Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells

From the Division of Molecular and Cellular Pathology (N.B.D., N.J.B., J.E.M.-U., T.M.L.), Department of Pathology, and the Division of Biochemistry and Molecular Biology (P.-L.C., C.W.P.), Department of Nutritional Sciences, The University of Alabama at Birmingham

Correspondence to Thomas M. Lincoln, PhD, Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0019. E-mail lincoln{at}vh.path.uab.edu

	Abstract

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Abstract—Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-I or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Key Words: nitric oxide • phenotype • matrix protein • vascular disease • atherosclerosis • restenosis

	Introduction

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Vascular diseases such as atherosclerosis and restenosis following arterial injury or transplantation are associated with a remodeling of the vascular wall due to the thickening of the intimal layer of the vessel.^{1 2} One event in the process of intimal thickening is the migration of medial VSMCs to the intima. Coincident with VSMC migration is an increase in the rate of proliferation and an increase in the synthesis and secretion of extracellular matrix proteins by the cells. All three cellular events—migration, proliferation, and secretion of matrix proteins—contribute to expansion of the intima, resulting in the formation of a neointimal tissue.

The cellular mechanisms responsible for the change in the behavior of medial VSMCs in vascular diseases have been investigated extensively.^{3 4 5 6 7} Normally, medial VSMCs are highly differentiated and function as contractile cells to regulate the diameter of the vessel. In response to injury, however, the cells acquire the capacity to migrate and produce vast quantities of matrix proteins as part of their "wound healing" function. This change in cellular behavior is a result of alterations in the phenotypic properties of the cell, termed "phenotypic modulation."^{8 9 10 11 12 13} Phenotypic modulation is basically a change in the differentiation state wherein the cells modulate from a "contractile" phenotype to a "synthetic" or secretory phenotype. The two phenotypic states are distinguishable in morphology, gene expression, and function.

The mechanisms responsible for phenotypic modulation of VSMCs are not well understood. It is generally appreciated that growth factors, derived from adherent platelets, leukocytes, and even the VSMCs, are involved in the modulation of contractile VSMCs to synthetic VSMCs. On the other hand, the regulatory factors that prevent phenotypic modulation or restore contractile function to synthetic VSMCs are not known.

A number of studies have indicated that NO inhibits VSMC proliferation in vitro and that NO donor drugs or transduction of the cDNA encoding the enzyme that produces NO, NO synthase, inhibits neointima formation in vivo.^{14 15 16 17 18 19 20} These studies suggest that NO may be one factor that either prevents phenotypic modulation of VSMCs or induces the conversion of synthetic VSMCs to contractile VSMCs. NO acts on VSMCs by stimulating soluble guanylyl cyclase to elevate cellular cGMP levels.^{21 22} cGMP, in turn, activates a serine/threonine protein kinase, PKG. Other actions of NO exist as well, including binding to heme-containing proteins (eg, cytochrome p450 and cytochrome oxidase), reacting with superoxide to form peroxynitrite, and reacting with sulfhydryl groups in proteins to produce nitrosothiols.²³ Therefore, the signaling mechanisms of NO in VSMCs are complex, and it is not certain which, if any, NO signaling pathways may affect VSMC phenotype.

Our laboratory recently reported that synthetic PKG-deficient VSMCs, when transfected with cDNAs encoding the type I PKG, assumed a contractile morphology and demonstrated an increase in the expression of contractile proteins.²⁴ On the other hand, PKG-deficient synthetic VSMCs secrete large quantities of extracellular matrix proteins, such as osteopontin and thrombospondin.^{25 26 27 28 29 30} In the present study, we tested the hypothesis that PKG may reduce matrix protein production by examining the role of PKG in the regulation of expression of extracellular matrix proteins in rat aortic smooth muscle cells. We have used cell lines deficient in PKG expression and compared those with cells stably expressing PKG constructs, including a constitutively active PKG catalytic domain.

