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(Circulation Research. 1995;76:951-957.)
© 1995 American Heart Association, Inc.

Articles

A Subpopulation of Smooth Muscle Cells in Injured Rat Arteries Expresses Platelet-Derived Growth Factor–B Chain mRNA

Volkhard Lindner, Cecilia M. Giachelli, Stephen M. Schwartz, Michael A. Reidy

From the University of Washington, Department of Pathology, Seattle.

Correspondence to Volkhard Lindner, MD, PhD, University of Washington, Department of Pathology SJ-60, Seattle, WA 98195.

	Abstract

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Abstract Proliferation of smooth muscle cells (SMCs) and formation of a neointima are characteristics of the response of rat carotid arteries to balloon injury. Rat platelet-derived growth factor (PDGF)-B was cloned, thus allowing us to use species-specific probes to carry out in situ hybridization on the surface of injured arteries. A distinct population of luminal SMCs (7% to 10%) in the developing neointima expressed PDGF-B mRNA, but very few luminal SMCs still expressed PDGF-B (0.5%) when the lesion had stopped growing. Primary SMC cultures revealed expression of PDGF-B mRNA in 1.6% of SMCs derived from normal tunica media and in 11% of SMCs derived from the neointima. These data demonstrate that SMCs in the injured vessel wall are heterogeneous with regard to PDGF-B expression and that subculturing of these cells may give rise to cultures that are either positive or negative for PDGF-B expression. Furthermore, with abundant expression of the PDGF receptor ß-subunit expressed by intimal SMCs, our findings provide evidence that PDGF-B synthesized by these cells may be involved in intimal lesion formation via a paracrine or autocrine mechanism.

Key Words: platelet-derived growth factor • intima • migration • platelet-derived growth factor receptor • in situ hybridization

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The concept that platelet-derived growth factor (PDGF) plays a role in formation of the neointima after balloon catheter injury¹ was derived originally from studies showing that PDGF is a mitogen and chemoattractant for smooth muscle cells (SMCs) in culture.² This idea was modified with the discovery that PDGF-B is only a weak mitogen, at least in the rat, for SMCs in vivo.^{3 4} Nonetheless, in vivo studies did show that infused PDGF-BB can act as a chemotactic factor, stimulating the migration of medial cells across the internal elastic lamina and increasing the extent of neointimal formation after injury.^{3 4} These data, however, do not rule out a replicative role for PDGF-BB, which might be acting as a comitogen with other growth factors. A role for PDGF-B in intimal thickening was also demonstrated by Nabel et al,⁵ who transfected PDGF-B plasmid DNA into pig arteries and demonstrated intimal lesion formation.

In the rat, the process of neointimal formation in response to arterial balloon injury continues for several weeks,⁶ but adhesion of platelets to the denuded surface is seen only during the first few days after denudation.⁷ Thus, stimulatory effects on underlying SMCs due to PDGF released from adhering platelets may be limited to a few days after injury. Given the short half-life in circulation, any effects of PDGF on intimal lesion formation beyond this time may only be possible if stimulation of SMCs occurs via a paracrine or autocrine mechanism within the vessel wall. The possibility that such a pathway exists was initially provided by the finding of PDGF-A mRNA expression in the balloon-injured vessel wall.⁸ PDGF-A, however, is probably not a good candidate for an autocrine factor, since in vitro studies showed that PDGF-A is a poor mitogen⁹ and may even act as an inhibitor, rather than a stimulant, of migration.^{10 11} In contrast, PDGF–B chain is usually a good mitogen in vitro,² and the PDGF receptor ß-subunit, which can only respond to binding of the PDGF–B chain ligand, was also expressed at high levels in the intima. The levels of PDGF-B mRNA are not modulated after injury, and only low expression was detectable by Northern analysis in RNA extracted from the vessel wall.⁸ In addition, attempts to localize the cells expressing PDGF-B mRNA by in situ hybridization of vessel cross sections with a probe for mouse PDGF-B were unsuccessful. In summary, infused PDGF-B appears to be chemotactic rather than mitogenic, and evidence for an autocrine/paracrine loop in neointimal formation was still missing, since we did not see local expression of PDGF-B.

Evidence for a possible autocrine role of PDGF-B, however, did exist in vitro when SMCs from the neointima were grown in culture in which PDGF–B chain mRNA levels increased with passage number.¹² Expression of the same transcript in cultures derived from normal tunica media, however, was either absent or detectable only after many passages were performed.¹² This observation suggests that SMCs expressing PDGF-B mRNA represent a subpopulation with differences in phenotype that could account for many of the unusual properties of the neointima.

