Cip1

Atherosclerosis 161 (2002) 105– 112 www.elsevier.com/locate/atherosclerosis

Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21Waf1/Cip1 Robert H. Weiss a,b,c,*, Collette J. Randour a a

Di6ision of Nephrology, Department of Internal Medicine, TB 136 Uni6ersity of California, Da6is, CA 95616, USA b Cell and De6elopmental Biology Graduate Group, Uni6ersity of California, Da6is, CA 95616, USA c Department of Veterans’ Affairs Northern California Health Care System, Mather, CA 95655, USA Received 18 January 2001; received in revised form 26 June 2001; accepted 10 July 2001

Abstract Progressive fibrosis in major organs, including the heart, the kidney and the vascular tree, plays an important role in mediating chronic disease and atherosclerosis. Production of extracellular matrix proteins, in many cases regulated by the growth factor TGF-b is an essential component of this process. In a parallel manner to TGF-b, the cyclin kinase inhibitors (CKIs; which are induced by TGF-b) regulate transit through the cell cycle, and their effect on growth has been shown to be bimodal in the case of vascular smooth muscle (VSM) cells. Using an antisense oligodeoxynucleotide to the CKI p21Waf1/Cip1, developed in our laboratory and shown to specifically inhibit p21Waf1/Cip1 protein levels, we asked whether attenuation of the CKI p21Waf1/Cip1 by transfection of this oligodeoxynucleotide results in the abolition of TGF-b-mediated growth inhibition and/or diminished matrix protein production and secretion in the presence or absence of TGF-b. Specific inhibition of p21Waf1/Cip1 protein with the antisense oligodeoxynucleotide markedly reduces the production and secretion of the matrix proteins fibronectin and laminin, both in the presence and absence of TGF-b stimulation, in VSM cells as observed by Western blotting of cell lysate and conditioned medium. In addition, TGF-b-mediated cell growth inhibition, though attenuated by this oligo, is preserved. Due to the relative ease and safety of transfecting antisense oligodeoxynucleotides into VSM, we believe that this work unmasks a potentially powerful technique for inhibition of matrix protein synthesis in VSM and related cell lines, and may lead to new treatment strategies for atherosclerotic as well as other systemic diseases characterized by aberrant matrix protein secretion. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Extracellular matrix; Vascular smooth muscle; Cyclin kinase inhibitor

1. Introduction The extracellular matrix plays a key role in the progression of fibrosis in a variety of disparate diseases in multiple organ systems. In vascular smooth muscle (VSM) and related [1] glomerular mesangial cells, overexuberant secretion of matrix proteins is involved in the progression of the atherosclerotic state and is likely responsible for progression of renal disease as well (reviewed in Refs. [2,3]). The growth factor TGFb, which in VSM cells is generally growth inhibitory [4,5], causes fibrosis in a variety of tissues and has been * Corresponding author. Tel.: + 1-530-752-4010; fax: +1-530-7523791. E-mail address: [email protected] (R.H. Weiss).

linked to an increase in matrix protein production as an etiology for this pathologic process [6,7], such that antibodies to TGF-b1 suppress arterial intimal hyperplasia and restenosis [8] as well as experimental glomerulonephritis [9]. The early lesion of atherosclerosis is associated with migration and proliferation of VSM cells. Once these cells enter the proliferative state, they attain a synthetic phenotype, which causes them to secrete matrix proteins [2,10]. Excessive secretion of these proteins may lead to fibrosis, but the same proteins may also regulate the cell phenotype and cause it to either remain secretory or become proliferative [11]. TGF-b has been implicated in the regulation of this process through its role in switching VSM cell response between hyperplasia and hypertrophy [12].

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The cyclin kinase inhibitors (CKIs) are proteins which regulate transit through the cell cycle by means of their interaction with the cyclins and the cyclin dependent kinases (cdks). One arm of TGF-b signaling, likely related to its VSM cell growth inhibitory effect, involves induction of the CKIs p21 and p27 [13,14]. However, while the CKI proteins had initially been thought to confer solely inhibitory effects upon cell growth, we [15] and others [16] have recently shown that attenuation of expression of these molecules results in, or corresponds to, growth inhibition in VSM cells. Recently, as well, data has appeared showing that p21 ( − / − ) knockout mice are characterized by attenuated progression to chronic renal failure after partial renal ablation [17]. Other studies have shown that the related CKI p27 may mediate the switch from hyperplasia to the hypertrophic phenotype in VSM cells in response to TGF-b [18]. In this study, we used antisense oligodeoxynucleotide (oligo) techniques to determine whether the CKI p21 plays a role in the regulation of TGF-b-mediated growth inhibition as well as matrix protein synthesis and secretion in VSM cells. We now show that attenuation of p21 levels in VSM cells does not abolish the growth inhibitory effect of TGF-b, yet it markedly attenuates both synthesis and secretion of the matrix proteins fibronectin and laminin both in response to, and independently of, TGF-b. These results in vascular cells have profound implications for treatment of atherosclerosis and arterial restenosis and suggest a new focus for future research in pharmacological methods for inhibition of VSM cell growth and matrix protein production.

