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Eur J Cardiothorac Surg 2004;25:91-97
© 2004 Elsevier Science NL
a Department of Cardiac Surgery, University of Heidelberg, INF IIO, 69120 Heidelberg, Germany
b Pathology Institute, University of Heidelberg, INF IIO, 69120 Heidelberg, Germany
c Department of Cardiology, University of Heidelberg, INF IIO, 69120 Heidelberg, Germany
d Department of Anaesthesiology, University of Heidelberg, INF IIO, 69120 Heidelberg, Germany
Received 28 April 2003; received in revised form 18 September 2003; accepted 23 September 2003.
* Corresponding author. Tel.: +49-6221-436-651; fax: +49-6221-436-651
e-mail: falk-udo.sack{at}urz.uni-heidelberg.de
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Abstract |
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 Top Abstract 1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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grade 3A. Both, infections and rejections were detectable after the second postoperative week. The expression of PDGF AA/BB and PDGF receptors 
/ß was examined immunohistochemically. The infiltrating cells were characterised by using monoclonal antibodies against CD3, CD4, CD8, CD57 and CD68. Results: Only endothelial cells revealed a relevant expression of PDGF ligand and receptor. Prior to implantation there was no or only weak reactivity of single cells for all PDGF factors. One week after HTx a significantly increased immunoreactivity of all PDGF factors was observed in all groups. Two weeks after HTx the expression of PDGF AA in the infection group and the expression of all four PDGF factors in the rejection group remained significantly elevated. In contrast, in the group with an uneventful course there was no statistical difference in the expression of all the four PDGF factors. Compared with the uneventful group, there were significantly more CD3+ cells in the infection and rejection group at all three time points. Two weeks after HTx, the rejection group showed a significantly elevated number of CD3+ cells compared to the values before implantation. Two weeks after HTx there were significant more CD68+ cells in the infection and rejection group compared with before implantation. Conclusions: One week after HTx the peritransplant injury predominantly influences the endothelial expression of PDGF ligand and receptor. In the first postoperative week, expression of PDGF could be detected. The persistance of evaluated PDGF expression might be of prognostic value in terms of a risk for either infection or rejection. These patients should be carefully monitored.
Key Words: Heart transplantation • Platelet-derived growth factor • Ligand • Receptor
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1. Introduction |
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TopAbstract1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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and -ß) were identified [reviewed in Ref. 7]. These receptors are localised on the cell surface as monomeres [7]. For signal transduction a dimerisation is essential. These receptors bind the ligands with different affinity. Receptor binds all three isoforms (AA, AB, BB), ß binds PDGF AA as well as PDGF AB, while receptor ßß binds only PDGF BB [4]. The extracellular matrix is influenced by PDGF through stimulation of the collagen synthesis and the control of the collagenase gene expression [8]. PDGF has a strong mitogenic and chemotactic effect on smooth muscle cells and on fibroblasts and is involved in the development of arteriosclerosis and CAV [9]. An increased expression of PDGF and its receptors was demonstrated in acute rejection after heart or kidney transplantation [10]. Ischemic and reperfusion initiate a multifactorial cascade that leads to changes so that quiesent endothelium become activated. Activated endothelial cells express proinflammatory properties that include the induction of adhesion molecules and growth factors [11]. No systematic examination of the PDGF expression in the first 2 weeks after human HTx has been performed up to now. Therefore it was the aim of this study to clarify the effect of peritransplant injury and postoperative complications on the expression of the PDGF ligand and receptor during the first 2 weeks after HTx.
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2. Patient population, materials and methods |
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TopAbstract1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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The differentiation into three groups was performed retrospectively on the basis of different clinical courses during the first year after transplantation: (a) 18 patients with a clinical uneventful course without infections and rejections >grade 2; (b) 11 patients with histologically, immunohistochemically and/or serologically verified cardiac or systemic infections; and (c) 17 patients who had at least one rejection episode grade 3A in the first postoperative year (first rejection 108.8±106.7 (SD) days after transplantation, range: 23–358 days). The resulting groups were assigned as ‘uncomplicated course’, ‘later infections’ and ‘later rejections’. Routine follow up was the same in all three groups, including endomyocardial biopsies once a week for the first month, every 2 weeks during the 2nd postoperative month and monthly until the 6th postoperative months. The patient were seen routinely in our transplant ambulance with physical examination, echocardiography and laboratory testing in regards to infection parameters. At 1 year following HTx, another endomyocardial biopsy was performed.