	Materials and Methods

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Preparation and Culture of Rat Aortic VSMCs
Rat aortic VSMCs were isolated from the thoracic and abdominal aortas of Sprague-Dawley rats (150 to 200 g) as described previously.³¹ Briefly, rats were killed by CO₂ inhalation, and aortas were excised and placed in an isolation medium of DMEM containing 20 mmol/L HEPES, 1 mg/mL BSA, 5 µg/mL amphotericin B, and 50 µg/mL gentamicin. After a thorough cleaning, aortas were placed in digestion medium (isolation medium containing 1 mg/mL elastase and 130 U/mL collagenase) for 8 minutes. The tunica adventitia was removed, and the remaining medial layers were minced and further digested for 1 to 2 hours in digestion medium containing 200 U/mL collagenase until a single cell suspension was obtained. Cells were washed twice with isolation medium and plated in culture flasks. Cells were maintained in DMEM containing 5% FBS, 5% calf serum, and 50 µg/mL gentamicin and subcultured weekly. The routine subculturing procedure was to remove the cells using 0.05% buffered trypsin and to split the cells 1:5. Viability was assessed after each experiment using trypan blue exclusion. The VSMCs were transfected at passage 3 and used between passages 6 to 10 after the initial isolation of transfected colonies.

Isolation of PKG cDNAs and Cell Transfection
The bovine PKG-I cDNA was constructed using a PCR approach. Fresh bovine lung mRNA was isolated using a modified guanidinium isothiocyanate method³² and purified on oligo dT cellulose. The mRNA was converted to first-strand cDNA using Abelson Maloney virus reverse transcriptase, and the cDNA was used as the template for the construction of individual segments using PCR. The cDNA encoding the holoenzyme of PKG-I³³ was ligated into pBluescript IISK(+) vectors and sequenced. The catalytic domain of PKG-I (base pairs 999 to 2016) was constructed and characterized as described previously.³⁴ For transfecting rat aortic VSMCs, the coding sequences were removed using BamHI digestion (PKG-I) or Xho I (PKG-I catalytic domain) and ligated into the vector pcDNA1-neo. The vectors were grown in Escherichia coli, purified by Wizard Maxi Prep, and transfected into passage-3 rat aortic VSMCs as follows: Cells were transfected with 5 µg recombinant vector DNA or control vector DNA using 10 µL Transfectam reagent (Promega) with precipitation of the DNA-liposome complex for 15 minutes at room temperature. The precipitate was added to the cell monolayer, and cells were incubated for 6 hours at 37°C and 10% CO₂. The transfection was terminated by adding DMEM with 20% FBS. Stably transfected cells were selected using 500 µg/mL G418. After isolation of colonies from 96-well plates, the transfected cell lines were maintained in 250 µg/mL G418. For all subsequent experiments, cells were plated and allowed to attach overnight. Cells were then serum-deprived for 48 hours in DMEM containing 1 mg/mL BSA before experiments were performed, except where noted otherwise.

Northern Analysis
Total cellular RNA was isolated from the cultured cells using the RNA STAT-60 isolation reagent according to the manufacturer's instructions (Tel-Test, Inc). In brief, VSMCs were grown to confluence on 3.5-cm² plates and lysed directly in 1 mL RNA STAT-60. The cell lysate was passed through the tip of a pipette several times and allowed to sit at room temperature for 5 minutes. Chloroform (0.2 mL per milliliter STAT-60) was added, and the suspension was shaken vigorously. The suspension was centrifuged at 7000 rpm for 15 minutes at 4°C. The upper aqueous phase was transferred to a fresh plastic tube, and the RNA was precipitated with 0.5 mL isopropanol per milliliter STAT-60. After 15 minutes of incubation at room temperature, the mixture was centrifuged, and the RNA pellet was washed with cold 70% ethanol. RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and quantified using 260-nm/280-nm ratios. The final preparation was DNA free and had a 260/280 ratio of >1.8. RNA (15 µg) was resolved on 1% formaldehyde agarose gels, transferred to Nytran membranes, and UV–cross-linked. After prehybridization in Quickhyb (Stratagene), the membranes were incubated with ³²P-labeled cDNA probes (1x10⁶ cpm/mL hybridization solution) in 100 µg/mL salmon sperm DNA. Hybridization was carried out for 2 to 3 hours at 65°C. Membranes were then washed twice with 2x SSC in 0.2% SDS, twice at room temperature, and again at 50°C as needed. Blots were developed, and the films were scanned with a laser scanner (Hewlett Packard Scanjet 3C). Absorbance units were corrected to GAPDH RNA standards. The probes for mouse osteopontin and human thrombospondin-1, a 1.1-kb EcoRI fragment,³⁵ were generated as purified insert DNAs and labeled using a Random Prime DNA labeling kit (Stratagene).

