Published ahead of print on October 25, 2007, doi:10.1164/rccm.200703-506OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200703-506OC
Raised LIGHT Levels in Pulmonary Arterial HypertensionPotential Role in Thrombus Formation1 Research Institute for Internal Medicine, 2 Department of Cardiology, 3 Section of Endocrinology, and 4 Section of Clinical Immunology and Infectious Diseases, Medical Department, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, Oslo, Norway Correspondence and requests for reprints should be addressed to Kari Otterdal, M.Sc., Research Institute for Internal Medicine, Rikshospitalet-Radiumhospitalet Medical Center, University of Oslo, N-0027 Oslo, Norway. E-mail: kari.otterdal{at}medisin.uio.no
Rationale: Thrombus formation and inflammation are involved in the pathogenesis of pulmonary arterial hypertension (PAH), and LIGHT (Lymphotoxin-like Inducible protein that competes with Glycoprotein D for Herpesvirus entry mediator on T lymphocytes) has been shown to promote vascular inflammation. Objectives: We sought to investigate the role of the tumor necrosis factor superfamily ligand LIGHT in the pathogenesis of PAH. Methods: We studied 73 patients with severe PAH and 10 control subjects. LIGHT and pro- and antithrombotic markers were assessed by enzyme immunoassays. Measurements and Main Results: (1) Patients with idiopathic PAH (n = 21), patients with PAH related to risk factors or associated conditions (n = 31), and those with chronic thromboembolic PAH (n = 21) all had raised serum levels of LIGHT compared with control subjects (n = 10). (2) LIGHT levels in femoral artery were significantly related to mortality in the patients with PAH. (3) Immunostaining of LIGHT and its receptors was seen in alveolar macrophages, vascular smooth muscle cells, and endothelial cells in lungs from patients with PAH. (4) Thirteen patients received prostacyclin infusion (3 mo), and all showed hemodynamic improvement, accompanied by decreased LIGHT levels. (5) Prostacyclin abolished the release of LIGHT from activated platelets in vitro, suggesting that the decrease in LIGHT during prostacyclin therapy could involve direct effects on platelets. (6) LIGHT increased tissue factor and plasminogen activator inhibitor type 1 and decreased thrombomodulin levels in endothelial cells, inducing a prothrombotic state in these cells. Conclusions: Our findings suggest prothrombotic effects of LIGHT in PAH involving endothelium-related mechanisms, potentially contributing to the progression of this disorder.
Key Words: endothelium • inflammation • platelets • prostacyclin
Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure leading to a progressive right-sided heart failure (HF) and ultimately death or need for transplantation (1). The pathogenic hallmark of PAH is increased pulmonary vascular resistance due to vasoconstriction, vascular cell proliferation and remodeling, and thrombus formation (1). A number of mediators seem to be involved in these processes (1, 2), and increasing amounts of evidence suggest that inflammatory mechanisms could also contribute to the pathogenesis of PAH (3). Thus, elevated circulating levels of inflammatory cytokines (e.g., IL-1 and IL-6) and enhanced pulmonary expression of several chemokines have been found in patients with PAH (4–6). Moreover, we have recently demonstrated raised levels of soluble (s) CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, in PAH, possibly playing a pathogenic role by operating through an interaction between platelets and endothelial cells (7). We have recently suggested that LIGHT (Lymphotoxin-like Inducible protein that competes with Glycoprotein D for Herpesvirus entry mediator on T lymphocytes), another platelet-derived member of the TNF superfamily, could play a pathogenic role in atherogenesis and plaque destabilization, at least partly by its ability to promote vascular inflammation (8, 9). On the basis of these properties, we hypothesized that LIGHT also could be involved in the pathogenesis of PAH. In the present study, we investigated this hypothesis by differential experimental approaches, including clinical studies in patients with PAH and experimental studies in cell types with relevance to PAH (i.e., endothelial cells and platelets), particularly focusing on the ability of LIGHT to promote endothelium-mediated thrombus formation, representing an important pathogenic event in PAH.