2. Materials and methods

2.1. Materials TGF-b1 and mouse monoclonal anti-human p21Waf1/Cip1 were obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal anti-rat fibronectin and laminin antibodies were obtained from Chemicon (Temecula, CA). Anti-goat horseradish peroxidase-conjugated IgG was obtained from BioRad (Richmond, CA). Lipofectin® was obtained from Life Technologies (Rockville, MD). Reagents for the enhanced chemiluminescence system and [3H]thymidine were obtained from Amersham (Arlington Heights, IL). All other reagents, including mouse monoclonal a-actin antibody and protein A-Sepharose beads, were from Sigma (St. Louis, MO).

2.2. Cell culture, DNA synthesis, and proliferation assays Cultures of A10 aortic VSM cells were obtained from

American Type Culture Collection (Rockville, MD), were maintained as described [19], and were used between passages 15 and 25. The cells were growth-arrested by placing them in serum-free quiescence medium, exposed to TGF-b or 10% serum-containing medium as indicated, and [3H]thymidine incorporation assessed as previously described [20].

2.3. Oligodeoxynucleotide transfections Phosphorothioate antisense and random sequence control oligodeoxynucleotides were synthesized by Oligos Etc. (Wilsonville, OR). The p21Waf1/Cip1 antisense vector was designed around the start codon of rat p21Waf1/Cip1, with sequence 5%-GAC ATC ACC AGG ATC GGA CAT-3%. The scrambled random sequence control oligodeoxynucleotide was 5%-TGG ATC CGA CAT GTC AGA-3%. For the lipofection procedure, cells were grown to 60% confluence, washed with sterile phosphate-buffered saline, and the appropriate concentration of oligodeoxynucleotide was mixed with 6.6 ml of Lipofectin® per ml of Opti-MEM medium and was added to the cells for 4 h at 37 °C. Serum-free medium (without oligodeoxynucleotide) was added overnight, the media was changed in the morning and the cells were stimulated with TGF-b or serum as indicated.

2.4. Immunoprecipitations VSM cells were grown to confluence. After incubation under appropriate conditions, the cells were washed with ice-cold phosphate-buffered saline and immediately lysed in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM bglycerolphosphate, 1 mM Na3VO4, 1 mg/ml Leupeptin, 1 mM PMSF) at 4 °C, the cells were scraped off with a rubber spatula and the insoluble material removed by centrifuging at 10 000×g for 10 min at 4 °C. Protein concentration was determined by A595 and lysates containing equal amounts of protein were incubated with 4 ml anti-fibronectin antibody at 4 °C overnight. Protein A-Sepharose beads were added and the resulting mixture was incubated for an additional 2 h at 4 °C. The beads were centrifuged in a microfuge for 20 s, and the pellet was washed three times with cold lysis buffer. The supernatant was decanted, gel loading buffer was added to the precipitate, and the solution was boiled for 5 min, and centrifuged. The supernatant was electrophoresed on a 7.5% SDSpolyacrylamide gel with equal volumes of sample per lane. The proteins were electrophoretically transferred to nitrocellulose and probed with fibronectin antibody.

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2.5. Western blots Cells were grown to confluence in 6 cm culture dishes and serum deprived. After transfection and or treatment with appropriate agonist, conditioned medium was removed and saved and the cells were washed with phosphate-buffered saline and lysed in lysis buffer. Both supernatant and cell lysate were normalized to the lysate protein concentrations and Western blotted as described [19].

