© 2006 European Society of Cardiology
Granulocyte Macrophage-Colony Stimulating Factor receptor expression on human cardiomyocytes from end-stage heart failure patients
a Department of Cellular and Molecular Biology and Pathology "L. Califano", "Federico II" University, Via S. Pansini 5, Ed. 19, 80131 Naples, Italy
b Department of Bio-Morphological and Functional Sciences, University "Federico II" Naples Italy
c Department of Cardiothoracic and Respiratory Sciences, Second University of Naples Italy
d Department of Experimental and Clinical Medicine, University "Federico II" Naples Italy
e Inter-University Centre for Cardiovascular Research (G.I.M.E.C.), Second University of Naples Italy
* Corresponding author. Tel.: +39 081 7463016; fax: +39 081 7701016. E-mail address: lorposti{at}unina.it
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Background: In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death.
The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors.
Aims: To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy.
Methods and results: GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays.
Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues.
Conclusions: We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.
Key Words: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) • GM-CSF receptor • Cardiac regeneration • Ischaemic and dilated cardiomyopathy
Received March 16, 2005; Revised July 20, 2005; Accepted December 7, 2005
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Heart failure is a leading cause of morbidity and mortality in developed countries [1]. The progression of heart failure (HF) is characterized by left ventricular remodelling, mainly represented by left ventricle (LV) dilation and dysfunction with reduced ejection fraction (EF). In remodelling ventricles, the progression of HF is associated with structural changes involving the extra-cellular matrix (ECM) [2] and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death [3].
The role of wall stress and neurohormonal activity [4] in modifying ventricular remodelling is well established, but the mechanisms are not completely understood [5].
Recent studies in animal models as well as in adult human hearts have shown that myocytes can proliferate before reaching their final size and that their proliferation may be stimulated and modulated by several factors [6,7]. These observations seem to contradict the dogma that myocytes withdraw permanently from the cell cycle and are destined to die without any further replication, showing the ability of these cells to proliferate and develop new cardiac tissue [8,9].
Myocyte proliferation seems to be modulated by neoangiogenesis and by a remodelling of the ECM that interacts with cardiac cells. Growth factors and cytokines may play a central role by controlling both neoangiogenesis and ECM deposition.
Anversa et al. [10] recently demonstrated that the repair of cardiac and vascular tissues by stem cells, resident in the myocardium, is a natural process after injury. In patients with acute myocardial infarction, mononuclear cells (CD34+) and plasma levels of endothelial growth factors are also significantly increased [11]. ECM proteins and proteolytic enzymes (e.g. matrix metalloproteinase) could facilitate cell mobilization, while adhesion molecules expressed at the site of injury could mediate the attachment to endothelial cells [12].
The first part of the process appears to be stem cell mobilization. Chemokines, stem cells factor and Granulocyte-Colony Stimulating Factor (G-CSF), as well as other cytokines, could stimulate this process. In fact, in an animal model with transient ischaemia and nonlarge infarction, G-CSF has beneficial effects, accelerating absorption of necrotic tissue by macrophages and reducing granulation and scar tissue via the expression of MMPs [13]. G-CSF also increases the participation of mobilized stem cells, supporting the concept of myocardial repair after infarction [14]. Although there is mobilization induced by cytokine, the transdifferentiation of HSCs into non-haematopoietic cell types is not a common outcome of the HSC developmental program [15]. These observations emphasize the role of cytokines in the response to injury and their potential role for the treatment of acute myocardial infarction.
In particular GM-CSF has an important biological role as well as a potentially therapeutic one. It is a haematopoietic growth factor secreted by a large variety of cells (T and B lymphocytes, monocytes-macrophages, fibroblasts, endothelial cells), whose main functions are the induction and the control of proliferation and differentiation [16].
We have recently demonstrated the effects of GM-CSF on the differentiation of dermal fibroblasts [17] and human osteoblast-like SaOS-2 cells [18]. GM-CSF is able to exert its effects by binding a specific receptor, GM-CSFR, which consists of an alpha (P) and a beta (β) chain, with molecular weights of 80 and 120 kDa, respectively [19].
