Expression and Function of a Recombinant PDGF B Gene in Porcine Arteries
Abstract Platelet-derived growth factor (PDGF) B is a mitogen and chemoattractant for smooth muscle cells in vitro, and expression of a recombinant PDGF B gene in porcine arteries stimulates intimal thickening. To define the mechanisms by which PDGF B gene expression induces intimal thickening in vivo, we examined its effects on smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration in intimal lesions of pig arteries after direct gene transfer of a recombinant PDGF B gene. PDGF B gene expression was associated with rapid formation of an intima, including 3- to 10-fold increases in intimal thickness and intima-to-media area ratio 4 to 21 days after gene transfer compared with control transfected arteries. Intimal smooth muscle cell proliferation was detected at 2 days, peaked at 7 days (P<.01), and declined by 14 days, although the total number of intimal nuclei progressively increased to 21 days (P<.01). Calculations of expected-to-observed ratios of intimal cells, based on BrdC proliferation indexes, demonstrated that the increases in intimal cell number on days 2 through 7 could not be accounted for by proliferation alone, suggesting that recombinant PDGF BB acts to stimulate cell proliferation and migration of smooth muscle cells into the intima. Extracellular matrix deposition and procollagen synthesis were observed after 7 days (P<.01) and were associated with a decline in cell density in the intima, suggesting that extracellular matrix synthesis may contribute to progressive intimal thickening in response to PDGF B gene expression. There was minimal accumulation of inflammatory cells, including macrophages and CD3(+) lymphocytes, in transfected arteries. These data suggest that PDGF B gene expression promotes intimal expansion by both proliferation and migration of smooth muscle cells followed by synthesis of extracellular matrix and therefore acts through several mechanisms to play a role in the pathogenesis of intimal lesions in vivo.
- Received June 19, 1995.
- Accepted October 4, 1995.
PDGF is expressed at low levels in normal adult arteries, but its expression is increased after tissue injury,1 2 3 in which it has been implicated in vascular pathology.4 5 Previous studies of balloon catheter–injured arterial tissue,6 naturally occurring atherosclerosis,2 and experimentally induced atherosclerosis7 8 have demonstrated elevated expression of PDGF and PDGF receptor in these lesions, suggesting an association of PDGF with arterial lesion formation. Recent studies have suggested a role for the dimeric form PDGF BB in the development of intimal thickening after arterial injury. PDGF BB stimulates smooth muscle cell migration and intimal thickening in a rat carotid injury model,5 and PDGF BB has been localized to macrophages7 and other intimal cells3 in lesions of human atherosclerosis.
The delivery of recombinant genes into vascular cells at specific sites in the circulation permits the systematic analysis of gene function in the arterial wall in vivo. Previous studies from our laboratory suggest that the introduction of a PDGF B gene in porcine arteries promotes formation of a neointima de novo.9 These observations and findings of Jawien and coworkers5 and Ferns and colleagues10 , which demonstrate that the infusion of a polyclonal PDGF antibody inhibits development of an intimal lesion, provide evidence that this growth factor contributes to intimal lesion formation in vivo. While these studies suggest that PDGF BB is associated with intimal thickening, the mechanisms responsible for development of the intima by PDGF B expression have not been defined. Accordingly, the goals of this study were to examine the pathogenesis of intimal thickening in PDGF B gene–transfected arteries by analysis of smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration.
