| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 2006;81:1372-1378
© 2006 The Society of Thoracic Surgeons
a Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany
b Institute of Pathology, University of Heidelberg, Heidelberg, Germany
c Department of Cardiology, University of Heidelberg, Heidelberg, Germany
d Institute of Biostatistics, University of Heidelberg, Heidelberg, Germany
Accepted for publication October 31, 2005.
* Address correspondence to Dr Koch, Department of Cardiac Surgery, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany (Email: achim_koch{at}med.uni-heidelberg.de).
 |
Abstract |
|---|
 Top Abstract IntroductionPatients and MethodsResultsCommentReferences |
|---|
METHODS: Right ventricular endomyocardial biopsies were obtained from 29 heart transplant recipients before implantation, after one and two weeks, and after one, two, and three years after heart transplant, and immediately frozen in liquid nitrogen. The growth factor expression was examined immunohistochemically.
RESULTS: The PDGFs were mainly expressed in vascular structures and they were less pronounced in cardiomyocytes. Especially after the first week, a significant increase was found in the expression of PDGF A and B as well as PDGF-receptors 
and ß. In the yearly biopsies, PDGF expression was rarely found. The bFGF expression was merely weak in the later period three years after transplant and the aFGF was only expressed in the early phase. A comparison of recipients with short and long ischemic time did not reveal any significant differences in the intensity of expression.
CONCLUSIONS: The increased expression of PDGF and FGF in the first postoperative week can be interpreted as an unspecific reaction to peritransplant injury. This might be related to important reparative, angioprotective, and wound-healing processes shortly after the heart transplant had taken place. The weak expression in the later period appears to be linked to a stable transplant function and a direct influence by the immunosuppressive therapy.
|
Introduction |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsCommentReferences |
|---|
Growth factors belong to a group of polypeptides that enhance cell proliferation by binding to specific membrane receptors. Apart from the induction of cell proliferation they have a significant influence on cell differentiation and chemotaxis, and they also play a part in inflammatory and immunologic reactions.
Platelet-derived growth factor was first isolated in 1974 from human thrombocytes and its function is to stimulate the proliferation of fibroblasts and smooth muscle cells. The PDGF is a cationic, hydrophilic protein with a molecular weight of 30.000 Dalton. Two polypeptide chains are cross-linked by disulfide-bridges. So far, four different PDGF-chains have been described: PDGF-A, PDGF-B, PDGF-C, and PDGF-D [1, 2]. These chains can form five isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. The A and B chains have a 60% homology and are coded by two different genes on chromosome 7 and 22. All isoforms are synthetized as inactive progenitor molecules, which are activated by limited proteolysis after cellular secretion [3].
The PDGF can be produced in fibroblasts, endothelial cells, and smooth muscle cells. It has strong chemotactic effects on fibroblast and smooth muscle cells and can stimulate matrix production in connective tissue cells [4]. There is a substantial body of evidence for its involvement in the development of fibrosis, atherosclerosis, and immunologic processes after solid organ transplantation [5].
The two different PDGF receptors (PDGFR) are both tyrosine-kinases. The -receptor binds to PDGF A-, B-, and C-chains, whereas the ß-receptor shows an affinity to the B- and D-chains. After PDGF binds to the receptor, the receptor-ligand-complex activates different proteins (eg, protein kinase C) and certain growth-associated genes as c-Myc. Both receptor subunits are strongly mitogenic [1]. Human fibroblast chemotaxis is only stimulated by the ß-receptor; the -receptor seems to have inhibitory effects. The mitogenic activity depends on the isoform and the number of receptor subunits on the cell surface. However, an increased PDGFR expression was found in the vascular smooth muscle cells in heart transplants [6].
The fibroblast growth factors have the common ability to bind heparin, therefore they are sometimes also referred to as heparin-binding growth factors. The members of the FGF family regulate growth, proliferation, differentiation, migration, and survival in a variety of cells. The main representatives of the FGF family are the acidic FGF (aFGF) and the basic FGF (bFGF). In cardiac tissues FGF are also synthetized in mast cells. Presently, at least four different FGF receptors have been characterized. The central biologic effect of FGF is an influence on cell differentiation and proliferation of fibroblasts and smooth muscle cells. The bFGF especially induces synthesis of extracellular matrix and has a role in the organization of capillary endothelial cells. However, FGF seems to influence atherosclerosis and accelerated transplant vasculopathy [5, 7–10].
