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European Journal of Heart Failure 2008 10(4):352-359; doi:10.1016/j.ejheart.2008.02.010
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© 2008 European Society of Cardiology

Increased expression of LIGHT/TNFSF14 and its receptors in experimental and clinical heart failure

Christen Peder Dahla,b,*, Lars Gullestadb, Børre Fevanga, Are M. Holmc, Linn Landrøa, Leif Erik Vinged, Arnt E. Fianee, Wiggo J. Sandberga, Kari Otterdala, Stig S. Frølanda,f, Jan Kristian Damåsa,f, Bente Halvorsena, Pål Aukrusta,f, Erik Øiea,b,d and Arne Yndestada

a Research Institute for Internal Medicine, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway
b Department of Cardiology, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway
c Department of Respiratory Medicine, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway
d Institute for Surgical Research, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway
e Department of Thoracic and Cardiovascular Surgery, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway
f Section of Clinical Immunology and Infectious Diseases, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo Norway

* Corresponding author. Research Institute for Internal Medicine, Rikshospitalet-Radiumhospitalet Medical Centre, N-0027 Oslo, Norway. Tel.: +47 23072786; fax: +47 23073630. E-mail address: c.p.dahl{at}medisin.uio.no (C.P. Dahl).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Clinical and experimental studies suggest a pathogenic role for inflammation in chronic heart failure (HF). LIGHT is a member of the tumour necrosis factor superfamily involved in innate and adaptive immune responses.

Aims: We sought to investigate a potential pathogenic role of LIGHT in chronic HF.

Methods: We used various clinical and experimental approaches including studies in post-infarction HF rats and in vitro studies of endothelial cells and peripheral blood mononuclear cells (PBMC).

Results: Our main findings were: (i) LIGHT and its receptors (i.e., HVEM and lymphotoxin-β receptor) were regulated during experimental HF, with strong expression in the infarcted area accompanied by up-regulation of HVEM in cardiomyocytes and endothelial cells also in the non-ischaemic part of the left ventricle. (ii) Patients with chronic HF had significantly increased expression of LIGHT on CD3+ T-cells accompanied by increased expression of HVEM on monocytes and within the failing myocardium. (iii) LIGHT induced interleukin (IL)-6 expression in endothelial cells. In HF patients, but not in healthy controls, such an IL-6-inducing effect was also seen in LIGHT activated PBMC.

Conclusion: Our findings in both clinical and experimental HF may suggest a role for LIGHT signalling pathways in the progression of chronic HF involving IL-6-related mechanisms.

Key Words: Heart failure • Inflammation • Cytokines • Tumour necrosis factor superfamily • Leukocytes

Received August 27, 2007; Revised November 26, 2007; Accepted February 4, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Despite a state-of-the-art treatment, chronic heart failure (HF) is still a progressive disorder with high morbidity and mortality, indicating that important pathogenic mechanisms remain active and unmodified by the present treatment modalities [1]. Several lines of evidence suggest that persistent inflammation could represent one of these unmodified mechanisms, contributing to the myocardial remodelling process characterizing chronic HF [2,3]. However, although the participation of inflammatory mediators in the pathogenesis of chronic HF is widely recognized, the identification and characterization of the different mediators remains incomplete.

While the role of tumour necrosis factor (TNF){alpha} in HF has been extensively examined and several studies have demonstrated raised TNF{alpha} levels in this disorder [4], recent reports suggest that other ligands in the TNF superfamily could also be involved in the progression of chronic HF. Thus, some studies have found increased Fas/Fas ligand levels in HF patients [5], and we have reported enhanced systemic expression of CD40 ligand and CD27 ligand [6,7] as well as increased myocardial expression of receptor activator of nuclear factor (NF){kappa}B (RANK) ligand [8] in clinical HF.

LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D (gD) for HVEM, a receptor expressed by T lymphocytes; TNFSF14) is a cytokine in the TNF superfamily that is involved in innate and adaptive immune responses as well as in regulation of cell survival and proliferation [9]. LIGHT binds to three distinct members of the TNF receptor family, i.e., the herpes virus entry mediator (HVEM), the lymphotoxin-β receptor (LTβR), and the soluble decoy receptor 3 [9]. Studies in animal models indicate that LIGHT may be crucial for the development of various autoimmune disorders (e.g., inflammatory bowel disease and rheumatoid arthritis) through effects on T-cells and T-cell homing into inflamed tissues [10,11]. This cytokine has also been suggested to promote atherogenesis at least partly by inducing matrix metalloproteinase (MMP) activity in macrophages and inflammation in endothelial cells [12,13], and very recently LIGHT was shown to be involved in regulation of lipid homeostasis [14]. Based on these properties, we hypothesized that LIGHT could also be involved in the progression of chronic HF. Herein we investigate this hypothesis using various experimental approaches, including studies in clinical and experimental HF, as well as in vitro studies in endothelial cells and peripheral blood mononuclear cells (PBMC).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Rat model of experimental HF
Myocardial LIGHT expression was investigated at various time points during the development of HF in male Wistar rats (275 g) after ligation of the left coronary artery. The procedure generally resulted in transmural myocardial infarction (MI) of the left ventricular (LV) free wall, comprising 40% to 50% of the ventricular circumference as assessed by perimetry of LV tissue sections. Sham-operated rats underwent the same procedure except for ligation of the coronary artery. Assessment of haemodynamic function and tissue sampling procedures were performed as described previously [15]. The animal experiments and housing were in accordance with institutional guidelines and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Isolation of cardiomyocytes and endothelial cells from rat myocardium
Cardiomyocytes and non-cardiomyocytes were isolated from myocardial tissue of rats euthanized 56 days after MI or sham operation by retrograde perfusion of Tyrodes solution (Hepes 25 mM, 130 mM NaCl, NaH2PO4 10 mM, MgCl2 2.5 mM, KCl 27 mM) supplemented with 0.2% glucose, 10 mM 2, 3-butandione monoxime and 1 mg/ml Collagenase type II (Worthington Biochem. Corp., Lakewood, NJ). The undigested infarcted myocardium of HF rats was removed together with the right ventricle and the atria. Cardiomyocytes and non-cardiomyocytes were then sedimented and separated by differential centrifugation. Endothelial cells were isolated from the non-cardiomyocyte cell fraction using mouse anti-rat CD31 antibody (Fitzgerald, Concord, MA) and subsequent extraction using a secondary anti-mouse antibody coupled to paramagnetic microbeads (Miltenyi Biotec, Auburn, CA). The purity of the isolated cell fractions was determined by immuncytochemical analyses, a similar pattern was found in sham-operated and HF rats. More than 95% of the cells in the cardiomyocyte fraction were sarcomeric actin-positive cardiomyocytes (monoclonal anti-rabbit sarcomeric actin antibody, Dako, Glostrup, Denmark), and more than 95% of the cells in the non-cardiomyocyte fraction were vimentin-positive fibroblasts (anti-human vimentin antibody [V9, Zymed, CA]). Furthermore, more than 90% of the cells in the endothelial cell fraction were CD31-positive endothelial cells (mouse anti-rat CD31 antibody [Fitzgerald]).

2.3. HF patients
One hundred and twenty-one patients (92 men and 29 women; median age 55.9±12.9 years) with stable HF for more than 6 months who were in New York Heart Association (NYHA) functional classes II-IV, were consecutively recruited from the Department of Cardiology, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo. Patients with acute coronary syndromes during the last 6 months and patients with significant concomitant disease, such as infection, malignancy, or collagen vascular disease, were not included. The underlying cause of HF was classified as coronary artery disease (CAD, n=59) or idiopathic dilated cardiomyopathy (IDCM, n=62) on the basis of disease history and coronary angiography. Twenty sex- and age-matched healthy individuals were included as controls. Informed consent for participating in the study was obtained from all individuals. The study was approved by the local ethics committee. Blood samples for the study (platelet-poor EDTA plasma) were collected and stored as previously described [12].

2.4. Tissue sampling from human myocardium
Tissue aliquots from the failing myocardium were removed from still-beating hearts immediately on explantation from patients with end-stage HF (NYHA classes III-IV, LV ejection fraction <35%) undergoing cardiac transplantation. Tissues were snap-frozen in liquid nitrogen, and stored at –80 °C until use. Control (non-failing) human LV tissue was obtained from sex- and age-matched subjects whose hearts were rejected as cardiac donors for surgical reasons. The cause of death of all donors was cerebrovascular accident or trauma, and none had a history of heart disease. The myocardium from these subjects was kept on iced water for 1 to 4 h before tissue sampling was conducted as described above. Samples for Western blot were homogenized separately, and proteins were extracted with ice-cold lysis buffer that contained a protease inhibitor cocktail at a ratio of 500 µl/50 mg of frozen weight tissue. Extracts were incubated on ice for 15 min and centrifuged at 12,000 g for 15 min at 4 °C. The supernatants were retained and protein concentrations of the sample measured by the BCA method (Pierce Chemical, Rockford, IL).