Western Blot Analysis
Cells were harvested from 60- or 100-cm² culture dishes in 0.5 mL of ice-cold PEM buffer containing 20 mmol/L sodium phosphate, pH 6.8, 2 mmol/L EDTA, 15 mmol/L ß-mercaptoethanol, 0.15 mol/L NaCl, and a cocktail of protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL pepstatin A, 10 µg/mL leupeptin, and 5 µg/mL aprotinin). The cells were homogenized two times using a sonicator set at 50% for 10 seconds and clarified by brief centrifugation. The extracts were prepared for SDS-PAGE as described previously.³¹ After separation by SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose membranes (pore size, 0.2 µm), and the membranes were incubated in 0.5% nonfat dry milk in TBS for 1 hour at room temperature to block nonspecific protein binding. For detection of PKG, the membranes were incubated with rabbit anti-bovine PKG prepared by our laboratory.³¹ For detection of osteopontin, membranes were incubated with rabbit anti-rat osteopontin antiserum and mouse anti-human thrombospondin monoclonal antibody.³⁶ Proteins were visualized using the enhanced chemiluminescence system.

[³⁵S]Methionine Labeling and Immunoprecipitation
To determine levels of protein synthesis, [³⁵S]methionine labeling experiments were performed. Cells were plated and grown to confluence (5 days), rinsed twice, and incubated in methionine-free medium supplemented with 100 µCi/mL [³⁵S]methionine for 3 hours. After this incubation, the medium was removed, and radioactive methionine incorporation and immunoprecipitation were done. Cells were then rinsed several times in PBS, removed from the culture dishes with a rubber policeman, and sonicated in ice-cold PEM buffer containing the protease inhibitors as described above. After centrifugation to remove the membrane fraction, the supernatant fractions were collected for protein determination using the Bradford reagent, radioactivity, and immunoprecipitation. For immunoprecipitation, equal volumes of cell extract were mixed with an immunoprecipitation solution containing 50 mmol/L Tris-Cl, pH 7.4, 5 mmol/L EDTA, 1% NP-40 detergent, and 0.02% NaN₃. One hundred fifty microliters of precleared 10% protein-A Sepharose 6MB was added per milliliter of extract, and the suspension was incubated at 4°C for 8 hours. After centrifugation, the Sepharose beads were washed three times in 100 mmol/L Tris-Cl, pH 7.4, 150 mmol/L NaCl, 0.1% NP-40, and 0.25% deoxycholate, followed by two rinses in PBS. The pellets were suspended in 25 µL water and 25 µL SDS sample loading buffer and heated to 100°C for 5 minutes. Two microliters of the supernatant was removed to determine protein counts per minute, and the rest was subjected to SDS-PAGE.

TCA Precipitation
To examine secreted osteopontin and thrombospondin, the cells were grown to confluence in 60-mm plates. The medium was replaced with serum-free medium containing 2 mg/mL BSA for 24 hours before TCA precipitation. The medium was removed, and an equal volume of 40% TCA was added. The mixture was incubated at 4°C for 30 minutes and centrifuged at 10 000g for 10 minutes to remove precipitated protein. The pellet was washed three times with 10% ice-cold TCA and once with absolute ethanol and dissolved in SDS-PAGE sample loading buffer.