Recently, elevated GM-CSF plasma levels have been observed in patients with severe heart failure correlated with the neurohormonal activation characterizing this syndrome [20].
Kohno et al. [21] proved that GM-CSF is an important factor in the initial steps of monocyte transendothelial migration, since it activates RhoA family and integrins which are essential for cell adhesion, lateral migration and diapedesis across the endothelial cells in the vascular wall. GM-CSF also transactivates the MMP-1 and MMP-9 gene expression via AP-1 motifs.
On the basis of these observations, we investigated the expression of GM-CSFR in healthy and end-stage human cardiac tissues to assess the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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The patient population comprised 8 men and 2 women (mean age 46±11.02, range 26-64 years) with end-stage heart failure, selected for transplantation according to published guidelines [22]. Aetiology was coronary artery disease (CAD) in 6 patients, and idiopathic dilated cardiomyopathy (IDCM) in 4 patients. The mean interval between symptom onset and transplantation was 34±17.18 months (range 1-60). Mean indexed pulmonary vascular resistance was 3.53±1.57 (range 1.0-5.8). Mean time on the transplant waiting list was 3.65±2.64 months (range 0.26-7.48). One patient deteriorated to status 1 and was kept on high inotropic support while all the other patients were in stable haemodynamic compensation, and were maintained on medical therapy (ACE-inhibitors, diuretics, β-blockers, digoxin and warfarin) (Table 1). Eight normal hearts from patients who had died due to non-cardiac causes, which were used for transplantation, were also studied.
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Our study conforms to the principles outlined in the Declaration of Helsinki [23]. The study was approved by the hospital ethics committee and informed consent was obtained from all transplanted subjects.
2.1. Western blotting
All cardiac fragments were homogenized in phosphate buffered saline (PBS) containing 1% Triton X-100 (Biorad, Richmond, CA) and protease inhibitors; as positive control, GM-CSFR expressing U937 cells (human promonocytes) [24] were homogenized in the same buffer. Tissue and cell lysates were collected and centrifuged at 14,000 rpm for 20 min at 4 °C. Supernatants were harvested and protein concentration in tissue lysates was determined by a colorimetric assay (Biorad); 1 µl of each sample was diluted in 800 µl of PBS plus 200 µl of the Biorad protein reagent and read in the spectrophotometer at 595 nm. For each sample, 100 µg of total proteins was incubated for 5 min at 90 °C in Laemmli sample buffer and separated by electrophoresis on 10% sodium-dodecyl sulphate-polyacrylamide gels. Gels were electroblotted on PVDF filters (Millipore, Bedford, MA); membranes were blocked with 5% fat-free dry milk, 1% ovalbumin, 5% fetal calf serum (FCS) and 7.5% glycine for 30 min at room temperature. After three washes in washing solution (PBS/0.1% ovalbumin, 0.1% fat-free dry milk, 1% FCS), membranes were incubated overnight at 4 °C with a mouse monoclonal antibody (mAb) against the GM-CSFRP chain (Serotec, Oxford, UK). After four 5 min washes, at room temperature, with washing solution, membranes were incubated for 30' at room temperature with horseradish peroxidase-conjugated goat anti-mouse antibody (Biorad), diluted to 1:3000 in PBS.
After three washes in washing solution and three final washes in T-TBS (50 mM Tris, pH 7.5; 0.5M NaCl; 0.2% Tween 20), membranes were stained with an enhanced chemoluminescence (ECL) detection kit (Amersham, Little Chalfront, England). Membranes were incubated overnight at 4 °C also with an anti-P-sarcomeric actin antibody (Sigma, St. Louis, Missouri, USA) as an internal standard. A quantitative analysis was performed with a Scanner Densytometre (Snapscan 1212, AGFA).