3. Results TGF-b has a bimodal effect on mitogenesis, being stimulatory or inhibitory depending on cell confluency and cell type [21,22]. These properties are remarkably similar to those of the CKI proteins, which themselves have been recently been described as positive and negative regulators of cell cycle progression [23] and mitogenesis [15]. TGF-b is largely growth inhibitory in vivo (reviewed in Ref. [24]), and we have confirmed this property in early passage rat VSM cells [25] and late passage rat mesangial cells [26]. To determine whether A10 VSM cells behave similarly, we serum-starved these cells for 24 h prior to stimulation with from 0.1 to 10 ng/ml TGF-b and examined their ability to incorporate [3H]thymidine into DNA, an assay which we have shown to correlate with cell number [15]. At all concentrations tested, DNA synthesis was significantly reduced with the addition of TGF-b for 24 h (Fig. 1). To determine whether this growth inhibitory effect persists when the cells are stimulated to enter G1 with the

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addition of serum (as would be the situation in vivo in the vasculature), we examined the effect of the addition of 10% serum-containing media on cells exposed to TGF-b at the same concentrations and found similar growth-inhibitory results (Fig. 2). We have recently described a phosphorothioate modified antisense oligo which specifically inhibits the CKI p21 in A10 VSM cells and leads to growth inhibition [15]. This effect, although not universal among cell lines [27], has been recently demonstrated in another early passage VSM cell line [16]. Using a random sequence phosphorothioate oligo as a control in these experiments, we examined the effect of the antisense oligos to p21 on p21 and a-actin protein expression in A10 VSM cells. There was marked inhibition of p21 protein level, but no effect on the level of a-actin protein level, after transfection of 400 nM antisense p21 oligodeoxynucleotide (Fig. 3), demonstrating specificity of this oligo on attenuation of steady state p21 levels. As an additional control, we have previously shown in parallel experiments that transfection with lipofectin alone lacks inhibitory effects on p21 protein levels [15]. Since TGF-b signals through induction of the CKIs p21 and p27 and since both TGF-b [25] and p21 [15] have been shown under some but not all conditions to decrease DNA synthesis in VSM cells, we next asked whether transfection of the antisense p21 or random sequence oligo abolishes the inhibitory effect of TGF-b on serum-stimulated VSM cell proliferation. Cells were transfected with the appropriate oligo for 4 h. The cells were then serum-starved overnight and subsequently stimulated with 10% serum-containing media. Two hours after serum addition, TGF-b was added at con-

Fig. 1. TGF-b decreases mitogenesis in serum-starved VSM cells. A10 VSM cells were grown to confluence in 24-well culture dishes and serum-starved for 24 h. TGF-b1 was added at the concentrations indicated for another 24 h. DNA synthesis was assessed by [3H]thymidine incorporation as described in Materials and Methods and is expressed as mean 9SEM of three wells per data point. (*) Indicates significance difference from control (no TGF-b). The experiment shown is representative of three separate experiments.

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Fig. 2. TGF-b decreases 10% serum-stimulated mitogenesis in VSM cells. A10 VSM cells were grown to confluence and serum-starved as in Fig. 1. The growth medium was changed to 10% serum-containing medium and, 2 h later, TGF-b was added at the indicated concentrations (in ng/ml) for 24 h. DNA synthesis was assessed by [3H]thymidine incorporation and is expressed as mean 9 SEM of three wells per data point; absolute counts differ slightly from the experiment in Fig. 1 due to differences in confluency of the cells. (*) Indicates significance difference from serum alone. The experiment shown is representative of two separate experiments.

centrations from 0.1 to 10 ng/ml for 24 h. While DNA synthesis of cells transfected with antisense p21 was markedly inhibited relative to cells transfected with control oligodeoxynucleotide (as we have previously shown when comparing antisense p21 with sense p21 oligodeoxynucleotide [15]), the inhibitory effect of TGF-b at 10 ng/ml remains under both conditions (Fig. 4). This shows that the growth inhibitory effect of TGF-b still occurs in the setting of attenuated levels of p21, suggesting the existence of an alternate pathway for TGF-b-mediated growth inhibition largely independent of p21 induction and consistent with data from several laboratories [15,28] showing an assembly factor role of p21. TGF-b is thought to mediate vascular intimal hyperplasia and restenosis, as well as glomerular fibrosis, through its induction of extracellular matrix protein secretion by VSM cells and renal mesangial cells. Since the CKIs, which are induced by TGF-b, are important in regulating cell cycle transit [23], as well as in the switch between hypertrophic and hyperplastic phenotypes [18], we asked whether the CKI p21 influences matrix protein secretion in VSM cells. Due to their established roles in vascular fibrosis, levels of the representative matrix proteins laminin and fibronectin were examined in both TGF-b stimulated and non-stimulated lysate and conditioned media of cells in which p21 expression had been attenuated. The cells were transfected with either antisense p21 or random sequence control oligodeoxynucleotide and then either left unstimulated or stimulated with TGF-b at for 0.1– 10

ng/ml for 4 h. Conditioned media and cell lysate were collected, and, prior to electrophoresis, both medium and lysate volumes were normalized for the protein content in total cell lysate to exclude any skewing of the data due to cell proliferation. The proteins were electrophoresed and immunoblotted with either fibronectin (after immunoprecipitation to eliminate extraneous bands which appeared in the absence of this procedure

Fig. 3. Transfection of VSM cells with antisense p21 oligodeoxynucleotide specifically inhibits p21 protein level. A10 VSM cells were grown to confluence in 6 cm dishes and transfected with either antisense p21 or random sequence control oligodeoxynucleotide (400 nM) as described in Section 2. The lysates, normalized for protein content, were Western blotted with either p21 or a-actin antibody. The experiment shown is representative of at least three separate experiments.

