Pulmonary hypertension (PH) is a rare disease characterized

Transcription

1 Plasma Levels of Receptor for Advanced Glycation End-Products and High-Mobility Group Box 1 in Patients With Pulmonary Hypertension Summary Satoshi Suzuki, 1 MD, Kazuhiko Nakazato, 1 MD, Koichi Sugimoto, 1 MD, Akiomi Yoshihisa, 1 MD, Takayoshi Yamaki, 1 MD, Hiroyuki Kunii, 1 MD, Hitoshi Suzuki, 1 MD, Shu-ichi Saitoh, 1 MD, and Yasuchika Takeishi, 1 MD An increase of pulmonary artery pressure in patients with pulmonary hypertension (PH) results in right ventricular failure and ultimately death. High-mobility group box 1 (HMGB1), a nuclear protein, acts as a pro-inflammatory cytokine by activating receptor for advanced glycation end-products (RAGE) in the extracellular environment. The purpose of this study was to examine the clinical significance of circulating levels of HMGB1 and RAGE in patients with PH. Plasma levels of HMGB1 and soluble RAGE were measured in 27 patients with PH (14 with pulmonary arterial hypertension [PAH], 13 with chronic thromboembolic pulmonary hypertension [CTEPH]) and 30 normal subjects as control. There was no difference in the plasma levels of HMGB1 between the PH patients and the control subjects. However, plasma levels of soluble RAGE were significantly higher in the patients with PH than in the controls (P < 0.001). Plasma soluble RAGE levels were higher in PAH (P < 0.001) and CTEPH (P < ) than in the controls. In addition, there was a statistically significant positive correlation between pulmonary artery pressure and plasma levels of soluble RAGE (r = 0.403, P < ). In the CTEPH patients, soluble RAGE levels were reduced after balloon pulmonary angioplasty (P < 0.001). Plasma levels of soluble RAGE, but not HMGB1, might be a novel marker that reflects the pathological condition in patients with PH. (Int Heart J 2016; 57: ) Key words: Pulmonary artery hypertension, Pattern-recognition receptor, Inflammation, Pathological marker Pulmonary hypertension (PH) is a rare disease characterized by elevated pulmonary arterial pressure (PAP) due to increased vasoconstriction and remodeling of the pulmonary microvasculature, and eventually leads to right ventricular failure and ultimately death. 1,2) Chronic thromboembolic pulmonary hypertension (CTEPH) is also recognized as a tragic disease if left untreated. 3) Development of PH involves the complex interactions of multiple effectors. The primary form of PH is classified as idiopathic pulmonary artery hypertension (PAH), while the secondary disease state, including congenital heart disease with systemic-to-pulmonary shunts, forms pulmonary vascular disease resembling idiopathic PAH in histological changes. This suggests a degree of commonality in the mechanisms of disease progression. 4) Although an updated classification of PAH has recently been reported, 5) the pathological condition leading to PH is different by each origin. Proteomic analysis in surgical samples of lung tissues from patients with PH has indicated increased expression of some proteins including receptor for advanced glycation endproducts (RAGE). 6) Moreover, protein and mrna levels of RAGE of pulmonary artery smooth muscle cells (PASMC) in PH patients are increased compared to that in healthy patients, and RAGE expression in PASMC is correlated with the severity of PH. 7) RAGE is one of the pattern-recognition receptor families that have multiple ligands, which are not only advanced glycation end-products (AGEs) but also S100/calgranulins, amyloid-β peptide, and high-mobility group box 1 (HMGB1). Editorial p.xxx HMGB1 is a nuclear protein that stabilizes nucleosome structure and regulates gene transcription. 8) Upon release into the extracellular environment, HMGB1 acts as a pro-inflammatory cytokine by binding to RAGE and toll-like receptors (TLRs). 9) Moreover, HMGB1, as well as RAGE, is ubiquitously expressed in various organs. A recent report revealed that HMGB1 is increased in patients with PAH, and HMGB1 contributes to the pathogenesis of PH via TLR-4. 