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(Circulation. 1997;96:1240-1249.)
© 1997 American Heart Association, Inc.

Articles

Expression and Localization of Platelet-Derived Growth Factor Ligand and Receptor Protein During Acute and Chronic Rejection of Rat Cardiac Allografts

Karl B. Lemström, MD, PhD; ; Petri K. Koskinen, MD, PhD

From the Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Finland.

Correspondence to Dr Karl Lemström, Transplantation Laboratory, PO Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. E-mail Karl.Lemstrom{at}Helsinki.Fi

	Abstract

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Background The molecular mechanisms of cardiac allograft vasculopathy (CAV) remain largely unknown. Using rat cardiac allografts, we examined by immunohistochemistry the expression and localization of platelet-derived growth factor ligand (PDGF-AA and -BB) and receptor (R and Rß) proteins during acute and chronic rejection.

Methods and Results In acute rejection, a prominent induction of both PDGF ligand and receptor proteins occurred in the interstitial mononuclear inflammatory cells (P<.05), most of which were ED1-immunoreactive. PDGF-Rß was also induced in the capillary endothelium (P<.01). In cardiac allografts with severe intimal thickening, PDGF-AA expression was localized to the media and intima, whereas PDGF-BB expression was less prominent and was detected mainly in interstitial ED1-immunoreactive inflammatory cells. Double staining revealed that intimal cells expressing PDGF-AA were -smooth muscle actin–positive but also -smooth muscle actin–negative myofibroblast-like cells and to a lesser extent, ED1-immunoreactive cells. Both PDGF-R and -Rß expression occurred in intimal, arterial endothelial, and interstitial mononuclear inflammatory cells. High-dose cyclosporin A (CsA) treatment significantly reduced both PDGF-AA and PDGF-R expression in intimal cells. Furthermore, linear regression analysis revealed that PDGF-AA, PDGF-R, and PDGF-Rß expression in intimal cells and PDGF-BB expression in interstitial mononuclear inflammatory cells correlated with intimal thickening.

Conclusions Alloimmune injury induces the expression of PDGF ligands, especially of PDGF-AA, in the graft vasculature and sufficient immunosuppression with CsA suppresses the expression of PDGF and inhibits the development of CAV. PDGF may have a substantial role in the regulation of smooth muscle cell migration and proliferation in an autocrine or paracrine manner during the development of CAV.

Key Words: transplantation • immunohistochemistry • arteriosclerosis

	Introduction

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Cardiac allograft vasculopathy has emerged as a major factor affecting graft survival in the long run.¹ We hypothesize that immune response induces persistent, low-grade damage to the vascular endothelium, which, in turn, induces vascular wall cells to secrete growth factors to repair the damage.^{2 3} This results in SMC replication in the vascular wall, migration of the SMCs from the media into the intima, and generation of an arteriosclerotic lesion throughout the entire length of the vessel wall. Studies of human and experimental ordinary atherosclerotic lesions have suggested a regulatory role for PDGF (reviewed in Reference 4⁴ ). The ligand consists of a disulfide-linked dimer of two polypeptides, the PDGF-A and PDGF-B chains, and can be expressed in the form of homodimers (PDGF-AA or -BB) or a heterodimer (PDGF-AB).⁴ It has been shown that SMCs⁵ and fibroblasts⁶ synthesize only the PDGF-A chain, whereas endothelial cells^{7 8} and macrophages⁹ synthesize both chains. Two separate PDGF receptors (PDGF-R and -Rß) have been identified (reviewed in Reference 10¹⁰ ). These receptors exist as monomers on the cell surface, but signal transduction by PDGF requires receptor dimerization.¹¹ PDGF-Rß binds only the PDGF-B chain, whereas PDGF-R binds both A and B chains.