The inability to identify cells expressing PDGF–B chain in vivo raises concerns that the unique properties found in cultured intimal cells^{12 13} could be reflective of culture conditions rather than the properties of these cells in their original tissue environment. Thus, the present report sought to reexamine cells expressing PDGF-B chain by a new method for in situ hybridization using an en face technique. This method allowed us to demonstrate that a small population of SMCs in the neointima of injured rat arteries expresses PDGF-B and that this proportion, though small, is elevated during the period of most marked formation of the neointima. We will discuss the possibility that these in vivo PDGF-B–producing cells could play a key role in forming the neointima and that the appearance of PDGF–B chain in cultured neointimal cells may represent an amplification of a preexisting subpopulation found in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arterial Injury Model
Male Sprague-Dawley rats (400 g, 3 to 4 months old, Bantin & Kingman, Edmonds, Wash) were used in all the experiments. All surgical procedures were carried out with the animals under general anesthesia (2.2 mg/kg body wt IP xylazine [AnaSed, Lloyd Laboratories] and 50 mg/kg body wt IP ketamine [Ketaset, Aveco Co, Inc]). Right and left carotid arteries were denuded with a 2F Fogarty balloon catheter as previously described.⁶ These vessels were used to study SMCs that had migrated onto the denuded surface from the underlying tunica media 4 days after denudation, where they formed an intimal lesion over the course of several weeks. This model allowed us to study luminal SMCs at early times after injury, when cells were replicating (up to 2 weeks after injury), and at late times, when proliferation had stopped (6 weeks). Deendothelialized segments of arteries were identified by intravenous injection of Evans blue (0.3 mL of 5% solution in saline) 10 minutes before the animals were killed. All animals were perfusion-fixed with phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde. For in situ hybridization and immunostaining, rats were killed at the indicated times after injury (between 4 days and 6 weeks). Denuded carotid arteries were divided into three segments, each measuring 8 to 10 mm in length. This allowed tissue to be used from the same animal for in situ hybridization with different probes as well as for immunostaining. For each time point, segments were also obtained for embedding in paraffin to carry out in situ hybridization on cross sections.

For colocalization of replicating and PDGF-B–expressing SMCs on the luminal surface of an 8-day neointima, 3 rats were injected intraperitoneally with a single dose of 5-bromo-2'-deoxyuridine (BrdU, 25 mg/kg, Boehringer Mannheim) 1 hour before death. Immunostaining on en face preparations with an antibody against BrdU was carried out as previously described.¹⁴ Replication of luminal SMCs was expressed as a percentage of all luminal SMCs.

Cell Culture
Two independent isolations of neointimal SMCs from carotid arteries injured 2 weeks earlier and medial SMCs from uninjured carotid arteries were grown in Waymouth MB 752/1 medium (GIBCO/BRL) supplemented with 10% bovine serum (Hyclone Laboratories Inc) as previously described.¹⁵ Cells were grown in primary cultures on chamber slides (Lab Tek, Nunc, Inc) until they were confluent, 10 days after plating. The cells were then stored in phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde until they were processed for in situ hybridization. Colocalization of PDGF-B mRNA expression and DNA synthesis was carried out in previously characterized rat SMC cell lines,¹⁶ of which one did (WKY12-22) and one did not (WKY3M-22) express PDGF-B mRNA. These cells were grown to confluence on chamber slides using Waymouth MB 752/1 medium and 10% bovine serum and were then switched to serum-free medium. Twenty-four hours later, the cells were restimulated with 10% bovine serum. One hour before fixing the cells with phosphate (0.1 mol/L, pH 7.4)–buffered 4% paraformaldehyde at 0, 6, 12, and 24 hours after serum stimulation, the cells were pulsed with BrdU (30 µmol/L). Immunostaining for BrdU was carried out as previously described.¹⁴

DNA Probes and Cloning of Rat PDGF-B
A cDNA library from cultured rat newborn aortic SMCs¹⁷ was screened for sequences related to the human PDGF-B cDNA clone, pSM-1,¹⁸ by using the hybridization conditions previously described.¹⁷ Twenty cDNA clones remained positive after three rounds of selection, with the largest clone (3-4a) containing a 3-kb insert representing a nearly full-length clone. The putative translated region was sequenced by the dideoxynucleotide chain termination method (Sequenase, USB), and each sequence was confirmed by sequencing the complementary strand. Sequence analysis was performed by using PCGENE sequencing software (Intelligenetics). Multiple alignments were performed by using the CLUSTAL program. Other DNA probes used for in situ hybridization were a 0.6-kb Pst I fragment of the extracellular domain of the rat PDGF receptor ß-subunit.¹⁹