Plasmids and Cell Transfection
A PDGF B plasmid expression vector (pSV-PDGFB) was prepared by ligating the Sal I–Xba I fragment of the v-sis gene into the Xho I–BamHI cloning site of the pSVL vector (Pharmacia, LKB Biotechnology).9 The v-sis gene is a homologue of the human PDGF B gene.11 12 13 This vector contains the SV40 late promoter to direct expression of the PDGF B gene. The efficiency of this vector has been tested previously in porcine endothelial and smooth muscle cells.9 A plasmid expression vector encoding hpAP was constructed by cloning a heat-stable hpAP gene14 into the RSV-β globin vector15 digested with Bgl II and HindIII (pRSV-hpAP). This vector utilizes an RSV promoter and β-globin enhancer to regulate expression of hpAP. This gene was selected as a reporter because the protein is heat stable above 65°C and can be distinguished from endogenous alkaline phosphatase by heat inactivation. Additional control studies were done with a plasmid expression vector encoding streptavidin (SA). This plasmid was constructed by ligating the BamHI fragment of a Streptococcus avidini gene into a Bgl II–HindIII cloning site of an RSV-β globin vector (pRSV-SA).15 Expression of the vectors encoding PDGF and reporter genes were tested in 293 cells and porcine endothelial cells by use of 5 μg DNA and 12 μg DOTMA/DOPE (GIBCO BRL), and immunohistochemistry was performed 2 days later. Approximately 70% to 80% of 293 cells and 15% of endothelial cells displayed a reaction product indicating PDGF, hpAP, or SA protein expression. This dose and ratio of DNA to liposomes did not result in cell death, similar to previous findings from our laboratory.16 17 No differences in gene expression were observed between plasmids.
In Vivo Gene Transfer
Direct gene transfer was performed in the right and left iliofemoral arteries of 26 domestic Yorkshire pigs, 12 with a PDGF B gene (pSV-PDGF B) and 14 with a reporter gene (pRSV-hpAP or pRSV-SA). After sedation with telazol (6 mg/kg body wt) and xylazine (2.2 mg/kg), the pigs were intubated and anesthetized with 1% halothane. Under sterile surgical techniques, a double-balloon catheter was inserted into the iliofemoral artery.18 The arterial segment was rinsed with 5 mL saline solution and 5 mL Opti-MEM (GIBCO BRL) to clear the vessel of blood. DNA liposome conjugates were prepared 10 minutes before the insertion of the catheter by mixing 12 μg DOTMA/DOPE (1 mg/mL) in 0.2 mL Opti-MEM at room temperature. Plasmid DNA (5 μg) (stock concentration >1 mg/mL) was added to the liposomes and left at room temperature for 5 to 10 minutes. Opti-MEM (0.5 mL) was added to the DNA liposome solution, and the complexes (0.7 mL) were instilled into the arterial space between the two balloons for 20 minutes at 150 mm Hg pressure. Previous studies from our laboratory demonstrated that direct gene transfer into an artery at this pressure produces minimal arterial injury.9 After gene delivery, the catheter was removed, and arterial flow was restored. Animals were killed 1, 2, 4, 7, 14, and 21 days after gene transfer. At each time point, 4 pigs were studied: 2 experimental pigs transfected with pSV-PDGF B (n=4 arteries) and 2 control pigs transfected with pRSV-SA gene (n=4 arteries). When the pigs were killed, arteries were serially sectioned into five rings in an identical manner. Sections 1, 3, 4, and 5 were fixed with 10% buffered formaldehyde or methyl Carnoy’s solution (60% methanol/30% ethanol/10% glacial acetic acid [vol%]) and embedded in paraffin. Section 2 was rapidly frozen in liquid nitrogen and stored at −80°C for DNA and RNA isolation. Two additional pigs underwent gene transfer of pRSV-hpAP into the right and left iliofemoral arteries (n=4 arteries), and these arteries were analyzed for reporter gene expression 4 days later. All animal studies were performed within the guidelines of the National Institutes of Health and with the approval of the University of Michigan Committee on the Use and Care of Animals.