Microscopy
To examine cells for extracellular thrombospondin-1 deposition, cells were subjected to immunocytofluorescence. Cells were plated on glass coverslips (50 000 cells per 1-cm² coverslip) and grown in DMEM containing 5% calf serum and 5% FBS in the presence of 250 µg G418. For thrombospondin immunocytofluorescence, the medium was changed to DMEM in the absence of serum, and the cells were incubated for 2 hours at 37°C. After this incubation, the cell monolayer was washed once with PBS and fixed in 3% formaldehyde at pH 7.4 for 10 minutes at room temperature. The cells were then washed three times in PBS, blocked with 1% BSA in PBS for 1 hour at room temperature, and washed once with PBS. The cells were then incubated for 1 hour with mouse anti-human thrombospondin-1 monoclonal antibody (diluted 1:1000) for 1 hour at room temperature. After antibody treatment, the monolayers were washed three times in PBS and incubated with a 1:50 dilution of goat anti-mouse conjugated with FITC for 45 minutes at room temperature. The monolayers were finally washed once with PBS and mounted on a microscope slide in one drop of mounting medium. The cells were examined for thrombospondin-1 using a Zeiss fluorescent microscope. For phase-contrast microscopy, unstained cells were visualized at x800 using an inverted phase microscope (Zeiss).

Materials
Elastase and collagenase (CLS III) were from Worthington Biochemicals. Bovine serum (fetal and calf), DMEM, G418, and PDGF-BB were purchased from GIBCO. Transfectam and Magic Maxi Prep were from Promega. Abelson Maloney virus reverse transcriptase and pcDNA1-neo were purchased from Invitrogen. RNA-STAT 60 was from Tel-Test B. Nytran membranes for Northern analyses were from Schleicher & Schuell, and the Quickhyb and Prime It II kits were purchased from Stratagene. Goat anti-mouse conjugated with FITC was from Jackson Immunoresearch Labs, and mounting medium was from Vector Labs Inc. Radioactive [³⁵S]methionine was purchased from ICN Radiochemicals, Inc. The antiserum to osteopontin was generously provided by Dr Pi-Ling Chang and that to thrombospondin was from Dr Joanne Murphy-Ullrich. All other reagents were of the highest grade and were purchased from either Sigma Chemical Co or Fisher Biochemicals.

	Results

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Characterization of PKG-Expressing Cell Lines
Our laboratory has shown that rat aortic VSMCs when passaged several times (ie, three or four passages) cease to express PKG-I or PKG-Iß when analyzed using either Northern analysis or Western analysis.^{31 37} Therefore, transfection of PKG cDNAs into cells deficient in PKG has been a useful approach to study the effects of PKG on VSMC function. In order to establish VSMCs expressing PKG, the cDNA encoding PKG-I was subcloned into pcDNA1-neo expression vector and transfected into rat aortic VSMCs as described in "Materials and Methods." The constitutively active catalytic domain of PKG-I, residues 334 to 671, was subcloned into the same vector to create cells expressing an active PKG, thus bypassing upstream signaling pathways (eg, NO) necessary for cGMP generation. After G418-based selection, several clonally derived cell lines expressing each construct were initially screened for PKG activity. These clonal cell lines were routinely compared with control cell lines, which were also clonally derived but transfected with empty vector and isolated by G418 resistance screening.

Fig 1 is a Western blot illustrating the expression of immunoreactive PKG compared with control transfectants (ie, plasmid only), which contained no detectable PKG. Extract protein from freshly isolated rat aorta (lane 4) and the PKG-expressing cell lines (lanes 2 and 3) contained comparable levels of immunoreactive protein. All cell lines, control and experimental, were treated with 5 ng/mL PDGF, since we observed that PDGF inhibited apoptosis in cells expressing PKG (data not shown). The phosphotransferase activity (measured as previously described³⁴ ) of the recombinant PKG expressed in the transfected cell lines was determined to be 4.5 nmol · min^-1 · mg protein^-1 in the presence of 1 µmol/L cGMP in extracts made from cells expressing the PKG-I holoenzyme; a similar level of phosphotransferase activity was observed in extracts derived from cells expressing the catalytic domain of PKG, except that the kinase activity was not dependent on the addition of cGMP.

View larger version (56K): [in this window] [in a new window]   Figure 1. Expression of PKG in transfected VSMCs. Cells were transfected with empty pcDNA1-neo (lane 1), pcDNA1-neo/catalytic domain (lane 2), or pcDNA1-neo/PKG-I (lane 3), as described in "Materials and Methods." Lane 4 is rat aorta extract protein; lane 5 is 10 ng of bovine lung PKG-I standard. In lanes 1 to 4, 50 µg of protein was applied to the gel, and Western blot analysis was performed using rabbit anti-bovine PKG antibody. Mr values are as follows: catalytic domain=39 kD and PKG-I=79 kD.