We previously demonstrated an inverse correlation between mean CsA levels and intimal thickening of epicardial and intramyocardial arterioles.¹² In addition, these occluded epicardial arteries significantly expressed P-selectin and VCAM-1 on the endothelium and were linked with perivascular inflammation of W3/25⁺ helper T cells and OX42⁺ macrophages, suggesting the possible role of delayed-type hypersensitivity reaction–like response in this process.^{12 13} Here, we investigated the effect of alloimmune response and CsA immunosuppression on the expression of PDGF ligand and receptor proteins in the development of CAV in rat cardiac allografts. We made an attempt to quantify and identify cells that stained positively for PDGF ligand and receptor. Syngeneic and allogeneic grafts were harvested 5 days after transplantation in the acute rejection model.¹⁴ In the chronic rejection model, in which the grafts were removed at 3 months, allograft recipients were treated with conventional triple-drug immunosuppression and three different dosages of CsA to dissect the effect of CsA on PDGF ligand and receptor expression in the development of CAV.¹²

	Methods

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Intra-Abdominal Heterotopic Cardiac Transplantations
Inbred DA (AG-B4, RT1^a) and WF (AG-B2, RT1^u) rat strains (200 to 300 g) (Laboratory Animal Center, University of Helsinki, Finland) were used, and transplantation procedures were performed as described.^{12 14 15} Some of the cardiac samples used in this study were originally procured in previous experimental sets.^{12 14}

Immunosuppressive Regimens
In the acute rejection model, transplant recipients were left nonimmunosuppressed, whereas in the chronic rejection model, they received triple-drug immunosuppression. Perioperatively, the rats received CsA (Sandimmun; Sandoz Pharma AG) 15 mg/kg SC as a single dose. For the injection, 50 mg/mL of CsA infusion substance was dissolved in Intralipid 200 mg/mL (KabiVitrum) at a final concentration of 3 mg/mL. Thereafter, CsA (Sandimmun mixture 100 mg/mL, Sandoz) at a dose of either 5, 10, or 20 mg·kg^-1·d^-1 was given with regular rat food. Methylprednisolone 0.5 mg·kg^-1·d^-1 (Solu-Medrol 40 mg/mL; Upjohn) and azathioprine 2 mg·kg^-1·d^-1 (Imuran; Wellcome) were administered in drinking water.¹²

Histology
At least two midsections of the allografts were fixed in 10% phosphate-buffered formalin overnight, routinely processed, and embedded in paraffin. Cross sections of cardiac allografts 4 µm thick were stained with Mayer's hematoxylin and eosin for general evaluation, with Masson's trichrome for fibrosis, and with Weigert–van Gieson stain for elastin. The slides were examined by light microscopy by two observers in a blind review, and the score assigned was determined by consensus of the observers. In this study, only epicardial arteries and intramyocardial arterioles were evaluated for histological changes attributable to chronic rejection. The changes in intimal thickness were scored as mild (score 1; <25% occlusion of the lumen) when the intima was readily discernible, moderate (score 2; 25% to 50% occlusion), and severe (score 3; >50% occlusion) when the lumen was encroached upon.¹² As the final score for intimal thickening, the mean score of epicardial arteries and intramyocardial arterioles is given.

Single Immunostaining
Serial frozen sections (4 to 6 µm) were cut, air-dried onto silane-coated slides, fixed in acetone for 20 minutes at –20°C, and stored at –20°C until used. Before immunostaining, the slides were refixed with chloroform and then air-dried. After incubation with 1.5% nonimmune goat serum (S-1000; Vector Laboratories), frozen sections of cardiac allografts were incubated with a primary antibody diluted in PBS with 1% BSA and 3% goat serum at +4°C for 12 hours. With intervening washes in Tris-buffered saline, the following steps were performed: biotinylated goat anti-rabbit antibody with 3% rat serum at RT for 30 minutes; avidin–biotinylated horseradish complex (Vectastain Elite ABC Kit, Vector Laboratories) in PBS at RT for 30 minutes; then the reaction was revealed by chromogen 3-amino-9-ethylcarbazole (Sigma) containing 0.1% hydrogen peroxidase, yielding a brown-red reaction product. The specimens were counterstained with hematoxylin, and coverslips were aquamounted (Aquamount; BDH Ltd).