Analysis of Gene Transfer and Expression
The presence of recombinant PDGF B DNA from transfected vessels was analyzed by PCR. Primers were synthesized from v-sis sequence in regions not homologous to porcine PDGF sequence to generate a 384-bp fragment: sense (25-mer), TCA TCC TCT TAA GCT GCG TAT TCG G; antisense (25-mer), ACA CCA GGA AGT TGG CAT TGG TGC G. Alkaline phosphatase gene transfer was analyzed by oligonucleotide primers as follows: sense (30-mer), TGG GGC CCT GCA TGC TGC TGC TGC TGC TGC; antisense (30-mer), TGG AAG TTG CCC TTG ACC CCG CAC AGG TAG; and streptavidin gene: sense (25-mer), TTC CCT GAC CAC GGT CTC GAT TAC G; antisense (25-mer), GCA GCC ACT GGG TGT TGA TCC TCG C. Genomic DNA was prepared from arteries by proteinase K digestion and phenol and chloroform extraction.19 The PCR was performed with 100 ng genomic DNA for 35 cycles of denaturation (94°C, 1 minute), annealing (60°C, 2 minutes), and polymerization (72°C, 2 minutes) in an automated DNA thermal cycler (Perkins-Elmer Cetus). Samples were analyzed by ethidium bromide staining in a 1% agarose gel. The sensitivity of the PCR analysis is approximately one copy of recombinant gene per 105 genomes.
Expression of recombinant PDGF B RNA was detected by RT-PCR. Total cellular RNA was obtained from arterial samples by acid guanidinium isolation.20 Nucleic acids were extracted and treated with DNAase I (30 U, 10 U/μL) (RNase-free) (Boehringer Mannheim Biochemicals) in buffer (in mmol/L: Tris-HCl 40, pH 7.9, NaCl 10, MgCl2 6, and CaCl2 0.1) at 37°C for 30 minutes to eliminate contamination. Samples were analyzed in the presence or absence of reverse transcriptase, 15 U (Promega) with oligo (dT). Primers for PDGF B, alkaline phosphatase, and streptavidin sequence were generated as described. The PCR was performed with 35 cycles of denaturing, annealing, and polymerization as described. Positive and negative controls were performed with each PCR.
Expression of recombinant hpAP protein was detected by histochemical analysis of transfected arteries as described.21 Briefly, formalin-fixed paraffin-embedded specimens were sectioned and incubated in PBS at 65°C for 60 minutes to inactivate endogenous alkaline phosphatase. Sections were incubated in PBS containing a chromogenic substrate of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (1 mg/mL) (GIBCO BRL) and nitro blue tetrazolium chloride (1 mg/mL) (GIBCO BRL) for 19 hours. This substrate yielded a dark purple stain in the presence of alkaline phosphatase. Sections were rinsed with PBS and counterstained with methylene green.
Immunohistochemical studies were performed with mAb to human PDGF BB, smooth muscle α-actin, vWf, BrdC, type I procollagen, porcine macrophages, and human CD3(+) lymphocytes. Serial sections of methyl Carnoy– or formalin-fixed, paraffin-embedded arteries (6 μm) were placed onto poly-l-lysine–coated slides, deparaffinized in three changes of xylene, and rehydrated in 100%, 95%, and 75% ethyl alcohol. Incubation in 0.3% hydrogen peroxide for 30 minutes exhausted endogenous peroxidase activity. For each antibody, the dilution that yielded optimal specific staining was determined in pilot experiments.