In Situ Hybridization and Immunostaining
In situ hybridization and immunostaining were carried out on cross sections and on en face preparations of vessel segments as recently described.¹⁴ After hybridization the slides were coated with autoradiographic emulsion (Kodak, NTB2), exposed for 3 weeks, and then developed (Kodak, D-19). Preparations were observed under the light microscope by using dark-field, bright-field, and a combination of epiluminescence and bright-field illumination (reflective light).

A monoclonal antibody recognizing rat macrophages and monocytes (ED1, Bioproducts for Science Inc) and smooth muscle -actin (HHF-35, a generous gift from Dr A. Gown) was used to identify macrophages and SMCs on en face preparations. A staining protocol was followed as previously described.^{20 21}

Quantification of PDGF-B–Positive SMCs
The nonspecific background level of hybridization was determined in two ways: (1) by hybridizing specimens with labeled sense probes and (2) by hybridizing adult medial SMC cultures that were known not to express PDGF-B mRNA by Northern analysis. In almost all cases these negative controls showed fewer than five grains per cell. PDGF-B–expressing cells were easily identified after hybridization with the antisense probe by the presence of an abundance of silver grains, which made grain counts almost impossible. The distribution of silver grains was therefore considered to be bimodal. A total of 56 arterial segments derived from 28 different animals were studied, of which 21 and 35 were hybridized with the sense and antisense probes, respectively. Analysis of SMCs in primary cultures was carried out in an identical fashion. Twenty-three fields for medial SMC cultures and 39 fields for intimal SMCs cultures were analyzed at x400 magnification. Statistical analysis on the in vivo data was performed by using Fisher's test for multiple comparisons, and the t test (unpaired, two tailed) was used to compare cultured medial and intimal SMCs.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of Rat PDGF-B cDNA
A neonatal rat SMC library was screened with a human cDNA clone for PDGF-B (pSM-1).¹⁸ Of the 20 clones identified in this manner, the largest one, 3-4a, was chosen for sequence analysis. As shown in Fig 1, the translated region of clone 3-4a showed high homology to the published amino acid sequences for porcine, human, and feline PDGF-B.^{18 22 23} The degree of homology with murine PDGF-B was 94% at the nucleic acid level.²⁴ When used for Northern blot analysis, 3-4a detected an 3.5-kb transcript in cultured rat pup SMCs that was indistinguishable from the band detected by pSM-1 (not shown). Thus, 3-4a most likely represents the rat homologue of PDGF-B.

View larger version (33K): [in this window] [in a new window]   Figure 1. A, Nucleotide and translated amino acid sequence of mature rat platelet-derived growth factor (PDGF)-B obtained from sequence analysis of clone 3-4a. B, Multiple alignment of mature PDGF-B amino acid sequence from rats, cats,23 and humans.22 The asterisks (*) and dots (.) indicate that positions are either perfectly or well conserved, respectively. Sequence data from this article have been deposited with the EMBL/GenBank libraries under accession No. 40991 (pending).

Expression of PDGF-B mRNA in Injured Arteries
The normal rat carotid artery only rarely contains intimal SMCs. Removal of the endothelium from the carotid artery induces proliferation of the underlying medial SMCs, with migration of SMCs into the intima a few days later.⁶ These neointimal cells can remain uncovered by regenerating endothelium for weeks and months after injury. Thus, after denudation intimal SMCs can be studied on the luminal surface.

Previous studies using Northern analysis of total RNA extracted from rat carotid arteries demonstrated that the transcript for PDGF-B was expressed at low levels,⁸ with no change in response to balloon injury. Moreover, in situ hybridization carried out on cross sections of arteries with a murine probe⁸ failed to localize PDGF-B mRNA to specific cells. Using the labeled rat probe on cross sections, we could identify occasional cells expressing PDGF-B mRNA, and these cells were only seen on the luminal surface of the neointima (Fig 2a). To obtain more convincing evidence for the expression of PDGF-B mRNA by luminal SMCs, we used an en face approach that examined the entire cell population present on the luminal surface. With this technique, denuded carotid arteries were studied at different times after injury. A small number of SMCs on the luminal surface at 5 days after injury were found to express the mRNA for PDGF-B (Fig 2b). Occasionally, these cells were arranged in clusters suggesting either expansion from a common precursor or convergence of groups of migrating cells (Fig 2d).