The following primary affinity-purified rabbit polyclonal antibodies were used: an IgG antibody to human recombinant PDGF-AA, recognizing human and rat PDGF-AA (a dilution of 6.7 µg/mL; ZP-214, Genzyme); an IgG antibody to recombinant human PDGF-BB, recognizing human and rat PDGF-BB (a dilution of 10 µg/mL; ZP-215, Genzyme); an IgG antibody raised against a peptide corresponding to amino acids 1065 to 1084 mapping at the carboxy terminus of PDGF-R of human origin, reacting with mouse, rat, and human PDGF-R (a dilution of 0.5 µg/mL; sc-338, Santa Cruz Biotechnology); and an IgG antibody raised against a peptide corresponding to amino acids 1082 to 1101 mapping at the carboxy terminus of PDGF-Rß of human origin, reacting with mouse, rat, and human PDGF-Rß (a dilution of 0.5 µg/mL; sc-339, Santa Cruz Biotechnology).

Specificity controls were performed with the same Ig concentration of species- and isotype-matched antibodies: mouse monoclonal IgG1 antibody (X931, Dako) and rabbit polyclonal Ig fraction (X936, Dako) for monoclonal and polyclonal antibodies, respectively. Additional control for the specificity of PDGF-AA (recombinant human PDGF-AA homodimer, Genzyme), PDGF-BB (recombinant human PDGF-BB homodimer, Genzyme), PDGF-R (control peptide, Santa Cruz), and PDGF-Rß (control peptide, Santa Cruz) staining involved the use of a working dilution of the affinity-purified polyclonal antibody after overnight incubation with a 20-fold molar excess of corresponding peptide antigen.

Double Immunostaining
To identify which cell types expressed PDGF ligands and receptors, double immunohistochemistry was applied on representative frozen sections. After staining for PDGF ligands or receptors with the peroxidase ABC method described above, cardiac frozen sections were washed in Tris-buffered saline, and avidin-biotin complex from the first step was blocked by incubation of the sections with an excess of avidin and biotin at RT for 2x15 minutes (Avidin/Biotin Blocking Kit, SP-2001, Vector Laboratories). After application of primary antibody at +4°C for 30 minutes, the following steps were performed: biotinylated horse anti-mouse antibody with 3% rat serum for monoclonals (AK-5002; Vector Laboratories) or biotinylated goat anti-rabbit antibody with 3% rat serum for polyclonals (AK-5001; Vector Laboratories) at RT for 30 minutes; and alkaline phosphatase avidin-biotin complex in PBS at RT for 30 minutes; then the reaction was visualized by Vector blue in 100 mmol/L Tris-HCl, pH 8.2 (Alkaline Phosphatase Substrate Kit, SK-5300, Vector Laboratories). Sections were counterstained with hematoxylin. Double-stained cells showed mixtures of brown-red (PDGF ligand or receptor) and blue (cell subsets) tones. The following antibodies were applied: W3/25 (Sera Lab), a mouse IgG1 antibody to rat T helper cells (CD4 equivalent); ED-1 (Serotec), an IgG1 antibody to rat monocytes and macrophages; a mouse IgG2a antibody to -smooth muscle actin (Biomakor); and a rabbit anti-human antibody to von Willebrand factor (A082, Dako A/S).

Quantification of Immunostaining
The blinded analysis was done semiquantitatively by scoring the intensity of staining from 0 to 3 as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate intensity with multifocal staining; and 3, intense diffuse staining of the cells analyzed.

Statistical Analyses
All data are given as mean±SEM. A nonparametric test was chosen because of small sample sizes and inability to determine whether the samples were normally distributed.¹⁶ Total variation between the groups was analyzed by Kruskal-Wallis one-way analysis (Statview 512+, BrainPower Inc). The rank sums obtained by the Kruskal-Wallis test were used for the Dunn test at the significance level of 5% or 1% (Medstat, Astra Group A/S) to find out which of the groups differed significantly from the others. In addition, linear regression analysis was applied to evaluate the possible relation of growth factor ligand and receptor expression to intimal thickening.¹⁷ Values of P<.05 were regarded as statistically significant.

	Results

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The specificity controls of immunohistochemical stainings included omission of primary antibody, substitution of the primary antibody with nonimmune mouse or rabbit serum, and preabsorption of the primary antibody with the corresponding peptide antigen. None of these controls showed any immunoreactivity (see Fig 2, left).