All patients received the same immunosupressive treatment during the first two postoperative weeks. For induction therapy, anti-thymocyte globuline (IMTIX-Sangstat) was i.v. administered in a dosage of 1.5 mg/kg per bwt starting at day 1. By means of flow-cytometry, T-lymphocyte subpopulations were determined daily. With a total lymphocyte count below 100 cells per µl, no further ATG was given. Additional immunosuppression consists of a triple therapy with methyl prednisolone, azathioprine and cyclosporine A. Cyclosporine A was adjusted to whole blood levels between 200 and 280 ng/ml during the first year. Azathioprine was given in a dosage of 0.5–1.0 mg per kg/bwt. Steroid were tapered to a maintenance dosage of 10 mg per day for the first postoperative year.
2.2. Myocardial and endomyocardial biopsies from cardiac allografts
A total of 114 biopsies from 46 heart transplant recipients (mean age: 52.2±12.1 (SD) years old, range: 15–70 years) were investigated. All allografts were perfused with cardioplegia solution CustodiolTM (Dr F. Köhler Chemie, GmbH, Gresbade-Hahnlein/Germany) before being harvested. Right ventricular myocardial biopsies (trabecula, harvested with scissors) from the allograft were obtained immediately before implantation. Endomyocardial routine surveillance biopsies were obtained 1 and 2 weeks after HTx using Konno bioptomes. The samples were immediately fixed in Tissue TekTM using liquid nitrogen.
2.3. Immunohistochemistry
The samples were cut (3 µm), put on slides, air dried, fixed in acetone for 10 min and stored at 70°C until use. Before immunostaining the slides were refixed with acetone and air dried. All steps were performed at room temperature. After incubation with bovine serum albumin (20 mg/ml) and 
-globulin (1 mg/ml) for 15 min, the sections were incubated with a primary antibody in a humid chamber for 1 h. After washing with Tris buffered saline (TBS) (0.15 M NaCl; 0.05 M Tris) and incubation with goat serum (diluted with TBS) for 15 min, incubation with biotinylated goat anti-rabbit or mouse antibody (DAKO, Hamburg, Germany) followed for 30 min (humid chamber). After a further washing with TBS, the cells were incubated with streptavidin alkaline phosphatase complex (Bio Genex, San Remo, CA, USA) (30 min, humid chamber), and stained with Fast Red (DAKO) yielding a red reaction product. The specimens were counterstained with hematoxylin. The following primary antibodies, diluted with antibody diluent (Zymed, San Francisco, CA, USA) were used: polyclonal rabbit-antibodies PDGF AA (ZP-214, Genzyme, Cambridge, MA, USA); PDGF BB (ZP-215, Genzyme), PDGFR- (sc-338, Santa Cruz Biotechnology). PDGFR-ß (sc-339, Santa Cruz Biotechnology) and monoclonal mouse antibodies CD3, 4, 8, 57, and 68 (DAKO). To establish the antibodies, cryosections from transmural myocardial samples and from an arteriosclerotically altered coronary artery of an explanted heart were used. Each series of immunohistochemical investigation was accompanied by positive and negative controls. For positive controls the same samples were used as for establishing. For negative controls antibody diluent (Zymed) was used instead of primary antibody.
2.4. Quantification of immunostaining
The blind analysis was done by two investigators separately from each other using a semiquantitative score for PDGF staining. The intensity of staining was classified from 0 to 3: 0, no visible staining; 1, weak staining of occasional cells; 2, moderate intensity, multifocal; and 3, strong intensity in more than 75% of the cells analysed. The respective infiltrating cells were counted and then referred to the morphometrically determine section area.