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Fig. 4. The TGF-b growth inhibitory effect is preserved despite attenuated p21 levels. A10 VSM cells were grown to confluence, transfected as described with either antisense (solid bars) or random sequence control (hatched bars) oligodeoxynucleotide, and serum-starved overnight. Subsequently, the cells were treated with 10% serum containing medium and/or TGF-b at the indicated concentrations (in ng/ml), and DNA synthesis assessed; absolute counts differ slightly from other experiments due to differences in starting confluency of the cells. (*) Indicates significance difference from serum alone. The experiment shown is representative of two separate experiments.

[26]) or laminin antibody. Production and secretion of laminin was markedly reduced after attenuation of p21 in both the absence (concentration ‘0’) and presence of TGF-b (Fig. 5). Furthermore, TGF-b actually decreased laminin secretion at higher doses in both random sequence and antisense p21 oligodeoxynucleotide transfected cells (Fig. 5). Fibronectin production and secretion into the medium was similarly decreased after p21 attenuation, both in the presence and absence of TGF-b stimulation, yet in this case TGF-b induced fibronectin production in both transfections with a maximal level in lysate when TGF-b was administered at higher doses (Fig. 6).

permissive effect of p21 for VSM cell growth is contradictory to the TGF-b-mediated increase in p21, which is purported to cause growth inhibition. In this situation (depicted in Fig. 4), we believe it likely that p21 is exerting both its positive and negative effects [31] on VSM cell growth: TGF-b is still able to cause growth inhibition (presumably by increasing CKI levels) despite an overall attenuation of p21 levels lowering DNA synthesis under all conditions. However, the setting in which p21 can exert both growth stimulation or inhibition, and the mechanism underlying this pleiotropic effect, is unknown and is the subject of continuing investigation in our laboratory.

4. Discussion Matrix proteins are secreted by a variety of cells and are important for structural integrity in the normal environment, yet these same proteins may be detrimental when they occur in abundance in the disease setting (reviewed in Ref. [29]). These proteins can also act as cell cycle control elements in atherosclerotic disease [2], and the specific matrix proteins laminin and fibronectin, studied here, are important in modulating the switch from contractile to synthetic phenotype in VSM cells (reviewed in Ref. [10]). Consistent with our recent report [15] and with the data shown in Fig. 4 of this study, other investigators have recently demonstrated a paracrine effect of p21 induction on promotion of mitogenesis in a variety of cell lines [30]. It may seem at first glance that the

Fig. 5. Antisense p21 oligodeoxynucleotide decreases TGF-b-mediated laminin production and secretion. A10 VSM cells were grown to confluence in 6 cm dishes and transfected with either antisense p21 or random sequence control oligodeoxynucleotide (400 nM) as described in Section 2. After stimulation with TGF-b for 24 h at the indicated concentrations, the cells were lysed. Both lysate and conditioned medium were normalized for lysate protein concentration and Western blotted with laminin antibody. The experiment shown is representative of three separate experiments.

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Fig. 6. Antisense p21 oligodeoxynucleotide decreases TGF-b-mediated fibronectin production and secretion. A10 VSM cells were grown to confluence in 6 cm dishes and transfected with either antisense p21 or random sequence control oligodeoxynucleotide (400 nM) as described in Section 2. After stimulation with TGF-b for 24 h at the indicated concentrations, the cells were lysed. Both lysate and conditioned medium were normalized for lysate protein concentration and then immunoprecipitated and subsequently Western blotted with fibronectin antibody. The experiment shown is representative of three separate experiments.

