Arthritis Care & Research Vol. 67, No. 12, December 2015, pp 1702–1711 DOI 10.1002/acr.22630 C 2015, American College of Rheumatology V

ORIGINAL ARTICLE

Association of Anemic Hypoxia and Increased Pulmonary Artery Systolic Pressure in Patients With Systemic Lupus Erythematosus KI-JO KIM,1 IN-WOON BAEK,2 CHONG-HYEON YOON,3 WAN-UK KIM,4

AND

CHUL-SOO CHO2

Objective. Pulmonary arterial hypertension (PAH) is a rare but serious complication of systemic lupus erythematosus (SLE). Chronic hypoxia is known to cause PAH resulting from pulmonary vascular remodeling. We investigated the association between anemic hypoxia and PAH in SLE patients. Methods. Systolic pulmonary artery pressure (PAP) was measured in 132 SLE patients by echocardiography. Increased PAP was defined as resting PAP > 40 mm Hg. Oxygen delivery (DO2) was estimated as the product of cardiac output and arterial oxygen content. Results. Of 132 patients, 17 (12.9%) had increased PAP, and these patients had significantly lower DO2 values than patients with normal PAP (P 5 0.002). The DO2 values inversely correlated with PAP values (g 5 20.308, P < 0.001) and plasma N-terminal pro–brain natriuretic peptide levels (g 5 20.323, P 5 0.001), but positively correlated with hemoglobin levels (g 5 0.402, P < 0.001). Compared to those with normal PAP, patients with increased PAP had significantly longer durations of anemia over the preceding 6–24 months. Patients with anemia of longer durations (‡3 months) in the preceding 6 months had a higher risk of increased PAP compared to those with shorter durations (P < 0.001). When SLE patients were divided into 3 groups according to hemoglobin and PAP, serum interleukin-6 (IL-6) levels increased across groups with higher PAP (P 5 0.001 for trend), but decreased across tertiles of hemoglobin levels (P 5 0.008 for trend). Conclusion. Our data indicate an association between chronic anemic hypoxia and increased PAP in SLE patients and suggest that increased IL-6 might participate in this process.

INTRODUCTION Pulmonary arterial hypertension (PAH) is a rare but serious cardiopulmonary manifestation of systemic lupus erythematosus (SLE) that carries a lower survival rate when compared to those without PAH (5-year survival rate 86% versus 96%) (1,2). The prevalence of PAH in SLE patients Study SC13IRMI0095. Supported by grants from the Rheumatology Research Foundation. 1 Ki-Jo Kim, MD: St. Vincent Hospital, The Catholic University of Korea, Suwon, Republic of Korea; 2In-Woon Baek, MD, Chul-Soo Cho, MD, PhD: Yeouido St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea; 3 Chong-Hyeon Yoon, MD, PhD: Uijeongbu St. Mary’s Hospital, The Catholic University of Korea, Uijeongbu, Republic of Korea; 4Wan-Uk Kim, MD, PhD: Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea. Address correspondence to Chul-Soo Cho, MD, PhD, Division of Rheumatology, Department of Internal Medicine, The Catholic University of Korea, Yeouido St. Mary’s Hospital, 10, 63-ro, Yeongdeungpo-gu, Seoul, 150-713, Republic of Korea. E-mail: [email protected]. Submitted for publication December 21, 2014; accepted in revised form May 19, 2015.

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is estimated to be between 0.5% and 14%, depending on the study population, study design, diagnostic modality, and criteria (3–7). A few studies have reported risk factors associated with the presence of PAH in patients with SLE. Some studies found a higher prevalence of Raynaud’s phenomenon (4,8,9) and antiphospholipid antibody (3,8,10–12), and shorter disease duration (4) in SLE patients with PAH compared to those without; however, these associations were not confirmed in other studies (6,7). Therefore, the clinical variables related to PAH in SLE patients and its underlying pathogenic mechanisms remain to be determined. PAH is characterized by vascular remodeling of the small pulmonary arteries leading to a progressive increase of pulmonary vascular resistance and right ventricular failure (13). Hypoxia is considered to play a critical role in this process. Elevation of pulmonary artery pressure (PAP) is seen in people who live at high altitude; the prevalence of PAH in people living at high altitude is much higher than that of sea-level residents (5–18% versus 0.0015–0.005%) (14,15). PAH is a common consequence of chronic hypoxic lung disease, including chronic obstructive pulmonary diseases (COPDs), interstitial lung disease, and cystic fibrosis (16). Hypoxia elicits pulmonary