Study Population We studied 73 patients with severe PAH in New York Heart Association (NYHA) functional classes III–IV (Table 1). The diagnosis of PAH was defined as mean pulmonary artery pressure (MPAP) of more than 25 mm Hg at rest, with a normal pulmonary capillary wedge pressure (PCWP;  12 mm Hg), indicating precapillary pulmonary hypertension. The study population was divided into three groups according to type of PAH (10): (1) patients with idiopathic PAH (IPAH; n = 21), diagnosed by the criteria of the National Institutes of Health Registry of Primary Pulmonary Hypertension; (2) patients with PAH related to risk factors or associated conditions (APAH; n = 31 [collagen vascular disease, n = 17; venoocclusive disease, n = 4 (all verified by autopsies); liver cirrhosis, n = 3; HIV infection, n = 2; congenital heart disease with right-to-left shunt, n = 5]); and (3) patients with chronic thromboembolic pulmonary hypertension (CTEPH) (n = 21) verified with pulmonary angiograms. Ten sex- and age-matched individuals (4 men and 6 women, 51 ± 15 yr) undergoing right-sided heart catheterization during electrophysiological studies served as control subjects. All control subjects had paroxysmal supraventricular arrhythmias, but otherwise normal hemodynamic function and myocardial structure. We also studied 31 patients with chronic HF (NYHA class II–IV; 15 with idiopathic and 16 with ischemic cardiomyopathy; 25 men and 6 women, 55 ± 11 yr) with stable HF for more than 6 months (median disease duration, 3 yr; range, 0.5–8 yr), characterized by pulmonary hypertension secondary to left ventricular failure (i.e., MPAP > 25 mm Hg [mean ± SD: 38 ± 7 mm Hg]) at rest combined with a PCWP of greater than15 mm Hg (range, 18–40 mm Hg), and a matched control group of 20 healthy individuals (17 men and 3 women, 56 ± 7 yr). The investigation conforms to the principles outlined in the Declaration of Helsinki. The regional ethics committee of Helseregion 2 approved the study, and informed consent was obtained from each subject.
Blood Sampling Protocol Blood samples were collected from the pulmonary artery, femoral artery, and peripheral vein (patients with chronic HF and their matched control group) into pyrogen-free blood collection tubes without any additives. The tubes were immediately immersed in melting ice and allowed to clot before centrifugation at 1,500 g for 10 minutes. Serum samples were stored at –80°C and thawed only once.
Endothelial Cell Culture
Platelet Preparation and Stimulation
Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction
Immunohistochemistry
Enzyme Immunoassays
Statistical Analysis
Serum Levels of LIGHT in Patients with PAH Patients with PAH, classified as IPAH (n = 21), APAH (n = 31), and CTEPH (n = 21), all had significantly raised serum levels of LIGHT as compared with control subjects (n=10) (Figures 1A and B). The differences were evident both in mixed venous (i.e., pulmonary artery) and in arterial blood (i.e., femoral artery). Patients with PAH (15.4 ± 1.4 vs. 13.1 ± 1.6 pg/ml, P < 0.05) and control subjects (5.2 ± 0.4 vs. 2.6 ± 0.4 pg/ml, P < 0.05) had significantly higher LIGHT levels in the femoral than in the pulmonary artery. Anticoagulant therapy could potentially attenuate the release of LIGHT from platelets, but we found no difference in LIGHT levels between patients using warfarin versus those who did not (pulmonary artery: 13.7 ± 2.1 vs. 13.3 ± 2.0 pg/ml, P = 0.36; femoral artery: 15.7 ± 2.8 vs. 16.1 ± 2.2 pg/ml, P = 0.70). There was no significant correlation between LIGHT levels and the magnitude of pulmonary hypertension in the patients with PAH (data not shown).
For comparison, we also analyzed LIGHT levels in peripheral venous blood from 31 patients with chronic HF with raised pulmonary pressure secondary to left ventricular failure (see METHODS), and interestingly, serum levels of LIGHT in these patients with pulmonary venous hypertension were comparable to those in 20 sex- and age-matched control subjects (Figure 1C), suggesting that the raised LIGHT level in PAH is not merely a marker of increased pulmonary pressure. In fact, the patients with pulmonary hypertension secondary to left ventricular failure all had an MPAP of greater than 25 mm Hg with a mean ± SD level of 38 ± 7 mm Hg, which is similar to the group of patients with CTEPH (MPAP, 42 ± 10 mm Hg).
LIGHT Levels in Relation to Mortality in Patients with PAH
Pulmonary Localization of LIGHT and Its Corresponding Receptors To further characterize the regulation of LIGHT in PAH, we examined the expression of LIGHT and its corresponding receptors (herpes virus entry mediator [HVEM] and lymphotoxin-β) in pulmonary tissue samples from autopsies of seven patients with PAH by immunohistochemistry. As shown in Figure 3, LIGHT immunoreactivity was seen within PAH lungs, with particularly strong immunostaining in alveolar macrophages, with some immunostaining in vascular smooth muscle cells, endothelial cells, and respiratory columnar epithelial cells; a similar pattern also seen for LIGHT's corresponding receptors. The autopsy material was unreliable, and any firm conclusion concerning thrombus formation and platelet-related LIGHT immunostaining could not be made.