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs of balloon-injured rat carotid arteries. a, In situ hybridization with a [35S]UTP-labeled antisense riboprobe for rat platelet-derived growth factor (PDGF)-B was carried out on a cross section 14 days after denudation. Arrow indicates a PDGF-B–positive luminal smooth muscle cell (SMC). b, En face preparation hybridized with the same probe 5 days after injury is shown. Note that some SMCs that have migrated onto the luminal surface express PDGF-B mRNA (arrows), whereas others are negative. Denuded areas indicate that migration of SMCs from the media into this space has not yet occurred. c, En face preparation with immunostaining for replicating SMCs after a single injection of 5-bromo-2'-deoxyuridine shows that a large number of luminal SMCs are replicating at 8 days after injury. d, Fourteen days after injury, clusters of PDGF-B–expressing SMCs are frequently found on the denuded surface. e, In situ hybridization with an antisense riboprobe for rat PDGF receptor ß-subunit 8 days after injury shows high expression levels of this receptor subunit in all SMCs. f, Hybridization with a labeled sense probe for PDGF-B (14 days after denudation) shows only background hybridization. g, In situ hybridization with labeled antisense PDGF-B probe on SMC cultures derived from normal adult aorta shows no expression (passage 10). h, Cultures from pup aorta, however, demonstrate expression of PDGF-B mRNA when hybridized with the antisense probe (passage 10). In situ hybridization is viewed under reflective light, with silver grains appearing blue. Original magnification x400.

The percentage of PDGF-B–positive cells on the luminal surface ranged between 7.1% and 10.7% and did not change significantly up to 14 days after injury (Fig 3). We also carried out in situ hybridization on luminal SMCs of mature lesions (6 weeks after denudation), in which cell replication is an infrequent event.^{6 25} In these lesions only occasional PDGF-B–positive SMCs were found (0.5%). This value was significantly lower compared with all the other time points (P<.05).

View larger version (27K): [in this window] [in a new window]   Figure 3. Bar graph showing quantitative evaluation of smooth muscle cells (SMCs) expressing platelet-derived growth factor (PDGF)-B mRNA in vivo and in primary culture. The percentage of PDGF-B–positive SMCs on the luminal surface was determined on en face preparations at the indicated time points. The percentage of PDGF-B–expressing SMCs was similar at days 5, 8, 10, and 14 but was significantly lower 6 weeks after balloon injury (P<.05). Note that the far left column represents replication of luminal SMCs 8 days after balloon injury following a single injection of 5-bromo-2'-deoxyuridine (BrdU). SMCs derived from normal tunica media and 2-week-old neointima were grown to confluence in primary culture. Significantly higher numbers of PDGF-B–expressing cells were found in the neointimal cell cultures. Data represent mean±SEM; n indicates the number of animals studied per time point. Twenty-three and 39 fields were analyzed under x400 magnification for medial and intimal SMC cultures, respectively.

We used an antibody (ED1) that recognizes rat macrophages to eliminate the possibility that the PDGF-B–expressing cells on the luminal surface were macrophages, since these cells have been reported to express PDGF-B^{26 27} under certain conditions. Macrophages were not detected on the luminal surface of injured vessels at the time points up to 2 weeks (data not shown). The identity of the cells on the luminal surface as SMCs was further verified by staining with an antibody against SMC -actin (data not shown).

In agreement with previous studies, we found high expression levels of PDGF receptor ß-subunit in all luminal SMCs at the time points studied (Fig 2e).⁸

The possibility that expression of PDGF-B mRNA is linked to cell replication prompted us to determine whether there is a correlation between PDGF-B expression and SMC replication. A single injection of BrdU was given to rats 8 days after balloon injury of the carotid artery, and the number of replicating SMCs on the denuded luminal surface was quantified with an antibody against BrdU. Unfortunately, we were unable to perform both immunostaining for BrdU and in situ hybridization on the same specimens (Fig 2c). The replication index (percentage) for luminal SMCs (Fig 3), however, was approximately four times higher than the percentage of luminal SMCs expressing PDGF-B mRNA.

