Osteopontin Expression in Platelet-Derived Growth Factor–Stimulated Vascular Smooth Muscle Cells and Carotid Artery After Balloon Angioplasty
Osteopontin (OPN), an arginine-glycine-aspartate (RGD)–containing adhesive glycoprotein, is constitutively expressed in rat aorta and carotid arteries and is markedly elevated in response to vascular injury. OPN is chemotactic for vascular smooth muscle cells (SMCs), suggesting a role in vascular remodeling. However, the mechanism for the regulation of OPN expression is poorly understood. In the present study, the effect of platelet-derived growth factor (PDGF) on OPN mRNA expression was investigated in cultured rat aortic SMCs (RASMCs). When RASMCs were stimulated with 1 nmol/L PDGF, a 2.4-fold increase in OPN mRNA expression was observed at 3 hours (P<.05) that peaked at 14 hours with a 6.7-fold increase (P<.001). This induction was blocked by a monoclonal anti-PDGF antibody. Further studies revealed that OPN mRNA expression was induced by PDGF-AB or PDGF-BB but not by PDGF-AA, indicating that only the β-type PDGF receptor mediates this response. Compared with basic fibroblast growth factor, epidermal growth factor, transforming growth factor-β, and interleukin-1β, PDGF was the most potent factor studied to induce OPN mRNA expression in RASMCs. Immunohistochemical studies demonstrated the elevation of OPN protein in PDGF-stimulated RASMCs. The temporal expression of OPN mRNA after rat carotid artery balloon angioplasty as assessed by both reverse transcription–polymerase chain reaction and Northern blot analysis revealed a 1.5-fold increase at 6 hours (P<.01) that peaked at 1 and 3 days with a 3.1-fold increase (P<.001). Immunohistochemical studies of carotid artery after angioplasty localized OPN expression in the medial SMCs at 1 day, ie, at a time of significant platelet adherence to the injured vessel, and thereafter to the intimal lesion during neointimal formation. These data suggest that OPN expression in vascular SMCs is regulated by PDGF through the β-type PDGF receptor in vitro, and possibly in vivo, in situations that involve PDGF released from platelets or other cellular sources, such as blood vessels after angioplasty injury.
- Received September 25, 1995.
- Revision received April 2, 1996.
The highly acidic, secreted 41-kD phosphoglycoprotein OPN contains the adhesive motif arginine-glycine-aspartate (RGD). OPN was originally identified in bone matrix and subsequently found in kidney, brain, placenta, and blood vessels.1 2 OPN mRNA is expressed in rat aorta and carotid arteries and is markedly elevated after balloon angioplasty, suggesting a previously unappreciated role for this matrix protein in vascular remodeling.3 OPN expression is elevated during neointimal formation and is a component of human atherosclerotic plaque4 ; furthermore, the expression of OPN mRNA and protein has been demonstrated in human coronary atherosclerotic plaque but not in undiseased vessels.5 OPN is chemotactic for vascular SMCs and involved in SMC migration, mainly via αvβ3 integrins on the surface of SMCs.6 7 8 Taken together, these data implicate a potential involvement of OPN in vascular biology and pathophysiology. However, little is known of the molecular mechanisms that regulate OPN expression in blood vessels.
PDGF has been proposed as a factor that may play a critical role in vascular remodeling after acute vascular wall injury and in conditions, such as atherosclerosis, under which it might promote SMC proliferation and migration.9 The present study investigated the effects of PDGF on OPN mRNA and protein expression in cultured RASMCs. Since PDGF consists of disulfide-linked dimers of two homologous polypeptide chains designated as A and B, and different dimeric forms of PDGF, ie, PDGF-AA, PDGF-AB, and PDGF-BB, play different roles in SMC function,10 11 12 the effects of these PDGF dimers on OPN mRNA expression in RASMCs were also examined. Moreover, the effects of the cytokine IL-1β and the growth factors bFGF, EGF, and TGF-β1, which are known to play roles in vascular remodeling, on OPN mRNA expression in RASMCs were also evaluated. In addition, OPN mRNA expression in rat carotid artery after balloon angioplasty was evaluated quantitatively by using RT-PCR, which was validated by using Northern blot analysis. Finally, the corresponding cellular components expressing OPN after angioplasty were analyzed by using immunohistochemical methods.
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.
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⇓).
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⇓).
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⇓).
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⇓).
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⇓).
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⇓⇓).
The present study demonstrated that PDGF plays a significant role in the upregulation of OPN mRNA and protein in cultured vascular SMCs. The increase in OPN mRNA expression in SMCs was induced by PDGF-AB and PDGF-BB but not PDGF-AA. Since PDGF-α and PDGF-β receptors transmit different signals into rat vascular SMCs, ie, PDGF-AB and PDGF-BB stimulate inositol 1,4,5-triphosphate release, [Ca2+]i increase, and pHi changes but PDGF-AA only induces the production of diacylglycerol,10 the effect of PDGF on OPN expression in RASMCs suggests that this induction was mediated through the PDGF-β receptor and its signal transduction pathway. Interestingly, the differential effects of the PDGF dimers on SMC functions have been recently noted: PDGF-AB and PDGF-BB appear to play a role in SMC proliferation and migration, whereas PDGF-AA stimulates proliferation and inhibits SMC migration.10 11 12 Therefore, the upregulation of OPN by PDGF-AB or PDGF-BB is in accordance with their function in SMC migration. No effect of PDGF on OPN mRNA expression was observed in SMCs that were treated with PDGF for 1 to 3 days.4 Based on the data from the present study, the OPN mRNA may already have decreased to a basal or low elevated level at that time.
