Osteopontin Expression in Platelet-Derived Growth Factor–Stimulated Vascular Smooth Muscle Cells and Carotid Artery After Balloon Angioplasty

Materials

Recombinant human PDGF-AA, PDGF-AB, PDGF-BB, bFGF, EGF, TGF-β1, and IL-1β were purchased from either Boehringer Mannheim Corp or GIBCO BRL. [α-³²P]dATP (3000 Ci/mmol) and [γ-³²P]ATP (5000 Ci/mmol) were obtained from Amersham Corp, Taq polymerase was purchased from Perkin-Elmer Corp, and the random priming DNA labeling kit was from Boehringer Mannheim. Oligodeoxynucleotides were synthesized by the oligonucleotide synthesis center at the Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.

Cell Culture, Growth Arrest, and Stimulation With PDGF and Other Growth Factors

RASMCs were isolated and cultured.¹³ Briefly, RASMCs were isolated from medial explants of the thoracic aorta of male Sprague-Dawley rats (300 to 350 g; Charles River) and cultured in Dulbecco's modified Eagle's medium (GIBCO BRL) supplemented with 10% fetal bovine serum and gentamicin (50 μg/mL). SMCs were allowed to grow out from the tissue, which was subsequently removed. After confluence was reached, cells were harvested by brief trypsinization and subcultured in T-75 flasks. RASMCs under six passages were used.

To evaluate the purity of the SMCs, cells were stained with monoclonal antibodies specific to SMC α-actin (from hybridoma cells, clone asm-1; Boehringer Mannheim).^{14 15} Cells were grown to subconfluence on chamber slides, washed with PBS, fixed in methanol for 15 minutes on ice, and air-dried. Slides were rinsed in PBS for 15 minutes. Endogenous peroxidase was blocked with 3% H₂O₂ in buffer for 15 minutes. Nonspecific binding was blocked by using normal goat serum in PBS for 15 minutes. Slides were incubated in monoclonal anti–α-actin (Sigma) at a 2.5 mg/mL concentration in PBS for 45 minutes, followed by biotinylated anti-mouse and avidin-biotin complex for 30 minutes each. The antibody binding was detected with 3′3-diaminobenzidine, which generated a brown reaction product. Slides were then counterstained with hematoxylin, dehydrated, cleared, and placed on coverslips by using a xylene-based mounting medium. The number of positive immunoreactive cells was counted by using a Nikon light microscope (×40 objective). The average percentage of positive cells was 96.8% from five individual slides.

RASMCs were grown to confluence, serum deprived for 48 hours, and then treated with either a growth factor or a cytokine for varying times (0 or 30 minutes or 1, 3, 6, 14, or 24 hours) or concentrations (0 to 100 nmol/L) (see figure legends for details).

Left Common Carotid Artery Balloon Angioplasty

Left common carotid artery balloon angioplasty was performed on male Sprague-Dawley rats (400 g; Charles River) under sodium pentobarbital anesthesia (65 mg/kg IP).¹⁶ Following an anterior midline incision, the left external carotid artery was identified and cleared of adherent tissue to allow the insertion of a 2F Fogarty arterial embolectomy catheter (Baxter Healthcare Corp). The catheter was guided a fixed distance (5 cm) down the common carotid arteries until the tip of the catheter was proximal to the aortic arch, at which point the balloon was inflated and withdrawn to its point of insertion. This procedure was performed three times, after which the catheter was removed, and a suture was tied around the external carotid artery to prevent exsanguination. The wound was closed with 9-mm Autoclips (Clay Adams). Throughout the surgical procedure, body temperature was maintained at 37±1°C by using a K-20-F water blanket (American Hamilton).

Animals were allowed to recover from surgery and were housed in pairs in Plexiglas cages on a 12-hour light/dark cycle with access to standard laboratory chow and drinking water ad libitum. All surgical interventions were performed in accordance with the guidelines of the Animal Care and Use Committee, SmithKline Beecham, and the American Association for Laboratory Animal Care.