2.5. Statistical analysis
All data are given as mean±SEM if not indicated otherwise. To test for statistical differences within one group the ‘Friedman Test‘ was applied followed by the ‘Wilcoxon matched pairs signed rank test‘. Values of P<0.05 were regarded as statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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and -ß was mainly confined to endothelial cells (EC) (Figs. 1A,B)
. In the few arteries sectioned no or only a weak expression of the four factors examined was found in smooth muscle cells of the media. No or only a slight cardiomyocyte expression was found for the four factors examined.
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3.3. PDGFR- expression
Before implantation there was no or only weak staining in single EC (Fig. 2C). One week after HTx the PDGFR- expression of EC showed a significant increase in all groups compared to the values before implantation: (group with uneventful course: P<0.01; infection group: P<0.016; rejection group: P<0.005). Two weeks after HTx the PDGFR- expression in EC decreased in the infection group to values not significantly different from the values before implantation. In the rejection group the expression in EC was still significantly (P<0.005) increased compared to the values before implantation.
3.4. PDGFR-ß expression
Before implantation there was only a weak intensity in the PDGFR-ß expression in EC (Fig. 2D) in all three groups. One week after HTx the PDGFR-ß expression in EC increased significantly in all groups compared to the values before implantation: (group with uneventful course: P<0.017; infection group: P<0.02; rejection group: P<0.002). Two weeks after HTx the PDGFR-ß expression in EC remained significantly increased only in the rejection group compared to the values before implantation (P<0.003).
3.5. Infiltrating cells
In the uneventful, infection and rejection group there were only a few CD4, 8, 57+ cells at all three time points investigated. Before and 1 week after HTx there were only a low number of CD3+ cells in the infection and rejection group. Two weeks after HTx the number of CD3+ cells was only significantly elevated in the rejection group compared to before implantation. At all three time points there were significantly (P<0.002, respectively) more infiltrating CD3+ cells in the infection and rejection group compared to the uneventful group. Before implantation the number of CD68+ cells was in all three groups equally low. One week after HTx the number of CD68+ cells increased significantly in the infection and rejection group compared to the values before implantation (P<0.008 and P<0.03). Two weeks after HTx the number of CD68+ cells was still significantly increased in the infection and rejection group compared to before implantation (P<0.03, respectively). In the infection group there was significantly more CD68+ cells in the first and second week after HTx than in the uneventful and rejection group.
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4. Discussion |
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TopAbstract1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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Before implantation PDGF AA was expressed only in some specimens in a few endothelial cells (Fig. 1). This result corresponds to the fact that no or only a weak expression of PDGF AA could be found in normal non-transplanted hearts [2,17]. During the first week there was a significant increase of the endothelial PDGF AA expression in all groups. This is consistent with earlier findings of other authors demonstrating an increased expression of PDGF AA shortly after HTx compared with non-transplanted hearts [2,17]. In this study only a slight cardiomyocyte PDGF AA expression could be detected, whereas an increased PDGF AA expression in cardiomyoctes was shown by other authors. One possible reason could be that in their study no time course was investigated, they pooled samples obtained 1 week up to a few months after transplantation.
In contrast to earlier investigations, our study demonstrated no PDGFR- in the biopsies before implantation in non-transplanted hearts [10]. Our study revealed a slight endothelial PDGFR- expression before implantation in the groups with complications. This discrepancy could be explained by changes in the donor heart, induced by brain death and intensive care resulting in cell stimulation. In the present study there was a significant increase of the PDGFR- expression 1 week after HTx. This finding is concordant with the result of another study [17].
In a previous study PDGF BB could not be found in non-transplanted hearts [10]. In this study only a trace of endothelial PDGF BB expression was seen before implantation in the infection group. In the first week after cardiac transplantation PDGF BB expression was significantly increased in all groups, but at a lower level compared with PDGF AA. Other authors also demonstrated a lower increase of PDGF BB compared with PDGF AA in transplanted hearts [10]. A recent study showed that the PDGF BB expression was increased shortly after HTx in endothelial cells [14], which were stimulated in vitro with mononuclear cells from transplant patients. In endomyocardial biopsies from patients with rejections <grade 2 a slight PDGF BB expression could be found in endothelial cells [18]. This expression increased during severe rejections. PDGF BB could be detected in infiltrating monocytes, in renal arteries and smooth muscle cells of the intima and media with and without rejection [10]. In contrast to these findings another study did not find PDGF BB either in normal hearts or in allografts [17].