2.5. Cell culture experiments
Freshly isolated PBMC, obtained from heparinized blood by Isopaque-Ficoll (Lymphoprep, Nycomed, Oslo, Norway) gradient centrifugation, were incubated in flat-bottomed 96-well trays (106/ml; Costar, Cambridge, MA), in medium alone (RPMI 1640 with 2 mM L-glutamine [Gibco, Grand Island, NY]) supplemented with 10% fetal-calf serum (Sigma, St Louis, MO) or stimulated with different concentrations of recombinant human LIGHT (rhLIGHT; R&D Systems, Minneapolis, MN). Primary human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cord veins, cultured as described elsewhere [16], and stimulated with different concentrations of rhLIGHT (R&D Systems). At different time points, cell-free supernatants (PBMC and HUVEC) and cell pellets (HUVEC) were harvested and stored at –80 °C. The endotoxin levels of all stimulants and culture media were <10 pg/ml (Limulus Amebocyte Assay).

2.6. Real-time quantitative RT-PCR
Total RNA was extracted using RNeasy columns (Qiagen, Hilden, Germany) (HUVECs) or acid-phenol extraction in the presence of chaotropic salts (TRIzol, Invitrogen, San Diego, CA) and subsequent isopropanol-ethanol precipitation (rat myocardium). All total RNA samples were subjected to DNase I treatment (RQI DNase; Promega, Madison, WI), and stored in RNA storage solution (Ambion, Austin, TX) at –80 °C. Sequence-specific PCR primers were designed with Primer Express software version 1.5 (Applied Biosystems, Foster City, CA; Table 1). Quantification of mRNA was performed with ABI Prism 7500 (Applied Biosystems) [17]. Gene expression of the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S (Table 1) was used for normalization.


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  		Table 1 Characteristics of the real-time PCR assays used in the study

 
2.7. Immunohistochemistry
Immunohistochemical analysis was performed on LV myocardial tissue from human cardiac explants and donor hearts and from rat HF and control hearts as previously described [15]. The antibodies used were two different purified polyclonal goat anti-human LIGHT IgGs (R&D System and Santa Cruz Biotech., Santa Cruz, CA), two different purified polyclonal goat anti-human LTβR IgGs (R&D Systems and Santa Cruz Biotech.), and purified polyclonal mouse anti-human HVEM (R&D Systems) and goat anti-human HVEM (Santa Cruz Biotech.) IgG. The immunoreactivities were amplified by the avidin-biotin-peroxidase system (Vectastain Elite kit; Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as the chromogen in a commercial metal-enhanced system (Pierce Chemical). The sections were counterstained with haematoxylin. Omission of the primary antibody and use of specific blocking peptides (Santa Cruz Biotech.) served as negative controls.

2.8. Western blotting
Western blotting was performed as described previously [18], with equal amounts of protein being separated from each sample by two-dimensional polyacrylamide gel electrophoresis (10%) before being transferred to poly (vinylidene fluoride) membranes. Filters were incubated with the same antibodies against LIGHT, HVEM, and LTβR as used for immunohistochemistry. Proteins were detected by enhanced-chemiluminescence with horseradish peroxidase-labelled anti-goat IgG (Santa Cruz Biotech.) or anti-rabbit IgG (Cell Signaling, Beverly, CA). All proteins were normalized against GAPDH (Chemicon International, Temecula, CA). The enhanced-chemiluminescence-exposed films were detected by using Kodak 440 CF imaging station (Boston, MA). The software Total Laboratory v.1.10 (Phoretix, Newcastle, UK) was used for quantification.

2.9. Flow cytometry
Cryopreserved PBMC were thawed as described previously [19]. Staining was performed using fluorescein isothiocyanate-conjugated anti-CD14, phycoerythrin-conjugated anti-CD3 and allophycocyanin-conjugated anti-CD3 from Becton Dickinson (San Diego, CA), and phycoerythrin-conjugated anti-LIGHT and anti-LTβR, and allophycocyanin-conjugated anti-HVEM from R&D systems. Isotype controls were used as appropriate. Flow cytometry was performed using a FACSCalibur instrument with CellQuest software (Becton Dickinson).