Effects of PKG Expression on Extracellular Matrix Protein Production
The effects of PKG-I and the catalytic domain of PKG were examined on the production of two matrix proteins known to exist in arterial lesions, osteopontin and thrombospondin-1. As shown in Fig 2, cells expressing either PKG-I and treated with 20 µmol/L 8-CPT-cGMP or the catalytic domain demonstrated a marked reduction in cell extract osteopontin (panel A) or thrombospondin-1 (panel B). The PKG-deficient control-transfected VSMCs, which exist primarily as the synthetic phenotype, produced large amounts of osteopontin and thrombospondin-1 in the presence of serum compared with the PKG-expressing cells, which exist primarily as the contractile phenotype. The cGMP analogue was used to selectively activate PKG in the holoenzyme PKG-I–transfected cells. There were no effects of 20 µmol/L 8-CPT-cGMP on matrix protein production in control-transfected cells. The Coomassie blue–stained gel used for the Western blot analysis is shown in Fig 2C. As can be seen in the panel, equal amounts of protein were loaded onto the gel for each treatment. In these studies, cells were grown in the presence of 5% FBS and 5% calf serum in order to stimulate the maximum production of extracellular matrix proteins.

View larger version (14K): [in this window] [in a new window]   Figure 2. Osteopontin and thrombospondin-1 production in transfected VSMCs. Cells were grown to confluence in the presence of 5% FBS+5% calf serum, cell lysates were prepared, and Western blot analysis was performed on 100 µg of protein per lane, as described in "Materials and Methods." A, Western blot analysis for osteopontin in cell extracts. B, Western blot analysis for thrombospondin-1 in cell extracts. C, Coomassie blue–stained gel demonstrating protein-loading conditions. In all panels, lane 1 represents extracts from control-transfected cells; lane 2, extracts from PKG-I–transfected cells treated with 20 µmol/L 8-CPT-cGMP for 48 hours; and lane 3, extracts from catalytic domain–transfected cells.

As shown in Fig 2, the Western blot analysis suggests that 48 hours after plating, both matrix proteins are being produced in greater amounts in the control-transfected cells compared with the PKG-transfected cells. It was possible, however, that the effects of PKG were due to a general reduction in protein synthesis inasmuch as PKG has been reported to inhibit growth of VSMCs. To examine the effects of PKG protein synthesis, cells were labeled with[³⁵ S]methionine for 3 hours, after which overall counts per minute per milligram protein was determined. There were no significant differences between control and PKG-expressing cells (control, 440 165±22 428 cpm/mg protein; PKG-I, 452 012±15 120 cpm/mg protein; n=3; P>.05), suggesting that there was not a generalized depression of protein synthesis, which could have accounted for the reduction in matrix protein synthesis. Furthermore, osteopontin immunoprecipitated from cell extracts accounted for 10 815 cpm/20 million cells from the control-transfected cultures compared with 2884 cpm/20 million cells from the PKG-I–transfected cultures.

The data described above have been obtained in three separate experiments using passaged PKG-deficient cells, which were clonally derived by stable transfection with control vectors or with vectors containing PKG-I holoenzyme or the catalytic domain. In every instance, the control-transfected cells (in each case the cells were PKG deficient, as determined by Western blot analysis) produced more osteopontin and thrombospondin-1 than did PKG-expressing cells.

Effects of PKG Expression on Extracellular Matrix Protein mRNA
These effects of PKG on osteopontin and thrombospondin-1 steady-state mRNA levels are shown in Figs 3 and 4. A representative Northern blot for steady-state osteopontin mRNA (Fig 3A) as well as an analysis using densitometric methods (Fig 3B) suggests that there was no statistically significant decrease in osteopontin mRNA levels in PKG-I–and catalytic domain–expressing cells. Similarly, for thrombospondin-1 (Fig 4A and 4B), there were no statistically significant differences in the levels of steady-state mRNA levels among the control, PKG-I–expressing, and catalytic domain–expressing cells. In both instances, the mRNA levels were normalized to GAPDH mRNA and were controlled for loading using ribosomal RNA standards. Inasmuch as the protein levels were reduced severalfold, these results suggest that a decrease in steady-state mRNA for osteopontin and thrombospondin-1 did not account for the mechanism of action of PKG.

