Tumor necrosis factor-alpha is expressed in donor heart and predicts right ventricular failure after human heart transplantation.

BACKGROUND-Myocardial failure is an important problem after heart transplantation. Right ventricular (RV) failure is most common, although its mechanisms remain poorly understood. Inflammatory cytokines play an important role in heart failure. We studied the expression of tumor necrosis factor (TNF)-alpha and other cytokines in donor myocardium and their relationship to the subsequent development of RV failure early after transplantation. METHODS AND RESULTS-Clinical details were obtained, and ventricular function was assessed by transesophageal echocardiography in 26 donors before heart retrieval. A donor RV biopsy was obtained immediately before transplantation, and each recipient was followed for the development of RV failure. Reverse transcriptase-polymerase chain reaction was performed to detect TNF-alpha, interleukin-2, interferon-gamma, and inducible nitric oxide synthase expression. Eight of 26 recipients (30.8%) developed RV failure. Seven of these 8 (87.5%) expressed TNF-alpha, but only 4 of the 18 (22.2%) who did not develop RV failure expressed TNF-alpha (P<0.005). As a predictor of RV failure, TNF-alpha mRNA had a sensitivity of 87.5%, a specificity of 83.3%, a positive predictive value of 70%, and a negative predictive value of 93.7%. Western blotting demonstrated more TNF-alpha protein in the myocardium of donor hearts that developed RV failure (658+/-60 versus 470+/-57 optical density units, P<0.05). Immunocytochemistry localized TNF-alpha expression to cardiac myocytes. Reverse transcriptase-polymerase chain reaction detected interferon-gamma in 2 (7.7%), interleukin-2 in 1 (3.8%), and inducible nitric oxide synthase mRNA in 1 (3.8%) of the 26 donor hearts, none of which developed RV failure. CONCLUSIONS-TNF-alpha expression in donor heart cardiac myocytes seems to predict the development of RV failure in patients early after heart transplantation.

Elevated tumor necrosis factor-alpha and interleukin-6 in myocardium and serum of malfunctioning donor hearts.

BACKGROUND: Myocardial dysfunction is a common and important problem in donor hearts. The mechanisms responsible remain unclear. We have studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6) in the myocardium and serum from donors with myocardial dysfunction (unused donors) and compared them with donors with good ventricular function (used donors) and patients with advanced heart failure (HF). METHODS AND RESULTS: Clinical details and ventricular function were assessed in 46 donors (31 used, 15 unused). Real-time reverse transcription-polymerase chain reaction, Western blotting, and immunocytochemistry were performed on myocardium and immunoassays on serum. TNF-alpha mRNA was 1.6-fold higher in unused than in used donors (P:<0.005) and 1.74-fold higher than in 36 patients with HF. IL-6 mRNA was 2.4-fold higher in unused than in used donors (P:<0.0001) and 4.67-fold higher than in HF (P:<0.0001). Western blotting showed higher TNF-alpha in unused (218. 3+/-6.4, n=4 versus 187.3+/-5.4, n=3 OD units) than used donors (P:<0.05). TNF-alpha expression was localized to cardiac myocytes. Serum TNF-alpha was higher in unused (8.72+/-1.3 pg/mL, n=13) than in used (6.12+/-0.8 pg/mL, n=25, P:<0.05) donors and HF (4.0+/-0.4 pg/mL, n=17, P:<0.005). Serum TNF-alpha receptors did not differ between unused (4.3+/-0.8 and 8.6+/-1.6 ng/mL, n=10) and used (3. 5+/-0.4 and 6.5+/-1.1 ng/mL, n=24) donors. There was a trend for higher serum IL-6 in unused (16.5+/-2.9 pg/mL, n=9) compared with used (13.9+/-1.6 pg/mL, n=26, P:=NS) donors. CONCLUSIONS: This study documented an increase in the expression of TNF-alpha and IL-6 in the myocardium of all donor hearts that was more marked in the dysfunctional (unused) donor hearts. This was accompanied by similar changes in the serum. This might have important therapeutic implications.

Parathyroid hormone related peptide gene expression in human fetal and adult heart.