Anemic Hypoxia With PAH in SLE

Significance & Innovations  Factors associated with pulmonary arterial hypertension (PAH) in systemic lupus erythematosus (SLE) patients and its underlying pathogenic mechanisms remain to be established. Oxygen delivery (DO2) is decreased in SLE patients with increased pulmonary artery pressure (PAP) and the value of DO2 is inversely correlated with PAP levels, but positively correlated with hemoglobin levels. Moreover, anemia of longer duration significantly increases the risk of PAP in SLE patients.  Increased serum interleukin-6 (IL-6) is associated with increased PAP levels, but with decreased hemoglobin levels in SLE patients. Chronic exposure to anemic hypoxia is responsible for the increased PAP in SLE patients, and IL-6 could play a contributory role in the development of PAH associated with anemia.

vasoconstriction by increasing potent vasoconstrictors such as angiotensin II, endothelin 1, serotonin, and thromboxane, some of which are expressed at high levels in pulmonary endothelial cells and smooth muscle cells (17). In addition, local hypoxia induces hypoxia-inducible factor activity, which activates the transcription of genes encoding multiple factors such as erythropoietin (EPO), hepatocyte growth factor, stem cell factors, stromal cell–derived factor 1a and vascular endothelial growth factor, and platelet-derived growth factor (18,19), which promotes the proliferation of fibroblasts and smooth muscle cells in situ and/or recruits circulating progenitor cells into hypoxic areas. All of these events act in concert to contribute to the structural remodeling of pulmonary vascular beds (17). Hypoxia results from conditions in which oxygen delivery (DO2) fails to satisfy the metabolic demand for oxygen. There are several types of tissue hypoxia caused by a decrease in oxygen saturation (hypoxic hypoxia), cardiac output (ischemic hypoxia), or hemoglobin concentration (anemic hypoxia), or an increase in the metabolic demands of the body (20). DO2 is the amount of oxygen delivered or transported to tissues in 1 minute (ml/minute/kg) and is dependent on pulmonary gas exchange, hemoglobin level, oxygen saturation, and cardiac output (20,21). PAH is reported to be associated with several kinds of anemia, including hemoglobinopathies (sickle cell anemia, thalassemias) and paroxysmal nocturnal hemoglobinuria (PNH) (22). Iron deficiency is highly prevalent (43–63%) in patients with idiopathic PAH (23,24). Moreover, anemia is an independent risk factor for PAH in patients with systemic sclerosis (25) and is associated with a higher prevalence of PAH in patients with chronic kidney disease (26). Given that anemia is found in approximately 50% of SLE patients (27) and is a cause of tissue hypoxia, it is speculated that anemic hypoxia is involved in the development of PAH in SLE patients. Therefore, we simultaneously measured DO2 and systolic PAP by echocardiography in 132 patients with