Primary antibodies were diluted in PBS with 1% BSA and applied to the slides for 1 hour. The following primary antibodies were used: a monoclonal mouse anti-human PDGF BB IgG2b antibody, 1:400 dilution (Promega); a monoclonal mouse anti–smooth muscle α-actin antibody, 1:500 dilution (Boehringer Mannheim Biochemical); a monoclonal goat anti-human vWf antibody, 1:10 000 dilution (Atlantic Antibodies); a monoclonal mouse anti-BrdC antibody, 1:1000 dilution (Amersham Life Sciences); a monoclonal SP1.d8 aminopeptide-specific type I procollagen antibody, 1:500 dilution (Developmental Studies, Hybridoma Bank, University of Iowa); a monoclonal 1912 carboxypeptide–specific type I procollagen antibody, 1:1000 dilution (Chemicon International, Inc); a monoclonal mouse anti-porcine macrophage IgG2b antibody, 1:100 dilution (ATCC HB 142.1, American Type Culture Collection); and a monoclonal rabbit anti-human CD3 antibody, 1:100 dilution (DAKO). The monoclonal mouse anti-human PDGF BB IgG2b antibody primarily detects PDGF BB homodimer but potentially can stain human PDGF AB heterodimer and AA homodimer and porcine PDGF. Control experiments were performed using a purified mouse IgG2b antibody, 1:100 dilution (Promega) or goat serum. These control first antibodies did not stain the arterial specimens. After washes in 0.05 mol/L Tris-buffered saline, a biotinylated secondary antibody, horse anti-mouse IgG2 or horse anti-rabbit IgG2, 1:400 dilution (Zymed Laboratories, Inc), was applied at room temperature for 30 minutes. Slides were developed with either a streptavidin–horseradish peroxidase complex (Vector Laboratories) or a Vectastain ABC–alkaline phosphatase reagent (Vector Laboratories). The streptavidin–horseradish peroxidase complex, 1:5000 dilution, was applied for 30 minutes at room temperature, followed by a diaminobenzidine substrate (Sigma) in 0.045% nickel chloride (room temperature, 10 minutes) to produce a gray-black reaction and by counterstain with methyl green. Biotinylated anti-mouse immunoglobulin, 1:200 dilution (Vector Laboratories), was applied for 30 minutes, followed by another 30-minute incubation in a Vectastain ABC–alkaline phosphatase reagent (Vector Laboratories). The substrate 3-amino-9-ethylcarbazole yields a red reaction product, and sections were counterstained in methyl green.
Measurement of Cell Proliferation and Morphometry
To determine the number of proliferating cells, arteries were labeled with BrdC, and immunohistochemistry was performed. Animals were injected with BrdC (25 mg/kg) 1 hour before they were killed. Total nuclei and BrdC-positive nuclei were counted in the intima of experimental and control arteries by a microscope-based video image analysis system (Image One Systems, Universal Imaging Corp). Eight high-power fields (magnification ×400) were counted in four sections from each artery. A proliferation index was calculated as the ratio of labeled cells to total number of cells. The percentage of intimal smooth muscle cells that were PDGF B positive was determined from multiple sections of double-immunostained PDGF B–transfected arteries by use of antibodies to smooth muscle α-actin and PDGF B. The numbers of positive cells per high-power field were counted by the video imaging system. Eight high-power fields were counted from four sections in each artery. The percentage of PDGF B cells that were BrdC positive was measured in a similar manner from sections double-immunostained with antibodies to PDGF B and BrdC.
Measurements of intimal and medial thickness, area, and total cell number were determined on four sections from each artery by use of Image One Systems. Intimal and medial boundaries were determined by digital planimetry. Intimal and medial areas in each section were measured, and I/M area ratios from the four sections were averaged to derive an I/M area ratio for each artery.
Calculations of an expected-to-observed ratio of the number of intimal cells were made in PDGF arteries for days 1 through 21 after gene transfer. These calculations were based on BrdC proliferation indexes and assumed a cell doubling rate of 4 days in pig arteries.21
Results are expressed as mean±SEM. Comparisons of intimal thickness, I/M area ratio, number of intimal nuclei, BrdC labeling index, number of nuclei per square micrometer, percent procollagen-positive cells, and percent trichrome-positive cells between PDGF B and control arteries were made by a two-tailed, unpaired t test. Changes in variables within experimental groups between time points were examined by ANOVA with Dunnett’s t test.22 23 A polynomial regression analysis was performed on intimal smooth muscle cell numbers from days 1 through 21. Statistical significance was assumed if a null hypothesis could be rejected at the P=.05 level.
Endothelial and Smooth Muscle Cells Express Recombinant Genes After Liposome-Mediated Gene Transfer
To determine the cell types transfected after liposome-mediated gene transfer, an hpAP expression vector was introduced into normal porcine arteries. Expression of recombinant hpAP protein was observed in cells in the intimal and luminal regions of the media 3 days after gene transfer (Fig 1A⇓ and 1B⇓). These cells were identified as smooth muscle and endothelial cells by immunohistochemical analysis with an mAb specific for smooth muscle α-actin (Fig 1C⇓) and vWf (Fig 1D⇓). Alkaline phosphatase staining was not observed in control arterial sections transduced with an S. avidini gene (Fig 1A⇓). In addition, morphological examination of these artery segments revealed no cell dropout or necrosis, suggesting that liposomal transfection at this dose and ratio of DNA to liposomes did not result in significant vascular cell toxicity. These studies demonstrate that both smooth muscle and endothelial cells express recombinant genes after DNA liposome transfection and are likely targets of recombinant PDGF B gene expression.