OBJECTIVE: The aim was to investigate the expression of parathyroid hormone related peptide (PTHrP) gene in the human fetal and adult heart. METHODS: Molecular biological techniques were employed as well as immunocytochemistry and western blot analysis using rabbit polyclonal anti-PTHrP(1-34) and anti-PTHrP (56-86) on normal human fetal and adult heart tissues. Northern blot analysis of both normal human fetal and adult heart total RNA, using a human full length cDNA probe, and polymerase chain reaction analysis of normal human fetal and adult heart cDNAs with exon specific oligonucleotides were carried out. RESULTS: Positive staining was detected with both anti-PTHrP(1-34) and anti-PTHrP(56-86) in fetal heart at 12 weeks of gestation. In both fetal and adult hearts, multiple putative PTHrP proteins were observed with apparent molecular mass of 14-125 kDa. Multiple hybridising PTHrP mRNA isoforms (1.4, 2.1, 3.2, and 4.5 kb) were detected in both fetal and adult heart total RNAs. The fetal and adult heart cDNAs amplified from the cDNA libraries showed the presence of the 5' non-coding exon II and coding exons III-IV but not the 5' non-coding exon Ic. CONCLUSIONS: PTHrP is expressed in normal human fetal and adult hearts suggesting that it has a function as an endogenous modulator of the cardiovascular system.

Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes.

OBJECTIVE: We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS: The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS: Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS: The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

Parathyroid hormone-related peptide can regulate the growth of human lung cancer cells, and may form part of an autocrine TGF-alpha loop.

Parathyroid hormone-related peptide (PTHrP) and transforming growth factor-alpha (TGF-alpha) were found to stimulate proliferation of human lung cancer cells (BEN-57). TGF-alpha stimulated PTHrP secretion from these cells. The polyclonal antisera raised against PTHrP significantly inhibited the growth of BEN-57 cells, and also the proliferation induced by TGF-alpha. Treatment of cells for up to 10 days with either a PTHrP receptor antagonist (PTHrP(7-34)) or PTHrP antiserum significantly inhibited the subsequent growth of these cells. We suggest that PTHrP may be a component of a complex autocrine loop involving TGF-alpha.

Parathyroid hormone-related peptide in normal human fetal development.

Parathyroid hormone-related peptide (PTHrP) has been detected in fetal serum and amniotic fluid. Using a combination of immunocytochemistry and molecular biology we have detected the peptide and its mRNA in a variety of fetal tissues throughout gestation. Tissue-specific mRNA isoforms were observed, the pattern of hybridization of which changed throughout gestation. In addition, the intensity and pattern of immunocytochemical localization of the peptide was found to vary over the time-period studied (8-30 weeks). PTHrP is expressed by a variety of tumours associated with the syndrome of humoral hypercalcaemia of malignancy and probably accounts for the hypercalcaemia by virtue of its limited amino acid homology with parathyroid hormone. These data demonstrate for the first time that PTHrP, a tumour-related peptide, is expressed during normal human fetal development, and suggest the possibility that it may function to regulate fetal calcium balance and growth in utero.

Parathyroid hormone-related peptide in the human fetal uro-genital tract.

Using a polyclonal antiserum raised against the first 34 amino acids of human parathyroid hormone-related peptide (PTHrP), we have localized PTHrP throughout the uro-genital tract of the human fetus aged between 8 and 40 weeks. Staining was present in the developing mesonephros, metanephros, gonads and in both the adrenal cortex and medulla. In particular, the developing mesonephric and metanephric renal tubules were intensely positive. Using Northern hybridization analysis we have detected a complex pattern of PTHrP mRNA transcripts ranging in size from 1.4 to 4.5 kb in early second trimester human fetal kidney. The presence of PTHrP in the mesonephros and metanephros provides evidence for a role for PTHrP in the regulation of fetal calcium metabolism. However, its presence in the gonad and adrenal gland invites the possibility of a wider role for PTHrP.

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts.

BACKGROUND: Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. METHODS: MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. RESULTS: Morphometric analysis showed that 66.3% +/- 3% to 69.6% +/- 6% of cells in group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta significantly inhibited gene transfer, whereas insulin-like growth factor-I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. CONCLUSION: MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.

Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma.

Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (3-34)NH2.

Rapamycin (sirolimus) inhibits heart cell growth in vitro.

The success of infant and neonatal heart transplantation has increased dramatically since the mid-1980s. This success is due both to improved medical and surgical techniques and better pharmacological management of rejection episodes. We report here the results of in vitro studies designed to investigate the effect of rapamycin (a macrolide antibiotic with potent antirejection activity) on the proliferation of fetal cardiac myocytes. Our data suggest that rapamycin inhibits the proliferation of these cells, which is an effect that appears to be irreversible. This inhibition is relevant to the use of rapamycin in the treatment of rejection episodes in the infant and neonate, in which cardiac myocyte development is incomplete.

Cyclin-dependent kinase inhibitor expression in human heart failure. A comparison with fetal development.