Expression of PDGF-B mRNA in Primary Cultures
SMC cultures derived from normal adult media generally give rise to cultures that do not express PDGF-B mRNA, whereas cultures derived from the neointima or pup aorta do express this mRNA,^{12 13} and expression levels increase with passage number. These differences in PDGF-B mRNA expression may be the result of different levels of expression by the entire cell population or may reflect differences in the percentage of cells expressing PDGF-B mRNA in the culture. To discriminate between these two possibilities, we determined the percentage of PDGF-B–positive cells in cultures derived from 2-week-old intima and from uninjured tunica media. Intimal SMCs were isolated by enzymatic digestion from the entire intima and grown until confluent for 10 days, and 10.9% of these cultured intimal SMCs expressed PDGF-B (Fig 3). However, primary cultures obtained from the tunica media of uninjured carotid arteries showed that only 1.6% of these SMCs expressed PDGF-B, which was significantly lower (86%, P.01) than the value determined for intimal SMCs (Fig 3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cultured SMCs can synthesize PDGF–B chain mRNA and may do so at a high level. However, the ability to make PDGF-B in vitro has consistently been seen only in cultured SMCs derived from 2-week-old rats, ie, rat pups, or from the neointima of balloon-injured carotid arteries (Fig 2g and 2h).¹² In contrast to the in vitro data, however, PDGF–B chain mRNA has only been seen in the vessel wall at low levels by Northern blotting, and there was no evidence of its presence by in situ hybridization carried out on arterial sections. Thus, it seemed that either the in vitro phenomenon was a property only of cultured cells or that detection in vivo might be limited with the previous techniques used. Using an en face approach, we report that expression of PDGF-B mRNA by individual SMCs can be detected in vivo. This finding shows that PDGF-B expression by SMCs is not a phenomenon limited to SMCs in culture; instead, it demonstrates that SMCs in the vessel wall are heterogeneous with regard to PDGF-B expression.

There are several explanations as to why these PDGF-B SMCs have not been previously detected by in situ hybridization on cross sections of arteries.⁸ With the cloning of rat PDGF-B cDNA, a species-specific probe was available that remained hybridized during the high-stringency wash of the specimens, which includes a ribonuclease digestion step. Using this probe for in situ hybridization, we did indeed find very few luminal SMCs on some cross sections that expressed PDGF-B. With the en face technique used in the present study, we were able to demonstrate more convincingly that PDGF-B–positive SMCs are present on the luminal surface. Because this technique allowed us to examine the entire luminal cell population, it was possible to quantify the frequency of PDGF-B–expressing cells. To analyze a similar number of cells on cross sections would require a large number of sections; this is especially true when only very few positive cells are present, eg, at 6 weeks after denudation.

PDGF-B is one of several mRNAs that showed constitutive differences in expression, ie, higher in pup or intimal cells than in medial unmanipulated cells.^{12 17 28 29} Both the PDGF-B–expressing pup/intimal phenotype (Fig 2g) and the nonexpressing adult medial SMC phenotype are stable in culture.¹⁶ The presence of cells with these properties in vitro led us to propose that vascular SMCs within injured vessels are a heterogeneous population, containing at least two different subpopulations. PDGF-B mRNA, however, has been localized only in cultured SMCs until now. Interestingly, expression levels for PDGF-B in intimal SMCs in vitro increased with passaging until steady levels were reached after approximately six passages.¹² Our data on the percentage of PDGF-B–positive SMCs in primary cultures of medial and intimal SMCs relate to this issue and demonstrate that the PDGF-B–expressing cells in the in vivo intima give rise to the PDGF-B–producing population of cultured cells derived from intimal cells. Serial passaging of the cultured cells may change the ratio of PDGF-B–expressing cells. For medial SMC cultures with a low frequency of PDGF-B–positive cells (1.6%), one possibility is that subculturing may cause a further decrease in PDGF-B–expressing cells and that they may eventually be lost. Alternatively, after long-term subculture and expansion of the PDGF-B–expressing population, significant levels of PDGF-B mRNA may be found only after extensive subculturing. These two possibilities provide an explanation for the observations made earlier¹² : variable amounts of PDGF-B mRNA were found in higher passage numbers of medial SMCs. However, these PDGF-B–positive SMCs are less likely to be lost from cultures of intimal SMCs in which the percentage of PDGF-B–positive cells is sevenfold higher than in the corresponding medial SMC cultures. The increase in PDGF-B expression with successive passage numbers of these cells seems to suggest that the increase in the PDGF-B–positive population of SMCs might possibly be the result of a growth advantage under the culture conditions.