Transfected Arteries Express Recombinant PDGF B DNA, mRNA, and Protein
To investigate the function of a PDGF B gene in porcine arteries in vivo, a PDGF B expression plasmid was transfected into iliofemoral arteries of 12 pigs by direct gene transfer. Twelve control pigs were transfected with a plasmid expressing an S. avidini gene. To examine the transfer of plasmid DNA in arteries, samples of PDGF B gene–transfected arteries were analyzed by PCR. PDGF B plasmid was detected in PDGF B arteries 4 and 21 days (Fig 2A⇓, lanes 1 and 2) after gene transfer but not in S. avidini gene–transfected arteries at 4 days (Fig 2A⇓, lane 3). To confirm expression of PDGF B, mRNA was analyzed by RT-PCR. PDGF B RNA was detected in samples incubated with reverse transcriptase but was not detected in its absence in transfected artery segments 4 and 21 days after gene transfer (Fig 2B⇓, lanes 1 through 4) compared with S. avidini gene–transfected arteries (Fig 2B⇓, lanes 5 and 6). Transfer of plasmid DNA and mRNA expression were confirmed in control arteries at similar time points (data not shown).
To examine PDGF B protein expression, immunohistochemical studies were performed with an mAb to human PDGF BB. Porcine arteries transfected with a PDGF B gene demonstrated immunoreactive protein in the intima and media (Fig 3C⇓ through 3F), whereas PDGF B protein was not detectable in the intima and media of control arteries (Fig 3A⇓). Immunoreactive protein was detected throughout the intima at 4 days (Fig 3C⇓ and 3D⇓) and was observed predominantly in the luminal region of the intima at 21 days (Fig 3E⇓ and 3F⇓).
PDGF B Gene Expression Stimulates Intimal Thickening
To determine the effect of PDGF B gene expression, transfected arteries were analyzed by light microscopy at 1, 2, 4, 7, 14, and 21 days after gene transfer. PDGF B gene expression was associated with a significant increase in intimal thickness compared with control arteries at 2 days (15.6±1.7 versus 6.4±0.5 μm) through 21 days (174.6±10.1 versus 19.4±1.9 μm) (P<.01 for 2, 4, 7, 14, and 21 days) (Fig 4A⇓). The I/M area ratio was also significantly increased in PDGF arteries compared with control arteries from 4 days (0.12±0.01 versus 0.05±0.01) through 21 days (0.43±0.03 versus 0.07±0.01) (P<.01 for 4, 7, 14, and 21 days) (Fig 4B⇓). Fig 5⇓ demonstrates representative histology sections from PDGF B arteries (Fig 5B⇓, 5D, 5F, 5H, 5J, 5L) compared with control arteries (Fig 5A⇓, 5C, 5E, 5G, 5I, 5K) at all time points after gene transfer. At 2 and 4 days, a two- to three-cell-layer intima was present in PDGF B arteries (Fig 5D⇓ and 5F⇓), and this intima thickened rapidly by 21 days (Fig 5L⇓). In contrast, a control artery at 21 days shows a small layer of intimal cells (Fig 5K⇓).
