin. Blood 65: 79-84, 1985[Abstract/Free Full Text].

20.   Jaffe, E. A., J. T. Ruggiero, L. K. Leung, M. J. Doyle, McKeown, P. J. Longo, and D. F. Mosher. Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix. Proc. Natl. Acad. Sci. USA 80: 998-1002, 1983[Abstract/Free Full Text].

21.   Juliano, R. L., and S. Haskill. Signal transduction from the extracellular matrix. J. Cell Biol. 120: 577-585, 1993[Free Full Text].

22.   Koyama, H., E. W. Raines, K. E. Bornfeldt, J. M. Roberts, and R. Ross. Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of cdk2 inhibitors. Cell 87: 1069-1078, 1996[Medline].

23.   LaBaer, J., M. D. Garret, L. F. Stevenson, J. M. Slingerland, C. Sandhu, H. S. Chou, A. Fattaey, and E. Harlow. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 11: 847-862, 1997[Abstract/Free Full Text].

24.   Lawler, J., K. McHenry, M. Duquette, and L. Derick. Characterization of human thrombospondin-4. J. Biol. Chem. 270: 2809-2814, 1995[Abstract/Free Full Text].

25.   Lawler, J., H. S. Slayter, and J. E. Coligan. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J. Biol. Chem. 253: 8609-8616, 1978[Abstract/Free Full Text].

26.   Liau, G., J. A. Winkles, M. S. Cannon, L. Kuo, and W. M. Chilian. Dietary-induced atherosclerotic lesions have increased levels of acidic FGF mRNA and altered cytoskeletal and extracellular matrix mRNA expression. J. Vasc. Res. 30: 327-332, 1993[Medline].

27.   Majack, R. A., S. C. Cook, and P. Bornstein. Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Proc. Natl. Acad. Sci. USA 83: 9050-9054, 1986[Abstract/Free Full Text].

28.   Majack, R. A., S. C. Cook, and P. Bornstein. Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J. Cell Biol. 101: 1059-1071, 1985[Abstract/Free Full Text].

29.   Majack, R. A., L. V. Goodman, and V. M. Dixit. Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J. Cell Biol. 106: 415-422, 1988[Abstract/Free Full Text].

30.   Majack, R. A., J. Mildbrandt, and V. M. Dixit. Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet-derived growth factor. J. Biol. Chem. 262: 8821-8825, 1987[Abstract/Free Full Text].

31.   McPherson, J., H. Sage, and P. Bornstein. Isolation and Characterization of a glycoprotein secreted by aortic endothelial cells in culture: apparent identity with platelet thrombospondin. J. Biol. Chem. 256: 11330-11336, 1981[Abstract/Free Full Text].

32.   Morgan, D. O. Principle of cdk regulation. Nature 374: 131-134, 1995[Medline].

33.   Mumby, S. M., D. Abbott Brown, D. Raugi, and P. Bornstein. Regulation of thrombospondin secretion by cells in culture. J. Cell. Physiol. 120: 280-288, 1984[Medline].

34.   Oldberg, A., P. Antonsson, K. Lindblom, and D. Heinegard. COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins. J. Biol. Chem. 267: 22346-22350, 1992[Abstract/Free Full Text].

35.   Owens, G. K. Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev. 75: 487-517, 1995[Abstract/Free Full Text].

36.   Parry, D., D. Mahony, K. Wills, and E. Lees. Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors. Mol. Cell. Biol. 19: 1775-1783, 1999[Abstract/Free Full Text].

37.   Peter, M., and I. Herskowitz. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell 79: 181-184, 1994[Medline].

38.   Phan, S. H., R. G. Dillon, B. M. McGarry, and V. M. Dixit. Stimulation of fibroblast proliferation by thrombospondin. Biochem. Biophys. Res. Commun. 163: 56-63, 1989[Medline].

39.   Pickering, J. G., L. Weir, K. Rosenfield, J. Stetz, J. Jekanowski, and J. M. Isner. Smooth muscle cell outgrowth from human atherosclerotic plaque: implications for the assessment of lesion biology. J. Am. Coll. Cardiol. 20: 1430-1439, 1992[Abstract].

40.   Prater, C. A., J. Plotkin, D. Jaye, and W. A. Frazier. The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol. 112: 1031-1040, 1991[Abstract/Free Full Text].

41.   Qabar, A. N., Z. Lin, F. W. Wolf, K. S. O'Shea, J. Lawler, and V. M. Dixit. Thrombospondin 3 is a developmentally regulated heparin binding protein. J. Biol. Chem. 269: 1262-1269, 1994[Abstract/Free Full Text].