2.10. Enzyme immunoassays
Concentrations of LIGHT, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, and TNF{alpha} were measured in duplicates by enzyme immunoassays (R&D Systems).

2.11. Statistical analysis
For comparisons of two groups of individuals, the Mann-Whitney U test was used. When more than two groups were compared, the Kruskal-Wallis test was used. If a significant difference was found, the Mann-Whitney U test was used to determine the differences between each pair of groups. For comparisons within the same individuals, the Wilcoxon signed-rank test was used. Throughout, we report 2-tailed probability values that were considered significant when <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. LIGHT and its receptors in experimental HF
To characterize LIGHT and its receptors in HF, we first examined myocardial gene expression of LIGHT, LTβR, and HVEM by real-time RT-PCR in a rat model of post-infarction HF; 2, 7, and 28 days after induction of MI, comparing HF rats (n=5) with sham-operated rats (n=4). As shown in Fig. 1A, LIGHT mRNA levels were markedly elevated in the infarcted area throughout the study period (~10-fold increase after 2 days), with a more modest rise in the non-ischaemic part of LV. These changes in LIGHT levels were accompanied by increased expression of its corresponding receptors in the infarcted area, of which LTβR was significantly increased at all time points after MI (~10-fold increase after 7 days, Fig. 1B), while HVEM gene expression showed a more gradual increase reaching the highest levels 28 days post-MI (~15-fold increase), representing a chronic stage of HF in this model (Fig. 1C). The gene expression of HVEM was also increased in the non-ischaemic part of the LV, reaching statistical significance at the end of the observation period (~2.5-fold after 28 days, Fig. 1C). Gene expression of LTβR also increased in this part of the LV, but although significant, the increase was rather modest (Fig. 1B).


Figure 01
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  		Fig. 1 Gene expression of LIGHT (A), lymphotoxin-β receptor (LTβR) (B), and HVEM (C) in non-ischaemic left ventricle (LV) and infarcted area (INF) from rats 2, 7, and 28 days (d) after induction of myocardial infarction (MI). LV of sham-operated rats (hatched bars) served as control. mRNA levels were quantified by real-time RT-PCR and are presented relative to the gene expression of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are given as mean±SEM. *p<0.05 versus sham-operated rats.

 
Immunohistochemical analyses revealed the presence of myocardial LIGHT, HVEM, and LTβR immunoreactivities in cardiomyocytes, vascular smooth muscle cells, and endothelial cells of sham-operated rat hearts, with a similar pattern in the non-ischaemic part of LV in HF rats (data not shown). Strong LIGHT, HVEM, and LTβR immunoreactivities were also localized to surviving cardiomyocytes, vascular smooth muscle cells, and endothelial cells within the infarcted area, accompanied by LIGHT, HVEM, and LTβR immunostaining of infiltrating cells (i.e., leukocytes and fibroblasts/myelofibroblasts) in the acute phase 7 days post-MI, and for LTβR also in the more chronic phase (i.e., 28 days post-MI) (data not shown).

3.2. Gene expression of LIGHT and its receptors in endothelial cells and cardiomyocytes in experimental HF
To further characterize the expression of LIGHT and its receptors in the chronic phase of experimental HF, we examined the expression of these mediators in the non-ischaemic LV by separating this part of the LV from HF rats (n=7) and sham rats (n=7) into cardiomyocytes, endothelial cells, and fibroblasts, 56 days after the induction of MI (or sham operation). The up-regulation of HVEM in this part of the LV during the chronic phase of HF (i.e., ≥28 days after MI) seems to reflect enhanced expression within cardiomyocytes (1.0±0.12 versus 2.1±0.35; p=0.014) and endothelial cells (1.0±0.22 versus 2.5±0.50; p=0.014), but not within fibroblasts (1.0±0.08 versus 1.0±0.13). In contrast, we found no significant up-regulation of LIGHT or LTβR in any particular cellular subtype in this part of the LV after 56 days (data not shown).