2.2. RNA purification
Total RNA was isolated from all fragments of cardiac tissue by the phenol-guanidine method. In brief, samples were homogenized in 4.5 M guanidine tyocianate, 0.75 M Na citrate pH 7.0 and 10% sarcosyl. After several centrifugations and precipitations in isopropanol, RNA was eluted in sterile, nuclease-free, double distilled H2O, to a total volume of 60 µl. Total RNA concentration was determined by absorbance measurements in triplicate (GeneQuant RNA/DNA calculator). The quality of RNA in all specimens was confirmed with a 2% agarose gel which allow to visualize the 28S and 18S bands, markers of RNA integrity (data not showed).
2.3. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Contaminating DNA was digested with DNase, using a DNase Kit (Invitrogen, Grand Island, NY, USA), and 2 µg of total RNA were reverse transcribed with 100 U Super Script II RNAse H-Reverse Transcriptase (Invitrogen) in a volume of 40 µl, using 100 ng random hexamer primers (Roche, Basel, Switzerland) and 10 mM of dNTP mix, according to the manufacturer's instructions (Invitrogen).
Primer sequences for RT-PCR were as follows:
- GM-CSFR forward primer 5'-GCA TTC CTC CTG ATC CCA GA 3'
- GM-CSFR reverse primer 5'-CCT GGA GTC AAA CCT CAC ATT G 3'
- GM-CSFR reverse primer 5'-CCT GGA GTC AAA CCT CAC ATT G 3'
The amplification was established using a DNA Thermal Cycler (Perkin Elmer Cetus) for 35 cycles consisting of:
- Denaturation: 95 °C for 45 s,
- Annealing: 60 °C for 30 s,
- Extension: 72 °C for 30 s.
- Annealing: 60 °C for 30 s,
At the beginning of the reaction, a cycle at 95 °C for 5 min was carried out to activate Taq Polymerase.
2.4. Real-time quantitative PCR
A quantitative assay for the expression of GM-CSFRP chain mRNA was made using the "ABI Prism 7000 Sequence Detection System" (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using the TAQMAN probe 5'-(FAM)-AATCGGATCTGCGAACAGTGGCACC-(TAMRA)-3', at a concentration of 250 nM, and the previously described primers at a concentration of 900 nM; 50 ng of cDNA, was employed in a total volume of 15 µl TAQMAN Universal PCR Master Mix (2x).
All PCR amplifications were performed in triplicate. PCR cycling profile consisted in AmpErase uracil-N-glycosylase (UNG) incubation for 2 min at 50 °C, and AmpliTaq Gold Activation for 10 min at 95 °C, as pre-denaturation steps, and in 50 two-step cycles at 95 °C for 15 s and at 60 °C for 60 s.
The breakthrough cycle (threshold) was set above the mean baseline fluorescence, at a point where the amplification process had reached the exponential phase.
All quantifications were normalized to an endogenous control, Hypoxanthine Phosphoribosyl Transferase 1 (HPRT 1) (Assay-on-demand Gene Expression Products, Applied Biosystems). HPRT1 turned out to be a very useful internal control, since its cellular expression was apparently unaffected by the expression of GM-CSFRP chain mRNA.
2.5. Immunohistochemistry
All samples for immunohistochemistry were fixed in formaldehyde, embedded in paraffin and sliced into 4 µm thick sections. After de-paraffinization, slides containing two serial sections were processed for heat-induced epitope retrieval. When cooled, slides were washed in PBS and then incubated overnight at 4 °C with a rabbit polyclonal antibody anti-ki67 (Novocastra, Newcastle, UK). After washing, slides were incubated with fluoresceine-conjugated anti-rabbit IgG (Sigma, St. Louis, MO, USA) for 1 h at 37 °C. One section was incubated with a mouse anti-GM-CSFRP mAb (sc-456, Santa Cruz, CA) and the contiguous one with a mouse anti-P-sarcomeric actin mAb (Sigma) for 1 h at 37 °C. After a further wash in PBS, sections were incubated with the secondary antibody, rhodamine-conjugated goat anti-mouse IgG (Sigma), for 1 h at 37 °C.