The objective of the present study is therefore to describe the expression of PDGF A, PDGF B, PDGFR-, PDGFR-ß, and aFGF and bFGF in right ventricular myocardial biopsies over a period of three years in a collective of consecutive heart transplant recipients. The study poses the following question: Are there any differences in the growth factor expression in the postoperative specimen and the biopsies obtained immediately after explantation? Furthermore, the issue concerning the influence of different lengths of ischemic times on the expression of growth factors will be raised.
|
Patients and Methods |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsCommentReferences |
|---|
The patients were divided into two subcollectives: one with a short ischemic time defined as below 170 minutes and the other with an ischemic time above 180 minutes. A recipient was not included in the presence of an acute rejection episode (above grade 2 according to the grading system of the International Society for Heart and Lung Transplantation). The overall mean ischemic time was 181 (range, 77 to 272, median 183) minutes. The mean ischemic time in the short ischemia group was 133 (range, 77 to 167, median 150) minutes and in the long ischemia group was 220 (range, 182 to 272, median 224) minutes.
In the total collective 26 recipients were male and 3 female. The mean age was 45 (range, 9 to 65, median 54) years. The mean height was 171 (range, 118 to 200, median 172) cm and the mean weight was 68 (range, 19 to 103, median 70) kg. The underlying cardiac disease was a dilative cardiomyopathy in 16 recipients, an ischemic cardiomyopathy in 10, and a transposition of the great arteries, a tetralogy of Fallot, and a double outlet right ventricle with one case each.
The donor collective consisted of 11 female and 18 male donors of a mean age of 31 (range, 10 to 55, median 32) years. Their mean height was 171 (range, 140 to 186, median 175) cm and weight was 70 (range, 25 to 95, median 72) kg. The causes of death was cranial injury in 15 cases, intracranial bleeding in 6, a basilaris thrombosis in 2 cases, and a subarachnoidal bleeding, a carbon monoxide intoxication, a thalamus tumor, a stroke, a gun shot in the head, and a status asthmaticus with one case each.
All patients had an intravenous induction therapy with antithymocyte globulin (ATG; IMTIX-Sangstat, Lyon, France) at a dosage of 1.5 mg/kg body weight from postoperative day 1 to day 7. The additional immunosuppression consisted of a triple therapy with cyclosporine A (adjusted to blood levels of 200 to 280 ng/mL during the first year), azathioprine (0.5–1.0 mg per kg bwt) and methylprednisolone. Prednisolone was tapered to a maintenance dosage of 10 mg/day for the first postoperative year.
All allografts were harvested after they had been cardioplegically perfused with Custodiol (Dr. F. Köhler Chemie GmbH, Alsbach-Hähnlein, Germany). Right ventricular trabecules were obtained with scissors immediately before implantation of the donor heart. These specimens served as pretransplant normal controls. For this study, scheduled routine endomyocardial right ventricular biopsies were taken weekly for the first two weeks and then yearly for three years after transplantation using a Konno-biotome. All samples were embedded in a drop of Tissue-Tek (Tissue-Tek , Sakura, Zoeterwoude, Netherlands) frozen in liquid nitrogen for further processing. Special emphasis was placed on the avoidance of freezing artifacts. Further processing was carried out in the freezing-cut-technique. The samples were cut using a freezing-microtome (Frigocut, Reichert-Jung, Nußloch, Germany) at a temperature of -20°C. Each sample was evaluated carefully by light microscopy for artifacts, which would have rendered them useless for further processing. The samples were fixed in acetone and stored for immunohistochemical staining at -70°C in an area impermeable to light.