View larger version (134K): [in this window] [in a new window]   Figure 2. Single immunostaining for PDGF ligand and receptor protein expression in chronically rejecting rat cardiac allograft arteries 3 months after transplantation. Left, To verify specificity of immunohistochemical staining, primary antibody was preabsorbed with corresponding peptide antigen, and staining showed no immunoreactivity for (A) PDGF-AA, (C) PDGF-BB, (E) PDGF-R, and (G) PDGF-Rß. Right, In allografts with severe intimal thickening, (B) strong and moderate PDGF-AA expression was seen in media and intimal cells of arteries, respectively. Expression of (D) PDGF-BB, (F) PDGF-R, and (H) PDGF-Rß was mild. Vessel lumen is indicated by asterisk, and elastic lamina interna by dotted line. Mayer's hematoxylin counterstaining. Original magnification x40 (left) and x100 (right).

PDGF-AA Ligand Expression
As shown in Table 1, mild PDGF-AA expression was localized to the capillary endothelium and media cells of arteries in nontransplanted DA hearts (Fig 1A). In syngeneic grafts, some PDGF-AA was induced in the arterial endothelium. Acute rejection induced significant expression of PDGF-AA in the interstitial mononuclear inflammatory cells (P<.05) compared with syngeneic grafts (Fig 1C). Most of these inflammatory cells were ED-1 immunoreactive (not shown). In allografts with a severe form of chronic rejection (CsA 5 mg/kg) recorded as intense intimal thickening of the vessels, the intensity of PDGF-AA expression was strong in the media cells of arteries, moderate in intimal cells (Fig 2B), and mild in interstitial mononuclear cells, cardiomyocytes, and capillary endothelium (Table 2). High-dose CsA (20 mg·kg^-1·d^-1) significantly inhibited PDGF-AA expression in the media (P<.05) and intima (P<.01) of these vessels compared with CsA at a dose of 5 mg·kg^-1·d^-1. Linear regression analysis (Fig 3; Table 3) revealed that in long-surviving cardiac allografts, at 3 months, there was a clear correlation between intimal thickening and the expression of PDGF-AA in the media layer of arteries (r=.831; P<.001) and in intimal cells (r=.735; P<.001). As shown in Fig 4 (A through D), the cells expressing PDGF-AA in the dense (spindle-shape SMCs; probably these SMCs have stopped proliferating) arteriosclerotic lesions were almost exclusively -SMC actin–immunoreactive (Fig 4D), whereas the PDGF-AA–positive cells in the loose (round SMCs; probably proliferating) arteriosclerotic lesions were infrequently detected by -SMC (Fig 4A and 4B) or ED1 (Fig 4C) antibodies.

View this table: [in this window] [in a new window]   Table 1. PDGF Ligand and Receptor Protein Expression in Acute Cardiac Allograft Rejection in the Rat

View larger version (165K): [in this window] [in a new window]   Figure 1. Single immunoperoxidase staining for (A to C) PDGF-AA, (D to F) PDGF-BB, (G to I) PDGF-R, and (J to L) PDGF-Rß protein expression in nontransplanted DA hearts (left), in syngeneic hearts (middle), and in acutely rejecting cardiac allografts (right) 5 days after transplantation. In nontransplanted hearts, (A) PDGF-AA and (J) PDGF-Rß were constitutively expressed in media of arteries. B, E, H, and K, In syngeneic controls, PDGF protein and receptor expression was almost nonexistent. C, F, I, and L, In acutely rejecting cardiac allografts, a clear induction of both PDGF ligand and receptor proteins could be found in interstitial inflammatory infiltrate. Arrows indicate examples of positive immunoreactivity. Mayer's hematoxylin counterstaining. Original magnification x100.

View this table: [in this window] [in a new window]   Table 2. Effect of Cyclosporin A–Based Triple-Drug Immunosuppression on PDGF Ligand and Receptor Protein Expression in Long-term Surviving Cardiac Allografts 3 Months After Transplantation

View larger version (24K): [in this window] [in a new window]   Figure 3. Correlation of intimal thickening with PDGF-AA (left) and PDGF-BB (right) protein expression in arterial wall of chronically rejecting cardiac allografts as analyzed by linear regression analysis. Correlation coefficients (r) and P values are given. Intimal thickness was semiquantitatively scored from 0 to 3: 1, <25% of lumen occluded; 2, luminal occlusion 25% to 50%; and 3, >50% of lumen occluded. Immunohistochemistry was also scored from 0 to 3.