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PATIENTS AND METHODS Subjects. One hundred twenty-four patients with SLE were recruited from Yeouido St. Mary’s Hospital (Seoul, Korea). In addition, 8 SLE patients with increased PAP from St. Vincent’s Hospital (Suwon, Korea) and Uijeongbu St. Mary’s Hospital (Uijeongbu, Korea) were included in the study. Patients were excluded if they had previous or coexisting congenital heart disease, valvular heart disease, left-sided heart failure, chronic thromboembolic disease, or COPD. Patients who had suspected active infections, other hemolytic conditions (sickle cell anemia, thalassemias, and PNH), and scleroderma overlap syndrome or mixed connective tissue disease were also excluded. All patients fulfilled the revised criteria of the American College of Rheumatology for classification of SLE (28). As a disease control, 22 patients with COPD who have increased PAP (6 women and 16 men) were also included (median age 67 years, interquartile range 59–74 years). This study was carried out in accordance with the Helsinki Declaration and approved by the Institutional Review Board of Yeouido St. Mary’s Hospital, and written informed consent was obtained from all participants. Clinical and laboratory profiles. Clinical and laboratory data were obtained from each patient at the time of transthoracic Doppler echocardiography. In SLE patients, demographic and clinical characteristics, including disease duration, comorbidity, organ involvement, Raynaud’s phenomenon, and medication use, were retrieved from charts. Laboratory parameters included complete blood count, urinalysis, serum creatinine, lipid profiles, uric acid, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, plasma N-terminal pro–brain natriuretic peptide (NT-proBNP), complement (C3, C4, and CH50), antibodies against double-stranded DNA, Ro/SSA, La/SSB, Sm, RNP, cardiolipin, b2-glycoprotein I, and lupus anticoagulant. Laboratory data such as hemoglobin levels prior to echocardiogram were retrospectively collected from medical charts, which had been measured at 2–8-week intervals (median 4 weeks) depending on the patients’ condition as part of standard clinical care. Anemia was defined by World Health Organization (WHO) criteria as a hemoglobin level ,12 gm/dl for women and ,13.0 gm/dl for men, and severe anemia was defined by a decrease in hemoglobin level below 10 gm/dl for both sexes (29). Standard laboratory tests for anemia were used to assess the cause of anemia, including red cell indices, reticulocyte count, serum haptoglobin, serum iron, total iron binding capacity, serum ferritin, and vitamin B12 and folate levels. Patients with anemia were classified as having hemolytic anemia, iron deficiency anemia (IDA), and anemia of chronic disease (ACD) according to the predefined criteria, as reported previously (30,31). SLE disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (32).

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Table 1.

Demographic and baseline clinical data of patients*

Women, no. (%) Age, years BMI, kg/m2 Disease duration, years Smoking, no. (%) Diabetes mellitus, no. (%) Hypertension, no. (%) Raynaud’s phenomenon, no. (%) Interstitial lung disease, no. (%) Renal involvement, no. (%) Renal insufficiency, no. (%)† Medication Prednisolone (during the preceding 1 month), mg/month Hydroxychloroquine, no. (%) Mycophenolate mofetil, no. (%) Azathioprine, no. (%) Methotrexate, no. (%) Aspirin, no. (%) Statin, no. (%) SLEDAI Anti-RNP antibodies, no. (%) Lupus anticoagulant, no. (%) Anticardiolipin antibodies, no. (%)§ Anti-b2GPI antibodies, no. (%)¶ Anti-dsDNA antibody titer, IU/ml C3, mg/dl C4, mg/dl CH50, units/ml ESR, mm/hour CRP level, mg/liter Uric acid, mg/dl NT-proBNP, pg/ml Hemoglobin, gm/dl Hemoglobin ,10 gm/dl, no. (%)

Normal PAP (£40 mm Hg) (n 5 115)

Increased PAP (>40 mm Hg) (n 5 17)

103 (89.6) 35 (28–44) 21.2 (19.6–23.1) 7 (4–13) 7 (6.1) 2 (1.7) 20 (17.4) 15 (13.0) 8 (7.0) 54 (47.0) 5 (4.3)

16 (94.1) 35 (27–41) 19.7 (19.5–25.0) 7 (2–11) 0 (0.0) 0 (0.0) 3 (17.6) 3 (17.6) 0 (0.0) 10 (58.8) 1 (5.9)

150 (150–225)

225 (150–547.5)

95 (82.6) 22 (19.1) 21 (18.3) 23 (20.0) 24 (20.9) 18 (15.6) 5 (3–10) 45 (39.1) 23 (20.0) 23 (20.0) 8 (7.0) 63 (21–272) 71.7 (51.1–87.9) 12 (6.6–18.3) 34.7 (20.4–45.9) 31 (18–49) 1.1 (0.3–3.3) 4.8 (3.9–6.1) 69 (43–152) 11.8 (10.5–12.7) 18 (15.7)

12 (70.6) 2 (11.7) 6 (35.3) 3 (17.6) 5 (29.4) 6 (35.3) 8 (6–11)‡ 7 (41.2) 4 (23.5) 6 (35.3) 3 (17.6) 107 (26–190) 55.5 (38.8–76.9) 9.4 (5.5–11.3) 21.3 (9.0–37.4) 38 (16–58) 2.1 (1.4–3.5) 7.2 (4.9–8.4)# 930 (759–5,341)** 8.9 (8.7–10.8)** 11 (64.7)**