The aim of the immunohistochemical staining was the proof of PDGF and FGF antigens. The staining was carried out in the indirect biotine-streptavidine method in a humid chamber at room temperature. Each series of immunohistochemical investigations was accompanied by positive and negative controls. After fixation in acetone, the frozen cuts were washed in phosphate buffered saline (PBS) and blocked by application of albumin/
-globulin (-venin; Leukon, Vienna, Austria) for 15 minutes. Afterward, the samples were incubated with the primary antibody for 1 hour, washed twice in PBS solution, blocked with goat-donkey serum for 30 minutes, incubated in a biotin-marked goat anti-rabbit respective donkey anti-goat secondary antibody (30 minutes at room temperature) and washed again twice in PBS. Next the samples were incubated in alkaline phosphatase-marked streptavidin (Streptavidin AP, BioGenex, San Ramon, CA) for 30 minutes, washed twice in PBS, and stained in Fast-Red (DAKO, Carpinteria, CA) for 10-25 minutes. The cell nuclei were counterstained in Mayer's hemalaun (Merck, Darmstadt, Germany) for 3 minutes, rinsed in water, and covered with Glyergel (DAKO).
The following primary antibodies were used: PDGF polyclonal rabbit antibodies, PDGF A (Santa Cruz Biotechnology, Santo Cruz, CA), PDGF B (Oncogene Research, San Diego, CA), PDGF R- and PDGF R-ß (Santa Cruz Biotechnologies), and polyclonal goat antibodies aFGF, bFGF (Santa Cruz Biotechnologies). Biotin goat anti-Rabbit IgG (ZYMED, San Francisco, CA) and Biotin-long spacer (SP)-conjugated Affini-pure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) served as secondary antibodies.
A condition for quantitative analysis is a random sampling; the samples were harvested, embedded, and cut at random. The analysis of the samples was carried out blinded by two different investigators, who separately employed a semiquantitative scoring system. The intensity of staining was classified in single steps from 0 to 3: 0, negative, no visible staining; 1, antigen weakly positive; 2, antigen positive; 3, antigen strongly positive.
The data are given as mean value, median, minimal, maximal values, and standard error of the mean as appropriate. To test for statistical differences in the semiquantitative groups, the Dixon and Mood test was used and for quantitative values the Friedman test was used. To test between the groups, the Kruskal-Wallis test, followed by the Wilcoxon (Mann-Whitney) U test, were applied. For the correlation between the PDGF expression at different biopsy time points, the Spearman correlation coefficient (r) was used. The p values less than 0.05 were regarded as statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsCommentReferences |
|---|
|
Expression of PDGFR-
All three groups showed only a weak expression of PDGFR- before implantation followed by a significant increase during postoperative week one. During week two, values decreased to remain elevated compared with the initial values. The PDGFR- was not expressed during the following three years (Fig 2A).
|
and PDGFR-ß was not significantly different between the groups with short and long ischemic times (Fig 2B).
Expression of aFGF
The expression of aFGF was on a weak level in all three groups at any point in time with no significant differences (Fig 3A).
|
|
Comment |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsCommentReferences |
|---|
Apart from the development of fibrosis and hypertrophy the long-term prognosis after heart transplant is further influenced by the extent of accelerated graft atherosclerosis. The influence of growth hormones appears to be of significant importance in these processes [12].
The expression of PDGF was examined by different investigators as well by immunohistochemistry in situ hybridization at different points in time after heart transplant. The research group of Sack and colleagues [13] found PDGFs (PDGF AA/ BB, PDGFR /ß) expressed after heart transplant mainly on endothelial and smooth muscle cells of the vascular system, only weakly expressed on cardiomyocytes and not expressed in the interstitial space. Compared with the biopsies before implantation they found an increased expression after one week, and a significantly elevated expression after the second week, only in a group of recipients with a high grade rejection episode. These results are contrasted by the findings of Shaddy and colleagues [14], who found an increased expression of PDGF AA/ BB and AB mainly in the interstitial space and to a lesser extent on vascular structures. However, the significance of their study is limited by the lack of a systematic comparison of the time points of the biopsies and a differentiation in the strength of PDGF expression.
The research group of Schnabel and colleagues [6] compared the expression of PDGF AA/BB and PDGFR /ß by in situ hybridization on cryofixed endomyocardial biopsies before implantation and after one, two, and five years after heart transplant. They also found only a weak expression of PDGF A and B and PDGFR-ß before implantation. One year after the operation, PDGF was expressed significantly more on endothelial cells and up to the fifth year it remained on an elevated level. The authors also showed that the expression of PDGF A/B and PDGFR-ß was highest in intramural arteries with activated endothelial cells and medial hypertrophy.