View this table: [in this window] [in a new window]   Table 3. Correlation of Intimal Thickening With PDGF Ligand and Receptor Protein Expression in Interstitial Space of Cardiac Allografts 3 Months After Transplantation

View larger version (103K): [in this window] [in a new window]   Figure 4. Facing page. Double immunostaining (arrows) with immunoperoxidase (brown-red) and alkaline phosphatase (blue) technique for PDGF ligand and receptor proteins and cell type identification markers in cardiac allografts, respectively. A, PDGF-AA/-SMC actin double-positive cells in media and inner intima layer. Cells in loose (round SMCs; probably proliferating) arteriosclerotic lesions were infrequently detected by (B) -SMCs or (C) ED1 antibodies, whereas cells in late, dense arteriosclerotic lesions were (D) PDGF-AA/-SMC actin double positive. (E) PDGF-BB/ED-1 double-positive cells were observed in interstitial inflammatory infiltrates. Examples of PDGF-R/-SMC actin (F), PDGF-R/ED-1 (G), PDGF-Rß/-SMC actin (H), and PDGF-Rß/ED-1 (I) double-positive cells in allograft arteries. J, Staining with -SMC actin (blue) and PDGF-Rß (red) demonstrating that luminal endothelial lining is PDGF-Rß–immunoreactive. Mayer's hematoxylin counterstaining. Original magnification x100.

PDGF-BB Ligand Expression
No PDGF-BB expression was recorded either in nontransplanted DA hearts or in syngeneic grafts (Fig 1D and 1E; Table 1). During acute rejection, mild PDGF-BB induction could be demonstrated in the interstitial mononuclear inflammatory cells (P<.05) (Fig 1F), most of which were ED1-immunoreactive (Fig 4E). In cardiac allografts with a severe form of CAV (CsA 5 mg/kg), PDGF-BB expression was mild in the interstitial mononuclear cells (Table 2), whereas higher doses of CsA reduced it (P=NS). According to linear regression analysis (Table 3), intimal thickening significantly correlated with PDGF-BB expression in interstitial mononuclear cells (r=.515; P<.05). In addition, some PDGF-BB expression was found in the media and intimal cells of arteries with moderate to severe intimal thickening, but there was no correlation with intimal thickening (Figs 2D and 3).

PDGF-R Expression
PDGF-R expression was nonexistent in nontransplanted DA hearts and in syngeneic grafts (Fig 1G and 1H; Table 1). In acute rejection, PDGF-R was mildly induced in the interstitial mononuclear cells (P<.05) and in the capillary endothelium (P=NS) (Fig 1I). In cardiac allografts with severe intimal thickening (CsA 5 mg/kg), mild PDGF-R expression was observed in the intima, in interstitial mononuclear cells, and on the arterial endothelium, whereas only a trace was seen in the media cells of arteries, in cardiomyocytes, and on the capillary endothelium (Fig 2F, Table 2). In the groups with mild (CsA 10 mg/kg) and nonexistent intimal thickening (CsA 20 mg/kg), the arterial endothelial PDGF-R expression was totally abolished by CsA (P<.05). As demonstrated by linear regression analysis (Fig 5), PDGF-R immunoreactivity in the intima of arteries (r=.795; P<.001) and in the endothelium of arteries (r=.648; P<.01) correlated with enhanced intimal thickening. Double staining demonstrated that most of the SMC-like cells expressing PDGF-R cannot be detected by -SMC actin (Fig 4F), ED1 (Fig 4G), W3/25 (not shown), or von Willebrand factor (not shown), suggesting that proliferating SMCs do not express -SMC actin.

View larger version (24K): [in this window] [in a new window]   Figure 5. Correlation of intimal thickening with PDGF -receptor (left) and ß-receptor (right) protein expression in arterial wall of cardiac allografts as analyzed by linear regression analysis. For explanations, see Fig 3.