View larger version (31K): [in this window] [in a new window]   Figure 3. Steady-state mRNA levels for osteopontin (OPN) in transfected VSMCs. Cells were grown, and mRNA was prepared as described in "Materials and Methods." A, Representative Northern blot analysis for steady-state OPN mRNA levels. B, OPN/GAPDH mRNA ratios. In panels A and B (lanes and bars, respectively), 1 represents extracts from control-transfected cells; 2, extracts from PKG-I–transfected cells treated with 20 µmol/L 8-CPT-cGMP for 48 hours; and 3, extracts from catalytic domain (CAT-DOM)–transfected cells. The data are expressed as the mean±SD from three independent experiments. Using the two-tailed t test, significant differences were found between control and PKG-I cells (P<.05) and between control and CAT-DOM cells (P<.05).

View larger version (33K): [in this window] [in a new window]   Figure 4. Steady-state mRNA levels for thrombospondin-1 (TSPN) in transfected VSMCs. Cells were grown, and mRNA was prepared as described in "Materials and Methods." A, Representative Northern blot for TSPN mRNA levels. B, TSPN/GAPDH mRNA ratios. In panels A and B (lanes and bars, respectively), 1 represents extracts from control-transfected cells; 2, extracts from PKG-I–transfected cells treated with 20 µmol/L 8-CPT-cGMP for 48 hours; and 3, extracts from catalytic domain (CAT-DOM)–transfected cells. The data are expressed as the mean±SD from three independent experiments. Using the two-tailed t test, there were no significant differences in the mRNA levels between control and PKG-I cells (P>.02) or between control and CAT-DOM cells (P>.02).

Effects of PKG Expression on Secreted Osteopontin and Thrombospondin-1
The results described above are for cellular levels of osteopontin and thrombospondin-1. Both proteins are secretory products of synthetic smooth muscle cells, and it was of interest to determine whether the reduction in intracellular protein levels was reflected in the amount of protein secreted into the medium. This analysis was performed by precipitating protein from the serum-free medium using TCA, followed by Western blot analysis. As shown in Fig 5, both osteopontin and thrombospondin-1 were secreted into the medium of the control-transfected cells. In PKG-expressing cells, the levels of the protein were reduced, similar to the results obtained with intracellular levels.

View larger version (17K): [in this window] [in a new window]   Figure 5. Osteopontin and thrombospondin-1 secretion from transfected cells. Cells were grown to confluence and serum-deprived for 24 hours in medium containing 2 mg/mL BSA. Protein was precipitated from the culture medium using 20% TCA as described in "Materials and Methods" and resolved by electrophoresis. Western blot analysis was performed for osteopontin (A) or thrombospondin-1 (B). Lane 1 from each panel is the medium taken from control-transfected cells; lane 2 is medium taken from PKG-I–transfected cells. CAT DOM indicates the catalytic domain.

Immunocytofluorescence Microscopy
The results thus far suggest that PKG expression reduces intracellular protein production and secretion of osteopontin and thrombospondin. We next examined whether one of these proteins, thrombospondin-1, became incorporated into the matrix of cells. The monoclonal antibody used in the Western analysis described above has been shown previously to be useful for the analysis of extracellular thrombospondin by immuofluorescence.³⁶ As shown in Fig 6A, control-transfected cells incorporated heavy amounts of thrombospondin that stained brightly in the extracellular matrix (arrows). The control-transfected cells demonstrated a flattened and irregular shape (see Fig 6C), and the extracellular staining observed consisted of irregularly shaped and weblike material that surrounded individual cells. Staining was also visible in the cytoplasmic space of the cells as well. On the other hand, very weak extracellular thrombospondin staining was observed in cells expressing PKG-I (Fig 6B). Practically no cytoplasmic thrombospondin was stained in the cytoplasm, consistent with the immunoblot analysis. These differences in extracellular thrombospondin staining were consistent also with the changes in the phenotypic properties of the two cell lines. PKG-expressing VSMCs demonstrated a morphology characterized by elongated cells with centrally located nuclei growing in parallel arrays (Fig 6D). These morphological features provided the general "hill and valley" appearance. There was little evidence of secretory activity in these cells compared with control-transfected VSMCs (Fig 6C). The control-transfected cells, in contrast to those expressing PKG, were flattened, grew in irregular arrays, and demonstrated numerous secretory vesicles in the perinuclear regions.