Left common carotid arteries were isolated from rats immediately after exsanguination under sodium pentobarbital anesthesia (65 mg/kg IP). Vessels that included areas devoid of endothelium and containing endothelium were removed at 0 (control) and 6 hours and 1, 3, 7, and 14 days after surgery. Once isolated, vessels were immediately frozen in liquid nitrogen and stored at −70°C for RNA preparation. At each of the six times vessels were pooled from three rats; five separate pooled samples were analyzed in the present studies.

Isolation of RNA and Northern Blot Analysis

For RNA preparation, carotid arteries were homogenized, whereas cultured SMCs were directly lysed, in an acid-guanidinium-thiocyanate solution and extracted with phenol and chloroform.¹⁷

Total RNA samples (10 to 20 μg/lane) extracted from cultured RASMCs were resolved by electrophoresis, transferred to a GeneScreen Plus membrane (DuPont–New England), and subjected to Northern hybridization.^{13 18} For Northern blot analysis, rat OPN and rpL32 cDNA were generated by using RT-PCR (as described below) and uniformly labeled with [α-³²P]dATP by using a random-priming DNA labeling kit. Hybridization of each probe was performed overnight with 1×10⁶ cpm/mL of probe at 42°C in 5× SSPE (750 mmol/L NaCl, 50 mmol/L NaH₂PO₄, pH 7.6, and 5 mmol/L EDTA), 50% formamide, 5× Denhardt's solution, 2% SDS, 100 μg/mL polyA, and 200 μg/mL boiled salmon sperm DNA. The membranes were washed in 2× SSPE and 2% SDS at 65°C for 1 to 2 hours with a change every 30 minutes and then autoradiographed at −70°C with a Cronex Lightning-Plus intensifying screen for varying times depending on the signal intensity. The relative band intensities were measured by using a PhosphorImager with an ImageQuant software package (Molecular Dynamics). A probe was stripped from the membranes in 10 mmol/L Tris, pH 7.5, 1 mmol/L EDTA, pH 8.0, and 1% SDS for 20 minutes at 95°C and then washed in 2× SSPE for 10 minutes before rehybridization with the other probe. Because the expression of the rpL32 gene is relatively constant under the present experimental conditions,¹³ it was used to normalize the differences of the samples loaded in each lane.

RT and PCR

Total cellular RNA (3 μg/sample) isolated 0 and 6 hours and 1, 3, 7, and 14 days after carotid artery balloon angioplasty was subjected to RT in the presence of 200 U RNase H⁻ SuperScript II reverse transcriptase (GIBCO BRL) and 1 μg oligo(dT)_12-18 primer at 37°C for 60 minutes according to the manufacturer's specifications. The resultant cDNA products were extracted by using phenol-chloroform and precipitated by ethanol. The cDNA pellets were dried under speed vacuum, resuspended in a 120-μL mixture of 10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 7.5, and stored at −20°C until required for PCR amplification.

PCR primers (Table⇓) were synthesized according to published sequences for rat OPN¹⁹ and rpL32²⁰ cDNAs. For quantitative purposes, OPN cDNA was coamplified with rpL32 cDNA, the expression of which is not changed under these experimental conditions and which was thus selected as an internal control. To ensure that the amplification was in the linear range, pilot studies were performed that used 25 to 1600 ng RNA (RT products) and 25 to 40 cycles of PCR in the presence of different amounts of ³²P-labeled primers.^{21 22} By performing these initial experiments, the following conditions were chosen as standards for PCR reactions in a volume of 50 μL: 100 ng RNA (for RT), 2.5 U TaqAmpli polymerase (Perkin-Elmer Corp), and 30 cycles of amplification in the presence of 1×10⁶ cpm (10 ng) labeled antisense primer for OPN and 5×10⁴ cpm for rpL32 antisense primer together with 100 ng of each nonradioactive sense and antisense primer. Amplification was performed by using a thermocycler (Perkin-Elmer Corp).¹⁸ The initial PCR amplification was performed as follows: denaturation, 3 minutes at 94°C; annealing, 1 minute at 54°C; and extension, 3 minutes at 72°C. Subsequent cycles of PCR consisted of denaturation for 15 seconds at 94°C; annealing, 20 seconds at 54°C; and extension, 1 minute at 72°C. The PCR products (10 μL/sample) were separated electrophoretically by using a 6% polyacrylamide gel. The gel was dried and subjected to autoradiography at room temperature overnight. Quantification was performed by using PhosphorImage analysis. The signals of the OPN cDNA were expressed as the relative ratios to those of the rpL32 cDNA in each coamplified sample.