Slides were washed once again in PBS and then stained with propidium iodide for 10' at room temperature, washed again in PBS and mounted with Vecta-shield. The observation was performed with a confocal microscope LSM 510 (Zeiss) by three independent observers.
2.6. Statistical analysis
The data for molecular biology were calculated as the mean±SD. Statistical analysis for normally distributed parametric data was performed using the Student's t-test, and differences with a p-value <0.05 were considered to be significant.
For non parametric data obtained by Real-time analysis the differences were analyzed using the REST program (see below) using the Pair Wise Fixed Reallocation Randomisation Test. Randomisation tests with a pair-wise reallocation are seen as the most appropriate approach for this application. They are more flexible than non-parametric tests based on ranks (Mann-Whitney, Kruskal-Wallis) and do not suffer a reduction in power relative to parametric tests (t-test, ANOVA). A p-value <0.05 was considered significant.
For immunohistochemical data, a quantitative evaluation of Ki67+ and GM-CSFRP cardiomyocytes was performed by counting the number of positive cardiomyocytes per unit area of 100 mm2. The significance of the differences was determined with the analysis of variance, and differences with a p value <0.05 were considered to be significant.
2.7. REST program
Real-time PCR using fluorescence dyes significantly simplifies and accelerates the process of producing reproducible and reliable quantification of mRNA. This has led to the development of new kinetic RT-PCR methodologies that are revolutionising mRNA quantification.
Absolute quantification is very common, where an appropriate external calibration curve is used to determine the absolute mRNA copy number. However, relative expression can also be used according to several established mathematical models.
Relative expression is based on the expression ratio of a target gene versus a reference gene and is adequate for most purposes to investigate physiological changes in gene expression levels. The most convenient mathematical model, which includes an efficiency correction for Real-time PCR efficiency of the individual transcripts is:
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The relative expression ratio of a target gene is computed, based on its Real-time PCR efficiencies (E) and the crossing point (CP) difference (
) of an unknown sample versus a control (CPcontrol-sample).
A new software tool, REST© (relative expression software tool) calculates the relative expression ratio on the basis of PCR efficiency (E), crossing point deviation (CP) of the investigated transcripts and on a newly developed randomisation test. REST, which uses the Pair Wise Fixed Reallocation Randomisation Test, is used to facilitate better understanding of relative quantification analysis in Real-time PCR.
The software exhibits suitable reliability and reproducibility in individual runs, confirmed by high accuracy and low variation independent of huge template concentration variations.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1. Expression of GM-CSFRP chain in cardiac tissues
In order to identify the GM-CSF receptor P chain (GM-CSFRP), whole lysates obtained from fragments of cardiac tissue (normal and end-stage heart failure) were analyzed by Western blot with a monoclonal anti GM-CSFRP antibody.
A band with the expected molecular weight of 84 kDa was present, with variable intensity, in all cardiac lysates (Fig. 1). A quantitative analysis of the GM-CSFR bands was performed (Fig. 2). No statistically significant difference was observed in the expression of GM-CSFRP, between healthy and end-stage heart failure samples.
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3.2. Real-time PCR
RT-PCR on total RNA, from healthy and end-stage heart failure cardiac tissues did not show a significant degradation and the presence of an amplification product of 82 base pairs, corresponding to the P chain of the GM-CSF receptor was found. U937 human promonocytes cell line was used as a positive control for the expression of GM-CSFRP mRNA (Fig. 3).
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Quantitative analysis of mRNA GM-CSFRP expression by the TAQMAN probe showed a significant increase in the cardiac tissue from patients with ischaemic and dilated cardiomyopathy as compared to the healthy controls. These differences, analyzed by the REST program, were statistically significant (p<0.05) (Fig. 4).
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GM-CSFRP mRNA levels were significantly higher in the samples from patients with dilated cardiomyopathy than in those with ischaemic aetiology (p<0.05). However, no correlation was found between GM-CSFRP expression and the clinical indices reported in Table 1.