View larger version (99K): [in this window] [in a new window]   Figure 6. Immunocytofluorescence and morphology associated with PKG expression in VSMCs. Cells were grown as described in "Materials and Methods" and serum-deprived for 48 hours in the presence of 5 ng/mL PDGF. Extracellular thrombospondin-1 was visualized by immunocytofluorescence using FITC-tagged secondary antibody toward mouse monoclonal anti–thrombospondin-1, also as described in "Materials and Methods." A, Immunocytofluorescence microscopy of control-transfected cells. B, Immunocytofluorescence microscopy of PKG-I–transfected cells treated with 20 µmol/L 8-CPT-cGMP for 48 hours. C, Phase-contrast microscopy of control-transfected cells. D, Phase-contrast microscopy of PKG-I–transfected cells treated with 20 µmol/L 8-CPT-cGMP for 48 hours. The arrows in panel A depict areas of dense weblike thrombospondin secretion.

	Discussion

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The results in the present study demonstrate for the first time that the major downstream mediator of NO action, PKG, suppresses the production of extracellular matrix proteins in VSMCs. These studies were made possible by our previous findings that the expression of PKG in cultured VSMCs is reduced to undetectable levels after several passages.^{31 37} Therefore, we were able to examine directly the effects of PKG on the production of extracellular matrix proteins by comparing PKG-deficient cells with those stably expressing the enzyme. In addition, we have used the constitutively active catalytic domain of PKG-I to demonstrate that it is the phosphorylation by PKG itself, and not another signaling pathway activated by NO, that is responsible for the changes in the production of extracellular matrix proteins. As summarized elsewhere,²³ NO has several actions unrelated to PKG activation, including (1) reaction with superoxide to produce peroxynitrite, (2) stimulation of protein ADP-ribosylation, (3) binding to other heme proteins, such as cytochrome oxidase, and (4) elevation of cGMP to levels sufficient to activate protein kinase A. It is this latter mechanism that we proposed that underlies in part the capacity of NO and cGMP to inhibit proliferation of phenotypically synthetic VSMCs.^{24 38} Any or all of these effects of NO could inhibit VSMC extracellular matrix protein production. Therefore, in order to specifically examine the effects of PKG on these events, we used the active catalytic domain construct, which, when expressed in eukaryotic cells, produces a constitutively active PKG that does not require the binding of cGMP for activation yet retains the substrate specificity of the holoenzyme.³⁴ Not only does this construct restore PKG activity to the cell, but its expression also allows one to bypass NO signaling altogether.

Recent studies have indicated that osteopontin and thrombospondin are important marker proteins for the synthetic phenotype.^{25 26 27 28} Cultured VSMCs existing in the synthetic phenotype and neointimal cells in vivo express high levels of osteopontin as well as thrombospondin-1.^{29 30} Using the technique of subtractive cloning, Giachelli et al²⁵ and Shanahan et al²⁶ found that osteopontin expression is markedly upregulated in response to vascular injury. The reduction of matrix protein production by PKG suggests that activation of this enzyme in situ may inhibit neointimal formation and reduce the incidence of vascular lesions and subsequent hemodynamic disorders. The reduction in expression of osteopontin and thrombospondin-1 in response to PKG is further evidence that PKG inhibits phenotypic modulation to the synthetic phenotype.