Immunohistochemistry

The carotid arteries at 0, 1, 3, 7, and 14 days after balloon angioplasty from five rats of each time point were perfusion-fixed with 10% phosphate-buffered formalin, excised, and stored in formalin. After 24 hours, the artery was transferred to 70% ethanol and subjected to standard histological processing by using a vacuum infiltration processor (Miles). Prior to embedding, the middle third portion of the artery was divided into four equal cross-sectional segments. Five-micron sections were cut, stained with hematoxylin and eosin, and evaluated microscopically. Additional 5-μm sections were placed on Capillary Gap Plus Microscope Slides (BioTek Solutions Inc) for immunohistochemical evaluation of OPN expression. The avidin-biotin–peroxidase immunohistochemical technique was used to detect OPN in rat carotid arteries. The reagents for this assay, excluding the primary antibody, were purchased as a kit (BioTek), and staining was performed on the TechMate 1000 Immunostainer. Slides were deparaffinized, rehydrated, and placed in the TechMate 1000. Endogenous peroxidase was blocked with 3% H₂O₂ in a buffer for 15 minutes, and nonspecific binding was blocked with normal goat serum in PBS for 15 minutes. The mouse anti-rat OPN antibody MPIIIB10(1), developed by Michael Solursh and Ahnders Franzen, was obtained from the Departmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md, and the Department of Biological Sciences, University of Iowa, Iowa City, Iowa, under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development. All slides were incubated in this primary antibody at a 1:50 dilution in PBS for 45 minutes, followed by biotinylated anti-mouse and avidin-biotin complex for 30 minutes each. Anti-OPN binding was detected by using 3′3-diaminobenzidine, which generated a brown reaction product. Slides were then counterstained with hematoxylin, dehydrated, cleared, and placed on coverslips by using a xylene-based mounting medium.

Immunohistochemical detection of OPN expression in cultured RASMCs was similarly processed, except that RASMCs were grown on chamber slides and fixed in methanol as described above for α-actin staining.

PDGF-Induced Expression of OPN mRNA in Cultured RASMCs

Unstimulated, serum-deprived cultured RASMCs expressed only low, basal levels of OPN mRNA. After stimulation with 1 nmol/L PDGF, RASMC expression of OPN mRNA was increased 1.6-fold at 1 hour compared with time 0, elevated at 3 hours (2.4-fold increase, P<.05), reached a peak at ≈14 hours (6.7-fold increase, P<.001), and maintained a low elevated level at 24 hours (2.3-fold increase, P<.05) (Fig 1A⇓). The concentration response in OPN mRNA expression in RASMCs revealed that the highest induction was observed at 10⁻⁹ mol/L PDGF stimulation (5.7-fold increase after 6 hours versus vehicle, P<.001) (Fig 2B⇓).

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Figure 1.

A, Representative Northern blot. RASMCs were grown to subconfluence in T-75 flasks, deprived of serum for 48 hours, and stimulated with 1 nmol/L PDGF-AB for the indicated times. Total cellular RNA was isolated, resolved (10 μg/lane) by electrophoresis, transferred to a nylon membrane, and hybridized to a rat OPN or an rpL32 cDNA probe. mRNA size was determined by comparison with the migration of an RNA ladder (GIBCO BRL) and the number of kilobases marked on the right. B, Line graph shows quantitative OPN mRNA data in PDGF-AB–stimulated RASMCs after normalizing with rpL32 as shown in A. Relative levels (mean values of two experiments) of OPN mRNA expression in 1 nmol/L PDGF-AA–stimulated RASMCs are also illustrated. Data are mean±SE of three separate experiments analyzed by ANOVA followed by Fisher's protected least-squares difference. *P<.05, ***P<.001 vs vehicle.