The Effect of Recombinant Human LIGHT on Prothrombotic Mediators in HUVECs To map any pathogenic consequences of the raised LIGHT levels in PAH, we examined the ability of recombinant human (rh)LIGHT to modulate the expression of pro- (i.e., TF and PAI-1) and antithrombotic (i.e., TM and TF pathway inhibitor [TFPI]) mediators in endothelial cells. Although rhLIGHT dose-dependently decreased the expression of the antithrombotic mediator TM, it increased the expression of PAI-1, a potent inhibitor of fibrinolysis, with a similar pattern on mRNA and protein levels (Figure 4). Moreover, although rhLIGHT had no effect on gene expression of TF, it significantly increased the protein levels of this prothrombotic mediator (Figure 4), and notably, this increase in TF levels was not accompanied by any changes in its pathway inhibitor (i.e., TFPI; data not shown). Our findings may suggest an enhanced prothrombotic potential in endothelial cells when these cells are exposed to LIGHT. Indeed, when heparinized human plasma was added to the LIGHT-stimulated HUVEC cultures, we observed significantly enhanced thrombin formation as assessed by increased levels of the prothrombin fragments F1+2 in the supernatants (Figure 5).
Effect of Prostacyclin Treatment on Serum Levels of LIGHT in PAH Prostacyclin is used as a therapeutic agent in patients with PAH (13). In the present study, serum samples were available from 13 of the patients with PAH (IPAH, n = 9; APAH, n = 4) who received continuous prostacyclin infusion (mean epoprostenol dose, 26 ± 6 ng · kg–1 · min–1), before and after 3 months of therapy. All patients showed hemodynamic and functional improvement (data not shown), accompanied by a significant decline in serum levels of LIGHT, which was seen in all but two patients (Figure 6).
Effect of Prostacyclin on Platelets and HUVECs We have recently shown that, upon activation, platelets release significant amounts of LIGHT (9). To elucidate if the decline in serum LIGHT levels during prostacyclin therapy could involve direct effects on platelets, we examined the ability of prostacyclin (PGI2) to modulate the release of this cytokine from platelets activated by the thrombin-receptor activation peptide serine-phenylalanine-leucine-leucine-arginine-asparagine (SFLLRN). As shown in Figure 7, PGI2 nearly abolished the release of LIGHT when added to platelets 30 minutes before SFLLRN (10 and 100 µM) stimulation. In contrast, PGI2 did not significantly modulate the LIGHT-mediated changes in TF, PAI-1, and TM expression in HUVECs (data not shown).
In the present study, we demonstrate that patients with PAH have markedly elevated levels of the TNF superfamily ligand LIGHT in both arterial and venous blood, most probably reflecting both pulmonary (e.g., increased leukocyte activation when passing through the inflamed pulmonary circulation) and secondary systemic (e.g., systemic hypoxia and enhanced oxidative stress) pathology in this disorder. Moreover, our immunohistochemistry analyses may suggest that the pulmonary tissue in itself (e.g., alveolar macrophages) could contribute to the increased LIGHT levels in PAH. Furthermore, we show that rhLIGHT dose-dependently increased TF and PAI-1 and decreased TM levels in HUVECs, accompanied by enhanced thrombin formation in these cells. The pathogenesis of PAH is a complicated, multifactorial process characterized by vasoconstriction and smooth muscle cell proliferation of the pulmonary arterioles, as well as in situ thrombosis of the small pulmonary arteries (1, 2). Such prothrombotic mechanisms seem not only to be operating in CTEPH but also in the other forms of PAH (1, 2, 14). Our findings in the present study may suggest that LIGHT could contribute to thrombus formation in PAH by its ability to induce a prothrombotic state in endothelial cells. In contrast to the patients with PAH, serum levels of LIGHT in patients with venous pulmonary hypertension secondary to left ventricular failure were comparable to those in their matched control group, further indicating that the raised LIGHT level in PAH is not merely a marker of increased pulmonary pressure but rather represents important pathogenic processes in this disorder. Abnormal thrombotic or thrombolytic activity due to endothelial dysfunction and platelet activation has been suggested to be important in the pathogenesis of PAH. Data from animal and human studies of pulmonary hypertension provide evidence for increased platelet activation, decreased soluble TM levels, and a net loss of fibrinolytic activity with excessive release of PAI-1 in this disorder (14–17). These abnormalities are suggestive of a shift of pulmonary vascular microenvironment toward a procoagulant, prothrombotic, and antifibrinolytic pattern (14). In the present study, we show that patients with PAH had markedly increased serum levels of LIGHT. Moreover, LIGHT stimulation of HUVECs did not only induced the release of the prothrombotic and antifibrinolytic mediators TF and PAI-1, respectively, but also decreased endothelial production of its natural anticoagulants (i.e., TM). We have recently shown that LIGHT may transform macrophages into a prothrombotic phenotype (8), and our findings in the present study indicate that similar mechanisms could