Growth factors such as bFGF, EGF, and TGF-β and cytokines such as IL-1β are implicated in vascular injury response and tissue remodeling (for review, see Reference 23). The effects of selected growth factors, including bFGF and TGF-β, on OPN mRNA expression in SMCs at a late stimulated time, ie, at 1 to 3 days, have also been studied.4 Based on our time-response study for PDGF stimulation of SMCs, it was certainly worth examining the effects of these factors on OPN mRNA expression at the early time. Since the induction profiles differed slightly among these factors, a time-course comparison was applied. In addition to PDGF, TGF-β, an important regulator of cellular matrix protein, upregulated OPN mRNA expression in RASMCs by 4.42-fold at 14 hours. bFGF, which is involved in all three waves of neointimal formation (ie, replication of SMCs within the media, migration of SMCs from the media into the intima, and proliferation and migration of SMCs within the intima),23 only upregulated OPN mRNA expression in SMCs by 1.9-fold at 14 hours. It was interesting to observe that IL-1β, an important inflammatory mediator and mitogen that is upregulated after vascular injuries and during atherosclerosis development,24 25 also upregulated OPN mRNA expression in SMCs. PDGF was the most potent regulator in the present study to induce OPN mRNA expression in SMCs. The various effects on OPN mRNA expression in SMCs, as determined by maximal induction levels, were as follows: PDGF>TGF-β>IL-1β>bFGF≥EGF. Other factors, such as angiotensin II,4 may also be important in regulating OPN expression in SMCs.
Immunohistochemical detection of OPN expression in rat carotid artery after balloon angioplasty demonstrated that OPN was evident in SMCs as early as 1 day after vascular injury. This temporal expression was further supported by the analysis of OPN mRNA expression after angioplasty by using quantitative RT-PCR and Northern blot analysis. Using this method, we detected a significant upregulation of OPN mRNA at 6 hours after injury and a peak level at 1 and 3 days (Figs 6 and 7⇑⇑). These results are generally in agreement with work4 that has demonstrated a significant increase in OPN mRNA and protein expression after carotid artery angioplasty by using in situ hybridization and immunohistochemical methods. However, the peak expression of OPN in the injured vessel was shown 4 to 7 days after balloon angioplasty in that report.4 The cause of this discrepancy in the temporal expression profile of OPN is not clear, but it may be due to the difference in experimental conditions.
It was interesting to find that OPN expression after balloon angioplasty was located in SMCs, especially where the endothelium was denuded and a significant number of platelets were adhered. Our results suggested that upregulation of OPN expression in SMCs might be caused by the PDGF that was released mainly from adhered platelets. Moreover, as a potent chemoattractant for SMCs, PDGF could initiate SMC migration from the media to the intima, which has been proposed as the second wave of neointimal formation after angioplasty.23 In addition, OPN is chemotactic for SMCs6 7 8 and thus could further promote SMC migration. Therefore, PDGF-stimulated OPN expression in SMCs, as demonstrated in the present study, could be a potential mechanism in vascular remodeling after vascular injury. While the role of platelets and PDGF in SMC migration in neointimal formation after angioplasty has been demonstrated by administering anti-platelet IgG or anti-PDGF antibodies to experimental animals,9 26 the hypothesis regarding the effects of PDGF on OPN expression in vivo and the role of OPN in SMC migration and tissue remodeling after injury remains to be further demonstrated when specific neutralizing antibodies against PDGF and/or OPN become available. In addition to PDGF, other regulating factors, such as angiotensin II and TGF-β, which have different timing of effects on OPN expression in cultured vascular SMCs, may also contribute to the spatial and temporal distribution of OPN expression after angioplasty.
In summary, the demonstration of PDGF-induced expression of OPN mRNA in SMCs and the coordinated upregulation of PDGF and OPN in human atherosclerotic lesions and in the neointima after balloon angioplasty suggest that PDGF plays a critical role in OPN expression in SMCs that in turn contributes to SMC migration during pathogenesis.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|EGF||=||epidermal growth factor|
|PCR||=||polymerase chain reaction|
|PDGF||=||platelet-derived growth factor|
|RASMC||=||rat aortic smooth muscle cell|
|rpL32||=||ribosomal protein L32|
|SDS||=||sodium dodecyl sulfate|
|SMC||=||smooth muscle cell|
|TGF-β||=||transforming growth factor-β|
We thank the excellent technical assistant from L. Vickery-Clark for preparing the carotid artery specimens and Dawn Zimmerman for immunohistochemical staining.
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