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Figure 2.

RASMCs were deprived of serum and stimulated with 0, 10⁻¹⁰, 3×10⁻¹⁰, 10⁻⁹, or 10⁻⁸ mol/L PDGF for 6 hours. A, Northern blot hybridization was performed as described in Fig 1⇑ legend. B, Bar graph shows quantitative data (n=3). ***P<.001 vs vehicle (0).

The specificity of PDGF-induced expression of OPN mRNA in RASMCs was confirmed when PDGF was preincubated with an anti-PDGF antibody before the stimulation (Fig 3A⇓). Quantitative data revealed that >97% of the PDGF-induced OPN mRNA level was inhibited when PDGF was incubated with the antibody before its addition to the cells (Fig 3B⇓).

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Figure 3.

RASMCs were deprived of serum for 48 hours and treated for 6 hours with PBS (veh), 1 nmol/L PDGF, or 1 nmol/L PDGF preincubated with anti-PDGF antibody (PDGF+Ab) for 10 minutes on ice. A, Total cellular RNA was isolated and analyzed by using Northern blot hybridization as described in Fig 1⇑. B, Bar graph shows quantitative data.

Since there are three dimer forms of PDGF that play different roles in SMC function, further experiments were performed to compare the effect of PDGF-AA, AB, and BB on OPN mRNA expression in RASMCs. PDGF-AB and BB revealed similar levels of induction (both time and concentration dependent) in OPN expression (data of PDGF-BB not shown), whereas PDGF-AA showed hardly any effect (Fig 1B⇑).

Effects of Non-PDGF Growth Factors and IL-1β on OPN mRNA Induction in RASMCs

To determine whether other growth factors and cytokines present in serum also contribute to OPN induction, the effects of bFGF, EGF, TGF-β, and IL-1β on OPN mRNA expression in RASMCs were studied. The strongest induction of OPN mRNA was observed 14 hours after PDGF stimulation (a 6.7-fold increase over basal level) compared with a 1.9-fold induction at 14 hours by bFGF, a 1.9-fold increase at 6 hours by EGF, a 4.4-fold increase at 14 hours by TGF-β1, and a 2.2-fold increase by IL-1β at 6 hours (Fig 4⇓).

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Figure 4.

Serum-deprived RASMCs were challenged with 1 nmol/L of PDGF, bFGF, EGF, or IL-1β or 5 ng/mL TGF-β for 1, 3, 6, 14, or 24 hours. Total cellular RNA was isolated and analyzed by using Northern blot hybridization. The relative intensity (fold increase compared with vehicle) of OPN was determined by using PhosphorImage analysis and plotted after normalization with rpL32. Data are mean values of three experiments for PDGF (as shown in Fig 1⇑) and two experiments for other factors.

OPN Expression in PDGF-Stimulated RASMCs

By using immunohistochemical staining of OPN with the mouse anti-rat OPN antibody MPIIIB10(1), an increase in OPN expression was observed in PDGF-stimulated (24 hours) RASMCs, while only a basal expression was detected in unstimulated, serum-deprived cells (Fig 5⇓).

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Figure 5.

RASMCs were treated with vehicle (A) or PDGF (B) for 24 hours as described in Fig 1⇑ legend. Immunostaining was performed by using mouse anti-rat OPN polyclonal antibodies as described in “Methods.” Stronger immunoreactive signals (dark brown) were seen in the PDGF-stimulated RASMCs.