10) However, the relation of HMGB1 and RAGE in PH patients remains unclear. Thus, the aim of the present study was to examine the clinical significance of circulating levels of HMGB1 and RAGE in patients with PH. From the 1 Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan. Address for correspondence: Satoshi Suzuki, MD, Department of Cardiology and Hematology, Fukushima Medical University, 1 Hikarigaoka, Fukushima , Japan. ssatoshi@fmu.ac.jp Received for publication May 7, Revised and accepted September 1, Released in advance online on J-STAGE March 11, (This is Advance Publication.) All rights reserved by the International Heart Journal Association. 234

2 Vol 57 No 2 HMGB1 AND RAGE IN PH 235 Methods Study subjects and protocol: The plasma levels of soluble RAGE and HMGB1 were measured in 27 patients (mean age, 54.4 years old, 14 in PAH and 13 in CTEPH) admitted to Fukushima Medical University Hospital for diagnosis and treatment and whose mean PAP exceeded 25 mmhg. Thirty normal subjects, with no abnormalities detected on physical examination, electrocardiogram, chest x-rays, and echocardiography served as controls (mean age, 59.0 years old). The PAH patients consisted of 9 with idiopathic PAH and 5 with congenital heart disease with systemic to pulmonary circulation shunt flow (3 with atrial septal defect, 2 with ventricular septal defect). Written informed consent was obtained from all study subjects. The study complied with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Fukushima Medical University. We evaluated body height, body weight, blood samples examination, echocardiographic parameters, and medications at baseline. Estimated glomerular filtration rate (egfr) was measured by the Modification of Diet in Renal Disease (MDRD) formula. Echocardiography was performed blindly by an experienced echocardiographer using standard techniques. Plasma levels of soluble RAGE and HMGB1 were measured using commercially available ELISA kits (RAGE, R&D Systems, Minneapolis, MN; HMGB1, SHINO-TEST CORPORATION, Sagamihara, Japan). Two-dimensional echocardiographic images were acquired from the parasternal long and short axis and apical 4-chamber views. Left ventricular ejection fraction (LVEF) was calculated using a modified Simpson s method. Statistical analysis: Results are expressed as the mean ± standard deviation (SD) in normally distributed data, and skewed variables are presented as the median (inter-quartile range). Categorical variables are expressed as numbers and percentages. A P value of 0.05 was considered as statistically significant. The baseline characteristics among groups were determined by the unpaired Student t-test for continuous variables and by the chi-square test for discrete variables. If data were not distributed normally, the Mann Whitney U test was used. Comparison of plasma soluble RAGE levels before and after balloon pulmonary angioplasty was performed by a paired t-test. Statistical analysis was performed with a standard statistical program package (SPSS ver. 21.0, IBM, Armonk, NY, USA). Results Basic clinical characteristics: Comparisons of basic clinical characteristics between the PH patients and the control subjects are shown in Table I. There were more females in the PH group than in the normal control group. Body height, body weight, and body mass index were lower in the PH patients than in the normal control subjects. With respect to laboratory data, serum sodium and triglyceride levels were lower, and lactate dehydrogenase and median value of plasma brain natriuretic peptide (BNP) were higher in the PH patients than in the normal control subjects. In terms of echocardiographic data, left ventricular dimension was larger, and left ventricular systolic function was better in the PH patients than in the normal control subjects. However, the tricuspid regurgitation pressure gradient and inferior vena cava diameter were higher in the PH patients than in the normal control subjects. Diuretics and warfarin were administered more frequently in the PH patients than in the normal control subjects. There was no difference in plasma levels