* Values are the median (interquartile range) unless indicated otherwise. PAP 5 pulmonary artery pressure; BMI 5 body mass index; SLEDAI 5 Systemic Lupus Erythematosus Disease Activity Index; anti-b2GPI 5 anti–b2-glycoprotein I; anti-dsDNA 5 anti–double-stranded DNA; ESR 5 erythrocyte sedimentation rate; CRP 5 C-reactive protein; NT-proBNP 5 N-terminal pro–brain natriuretic peptide. † Defined as a serum creatinine level .2 mg/dl. ‡ P , 0.05 by Mann-Whitney U test or chi-square test. § Antibody positivity at $10 IU/ml. ¶ Antibody positivity at .10 IU/ml. # P , 0.01 by Mann-Whitney U test or chi-square test. ** P , 0.001 by Mann-Whitney U test or chi-square test.

To determine the presence or absence of chronic parenchymal lung disease, plain chest radiography was obtained for all patients, and high-resolution computed tomography (HRCT) was performed for further evaluation when the chest radiography demonstrated a suspicious parenchymal lung lesion. In addition, patients with increased PAP on echocardiography were further assessed by either HRCT or pulmonary function test, depending on patients’ compliance, to determine whether raised PAP was secondary to lung disease. In pulmonary function testing, patients with significant lung disease as defined by forced expiratory volume in 1 second (FEV1)/forced

vital capacity ratio ,70% or a FEV1 ,60% of the predicted value were excluded. Serum samples obtained from 90 patients with SLE were stored at 2708C and then interleukin-6 (IL-6) concentrations were measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). Measurement of PAP and cardiac output by Doppler echocardiography. Transthoracic echocardiography was performed on all patients to measure PAP. Cardiac morphology, flow velocity, and ejection fraction were evaluated by 2-dimensional, M-mode, and color Doppler echocardi-

Anemic Hypoxia With PAH in SLE ography. Left ventricular ejection fraction was assessed using the modified Simpson’s method. Cardiac output was the product of heart rate and stroke volume, which is the difference between left ventricular end-diastolic volume and end-systolic volume. The right ventricular to right atrial systolic pressure gradient was calculated using a modified Bernoulli equation: 4 3 (tricuspid regurgitant jet velocity)2. PAP was quantified by adding the Bernoulliderived pressure gradient to the estimated mean right atrial pressure. In our study, increased PAP was defined as resting PAP . 40 mm Hg, since a PAP of 40 mm Hg is generally accepted as the upper limit of normal in most studies (6,7,33,34). Assessment of DO2. DO2 was estimated by using the equation DO2 5 CO 3 (SaO2 3 1.39 3 Hb), where CO 5 cardiac output in liters per minute, SaO2 5 percentage of hemoglobin O2 saturation, Hb 5 hemoglobin concentration in grams per deciliter, and 1.39 5 the hemoglobin binding constant (20,21). SaO2 was obtained from arterial blood gas analysis or measured by pulse oximetry as reported previously (35). DO2 was further adjusted for body weight (kg) because oxygen needs differ relative to body size (36,37). In addition, severe anemia was defined as a hemoglobin concentration less than 10 gm/dl on the basis of a previous study showing that a hematocrit level of less than 30% was shown to be associated with high damage accrual in the disease course of SLE patients (38). Cumulative exposure to severe anemia was quantified over the preceding 6 to 24 months before entering the study. Complete blood counts, including hemoglobin concentration, were measured at 2–8-week intervals (median 4 weeks), depending on the patients’ condition. Statistical analyses. Results for continuous data of abnormal distribution are shown as medians with interquartile ranges (IQRs), and comparisons between groups were made using Mann-Whitney U tests. Categorical or dichotomous variables are expressed as percentages and

1705 were compared using the chi-square test or Fisher’s exact test. Continuous data across categorical variables were compared by analysis of covariance. Correlation between 2 variables was assessed using the Spearman’s rank correlation coefficient. Two-sided P values of less than 0.05 were considered statistically significant.