PDGF B Gene Expression Is Associated With Intimal Smooth Muscle Cell Proliferation
The intima of PDGF B arteries demonstrated a progressive increase in the number of nuclei, from 174±8 at 1 day to 1618±265 at 7 days and 2263±280 at 21 days (P<.05, 1 day versus 4, 7, 14, and 21 days, ANOVA with Dunnett’s t test) (Fig 6A⇓). There was a linear increase in intimal cells to day 7, but after day 7, the rate of cell accumulation reached a plateau (R2=.87). The total number of cell nuclei in the intima was up to threefold greater in PDGF B arteries compared with control arteries at 7, 14, and 21 days (P<.01) (Fig 6A⇓). The increase in cell nuclei was paralleled in the first week by an increase in cell proliferation. Cell proliferation was observed in the intima of PDGF B arteries 24 hours after injury (1.7±0.8% labeling index), was maximal at 7 days (7.5±1.0% versus 1.8±0.2%, control arteries, P<.01), but subsided at day 14 (0.2±0.04%) (Fig 6B⇓). Double immunostaining performed with mAbs to BrdC and smooth muscle α-actin identified smooth muscle cells as the major proliferative cell type in the intima (Fig 7A⇓ through 7C).
To examine the role of PDGF BB in intimal cell proliferation, double-label immunostaining with mAbs to PDGF BB, BrdC, and smooth muscle α-actin was performed. The percentage of smooth muscle cells expressing PDGF BB and the percentage of PDGF-positive cells that were proliferating were measured with the video image analysis system (Fig 7D⇑ and 7E⇑). Seven days after gene transfer, 33.8±7.9% of intimal smooth muscle cells were PDGF B positive, and 8.8±2.1% were PDGF B positive at 21 days. Intimal PDGF BB cells underwent proliferation: 10.3±1.7% of intimal PDGF B cells were BrdC positive at 7 days compared with 4.5±0.6% at 21 days. This percentage of BrdC-positive cells, however, is higher than the intimal BrdC labeling index at 7 (7.5±1.0%) and 21 (0.5±0.1%) days, suggesting a proliferative advantage to PDGF BB cells. These studies suggest a correlation between PDGF expression and intimal smooth muscle cell proliferation.
These observations suggested that PDGF B gene expression in porcine arteries was associated with early formation of an intima that progressed over a period of 3 weeks, compared with control arteries. Smooth muscle cells proliferated in the intima, peaking at 7 days. The total number of cells continued to increase in the intima from 7 to 21 days. To determine whether cell migration might contribute to the number of intimal cells during this time interval, an expected-to-observed ratio of intimal cells was calculated for PDGF arteries 1 to 21 days after gene transfer, based on BrdC labeling indexes. The expected-to-observed ratio was 0.51 at 2 days, 0.62 at 4 days, 0.42 at 7 days, 0.94 at 14 days, and 0.97 at 21 days. These data suggest that proliferation alone cannot account for the total number of observed intimal cells at 2 to 7 days, suggesting possible migration of cells into the intima. These observations are consistent with previous observations that PDGF BB stimulates migration of smooth muscle cells into the intima of injured arteries.5 10
PDGF B Gene Expression Is Associated With Extracellular Matrix Synthesis After 14 Days
The number of nuclei in the intima of PDGF B arteries progressively increased from 1 to 21 days. Cell density, expressed as the number of nuclei per square micrometer, decreased in the intima when the intimal area was greatest at 14 and 21 days (day 1, 0.18±0.01; day 4, 0.29±0.03; day 7, 0.36±0.04; day 14, 0.19±0.02; day 21, 0.15±0.02) (Fig 8A⇓). This decrease in density suggested that extracellular matrix deposition contributes to intimal thickening. To investigate this hypothesis, we examined type I procollagen synthesis and trichrome staining in PDGF B and control arteries. The percentage of cells in PDGF B arteries that stained with a type I procollagen antibody steadily increased from 1 day (6.3±0.1%) to 7 days (10.1±0.8%), 14 days (14.3±1.3%), and 21 days (12.3±0.9%) (P<.05, 1 day versus 7, 14, and 21 days, ANOVA with Dunnett’s t test [Fig 8B⇓]). Trichrome staining in the intima of PDGF B arteries also progressively increased from 1 day (1.2±0.2%) to 7 days (14.1±1.6%) and 14 days (19.3±1.9%) and was greatest at 21 days (42.8±5.2%) (P<.05, 1 day versus 7, 14, and 21 days, ANOVA with Dunnett’s t test), suggesting an accumulation of extracellular matrix at later times (Fig 8C⇓). The absolute number of cells that stained with a procollagen antibody was greater at 7, 14, and 21 days in PDGF B arteries compared with control arteries (P<.05) (Fig 8A⇓). These findings suggest that PDGF B gene expression promotes intimal expansion that includes matrix deposition at 2 and 3 weeks after gene transfer.