In a similar fashion to recent findings regarding the CKIs [23], TGF-b can be either stimulatory or inhibitory towards cell growth [21]. Despite its bimodal effect on cell proliferation, it is clear that TGF-b induces the pathologic appearance of matrix proteins, and this growth factor has therefore been implicated as a causative agent in a variety of diseases which are characterized by fibrosis [3,32]. However, in addition to its bimodal effects on mitogenesis, TGF-b also exhibits both positive and negative effects on cell matrix protein production, as we have shown in this study (Figs. 5 and 6). Furthermore, while aberrant secretion of matrix proteins is clearly involved in the process of fibrosis, there is also evidence that fibronectin [33] and laminin [11], the matrix proteins studied here, may actually be mediating the growth inhibitory effects of TGF-b. The CKIs p21 and p27 have long been known to be induced by TGF-b [13,14], and it has been assumed that expression of these CKI proteins link this growth factor to cell cycle arrest. The role of the CKIs in cell proliferation, however, is not limited to TGF-b signaling. It is likely that the cyclin/cdk/CKI system serves as a final common pathway in a number of cell growth regulatory stimuli. In addition to their roles in proliferative signaling, we and others have shown that in VSM, renal mesangial, and prostate carcinoma cells, p27 and p21 are induced by the growth inhibitory statins [34– 36]. While these proteins likely in some manner cause attenuation of DNA synthesis in that setting, we have also shown that p21 is required for the full mitogenic effect of PDGF and serum [15,27], a correlation which has recently been confirmed by another group [16]. This bimodal effect of p21 on cell proliferation is remarkably similar to the descriptions of these effects by TGF-b, such that our findings of p21 regulation of matrix protein secretion are not entirely unexpected.

While the switch from the hyperplastic to the hypertrophic phenotype in VSM cells has been attributed to p27 [12,18], the effect of the CKIs on matrix protein secretion by these cells has not been investigated. We now show that the ability of VSM cells to synthesize and secrete the matrix proteins laminin and fibronectin, both dependently and independently of TGF-b is mediated at least in part through p21. We have previously shown that, in mesangial cells, TGF-b at 10 ng/ml induced secretion of the matrix proteins fibronectin and laminin [26]. However, in the VSM cells used in the present experiments, higher concentrations of TGF-b were associated with decreased laminin synthesis and secretion into conditioned media. This may be due to the fact that continued stimulation of VSM cells by TGF-b causes them to remain in a synthetic phenotype associated with fibronectin secretion, whereas cells remain in the contractile phenotype when grown in the presence of the secreted laminin [11]. Whether the antisense oligo used in these experiments inhibit mRNA levels of p21 is not relevant to our results. While it is not at all clear in what manner antisense oligos do in fact ultimately inhibit protein levels (reviewed in Refs. [37,38]), the important issue relevant to our results is the finding of marked and specific inhibition of steady state p21 protein levels by the oligos we have investigated (Fig. 3). What is the significance of our findings to vascular biology? It is noteworthy that transfection of antisense oligodeoxynucleotides has been shown to be effective in vivo both with and without lipofection reagents [38], and in some situations antisense oligodeoxynucleotides have even proved effective under conditions as simple as tail vein oligodeoxynucleotide infusions [39]. Therefore the possibility of utilizing these compounds clinically to target VSM growth regulatory proteins important in atherosclerosis, vascular restenosis after angioplasty, and hemodialysis access stenosis cannot be discounted. There exist potential applications for matrix protein attenuating molecules in renal disease as well as the obvious applications to vascular disease. It has been demonstrated that, in the case of renal mesangial cells, p21 is required for glomerular hypertrophy in experimental diabetic nephropathy [40]. Whether p21 is allowing secretion of matrix proteins in this scenario such that its inhibition would diminish this response, remains to be determined. In VSM cells, p27 has been shown to have a similar role in the promotion of hypertrophy [18], yet the role of p21 and matrix protein secretion in this phenomenon in VSM cells is not known. In support of our data in VSM cells, others have also demonstrated a positive effect of overexpression of p21 on matrix protein production (including fibronectin), consistent with our data showing the converse effect in cells after attenuation of p21 [30].

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In the setting of angioplasty, VSM cells have been shown to modulate from a contractile to a synthetic phenotype after induction of intimal lesions by balloon catheterization [41,42]. This may in turn result in excess production of matrix proteins, with or without the requirement for TGF-b, leading to ultimate restenosis of the vessel. Attenuation of matrix protein production and secretion with antisense p21 transfection into VSM cells may therefore prove to be useful in the setting of renal or coronary artery angioplasty, whereby such oligos could be lipofected into angioplastied blood vessels at the time of balloon catheterization in an attempt to decrease both VSM cell proliferation and matrix production. Because similarities in pathophysiology exist between re-stenosis of arteries and hemodialysis graft stenosis, these oligos may also ultimately prove useful in hemodialysis access revision procedures.

[12]

[13]

[14]

[15]

[16]

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Acknowledgements

[18]

This work was supported by the Research Service of the U.S. Department of Veterans Affairs, grants from the National Kidney Foundation and the UC Davis Health System, and by a gift from Dialysis Clinics, Inc.

[19]

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