AIMS: Terminal differentiation of cardiac myocyte is associated with their permanent withdrawal from the cell cycle. In adult end-stage heart failure, significant numbers of myocytes express proliferating cell nuclear antigen yet fail to progress to cell division. Cyclin dependent kinase inhibitors are powerful inhibitors of the cell cycle and may play a direct role both in myocyte development and in preventing cell division in the adult. METHODS AND RESULTS: The expression of the CIP/KIP cyclin dependent kinase inhibitors p21, p27, p57 and the retinoblastoma protein was examined in acute (seen in brain dead transplant donors) and end-stage heart failure by Western blot analysis and compared to that seen in human and rat cardiac development. The expression of p21 showed a gradual increase during development in both rat and man, becoming maximal in adulthood p27. levels showed an initial rise with subsequent continual expression throughout life. p57 expression was detectable at only early stages in rat but persisted throughout life in man. In both acute and end-stage heart failure the levels of p21, p27 and p57 reverted to a pattern similar to that observed in human fetal heart: p21 and p27 declined while p57 expression was significantly increased. In contrast, retinoblast protein levels declined during human heart development but were unaltered in heart failure. CONCLUSIONS: The expression of p21, but not p27 or p57, is consistent with a role in the gradual withdrawal of cardiac myocytes from cell cycle during development. In adult heart failure cyclin dependent kinase inhibitor expression reverts to the fetal pattern but is insufficient to initiate cell cycle activation.

Two nuclear proteins bind to the promoter P3 region of the human parathyroid hormone-related peptide gene.

We have used a 5' regulatory sequence fragment of the human parathyroid hormone-related peptide (PTHrP) gene to identify nuclear DNA-binding proteins (DBP), using South-Western analysis. The PTHrP 549 bp DNA fragment was amplified from a human genomic DNA, and composed of 5' non-coding exon Ic, the intervening intron and 5' non-coding exon II. The DNA fragment bound very specifically to a 70 kDa and a 65 kD protein from the nuclear extract of human hepatocyte, HepG2, and keratinocyte, SVK-14, cell lines. This is the first evidence of a physical binding between nuclear protein and the human PTHrP gene. The 70 kDa and 65 kDa nuclear proteins may have a role in the regulation of human PTHrP gene expression.

An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes.

Developing cardiac myocytes divide a limited number of times before they stop and terminally differentiate, but the mechanism that stops their division is unknown. To help study the stopping mechanism, we defined conditions under which embryonic rat cardiac myocytes cultured in serum-free medium proliferate and exit the cell cycle on a schedule that closely resembles that seen in vivo. The culture medium contains FGF-1 and FGF-2, which stimulate cell proliferation, and thyroid hormone, which seems to be necessary for stable cell-cycle exit. Time-lapse video recording shows that the cells within a clone tend to divide a similar number of times before they stop, whereas cells in different clones divide a variable number of times before they stop. Cells cultured at 33 degrees C divide more slowly but stop dividing at around the same time as cells cultured at 37 degrees C, having undergone fewer divisions. Together, these findings suggest that an intrinsic timer helps control when cardiac myocytes withdraw from the cell cycle and that the timer does not operate by simply counting cell divisions. We provide evidence that the cyclin-dependent kinase inhibitors p18 and p27 may be part of the timer and that thyroid hormone may help developing cardiac myocytes stably withdraw from the cell cycle.

Expression of RGS3, RGS4 and Gi alpha 2 in acutely failing donor hearts and end-stage heart failure.

BACKGROUND: Regulators of G-protein Signalling (RGS) proteins have been shown to limit in vitro signalling of G proteins. In common with end-stage heart failure, we have recently shown that upregulation of the inhibitory G-protein, Gialpha, occurs in acutely failing donor hearts unused for transplantation due to severe myocardial dysfunction. In light of recent data on RGS proteins, we have evaluated mRNA and protein expression of RGS3, RGS4 and Gialpha2 in the myocardium from normal, end-stage failing and acutely failing unused donor hearts. METHODS AND RESULTS: Myocardial samples were obtained from end-stage failing hearts explanted prior to transplantation (n=19), unused donor hearts with ejection fractions < 30% (n=14) and used donor hearts with good function (ejection fraction > 60%) (n=4-7). mRNA levels were quantified using quantitative reverse transcriptase polymerase chain reaction. Levels of RGS3 and RGS4 mRNA were found to be significantly upregulated in unused donor and end-stage failing myocardium (P < 0.05 and 0.01, and P < 0.05 and 0.02, respectively) compared to non-failing hearts. Protein abundance of RGS3 and RGS4 was found to be higher in myocardium from end-stage failing hearts, and relative RGS4 expression higher in unused donor hearts. CONCLUSIONS: We show here that RGS3 and RGS4 mRNA and protein expression is upregulated in human heart failure. These observations suggest that RGS4 may be induced in the heart to regulate cell signalling pathways in response to hypertrophy, and support the existence of a negative feedback loop for the long-term regulation of hypertrophy.