3.3. LIGHT and its receptors in human HF
We next examined the expression of LIGHT, HVEM and LTβR by flow cytometry in cryopreserved PBMC from 14 HF patients (7 with ischaemic and 7 with idiopathic dilated cardiomyopathy; 13 in NYHA classes III-IV and 1 in NYHA functional class II) and in 14 healthy controls. T-cells (Fig. 2), but not monocytes (data not shown), showed significantly enhanced expression of LIGHT in HF patients with a similar pattern in ischaemic and non-ischaemic cardiomyopathies. Conversely, a significantly increased proportion of monocytes, but not T-cells (data not shown), expressed HVEM and LTβR in HF patients, although the difference in LTβR expression was rather modest (Fig. 2). In contrast to the increased proportion of LIGHT+ T-cells, there were no differences in plasma levels of LIGHT between HF patients (n=121) and sex- and age-matched healthy individuals (n=20) (13.4±0.7 pg/ml versus 11.9±1.0 pg/ml, for HF patients and controls, respectively; p=0.65), with no relation to aetiology or the severity of HF (data not shown). Finally, immunohistochemical analyses revealed the presence of myocardial LIGHT, HVEM, and LTβR immunoreactivities in cardiomyocytes, vascular smooth muscle cells, and endothelial cells of both non-failing and failing hearts (Fig. 3). However, we did use specific antibodies against the different cell types, and the data should be interpreted with some caution. When performing more quantitative estimates using Western blot analyses, HVEM (Fig. 3), but not LIGHT and LTβR (data not shown), was found to be strongly up-regulated in failing (n=6) as compared with non-failing myocardium (n=10) (~3.8-fold increase).


Figure 02
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  		Fig. 2 Expression of LIGHT in CD3+ T-cells (Panel A), HVEM in CD14+ monocytes (Panel B), and lymphotoxin-β receptor (LTβR) in CD14+ monocytes (Panel C) in heart failure (HF) patients and healthy controls. Bars represent mean±SEM values. The corresponding flow charts show expression of LIGHT, HVEM, and LTβR in CD3+ T-cells (LIGHT) and CD14+ monocytes (HVEM and LTβR) in one representative HF patient and one healthy control. *p<0.05 versus controls.

 


Figure 03
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  		Fig. 3 The expression of LIGHT and its receptors in human hearts. The upper panels show representative photomicrographs demonstrating LIGHT (A), HVEM (B), and lymphotoxin-β receptor (LTβR) (C) immunostaining of a failing explanted human heart. LIGHT, HVEM, and LTβR immunostaining was seen in cardiomyocytes (thick arrows) and in vascular smooth muscle and endothelial cells (thin arrows). Panel D is a negative control demonstrating no immunoreactivity when the primary antibody was omitted. All sections were counterstained with haematoxylin. Magnification x200. The lower panels show representative Western blot of HVEM and the housekeeper β-tubulin in explanted LV tissue obtained from HF patients (p) and donors (d). The bar graph presents the mean±SEM out of six donors and ten HF patients. *p<0.05 versus donors.

 
3.4. Potential effects of LIGHT in HF
The major finding of the study so far was the significant up-regulation of HVEM within the myocardium both in human and experimental HF, with enhanced expression in endothelial cells in the non-ischaemic part of LV as a prominent feature in experimental HF. To map any possible pathogenic consequences of this pattern, we examined the effect of rhLIGHT on the release of IL-6 in endothelial cells, a cytokine implicated in the myocardial remodelling process both during acute and chronic HF [20]. As shown in Fig. 4, rhLIGHT significantly increased IL-6 levels in HUVECs as shown both at the mRNA and protein levels. HVEM was also up-regulated on monocytes in HF patients and we therefore finally examined the ability of rhLIGHT to promote IL-6 release in these cells. While there was no increase in IL-6 levels in healthy controls (n=7), LIGHT induced a 2.5-fold increase in IL-6 release in PBMC from HF patients (n=11) after culturing for 20 h. This selectively inflammatory effect of LIGHT in PBMC from HF patients seemed in some degree to be restricted to IL-6. Thus, although we observed some LIGHT-mediated effects on IL-8, MCP-1, and TNF{alpha}, the increase did not reach statistical significance in either HF patients or controls (data not shown).