Another interesting question is whether the increased number of SMCs expressing PDGF-B mRNA in intimal cultures is due to a selective advantage of these cells in migrating from the media into the intima in response to balloon injury. Indeed, our recent data have shown that an infusion of PDGF-BB causes an increase in the accumulation of SMCs in the intima of injured rat carotid arteries,³ which occurs as a result of the increased migration of SMCs from the media into the intima. Thus, SMCs expressing their own PDGF-B and PDGF receptor ß-subunit may have an increased capacity to migrate, leading to a higher percentage of PDGF-B–positive SMCs in the intima. An alternative explanation is that PDGF-B–expressing SMCs have evolved as a consequence of vascular injury associated with balloon catheter denudation. The fact that we cannot detect PDGF-B mRNA expression in SMCs of the uninjured media lends support to this possibility. As a result of balloon injury, a situation is created in which SMCs in the neointima provide the luminal lining of the vessel wall. In that regard, it should be noted that PDGF-B–positive SMCs were found only on the luminal surface of denuded vessels. One difference between these luminal cells and the SMCs deeper in the intimal lesion is the fact that the luminal cells are exposed to shear stress. It is of interest that Resnick et al³⁰ have identified a fluid shear stress–responsive element in the PDGF–B chain promoter. However, it remains unclear why shear stress would induce PDGF-B mRNA only in some luminal SMCs and not in the entire luminal SMC population. In any case, PDGF-B synthesized by SMCs on the luminal surface might be stimulating intimal lesion formation via its chemotactic properties by recruiting SMCs into the intima.

It is also of interest that the number of SMCs in the intimal lesion expressing PDGF-B at 6 weeks after injury was significantly lower than at the earlier time points. From previous studies we know that the intimal lesion at these late time points is quiescent; ie, SMC replication is a rare finding, and the size of the lesion is no longer increasing.⁶ At the earlier time points (5 to 14 days), however, migration of SMCs from the media into the intima occurs, and intimal SMCs are dividing rapidly, causing the lesion to grow.^{6 14 31} Expression of PDGF-B mRNA does not appear to correlate with replication of intimal SMCs per se, since 40% of the luminal SMCs at 8 days after injury are replicating at any given time (Fig 3), yet no more than 10% of these cells express PDGF-B mRNA. Furthermore, as recently shown by Lemire et al,¹⁶ some SMC cell lines constitutively express PDGF-B mRNA (WKY12-22), whereas others do not (WKY3M-22, Fig 2f). Labeling of replicating SMCs with a 1-hour pulse of BrdU at various times after serum stimulation revealed that no more than 18% of the cells in the PDGF-B–positive cell line were replicating at any of the time points (data not shown), whereas all of the cells were expressing PDGF-B mRNA (Fig 2h). Together, these findings argue that PDGF-B mRNA is expressed only by a subpopulation of SMCs, and there appears to be a positive correlation between the time when growth of the intimal lesion occurs and the time when PDGF-B is expressed by SMCs.

The possible contribution of PDGF-B to the formation of the neointima is not clear. The predominant receptor expressed in the neointima is PDGF receptor ß-subunit,⁸ a molecule that can only bind PDGF-B. The present data demonstrate that a small portion of the cells in the intima, 10% at the surface of the neointima, express PDGF-B and could therefore serve as the source for an autocrine or paracrine agonist long after platelet release is over. It should be emphasized, however, that PDGF-BB is only a weak mitogen for SMCs in vivo³ ; therefore, the mitogenic effect of the growth factor may not be critical to the growth of the lesion. Furthermore, injection of neutralizing antibodies to PDGF did not inhibit replication of intimal SMCs, although they did inhibit lesion formation.⁴ A possible mechanism for PDGF as a stimulant of migration has recently been proposed by Yabkowitz et al,³² who demonstrated that PDGF-B–induced migration of SMCs is mediated via thrombospondin. In addition, PDGF-B has been found to induce integrins in vascular SMCs that are thought to be important in SMC migration,^{33 34 35} including ß₁ and ß₃ integrins.

Our data demonstrate the heterogeneity of vascular SMCs in injured arteries in vivo with regard to PDGF-B mRNA expression. Furthermore, they provide the basis for a potential PDGF autocrine/paracrine loop in neointimal formation.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-03174, HL-41103, and HL-42270.

Received March 8, 1994; accepted February 24, 1995.

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