A particularly interesting study of Zhao and colleagues [15] compared the results of immunohistochemistry and in situ hybridization with data from reverse transcriptase–polymerase chain reaction (RT/PCR) in right ventricular endomyocardial biopsies. The PCR showed that PDGF A was expressed in the majority of the biopsies after heart transplant whereas there was no expression in the biopsies of donor hearts before the beginning of the organ harvest. These results were concordant with the findings in immunohistochemistry and in situ hybridization, which located the PDGF A expression mainly in vascular structures.
Summarizing the immunohistochemical data of Sack and colleagues [13], Schnabel and colleagues [6], and Zhao and colleagues [15], the expression pattern of PDGF after heart transplantation seems to be mainly located in the vasculature; this finding was confirmed by the present study.
The significantly increased PDGF expression one week after heart transplant was predictable: PDGF has strong chemotactic characteristics on fibroblasts, smooth muscle cells, leukocytes, and monocytes, thus it plays a key role in inflammatory reactions shortly after heart transplantation. These characteristics influence three basic processes in wound healing: the migration of neutrophils, monocytes, and fibroblasts, the activation of macrophages and fibroblasts with consecutive de novo synthesis of cytokines, growth factors, and extracellular matrix proteins, and the reorganization of the interstitial collagen. The exact mechanisms for the secretion of PDGF after heart transplant are discussed controversially: ischemia and reperfusion appear to activate endothelial cells to secrete cytokines and growth factors (IL-2, INF-, and PDGF) [16].
During the second postoperative week the expression of all PDGFs remained on an elevated level. This finding also corresponds with the results of other authors [7, 13, 14] and can be interpreted as a further presence of immunologic processes in the transplanted organ.
The expression of PDGF shortly after heart transplantation is therefore well-described; however, another interesting finding of this study is the absence of the systematic expression of PDGF in the long-term follow-up. Only in a small number of biopsies was an expression of PDGF A and PDGFR-ß visible (Table 1). Compared with the extent of PDGF expression in the first two weeks after heart transplantation, the regression gains statistical significance for all PDGFs and receptors examined (Table 1). The low grade of PDGF expression in this study might be related to the recipient collective, which showed a clinically uneventful postoperative course with a low incidence of rejection episodes. In combination with chronic rejection, Schnabel and colleagues [6] described an increased expression of PDGFs on the protein and mRNA levels one year after heart transplantation in the neighborhood of intramural small arteries and areas with perivascular fibrosis.
|
Fibroblast Growth Factor (FGF)
In regard to the immunohistochemic expression, FGF is not solely stored intracellularly and in the extracellular matrix but is also present in higher amounts in endothelial and smooth muscle cells. This phenomenon is caused by the high affinity of FGF to heparan-sulfate proteoglycan. This is exactly the pattern in which FGF a and b were distributed in the present study. Shaddy and colleagues [14] and Zhao and colleagues [15] also found FGF expressed predominantly in vascular structures.
However, FGF has a strong mitogenic effect on fibroblasts, cardiomyocytes, endothelial cells, and smooth muscle cells (Table 2). They are important mediators of angiogenesis, myointimal proliferation, and repair processes in transplanted hearts. In an animal infarct model, aFGF and bFGF reduced infarct size and increased the number of small arterioles and capillaries [18–20]. The BFGF was shown to be a potent vasodilator mainly by release of NO and through a specific FGF-receptor (18). The processes inducing FGF production in transplanted hearts have not yet been completely examined, but a potential role of ischemia, reperfusion, and transplantation itself is discussed.
|
Summarizing, the results of the present study demonstrate that the right ventricular myocardial expression of PDGF A/B and their receptors /ß differed quantitatively and qualitatively in early and late periods after heart transplant. The ascertained high expression of all four proteins in the first week is related to important reparative processes, especially of vascular structures. The PDGF A/B also participate in this adaptation process during the second week. These results are in accordance with the findings of other authors. In a patient collective with a clinically uneventful further course, PDGF does not appear to play any important role in the long-term processes in the first three years after heart transplant. For further analysis the influence PDGF expression on rejection rate and early transplant vasculopathy or the inhibitory effect of immunosuppression as cyclosporine A on PDGF and FGF expression should be investigated in larger collectives.
|
References |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsCommentReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