Growth factor expression during rat development: a comparison of TGF-beta 3, TGF-alpha, bFGF, PDGF and PDGF-R.

At least part of the mechanism underlying fetal development appears to be the production of a number of growth factors considered important in the process of tumour formation. Using immunocytochemistry, we have investigated the temporal and spatial pattern of expression of some of the important growth factors, by the fetus. We describe here the cellular localization of transforming growth factor beta 3 (TGF-beta 3), platelet derived growth factor (PDGF) and its receptor (PDGF-R), TGF-alpha and basic fibroblast growth factor (bFGF) in the fetal rat from day 13 to 21 of gestation. Using antisera raised against an N-terminal portion of TGF-beta 3, immunoreactivity peaked around day 16 and was seen predominantly within epithelial cells. However, using antisera raised against the C-terminal of this molecule immunoreactivity was seen exclusively within the extracellular matrix underlying adjacent epithelia, and was maintained up until day 21 of gestation. Strong expression of TGF-alpha was seen in cells of most organs throughout the gestation period studied. Immunoreactivity for bFGF, PDGF and PDGF-R peaked around day 18 in both epithelial and mesenchymal cells of all major organ systems and then declined by day 21. These data suggest distinct roles for each factor during embryogenesis and tumorigenesis.

Parathyroid hormone-related peptide: expression in fetal and neonatal development.

Hypercalcaemia frequently complicates the clinical management of cancer. Many factors have been implicated in the pathogenesis of this humoral hypercalcaemia of malignancy, the most recent candidate being parathyroid hormone-related peptide (PTHrP). Until now, this peptide has been detected only in some normal and transformed adult tissues. In recent years, it has become apparent that tumours are capable of expressing and secreting factors previously elaborated only during fetal life. Many of these factors act to stimulate the growth of both tumour and fetal cells in an autocrine manner. The data presented here demonstrate that PTHrP is expressed in the human and rat fetus throughout gestation. Immunocytochemistry reveals a gestationally related, changing pattern of expression which is paralleled by changes in mRNA transcription. These data support the hypothesis that PTHrP may function as a fetal growth factor.

Vascular endothelial growth factor release following coronary artery bypass surgery: extracorporeal circulation versus 'beating heart' surgery.

AIMS: The aim of this study was to examine the circulating levels of vascular endothelial growth factor, following coronary artery bypass graft surgery performed using both standard cardiopulmonary bypass or the 'octopus technique' on the beating heart. BACKGROUND: Vascular endothelial growth factor has a number of effects that are beneficial in the setting of coronary artery bypass graft surgery including cardioprotection, potent angiogenic activity and amelioration of intimal hyperplasia. Hypoxia is a powerful stimulator of vascular endothelial growth factor expression yet the ability of ischaemia, occurring during coronary artery bypass graft surgery, to induce vascular endothelial growth factor production is unknown. METHODS AND RESULTS: Serum vascular endothelial growth factor levels were determined in patients undergoing coronary artery bypass graft surgery with standard cardiopulmonary bypass (CPB-CABG group; n=20), with off-pump coronary artery bypass; (OP-CABG; n=12) and in patients undergoing non-cardiac major surgery (n=6). The effect of hypoxia on vascular endothelial growth factor release by neonatal rat cardiac myocytes in vitro was studied. In the CPB-CABG group vascular endothelial growth factor levels were significantly increased to 78.5+/-39.3 and 110.5+/-16.3 pg. microl(-1)8 and 24 h post-operatively, declining to 14.9+/-9.9 pg. microl(-1)by 48 h to pre-operative values (14.4+/-8.6 pg. microl(-1)). Significantly higher vascular endothelial growth factor levels were also present in the OP-CABG group 3, 6 and 24 h post-operatively (levels 136. 6+/-29.3, 143+/-26.12 pg. microl(-1)and 93.5+/-20.1 pg. microl(-1), respectively). However, non-cardiac major surgery did not result in elevated vascular endothelial growth factor levels post-operatively (46.36+/-9.76 vs pre-surgery levels of 26.84+/-6.1 pg. microl(-1)). Either 15 min or 3 h of hypoxia stimulated vascular endothelial growth factor release from neonatal rat cardiac myocytes in vitro. Twenty-four and 48 h post hypoxia, levels of vascular endothelial growth factor were significantly elevated by approximately 17.5- and 48.5-fold respectively. CONCLUSIONS: These data demonstrate myocardial ischaemia secondary to CPB-CABG and OP-CABG to be a potent stimulator of vascular endothelial growth factor production, which may have implications for graft endothelialization and cardiovascular haemodynamics post-operatively.