In this study PDGFR-ß was already slightly visible in the specimens before transplantation in all groups. This is consistent with earlier findings demonstrating PDGFR-ß expression in non-transplanted hearts [10]. We demonstrated that in the first week the expression of PDGFR-ß increased significantly in all groups examined. In the second week only in the rejection group the expression remained significantly increased.
In our study we realised a dramatic increase in the expression of PDGF and its receptors in the first week after HTx, although there were no signs of rejection or infection at this time. It is well-known that ischemia and reperfusion lead to endothelial injury and activation [19]. The activated endothelial cells in man secrete for example basic fibroblast growth factor (bFGF), macrophage chemoattractant protein-I, interferon (IFN)-, interleukin (IL)-6 and PDGF [20]. We could also identify a uniform moderate bFGF expression in all groups at all the time points examined (unpublished results). Furthermore an increased expression of adhesion molecules and HLA II molecules following HTx was shown [21]. This causes an myocardial infiltration with mononuclear cells [20]. We could demonstrate an increase in CD68 positive interstitial cells in the biopsies from the first week in all groups. The number of these cells decreased in the second week in the normal group and remained increased only in the groups with complicated course (unpublished results). Furthermore, the endothelial cells expressed CD40 in allografts after HTx [22], an important costimulating molecule for lymphocytes. The antigen presentation mentioned above and the IL-1-production by infiltrating macrophages, lead to an activation and clonal expansion of T helper cells. The release of IL-2 and other cytokines by activated T helper cells caused recruitment, activation and clonal expansion of T cytotoxic cells, T suppressor cells, natural killer cells and B cells. Owing to the increased antigen presentation HLA II molecules on endothelial cells T helper cells are enhanced and activated. These activated T helper cells produce cytokines (e.g. IL-1, IFN-), which in turn lead to an increased expression of adhesion molecules. This results in an amplification of the infiltration of immunocompetent cells. Furthermore, the T cells force a stimulation of PDGF synthesis in endothelial cells [23]. IFN- leads to an activation of macrophages, which then produce an increased amount of cytokines (IL-6, IL-1, tumour necrosis factor (TNF)- and PDGF). The proliferative and mitogenic effect of many of these cytokines (e.g. IL-1, TNF-) appear to be due to PDGF induction [24]. During the second week after HTx the expression of all examined factors decreased in the normal and infection group (except PDGF AA in the infection group). This may reflect a different vulnerability of endothelial cells to peritransplant injury, which should generally exert reduced effects in the second week. In the rejection group the expression of all factors examined remained significantly increased after 2 weeks. We assume that further immunological/inflammatory processes persist, which permanently activate endothelial and interstitial cells. These activated cells express PDGF ligand and receptor increasingly.
In summary, enhanced upregulation of PDGF ligand and receptor early after human cardiac transplantation persists only in the group of patients presenting rejection episodes >grade 3A in the first year after transplantation. We propose that a permanent increased expression of PDGF ligand and receptor in human cardiac allografts shortly after transplantation may indicate patients with a elevated risk for later acute rejections. These patients need a intensive surveillance whereas the time intervals for surveillance biopsies might be prolonged in patients without this risk. Furthermore, besides the early identification of patients with an elevated risk for later rejections, the expression of PDGF ligand and receptor might be an early marker for the efficacy of new immunosuppressive regimens.
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Footnotes |
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References |
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TopAbstract1. Introduction2. Patient population, materials...3. Results4. DiscussionReferences |
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in human cardiac allografts. Transplantation 1995;59:605.[Medline]
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=P<0.01). (A) Endothelial PDGF AA expression; (B) endothelial PDGF BB expression; (C) endothelial PDGFR-