of HMGB1 between the PH patients and the control subjects (10.0 ± 4.7 versus 11.6 ± 8.2 ng/ml, P = ) (Figure 1A). However, the plasma levels of soluble RAGE were significantly higher in the patients with PH than in the controls (1,836.9 ± versus ± pg/ml, P < ) (Figure 1B). Comparisons among the PH patients: The PH patients were divided into two groups based on the primary disease: PAH and CTEPH. As shown in Figure 2, plasma soluble RAGE levels were higher in PAH (P < 0.001) and CTEPH (P < ) than those in the controls. We evaluated and compared the clinical characteristics of the PAH and CTEPH patients. As shown in Table II, the PAH patients were younger than the CTEPH patients (P = ). Anemia was more severe and the plasma BNP level was slightly higher in the PAH patients than in the CTEPH patients. Echocardiographic data did not show significant differences between the two groups. The use of warfarin was higher in the CTEPH patients (100%) than in the PAH patients (P = ). With respect to vasodilators for PH, there was no difference in the use of prostanoids between these two groups, whereas the use of phosphodiesterase-5 inhibitors or endothelin receptor antagonists was higher in the PAH patients than in the CTEPH patients (P = and P = , respectively). Next, we examined the correlation between the degree of tricuspid valvular regurgitation pressure gradient (TR-PG) and plasma levels of soluble RAGE and HMGB1. As shown in Figure 3, plasma soluble RAGE levels showed a significantly positive correlation with TR-PG (r = 0.403, P < ), but there was no significant correlation between plasma HMGB1 levels and TR-PG. We also examined the changes in plasma levels of soluble RAGE before and after balloon pulmonary angioplasty (BPA) in the CTEPH patients. BPA was undertaken in 11 of the 13 CTEPH patients, and plasma samples were collected just before and immediately after the BPA procedure. As shown in Figure 4, plasma levels of soluble RAGE were significantly decreased immediately after BPA in the 11 patients with CTEPH (3,005.4 ± 1,026.8 versus 2,206.1 ± pg/ml, P < 0.001). However, mean PAP before and after BPA and the degree of PAP change were not correlated with the soluble RAGE level (data not shown). Discussion In the present study, we have showed that plasma soluble RAGE levels might be more important for the assessment of PAH severity than HMGB1. HMGB1 is ubiquitously expressed in all vertebrate nuclei, and, when released into extracellular conditions, it binds to RAGE or TLRs, both of which are recognized as patternrecognition receptors. Activation of RAGE or TLRs by HMGB1 induces the release of pro-inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α, 11,12) and this inflammatory activation plays a prominent role in the develop-

3 236 SUZUKI, ET AL Int Heart J March 2016 Table I. Comparison of Clinical Characteristics Between Control and PH Patients Control (n = 30) PH (n = 27) Age (years) 59.0 ± ± Gender (male/female) 19 / 11 5 / Body height (cm) ± ± Body weight (kg) 73.7 ± ± Body mass index (kg/m 2 ) 27.3 ± ± Hypertension (n, %) 19 (63%) 13 (48%) Diabetes mellitus (n, %) 10 (33%) 9 (33%) Hyperlipidemia (n, %) 15 (50%) 15 (56%) Blood sample data White blood cell (/ml) 6,152 ± 1,995 6,352 ± 2, Hemoglobin (g/dl) 14.0 ± ± Hematocrit (%) 41.7 ± ± Total protein (g/dl) 7.01 ± ± Albumin (g/dl) 3.94 ± ± AST (IU/L) 26.5 ± ± ALT (IU/L) 26.1 ± ± LDH (IU/L) ± ± Total bilirubin (mg/dl) 0.83 ± ± BUN (mg/dl) 14.5 ± ± Serum creatinine (mg/dl) 0.87 ± ± Estimated GFR (ml/minute/1.73 m 2 ) 71.9 ± ± Na (meq/l) ± ± K (meq/l) 4.15 ± ± Uric acid (mg/dl) 6.00 ± ± Triglycerides (mg/dl) ± ± LDL cholesterol (mg/dl) ± ± HDL cholesterol (mg/dl) 54.1 ± ± Fasting blood glucose (g/dl) ± ± HbA1c (%) 5.51 ± ± BNP * (pg/ml) 17.3 (24.1) (301.4) hs-crp * (mg/dl) 0.05 (0.53) 0.11 (0.82) Echocardiography IVS (mm) 10.7 ± ± LVEDD (mm) 47.0 ± ± 7.2 < LVESD (mm) 30.9 ± ± 6.2 < LVFS (%) 34.7 ± ± LVEDV (ml) 91.0 ± ± 27.2 < LVEF (%) 62.7 ± ± Left atrial volume (ml) 55.4 ± ± Left atrial diameter (mm) 36.1 ± ± TR-PG (mmhg) 11.2 ± ± 26.9 < IVC (mm) 12.7 ± ± Medication (n, %) β-blocker 4 (13%) 6 (22%) Diuretics 1 (3%) 16 (59%) < Ca channel blocker 8 (27%) 6 (22%) ACE inhibitor 1 (3%) 4 (15%) ARBs 10 (33%) 6 (22%) Statin 7 (23%) 7 (26%) Warfarin 5 (17%) 19 (70%) < Vasodilator for PAH 0 (0%) 19 (70%) < AST indicates aspartate transaminase; ALT, alanine transaminase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; GFR, glomerular filtration rate; LDL, low-density lipoprotein; HDL, high-density lipoprotein; BNP, brain natriuretic peptide; hs-crp, high-sensitive C-reactive protein; IVS, interventricular septum thickness; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; LVFS, left ventricular fractional shortening; LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection fraction; TR-PG, tricuspid regurgitation pressure gradient; IVC, inferior vena cava; ACE, angiotensin-converting enzyme; and ARB, angiotensin II receptor blocker. * Skewed data are reported as median (inter-quartile range). P ment of PAH. 13,14) We have reported that cardiac-specific overexpression of HMGB1 in mice suppresses myocardial damage after myocardial infarction and progression of cardiac hypertrophy after transverse aortic constriction ) However, the clinical significance of HMGB1 and one of its receptors, RAGE, has yet to be clarified. Recent reports revealed that HMGB1 promotes the development of PAH, and the inhibition of HMGB1 attenuates PAH

4 Vol 57 No 2 HMGB1 AND RAGE IN PH 237 Figure 1. Comparisons of plasma levels of soluble receptor for high-mobility group box 1 (HMGB1) (A) and advanced glycation-end products (RAGE) (B) between patients with pulmonary hypertension (PH) and control subjects. # P < versus control. Figure 2. Comparison of plasma soluble RAGE level among 3 groups. # P < versus control. PAH indicates pulmonary artery hypertension; and CTEPH, chronic thromboembolic pulmonary hypertension. Figure 3. Correlation between the degree of tricuspid valvular regurgitation pressure gradient (TR-PG) and plasma levels of soluble RAGE (A) and HMGB1 (B).

5 238 SUZUKI, ET AL Int Heart J March 2016 Figure 4. Comparison of soluble plasma RAGE levels before and after balloon pulmonary angioplasty (BPA) in CTEPH patients. Table II. Comparison of Patient Clinical Characteristics Between PAH and CTEPH PAH (n = 14) CTEPH (n = 13) Age (years) 46.8 ± ± Gender (male/female) 2 / 12 3 / Body mass index (kg/m 2 ) 22.8 ± ± Blood sample data Hemoglobin (g/dl) 12.9 ± ± Hematocrit (%) 39.0 ± ± Albumin (g/dl) 3.70 ± ± BUN (mg/dl) 15.3 ± ± Serum creatinine (mg/dl) 0.70 ± ± Estimated GFR (ml/minute/1.73 m 2 ) 74.0 ± ± Na (meq/l) ± ± K (meq/l) 4.02 ± ± BNP * (pg/ml) 97.0 (124.2) (498.5) hs-crp * (mg/dl) 0.21 (1.04) 0.11 (0.39) Echocardiography IVS (mm) 10.0 ± ± LVEDD (mm) 38.0 ± ± LVFS (%) 38.4 ± ± LVEDV (ml) 52.4 ± ± LVEF (%) 67.1 ± ± Left atrial volume (ml) 46.0 ± ± TR-PG (mmhg) 71.2 ± ± IVC (mm) 15.5 ± ± Medication (n, %) β-blocker 4 (29%) 2 (15%) Diuretics 10 (71%) 6 (46%) Warfarin 6 (43%) 13 (100%) Prostanoids 6 (43%) 6 (46%) Phosphodiesterase-5 inhibitors 9 (64%) 2 (15%) Endothelin receptor antagonists 10 (71%) 3 (23%) Abbreviations as in Table I. P in rats given monocrotaline. 18,19) They demonstrated that anti- HMGB1 antibody attenuated the increase of inflammatory activation in bronchoalveolar lavage fluid, thickening of the pulmonary artery, and right ventricular systolic pressure and hypertrophy in monocrotaline-injected rats. 18,19) Thus, HMGB1 has a critical role in the progression of PAH. Bauer, et al reported the detailed mechanisms for this by in vitro and in vivo examinations. One report by Bauer, et al suggested that HMGB1 inhibited human pulmonary artery endothelial cell migration induced via TLR4 signaling by vascular endothelial growth factor stimulation and hypoxia. 20) Another examination by Bauer, et al reported that HMGB1 levels in idiopathic PAH patients significantly increased with the increase in severity of mean PAP. 9) In their report, PAH, right ventricular hypertrophy, and muscularized arterioles in the pulmonary artery were reduced by the HMGB1 neutralizing antibody in a hypoxia-induced mouse model. Also, the development of hypoxia-induced PAH was significantly attenuated in TLR4 knockout mice, but not in RAGE knockout mice, and exacerbation of hypoxia-induced PAH was suppressed in TLR4 knockout mice