RESULTS Characteristics of the study population. Among enrolled SLE patients, 17 (12.9%) had increased PAP . 40 mm Hg (range 41–90 mm Hg) and 115 (87.1%) had PAP # 40 mm Hg; 21 of 115 patients whose PAP was #40 mm Hg showed PAP of 30–40 mm Hg. Demographic and clinical characteristics of the patients are listed in Table 1. Of 79 patients with anemia defined by WHO criteria, 62 (78.5%) had ACD, 14 (17.7%) had IDA, and 3 (3.8%) showed evidence of hemolytic anemia. When patients were divided into 2 groups by PAP value (increased, .40 mm Hg versus normal, #40 mm Hg), sex, age, body mass index, disease duration, presence of diabetes mellitus or hypertension, and history of cardiovascular disease (coronary artery disease and stroke) were not significantly different between the groups. Among 7 patients with normal PAP, 6 patients were ever smokers and 1 patient was a current smoker, and all of the patients with increased PAP were identified as never smokers (Table 1). All patients with increased PAP showed no evidence of chronic parenchymal lung disease. Patients with increased PAP had significantly higher SLEDAI scores (P 5 0.032), and were being treated with higher doses of prednisolone over the preceding 4 weeks compared to those with normal PAP, although the latter did not reach statistical significance (P 5 0.090). Similarly, there was a tendency toward lower CH50 levels in patients with increased PAP compared to those with normal PAP (P 5 0.072). However, we did not find any difference between groups in prevalence of Raynaud’s phenomenon, renal involvement, or presence

Figure 1. Comparison of oxygen delivery (DO2) between systemic lupus erythematosus patients with increased versus normal pulmonary artery pressure (PAP). A, DO2 values were significantly lower in patients with increased PAP than in those with normal PAP (median 8.5 [interquartile range (IQR) 7.7–9.0] versus median 9.7 [IQR 8.2–12.0] ml/minute/kg; P 5 0.002). Scatter plots show correlation of DO2 with B, PAP, and C, plasma N-terminal pro–brain natriuretic peptide (NT-proBNP). DO2 values inversely correlated with PAP (g 5 20.308, P , 0.001) and plasma NT-proBNP levels (g 5 20.323, P 5 0.001).

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Figure 2. Comparison of oxygen delivery (DO2) variables between systemic lupus erythematosus (SLE) patients with increased versus normal pulmonary artery pressure (PAP). A, Hemoglobin levels were significantly lower in SLE patients with increased PAP compared to those with normal PAP (median 11.9 [interquartile range (IQR) 10.6–13.0] versus median 8.9 [IQR 8.7–10.1] gm/dl; P , 0.001). There was no difference in B, cardiac output (median 3.4 [IQR 2.7–4.0] versus median 3.5 [IQR 3.4–3.8] liters/minute), and C, percentage of hemoglobin O2 saturation (SaO2; median 98% [IQR 98–98%] versus median 98% [IQR 97.4–98%]), between groups (all P . 0.05). D, Scatter plot shows correlation of DO2 with hemoglobin levels. DO2 values positively correlated with hemoglobin (g 5 0.402, P , 0.001).

of antiphospholipid antibodies, which are reported to be associated with PAH (3,8–12). Notably, patients with increased PAP had a higher frequency of severe anemia (defined by hemoglobin levels below 10 gm/dl) and lower hemoglobin levels than patients with normal PAP (both P , 0.001). Furthermore, there was an inverse correlation between levels of hemoglobin and PAP (g 5 20.420, P , 0.001). This association remained significant after adjustment for estimated glomerular filtration rate (eGFR; g 5 20.438, P , 0.001). The levels of NTproBNP and uric acid were significantly higher in patients with increased PAP than those in patients with normal PAP (P , 0.001 and P 5 0.007, respectively). However, neither CRP level nor ESR appeared to be associated with increased PAP. Although statin therapy was more frequent in patients with increased PAP than in those with normal PAP (P 5 0.084), there was no difference between groups in use of medications, including hydroxychloroquine, mycophenolate mofetil, azathioprine, methotrexate, and aspirin.

Decreased DO2 in SLE patients with increased PAP and inverse correlation of DO2 with levels of PAP and plasma NT-proBNP. It is well known that chronic hypoxia results in pulmonary vascular remodeling, which is a common pathologic feature of PAH (17). Hypoxia occurs when DO2 fails to meet the metabolic demands of the tissues. Therefore, we sought to investigate whether decreased DO2 is associated with increased PAP in SLE patients. Weightadjusted DO2 was calculated as an index for assessing hypoxia (36,37). As shown in Figure 1A, DO2 values were significantly lower in patients with increased PAP than in those with normal PAP (median 8.5 [IQR 7.7–9.0] versus median 9.7 [IQR 8.2–12.0] ml/minute/kg; P 5 0.002). In linear correlation analysis, DO2 values were correlated inversely with PAP (g 5 20.308, P , 0.001) (Figure 1B) and plasma NT-proBNP levels (g 5 20.323, P 5 0.001) (Figure 1C), which is considered to be a biomarker of PAH (39). These correlations remained statistically significant after adjustment for confounders in our study (prednisolone dose, uric acid, and SLEDAI) and factors known to be