The production of osteopontin and thrombospondin-1 protein was suppressed rather dramatically by PKG. Considering that overall protein synthesis was not different between the two cell phenotypes, these results suggest that PKG specifically reduces the synthesis of these proteins. In addition, PKG expression reduces the secretion of osteopontin and thrombospondin into the medium and reduces the amount of extracellular thrombospondin incorporated into the matrix of the cultured cells. The mechanism of action of PKG is not known at this time, but it would appear that a suppression of the steady-state mRNA levels encoding these proteins plays a comparatively minor role. Our results suggest that the effects of PKG on protein production may involve more complex effects on mRNA processing and/or translation since the steady-state mRNA levels were not significantly reduced compared with the proteins themselves. It is interesting to speculate that PKG may control the translation of proteins, such as matrix proteins, as cells modulate between two differentiated phenotypic stages. This is in contrast to the transcriptional control mechanisms known to exist in the ontogenic development of smooth muscle cells from undifferentiated mesenchymal cells. We would suggest that the modulation of adult VSMCs between contractile and synthetic phenotypes does not represent changes in the state of differentiation but represents alterations between two differentiated phenotypes (ie, one for the control of tone and one for control of wound-healing processes). Thus, the gene expression control mechanisms in adult cell phenotypic modulation might be expected to be different than those that exist in embryological development. More studies will be needed to decipher the biochemical mechanisms by which PKG regulates the expression of these gene products.

The expression of PKG, unlike several other serine/threonine protein kinases, is subject to rapid changes in VSMCs, and the enzyme is virtually absent from synthetic phenotype cells.²⁴ We have found a similar situation to exist in vivo where PKG expression is suppressed in swine coronary arterial smooth muscle cells after stenting or balloon injury.³⁹ This suggests that PKG may be a critical control point in the regulation of VSMC phenotype both in vitro and in vivo. Restoration of PKG via transfection to cultured cells restores at least some of the characteristics of the differentiated phenotype.²⁴ This includes the inhibition of VSMC migration in response to PDGF^{24 40 41} and, in the present study, the suppression of extracellular matrix proteins that have been shown to be necessary to support migration and proliferation of VSMCs.^{25 26 27 28 29 30 40}

VSMC proliferation is also an early event occurring primarily in the first several days in response to injury in human and porcine models.^{1 2 4} Thickening of the intima, however, continues over the course of several weeks even in the absence of excessive cellular proliferation.^{41 42} This has led to the notion that increased migration of cells to the intima and the change in the phenotype of the cells from contractile to secretory is a major mechanism in the formation of the neointima. The expression of osteopontin and thrombospondin are critical for supporting increased cell migration in response to injury.^{29 30 40} It is likely in fact that the inhibition of VSMC migration reported by our laboratory²⁴ and others^{43 44} is secondary to the suppression of extracellular matrix protein production. Matrix protein suppression by NO-cGMP signaling pathways may be of major significance in the reduction of vascular lesions in light of evidence that PKG does not appear to be responsible for the inhibition of VSMC proliferation by NO.^{24 38}

In summary, we have demonstrated that PKG, the major receptor protein mediating NO and cGMP signaling pathways in vascular smooth muscle, inhibits production of the extracellular matrix proteins osteopontin and thrombospondin-1 in cultured VSMCs. Because these proteins are considered important phenotypic markers for VSMCs of the synthetic phenotype, the results provide new information on the role of the NO-cGMP-PKG signaling pathway in regulating VSMC function in disease. Reduction in production of extracellular matrix proteins may underlie the inhibition of migration of VSMCs and the maintenance of the contractile phenotype of these cells.

	Selected Abbreviations and Acronyms

 

8-CPT-cGMP = 8-chlorophenylthio-cGMP PCR = polymerase chain reaction PDGF = platelet-derived growth factor PKG = cGMP-dependent protein kinase TCA = trichloroacetic acid VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (HL-34646 and HL-53426) to Dr Lincoln. The authors are grateful for the technical assistance of Antonio Pallero. We thank Dr Peter G. Anderson for helpful discussions and Dr Trudy Cornwell for her assistance and advice with the cell culture work. We also wish to thank Dr Jack Lawler, Harvard Medical School, for the human thrombospondin cDNA used for these studies.

Received June 25, 1997; accepted October 29, 1997.

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