Lack of Inflammatory Cell Infiltration in the Intima and Effects of PDGF B Gene Expression on the Media
Macrophages were observed in the intima at 1 and 2 days after gene transfer in both PDGF B and control arteries but at a low frequency, two or fewer per high-power field (data not shown). T cells were not observed in samples from PDGF B or control arteries (data not shown). The medial cross-sectional area did not differ significantly between PDGF B and control arteries at any time. No significant increase in medial thickening was observed from 2 to 21 days after gene transfer in either group. Cell proliferation did not change in the media between 1 and 21 days in PDGF B or control arteries. These observations suggest that medial smooth muscle cells did not undergo significant proliferation after transfection with a PDGF B gene, and medial thickening was not present.
In this study, direct gene transfer was used to create somatic transgenic models to define gene function in arteries. We examined the pathogenesis of intimal thickening in porcine arteries after expression of PDGF B. Our findings show that recombinant PDGF B gene expression promotes rapid formation of an intima over a period of 3 weeks. The cellular composition of this intima is complex and changes with time. Within the first week after gene transfer, intimal smooth muscle cell proliferation is present. PDGF BB is expressed by these proliferating smooth muscle cells, suggesting that it can regulate cell proliferation in vivo. Intimal cell proliferation peaked at 1 week and declined 2 weeks after gene transfer. The total number of cells in the intima at 2 to 7 days could not be accounted for by proliferation alone, suggesting that migration of medial smooth muscle cells to the intima may contribute to the intimal lesion. These findings are consistent with previous observations in a different model, an injured rat carotid artery, that PDGF stimulates migration of medial smooth muscle cells to the intima.5 10 In addition, PDGF B gene expression initiates intimal expansion that includes extracellular matrix deposition at later time points.
The present findings suggest that PDGF B plays a major role in intimal lesion formation. Whether these effects are mediated directly or indirectly through autocrine or paracrine effects of PDGF BB is unknown. Evidence in support of a direct role for PDGF in smooth muscle cell accumulation includes mRNA studies showing that a PDGF B gene is expressed in porcine arteries for at least 3 weeks after gene transfer, and these same arteries developed intimal lesions, in contrast to control vessels in which intimal thickening was not observed. Double-labeling immunohistochemical studies show that smooth muscle cells that express PDGF BB undergo proliferation. In addition, it is possible that endogenous PDGF protein was produced in PDGF B gene–transfected arteries in response to the recombinant gene. Previous control experiments for the effects of mechanical injury alone in balloon-injured nontransfected porcine arteries suggested that expression of endogenous PDGF protein was dependent on the severity of vessel injury,9 and PDGF BB protein was not detected in arteries transfected with pRSV-SA in this study. Finally, factors responsible for extracellular matrix accumulation, including TGF-β, may be regulated in part by PDGF B expression. For example, TGF-β–induced increases in smooth muscle cell proliferation and matrix synthesis have been shown to be mediated by PDGF.24 25
PDGF B gene expression may promote both smooth muscle cell proliferation and migration in this animal model. Experimental studies in rats indicate a role for PDGF BB in smooth muscle cell migration after balloon injury. The intima of the normal rat carotid artery does not contain smooth muscle cells, but within 7 days after balloon injury, an intima develops that is composed of smooth muscle cells that have migrated from the media into the intima.26 27 Infusion of recombinant PDGF BB protein into balloon-denuded carotid arteries resulted in an increase in medial smooth muscle cell replication and migration of smooth muscle cells from the media into the intima.5 Likewise, treatment of nude rats subjected to balloon injury of the carotid artery with high doses of a PDGF antibody resulted in a reduction in intimal thickening but no change in thymidine labeling,10 suggesting that a predominant effect of PDGF BB in these arteries is migration of medial smooth muscle cells. Furthermore, recent studies suggest that PDGF BB induces migration and proliferation via different signaling pathways.28