42.   Raugi, G. J., J. S. Mullen, D. H. Barb, T. Okada, and M. R. Mayberg. Thrombospondin deposition in rat carotid artery injury. Am. J. Pathol. 137: 179-185, 1990[Abstract].

43.   Reed, M. J., L. Iruela-Arispe, E. R. O'Brien, T. Truong, T. LaBell, P. Bornstein, and E. H. Sage. Expression of thrombospondins by endothelial cells. Injury is correlated with TSP-1. Am. J. Pathol. 147: 1068-1080, 1995[Abstract].

44.   Roth, J. J., V. Gahtan, J. L. Brown, C. Gerhard, V. K. Swami, V. L. Rothman, T. N. Tulenko, and G. P. Tuszynski. Thrombospondin-1 is elevated with both intimal hyperplasia and hypercholesterolemia. J. Surg. Res. 74: 11-16, 1998[Medline].

45.   Ruoslathi, E., and Y. Yamaguchi. Proteoglycans as modulators of growth factor activities. Cell 64: 867-869, 1991[Medline].

46.   Savani, R. C., C. Wang, B. Yang, S. Zhang, M. G. Kinsella, T. N. Wight, R. Stern, D. W. Nance, and E. A. Turley. Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM. J. Clin. Invest. 95: 1158-1168, 1995.

47.   Schulze, A., K. Zerfass, D. Spitkovsky, S. Middendorp, J. Berges, K. Helin, P. Jansen-Dürr, and B. Henglein. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site. Proc. Natl. Acad. Sci. USA 92: 11264-11268, 1995[Abstract/Free Full Text].

48.   Schulze, A., K. Zerfass-Thome, J. Bergès, S. Middendorp, P. Jansen-Dürr, and B. Henglein. Anchorage-dependent transcription of the cyclin A gene. Mol. Cell. Biol. 16: 4632-4638, 1996[Abstract].

49.   Sherr, C. J. G1 phase progression: cycling on cue. Cell 79: 551-555, 1994[Medline].

50.   Spyridopoulos, I., and V. Andrés. Control of vascular smooth muscle and endothelial cell proliferation and its implication in cardiovascular disease. Front. Biosc. 3: 269-287, 1998.

51.   Stouffer, G. A., Z. Hu, M. Sajid, H. Li, G. Jin, M. T. Nakada, S. R. Hanson, and M. S. Runge. beta 3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells. Circulation 97: 907-915, 1998[Abstract/Free Full Text].

52.   Sylvester, A. M., D. Chen, K. Krasinski, and V. Andrés. Role of c-fos and E2F in the induction of cyclin A transcription and vascular smooth muscle cell proliferation. J. Clin. Invest. 101: 940-948, 1998[Medline].

53.   Tanner, F. C., Z.-Y. Yang, E. Duckers, D. Gordon, G. J. Nabel, and E. G. Nabel. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ. Res. 82: 396-403, 1998[Abstract/Free Full Text].

54.   Taraboletti, G., D. Roberts, L. A. Liotta, and R. Giavazzi. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell Biol. 111: 765-772, 1990[Abstract/Free Full Text].

55.   Ueno, H., S. Masuda, S. Nishio, J. J. Li, H. Yamamoto, and A. Takeshita. Adenovirus-mediated transfer of cyclin-dependent kinase inhibitor-p21 suppresses neointimal formation in the balloon-injured rat carotid arteries in vivo. Ann. NY Acad. Sci. 811: 401-411, 1997[Medline].

56.   Van Zanten, G. H., S. de Graaf, P. J. Slootweg, H. F. G. Heijnen, T. M. Connolly, P. G. de Groot, and J. J. Sixma. Increased platelet deposition on atherosclerotic coronary arteries. J. Clin. Invest. 93: 615-632, 1994.

57.   Wang, X.-Q., and W. A. Frazier. The thrombospondin receptor CD47 (IAP) modulates and associates with alpha 2beta 1 integrin in vascular smoth muscle cells. Mol. Biol. Cell 9: 865-874, 1998[Abstract/Free Full Text].

58.   Yang, Z.-Y., R. D. Simari, N. D. Perkins, H. San, D. Gordon, G. J. Nabel, and E. G. Nabel. Role of p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc. Natl. Acad. Sci. USA 93: 7905-7910, 1996[Abstract/Free Full Text].

59.   Zerfass-Thome, K., A. Schulze, W. Zwerschke, B. Vogt, K. Helin, J. Bartek, B. Henglein, and P. Jansen-Dürr. p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression. Mol. Cell. Biol. 17: 407-415, 1997[Abstract].

Am J Physiol Heart Circ Physiol 277(3):H1100-H1106
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