PDGF-Rß Expression
Nontransplanted DA hearts expressed PDGF-Rß in the media cells of arteries and on the capillary endothelium (Fig 1J; Table 1). In syngeneic grafts, PDGF-Rß expression was nonexistent (Fig 1K). During acute rejection, PDGF-Rß was induced on the endothelium of arteries (P=NS), in the interstitial mononuclear inflammatory cells (P<.05), in the perivascular inflammatory cells (P=NS), and in the capillary endothelial cells (P<.01) (Fig 1L). In cardiac allografts with severe CAV, PDGF-Rß expression was mild in the perivascular mononuclear cells, in intimal cells (Fig 2H), and in the capillary endothelium, whereas it was moderate on the arterial endothelium (Fig 4H and 4J) and in interstitial mononuclear cells. Increases in CsA dose decreased PDGF-Rß expression in the intimal and mononuclear inflammatory cells and on the capillary and arterial endothelium (P=NS). Furthermore, double staining revealed that the PDGF-Rß–expressing cells in the intima were also -SMC actin–immunoreactive (Fig 4H) and ED1-immunoreactive (Fig 4I). As analyzed by linear regression (Fig 5; Table 3), a significant correlation between intimal thickening and the expression of PDGF-Rß in the intimal cells (r=.753; P<.001) and on the arterial (r=.686; P<.01) and capillary endothelium (r=.502; P<.05) was observed.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of the present study demonstrate that during alloimmune injury, arterial SMCs expressed elevated levels of PDGF proteins, especially of PDGF-AA in the media and intima. In acutely rejecting cardiac allografts, significant induction of PDGF ligands and receptors occurred in the interstitial ED1-immunoreactive cells. In chronically rejecting cardiac allografts, PDGF-AA, PDGF-R, and PDGF-Rß expression in intimal cells as well as PDGF-BB expression in interstitial mononuclear inflammatory cells correlated with the development of intimal thickening. Some PDGF-BB expression was seen in the media and intimal cells of arteries, but there was no clear relation to intimal thickening. This study also shows that high-dose CsA treatment was concomitantly related to a significant reduction in PDGF-AA and PDGF-R expression in intimal cells and intimal thickening compared with low-dose CsA treatment, in which intimal thickening was maximal.

Double staining for PDGF-AA and cell identification markers demonstrated a different pattern of -SMC actin expression in the dense late arteriosclerotic lesions (spindle-shaped myofibroblast-like cells, which have probably stopped proliferating) compared with the loose ongoing lesions (round myofibroblast-like cells, which probably are proliferating). In the loose lesions, PDGF-AA–expressing cells were infrequently detected by -SMC actin and ED1 antibodies. To the contrary, PDGF-AA–positive cells in the dense arteriosclerotic lesions almost exclusively expressed -SMC actin. Double-staining for PDGF-R and different cell types demonstrated a pattern of expression similar to that seen with PDGF-AA. This is consistent with earlier findings demonstrating an inverse correlation between SMC proliferation and -SMC actin synthesis.^{18 19} In addition, it has been shown that PDGF reduces SMC -actin synthesis, although it induces cell proliferation.^{20 21}

The expression and localization patterns of PDGF in arteriosclerotic lesions are not quite clear, possibly because the specimens used in several studies have most often been obtained from end-stage arteriosclerotic lesions. The synthesis of clinical studies indicates that most PDGF-A is expressed in mesenchymal cells (SMCs and fibroblasts), whereas PDGF-B is detected in macrophages and endothelial cells.^{22 23 24 25} To the best of our knowledge, there is only one thorough experimental study on PDGF ligand and receptor mRNA expression in a rat carotid denudation model demonstrating elevated levels of PDGF-A chain and PDGF-Rß expression in the intima, whereas PDGF-R transcripts were increased mainly in the media. No clearly positive cells for PDGF-B could be found.²⁶ The results of the present study at protein levels are consistent with the results described above and suggest a more important role for PDGF-AA in the development of arteriosclerotic changes in CAV in vivo, although in vitro studies show that PDGF-BB is a more potent mitogen for SMCs than is PDGF-AA.²⁷