Determination of the role of PDGF BB in migration and proliferation in peripheral porcine arteries is complex, since the intima of normal porcine arteries contains rare, scattered smooth muscle cells.29 30 Direct measurement of smooth muscle cell migration is not possible, since the intima already contains smooth muscle cells; furthermore, we currently lack a method to label medial smooth muscle cells that may be responsive to autocrine or paracrine PDGF. Several lines of evidence suggest a possible role for PDGF B–induced migration in this study. We observed a greater number of smooth muscle cells in the intima of PDGF B arteries at 2 to 7 days than predicted from proliferation indexes, suggesting that migration may account for the increase in intimal cell number. Medial cells near the internal elastic lamina express hpAP and PDGF BB after gene transfer, and these PDGF BB cells are proliferating, as shown in double-labeling studies. However, regardless of the methods of statistical analysis, strong statements regarding mechanism cannot be made on the basis of correlation. Unfortunately, current methodology places some limitations on mechanistic studies, and inferential conclusions must be drawn.
The PDGF B/c-sis gene has been identified as the human homologue of the v-sis gene that was expressed in porcine arteries in this study. v-sis is a 271–amino acid protein encoded by an open reading frame within the simian sarcoma virus genome. Residues 1 through 51 are derived from the env portions of the open reading frame, whereas residues 52 through 271 are highly homologous (96%) to the PDGF B/c-sis gene product.12 13 The primers used for PCR analysis were specific for v-sis sequence and not porcine PDGF, and hence, it is unlikely that endogenous porcine PDGF was detected in the PCR. However, although we cannot exclude the possibility that expression of v-sis indirectly induced expression of porcine PDGF B/c-sis, we know of no data to suggest direct cross-induction between v-sis and c-sis. Hence, the findings are attributable to v-sis expression, although we cannot exclude coexpression of endogenous porcine c-sis.
Cell proliferation in the intima declined after 7 days, while the intima continued to expand. Synthesis of extracellular matrix protein was observed between 1 and 3 weeks and is likely to contribute to continued growth of the intima. Cell proliferation decreased despite continued expression of the recombinant PDGF B gene, suggesting endogenous regulation of gene expression. It is possible that extracellular matrix or other factors within the artery may negatively regulate smooth muscle cell proliferation, including TGF-β or other cofactors, and that these factors may counterbalance any direct effects of the PDGF B transgene on intimal smooth muscle cells. Few inflammatory cells, including macrophages and T cells, were observed in PDGF B arteries. These findings suggest that PDGF B gene expression, in the absence of balloon injury or hyperlipidemia, is not associated with recruitment of macrophages and T cells. In addition, it is unlikely that macrophages contributed a majority of PDGF BB in these nonatherosclerotic arteries. Taken together, these studies suggest a direct role for PDGF in the regulation of smooth muscle cell growth and repair in porcine arteries and provide a model in which PDGF ligand and receptor antagonists can be tested to dissect how the different forms of PDGF function in vivo.
Selected Abbreviations and Acronyms
|DOTMA/DOPE||=||N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium chloride/dioleoyl phosphatidylethanolamine|
|hpAP||=||human placental alkaline phosphatase|
|PCR||=||polymerase chain reaction|
|PDGF||=||platelet-derived growth factor|
|TGF||=||transforming growth factor|
|vWf||=||von Willebrand factor|
This work was supported by National Institutes of Health grants HL-43757 (Dr E. Nabel), HL-42119 (Dr D. Gordon), and CA-59327 (Dr G. Nabel) and a Grant-in-Aid from the American Heart Association (Dr D. Gordon). Dr E. Nabel is an Established Investigator of the American Heart Association. We gratefully acknowledge the technical assistance of Lingling Xu and manuscript preparation by Gail Reisdorph.
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