OPN mRNA Expression in Rat Carotid Arteries After Balloon Angioplasty

The temporal expression of OPN mRNA in rat carotid artery after balloon angioplasty was determined by using quantitative RT-PCR (Fig 6A⇓). The relative levels of OPN mRNA expression, after normalizing to rpL32 mRNA, are depicted in Fig 6B⇓. A basal level of OPN mRNA was observed in normal carotid artery. OPN mRNA levels increased rapidly after balloon angioplasty, showing a 1.5-fold increase at 6 hours (P<.01, n=5), reaching a maximal increase at 1 and 3 days (3.1-fold, P<.001, n=5), and decreasing to basal levels 14 days after injury. A similar induction profile of OPN mRNA expression was observed at the selected times by using Northern blot analysis (Fig 7⇓).

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Figure 6.

A, Representative autoradiograph of the PCR products for OPN mRNA expression after balloon angioplasty. RT-PCR was performed under standard conditions as described in “Methods.” The coamplified PCR products (10 μL/lane) were resolved by electrophoresis in a 6% polyacrylamide gel, dried, and autoradiographed. Each sample, at 0, 0.25, 1, 3, 7, or 14 days after angioplasty, was pooled from balloon-injured carotid artery of three animals. B, Bar graph shows signal intensity, which was quantified by using PhosphorImage analysis, of the amplified DNA bands for OPN and rpL32 shown in A. The ratios of OPN to rpL32 were determined based on each coamplified sample; relative levels (illustrated as ratios) for OPN are depicted. Data are mean±SE of five separate experiments (n=5, or 15 animals) for each time. **P<.01, ***P<.001 vs control (time=0) by ANOVA followed by Fisher's protected least-squares difference.

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Figure 7.

Northern blot analysis of OPN mRNA expression in rat carotid artery after balloon angioplasty. Total cellular RNA was prepared from 10 animals for each time (at 0, 0.25, 3, and 14 days after angioplasty), and 15 μg/lane was resolved by electrophoresis and subjected to Northern blot analysis as described in Fig 1⇑.

Immunohistochemical Analysis of OPN Expression in Rat Carotid Artery After Balloon Angioplasty

The temporal expression and spatial distribution of OPN protein in rat carotid artery after balloon angioplasty were studied by using immunohistochemical methods; representative fields are illustrated in Fig 8⇓. It was interesting to note that strong immunopositive signals demonstrating OPN expression were seen in medial SMCs adjacent to the internal elastic laminae at 1 day after balloon injury, where significant numbers of platelets adhered (Fig 8B⇓); later, the strong signals were located in the intimal SMCs (ie, at days 3 and 7, Fig 8C and 8D⇓⇓). At day 14, the immunopositive signal was mainly distributed at the innermost surface of the intimal lesion close to the lumen of the blood vessel (Fig 8E⇓). The highest intensity of staining was cytoplasmic. Positive staining was also observed extracellularly in the media and neointima, particularly close to the edges of cells. In general, staining was observed in selected cells in the media soon after injury, but at later times, when the neointimal and medial cells were positive, the positive neointimal cells exhibited a stronger staining intensity compared with their medial counterpart. As expected, the positive control chondrocytes (cartilage) and osteoblasts (bone; data not shown) showed a strong positive reaction, which was not the case when the serial sections were incubated with the preimmune sera (compare Fig 8G and 8H⇓⇓).

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Figure 8.

Cross sections of rat carotid artery at days 0 (A), 1 (B), 3 (C), 7 (D), and 14 (E) after balloon angioplasty were incubated with mouse anti-rat OPN polyclonal antibodies, and samples at day 7 were incubated with preimmune sera (F) as described in “Methods.” Positive (G) and negative (H) controls were performed on cartilages by using antisera and preimmune sera, respectively. Note the strong immunoreactive signal (dark brown) in the medial SMC layer 1 day after balloon injury (B) and later in the intimal SMC-like cells at 3 (C) and 7 (D) days. At day 14 (E), the immunopositive signal is mainly distributed at the intimal lesion close to the lumen. The sections were counterstained with hematoxylin (original magnification ×200).