A recent in vitro study showed that activation of PDGF-R leads to SMC proliferation but inhibits SMC migration, whereas activation of PDGF-Rß stimulates both the proliferative and migratory responses of SMCs.²⁸ Studies with a rat carotid denudation model and direct recombinant PDGF gene transfer into uninjured rat carotid arteries have established the biological role of PDGF in the generation of atherosclerotic lesions, but there is no clear evidence to show which PDGF ligands and receptors are key regulatory molecules in this process in vivo.^{29 30 31 32}

Although the role of PDGF in the development of ordinary atherosclerotic lesions has been well examined, less is known about its possible impact on the generation of CAV.^{33 34 35 36 37} On the basis of our previous results, we have hypothesized that continuous low-grade immunological damage and inflammatory response may be related to the release of cytokines and growth factors from these cells, resulting in the migration of SMCs from the media and their proliferation in the intima followed by intimal thickening.² Activated macrophages are capable of producing PDGF.^{25 38 39} Macrophages may penetrate into the subendothelium of arteries, and this may be facilitated by expression of adhesion molecules such as P-selectin and VCAM-1, as we have shown to occur in chronically rejected cardiac allografts.^{12 13} Conversely, T cells are not able to produce PDGF, but they may induce secretion of PDGF-like protein from endothelial cells.⁴⁰ Furthermore, cytokines such as transforming growth factor-ß, tumor necrosis factor-, and interleukin-1 released by macrophages and T cells may induce autocrine stimulation of SMCs by PDGF-AA.^{41 42 43} This autocrine stimulation may also operate in cardiac allografts, because intragraft levels of these cytokines are all increased in chronically rejecting cardiac allografts (References 13, 44, and 45^{13 44 45} and unpublished observations). Although PDGF-AA per se, compared with PDGF-BB, is a weak mitogen for SMC proliferation in vitro, we believe that PDGF-AA plays a major role in the development of intimal lesions, possibly in combination with other growth factors (PDGF-BB), serving as a permissive agent or mitogenic cofactor for full mitogenic effects, as suggested by others as well.⁴⁶

Study Limitations
There are certain limitations to this and previous studies by others aiming to localize PDGF ligand and receptor expression by immunohistochemistry, in situ hybridization, or polymerase chain reaction, because these methods are not quantitative and do not give the protein concentrations in the vessel wall, ie, they do not reveal the biological function of mediators investigated. The statistical analysis may also be misleading. The correlation of PDGF ligand and receptor expression to intimal thickening was analyzed by linear regression analysis, which shows the probability that the two phenomena are coincidental but does not mean "cause and effect." Therefore, further studies using specific PDGF receptor blockers or PDGF ligand and receptor antisense oligonucleotides will be needed to evaluate the biological role of mediators in the development of ordinary atherosclerosis and chronic rejection.

Because the patterns of anti-PDGF ligand and receptor immunostainings were completely blocked by an excess of recombinant ligand and receptor proteins and there was no immunostaining when the same Ig concentration of species- and isotype-matched nonimmune antibodies was used, we believe that our results represent a true pattern of this growth factor ligand and receptor expression. Our results are strongly suggestive for the role of PDGF ligand and receptor expression, especially of PDGF-AA, in the development of chronic rejection in rat cardiac allografts, although the association does not permit us to determine the biological function of PDGF in lesion formation. Certainly, a possibility exists that the staining intensity between PDGF-AA and PDGF-BB might be due to differences in the affinities and titers of the antibodies applied in this study.

	Selected Abbreviations and Acronyms

 

CAV = cardiac allograft vasculopathy CsA = cyclosporin A PDGF = platelet-derived growth factor RT = room temperature SMC = smooth muscle cell VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This work was supported by grants from the Finnish Foundation for Cardiovascular Research, Helsinki University Central Hospital, Technology Development Center, and University of Helsinki, Finland. The authors are grateful to Maria Sandström, RN, Tuula Lahtinen, RN, and Eriika Wasenius, RN, for their excellent technical assistance.

Received December 9, 1996; revision received February 13, 1997; accepted February 20, 1997.

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