6 Vol 57 No 2 HMGB1 AND RAGE IN PH 239 by the addition of exogenous HMGB1. Their results suggest that TLR4 might have more essential roles in the progression of PAH than RAGE, despite the fact that HMGB1 binds to both TLR4 and RAGE. Some other reports suggest an association of PAH with RAGE. 6,7,21) Furthermore, RAGE expression from PASMC increased with the severity of PAH but was not increased in organs other than the lung. 7) Moreover, it was demonstrated that RAGE inhibition decreased proliferation in PASMC in human tissue samples, and decreased the degree of PAH, right ventricular hypertrophy, and pulmonary artery remodeling in a monocrotaline-induced PAH rat model. From their results, they concluded that RAGE might have some critical roles in PAH progression through a signal transducer and activator of transcription 3, bone morphogenetic protein receptor 2, and peroxisome proliferator-activated receptor gamma. To trigger RAGE activation, however, S100A4 (known as Mts1, metastasin, and calvasculin) has a crucial role, which is one of the important ligands for RAGE as well as for HMGB ) Another report found that S100A4/Mts1-overexpressing female mice exhibit increased right ventricular pressure and pulmonary vascular remodeling. This suggests that S100A4 might have more essential roles in PAH progression through RAGE activation than HMGB1. Moser, et al reported that the soluble RAGE level was significantly higher in IPAH and CTEPH patients than in controls, and the HMGB1 level was higher in CTEPH patients, but not in idiopathic PAH patients. 25) From their report and our results, the essential role of HMGB1 and soluble RAGE might be different between idiopathic PAH and CTEPH patients. Few reports have demonstrated an association between RAGE, HMGB1 and CTEPH, therefore, the role of HMGB 1 and soluble RAGE in CTEPH patients might be an important research target in the future. In the present study, we demonstrated that HMGB1 did not show a significant increase in the PH patients compared to the control subjects, and soluble RAGE might be an essential marker that indicates the degree of both PH severity and therapeutic effects of balloon angioplasty in CTEPH. Study limitations: Several limitations of this study should be addressed. First, we could not check all ligands for RAGE such as AGEs, amyloid-β peptide, and S100A4, or the expression of TLRs. Also, it was not clear as to what represented the most important clinical significance for PH in all ligands for RAGE. Second, we demonstrated a significant reduction of soluble RAGE level after BPA in CTEPH patients, however, the hemodynamic effect after BPA was different in each patient. The significance of the soluble RAGE level reduction was not clear, therefore, this point should be clarified in future research. Third, the sample size was small and the study was conducted at a single center. Therefore, we cannot extrapolate our results to the general heart failure population. Hence, prospective studies with a larger population are needed. Conclusions: Plasma levels of soluble RAGE, but not HMGB1, were higher in the PH patients than in the controls, especially in those with PAH and CTEPH. The level of plasma soluble RAGE might be a novel marker that reflects the pathological condition in patients with PH. Disclosure Conflict of interest: Satoshi Suzuki and Akiomi Yoshihisa belong to an endowed department (affiliation with Fukuda Denshi Co., Ltd). The remaining authors have no such affiliations. References 1. Simonneau G, Robbins IM, Beghetti M, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2009; 54: S (Review) 2. Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet 2003; 361: (Review) 3. Riedel M, Stanek V, Widimsky J, Prerovsky I. Longterm followup of patients with pulmonary thromboembolism. 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