Figure 04
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  		Fig. 4 Effects of recombinant human LIGHT (100 ng/ml) on IL-6 expression in human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMC). Panel A shows gene expression of interleukin (IL)-6, quantified by real-time quantitative RT-PCR and relative to GAPDH, and Panel B shows the release of IL-6 protein from HUVEC measured at different time points (hours, h) after stimulation in 5 independent experiments. The comparison was made between the stimulated and the unstimulated levels in each experiment by the Wilcoxon rank sum test for paired data. Panel C shows the release of IL-6 protein from PBMC in 11 HF patients and 7 healthy controls after culturing for 20 h. Data are presented as mean±SEM. *p<0.05 and **p<0.01 versus unstimulated cells (Unstim).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study we show myocardial expression of LIGHT and its receptors in both experimental and clinical HF, with enhanced expression of HVEM as the most prominent finding. In human HF, HVEM was also up-regulated in circulating monocytes, accompanied by increased expression of LIGHT in circulating T-cells. Finally, we show that LIGHT markedly induced IL-6 expression in endothelial cells as well as in PBMC from HF patients.

Studies in animal models suggest that LIGHT signalling pathways may be crucial for the development of various autoimmune disorders such as inflammatory bowel disease, nephritis, diabetes mellitus, and arthritis [10,11]. However, while several studies have examined the pathogenic role of LIGHT in a variety of animal models [10], there are few reports on the role of this cytokine in human disorders. Recently, enhanced LIGHT expression was found in human atherosclerotic disorders, potentially promoting lipid accumulation, matrix degradation, and thrombus formation [12,13]. Herein we show increased expression of LIGHT and its receptors in human HF both within the failing myocardium (i.e., HVEM), and in circulating T-cells (i.e., LIGHT) and monocytes (i.e., HVEM and LTβR). This up-regulation did not seem to be restricted to those with ischaemic cardiomyopathy, suggesting that the increased expression of LIGHT in chronic HF is not merely a reflection of atherosclerosis in these patients. Moreover, our studies in experimental HF suggest enhanced myocardial HVEM and LIGHT expression not only in the infarcted area, but also within cardiomyocytes (i.e., LIGHT and HVEM) and endothelial cells (i.e., HVEM) of the non-ischaemic part of LV, further suggesting a link between these mediators and myocardial remodelling.

LIGHT seems to be predominantly expressed in lymphoid tissues and on the surface of immature dendritic cells and activated T-cells [9]. In the present study we show an increased proportion of LIGHT+CD3+ T-cells in HF patients, further supporting that T-cell activation is a characteristic feature of chronic HF [17]. Moreover, this up-regulation of LIGHT on T-cells was accompanied by increased expression of one of its receptors (i.e., HVEM) on both circulating monocytes and within the failing myocardium. Earlier studies have shown that HVEM mRNA is found in multiple organs and tissues, with particularly high levels in lymphocyte-rich organs [21]. Our findings in experimental HF suggest that this receptor can also be expressed in cardiomyocytes, with particularly high expression in the failing myocardium. LIGHT has been reported to promote several effects relevant to HF, such as production of inflammatory cytokines, matrix degradation, and increased production of reactive oxygen species in leukocytes, endothelial cells, and fibroblasts [13,16,22]; some of these effects seem to involve LIGHT-HVEM interaction. Furthermore, studies in animal models suggest that LIGHT-HVEM interaction may play a major role in inflammatory bowel disease, allograft rejection, and graft-versus-host disease [9]. Future studies should attempt to clarify whether LIGHT-HVEM interaction could also be involved in the progression of chronic HF.

IL-6 and related cytokines have been implicated in the development of myocardial remodelling through enhanced stimulation of their common receptor subunit gp130 that is expressed on cardiomyocytes [20]. Herein we show enhanced HVEM expression in endothelial cells in the non-ischaemic part of the LV in experimental HF, and with relevance to this finding, we found that LIGHT markedly induced IL-6 expression in HUVEC as assessed both at the mRNA and protein levels. Moreover, while LIGHT had no effect on IL-6 release in PBMC from healthy controls, it strongly increased the release of IL-6 in PBMC from HF patients, potentially at least partly reflecting increased proportion of LTβR+CD14+ and in particular of HVEM+CD14+ monocytes in HF patients. Independent of the mechanisms, our findings of a LIGHT-mediated IL-6 response in PBMC and endothelial cells, may further support a role for LIGHT in the progression of HF.

LIGHT has previously been shown to promote atherogenesis [12,13]. Our findings in the present study suggest that this TNF superfamily ligand could also be involved in the progression of chronic HF, independent of the presence of CAD. These pathogenic effects could involve interaction between T-cells and endothelial cells as well as IL-6-related mechanisms. However, future studies will have to delineate more precisely the role of LIGHT/HVEM/LTβR interaction in human HF.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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