3.3. Immunohistochemistry
The presence of GM-CSFRP on the surface of some cardiomyocytes was observed in all end-stage heart failure hearts (whether affected by ischaemia or by dilated cardiomyopathy) despite the great individual variability in the distribution of the immunopositivity. In contrast, immunopositivity for GM-CSFR was present in only four of the eight normal hearts; moreover, in positive hearts, it was present in fewer cells as compared to the end-stage heart failure samples. All GM-CSFR positive cells were strongly positive for P-sarcomeric actin, which is commonly considered to be a specific protein of cardiomyocytes (Fig. 5). The expression of Ki67 was evident both in normal and end-stage heart failure sections, but was not observed on the myocytes that were GM-CSFR positive, and there were fewer differences between end-stage heart failure and normal hearts (Fig. 6).
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GM-CSFR+ cardiomyocytes were 7.3±0.7% of the entire number of cardiomyocytes in 100 mm2 from end-stage heart failure samples and 1.003±0.004% in 100 mm2 from the four normal hearts that exhibited GM-CSFRP (p<0.05). Ki67+ cardiomyocytes were 1.4±0.6% in 100 mm2 from end-stage heart failure samples and 0.8±0.4% in 100 mm2 from normal hearts (difference not significant) (Table 2).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Recent studies have demonstrated that cytokines may play a pathogenic role in heart repair by inducing hypertrophy, and in promoting apoptosis or fibrosis [25,26]. In particular GM-CSF plasma levels are increased in patients with severe heart failure [20]. This cytokine may influence biological activities, growth and apoptosis through the activation of specific receptors and intracellular pathways, such as JAK-STAT [27] or HSP 70 (Heat Shock Protein 70) [28] activation, in the heart as well as in other organs.
On the basis of this observation, we studied the expression of GM-CSFR in cardiac tissues from end-stage heart failure patients and compared it with specimens from healthy subjects. Since the detection of GM-CSFR in cardiac tissues can be affected by the presence of other cell types, such as endothelial cells, fibroblasts and haematopoietic cells, immunofluorescence with anti GM-CSFRP mAb was performed to determine which cell type in our samples was responsible for the signal observed by Western blot and Real-time PCR. The data obtained by confocal microscopy showed localization of GM-CSFRP on the cardiomyocyte membrane.
GM-CSFRP positive cells were found in all end-stage heart failure cardiac samples at significantly higher numbers than in the normal control tissues, as confirmed by analysis of variance. The identification of the GM-CSFR positive cells as cardiomyocytes was confirmed by the co-expression of P-sarcomeric actin. We performed immunostaining with anti Ki67 Ab, to evaluate whether GM-CSFR positive cells were involved in proliferative processes. Immunofluorescence demonstrated that this was not the case, because all GM-CSFR immunopositive cells were Ki67 negative.
The data suggest a possible role of haematopoietic growth factors in the control of cardiac tissue remodelling.
The biological meaning of cardiomyocytes expressing the receptor for a hematopoietic growth factor is very interesting. We can only hypothesize that GM-CSF, like other growth factors, plays a role in ECM turnover, through modulation of MMPs activity as well as on the soluble cellular adhesion molecules [29] in the myocardium. In particular, the increased expression of GM-CSFR suggests an increased activity of GM-CSF that is able to modulate HSP 70 and to modify Collagen type I deposition. Also laminin plays an important role in ECM functions and modulates, through its receptor 67LR, GM-CSF signalling [30]. Several studies to evaluate ECM components and 67LR expression in the same cardiac tissue samples are currently ongoing.
Wieczorek et al. [31] suggested that growth factors, such as GM-CSF and some cytokines, induce release of stem cells from the bone marrow into peripheral blood during mobilization as well as during steady-state haematopoiesis, by signalling through gelatinase pathways.
These data are in agreement with a new role for GM-CSF in cardiac tissue repair through an increased expression of MMPs.
Further research on growth factors, in order to define the specific role of GM-CSF in end-stage heart failure, is now required.
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Acknowledgement |
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This study was supported by a grant for a National Research Project (PRIN) by MIUR-Italy.
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