be operating in endothelial cells. LIGHT has previously been suggested to induce vascular inflammation through its ability to increase chemokine release and expression of adhesion molecules in endothelial cells (9). If these LIGHT-mediated effects on endothelium-related inflammation and thrombus formation also are operating in vivo in patients with PAH within the pulmonary vasculature, they could potentially contribute to the pathogenesis of this disorder. However, measurement of circulating LIGHT levels may not necessarily reflect LIGHT levels within the lungs. Nonetheless, our immunohistochemical analyses of pulmonary tissue from patients with PAH showed strong LIGHT immunostaining in alveolar macrophages, with some immunostaining also in vascular smooth muscle cells and endothelial cells, with a similar pattern for LIGHT's corresponding receptors. Although these data do not prove increased pulmonary LIGHT expression in PAH, they show that LIGHT actually is present in the lungs of patients with PAH, potentially exerting harmful effects through interactions with its corresponding receptors. It may be argued that the LIGHT concentrations used in the in vitro experiments were much higher than the measured serum levels in patients with PAH. However, we have also previously shown significant LIGHT-mediated effects on the endothelium at 1 ng/ml (9), and compared with sCD40L, which also has inflammatory effects in PAH (7), LIGHT seems to be a more potent inducer of inflammation (9). It is not inconceivable that within an inflamed microenvironment, consisting of activated platelets, macrophages, and endothelium (all important cellular sources of LIGHT that may be operating within the lungs in patients with PAH), LIGHT levels could be comparable to those used in the present study, potentially inducing relevant pathophysiological effects. Recently, intravenous prostacyclin (i.e., epoprostenol) has been documented to improve exercise tolerance, hemodynamic measures, and survival in patients with PAH (13). Several reports have ascribed the benefit of prostacyclin therapy to platelet inhibition and improvement of endothelial dysfunction (14, 15, 18). In the present study, we show that the beneficial effect of prostacyclin therapy in PAH was accompanied by a significant decrease in serum levels of LIGHT. We have previously shown that, upon activation, platelets release significant amounts of LIGHT (9). Herein we show that PGI2 abolished the SFLLRN-mediated increase in LIGHT-release in platelet-rich plasma in vitro, suggesting that the decrease in LIGHT during prostacyclin therapy in PAH could involve direct effects on platelet activation. Interestingly, we have previously reported that prostacyclin therapy had no effect on sCD40L levels in the same PAH population (7). LIGHT is released from activated platelets in a gradual and long-lasting manner, showing a similar pattern as previously described for platelet release of sCD40L (9, 19). However, the difference in response to prostacyclin therapy suggests that the platelet-mediated release of these TNF superfamily ligands at least partly may be differently regulated. Nevertheless, enhanced levels of several platelet-derived vasoconstrictors and growth factors have previously been found in PAH (7, 20), and our findings suggest that these platelet-derived mediators also involve LIGHT. Recent studies suggest that inflammatory mediators, such as chemokines and TNF superfamily ligands (i.e., CD40L), could be involved in the pathogenesis of PAH, possibly also involving platelet-mediated inflammation (3, 7). LIGHT has previously been suggested to promote proliferation of vascular smooth muscle cells in graft arterial disease (21) and to induce vascular inflammation in relation to atherosclerotic disorders (9). Together with our demonstration of LIGHT-mediated prothrombotic effects in endothelial cells, these properties suggest that LIGHT should be added to the list of inflammatory and platelet-derived mediators that could contribute to disease progression in PAH. Although pulmonary vasoconstriction followed by remodeling of the pulmonary vessels are believed to be important primary events in the pathogenesis of PAH (1, 2), additional contributing responses, such as inflammation, which also include increased LIGHT levels, could contribute to the progression of this disorder, potentially representing part of a common final pathogenic pathway involved in all forms of PAH. However, although the present study may suggest a pathogenic role of LIGHT in PAH, further studies examining a larger number of patients as well as LIGHT expression within normal pulmonary tissue are needed before any firm conclusion can be drawn.
Supported by grants from the Norwegian Council on Cardiovascular Diseases, Research Council of Norway, the University of Oslo, Medinnova Foundation, Helse Sør, and Rikshospitalet-Radiumhospitalet Medical Center. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200703-506OC on October 25, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 29, 2007; accepted in final form October 22, 2007
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mRNA expression in lung biopsy specimens of primary pulmonary hypertension. Chest 1998;114(1, Suppl):50S–51S.