Anemic Hypoxia With PAH in SLE associated with PAH in previous studies, such as disease duration, presence of Raynaud’s phenomenon, antiphospholipid antibody, and renal failure (3,8–12). These data indicate that inadequate DO2 to tissues as measured by DO2 is closely associated with increased PAP in SLE patients. To identify the key determinant of decreased DO2 values in SLE patients, variables used in the equation of DO2 were compared between the 2 groups. Hemoglobin levels were significantly lower in SLE patients with increased PAP compared to those with normal PAP (median 11.9 [IQR 10.6–13.0] versus median 8.9 [IQR 8.7–10.1] gm/dl; P , 0.001) (Figure 2A), while no difference was noted in cardiac output (median 3.4 [IQR 2.7–4.0] versus median 3.5 [IQR 3.4–3.8] liters/minute) and SaO2 (median 98% [IQR 98–98%] versus median 98% [IQR 97.4–98%]) between groups (all P . 0.05) (Figures 2B and C). Moreover, DO2 values were significantly correlated with hemoglobin levels in SLE patients (g 5 0.402, P , 0.001) (Figure 2D). In our study, we included the 22 COPD patients with increased PAP as a disease control. DO2 values in COPD patients with increased PAP were significantly decreased as compared to those in SLE patients with increased PAP (median 8.5 [IQR 7.6–9.1] versus median 8.5 [IQR 7.7–9.0] ml/minute/kg; P 5 0.878), but DO2 values did not correlate with hemoglobin levels (P 5 0.229). Collectively, these findings suggest that decreased DO2 to tissues due to anemia is associated with increased PAP in SLE patients. Association of chronic anemic hypoxia with increased PAP in SLE patients. Although hypoxia is known to cause PAH in both animal and human studies (17), anemic hypoxia at a certain point in the disease does not necessarily represent increased PAP in SLE patients. Therefore, we calculated the cumulative duration of severe anemia (as defined by hemoglobin levels below 10 gm/dl) over the preceding 3, 6, 12, and 24 months to determine the association between duration of anemia and increased PAP. When patients were stratified into 3 groups based on PAP values (group 1, ,30 mm Hg; group 2, 30–40 mm Hg; and group 3, .40 mm Hg), patients in group 3 had a significantly longer duration of anemia than patients in groups 1 and 2 during the preceding 3, 6, 12, and 24 months, while there was no difference between groups 1 and 2 (Figure 3). In the preceding 6 months, patients with anemia of longer duration ($3 months) had a higher risk of increased PAP compared to those with shorter durations (,3 months; odds ratio [OR] 29.4 [95% confidence interval (95% CI) 8.2–105.6], P , 0.001). This difference was also observed in the preceding 12 months (OR 17.2 [95% CI 5.2–57.6], P , 0.001). However, remote exposure to anemia (6–12 months before) in patients who did not have recent anemia (within 6 months) did not influence the risk of increased PAP (data not shown). Association of serum IL-6 levels with levels of hemoglobin and PAP. Earlier studies showed that serum IL-6 levels were inversely correlated with hemoglobin levels in SLE patients (40) and the geriatric syndrome of frailty (41), both of which are characterized by chronic inflammatory states with increased serum IL-6. Moreover, IL-6 was known to promote the development and progression of

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Figure 3. Comparison of anemia duration in 3 groups according to their pulmonary artery pressure (PAP). Systemic lupus erythematosus patients were divided into 3 groups by PAP (group 1, ,30 mm Hg; group 2, 30–40 mm Hg; and group 3, .40 mm Hg), and cumulative durations of anemia between groups were compared by analysis of covariance. Patients in group 3 had a significantly longer duration of anemia than patients in groups 1 and 2 during the preceding 3, 6, 12, and 24 months; however, there was no difference between group 1 and group 2. * 5 P , 0.05; *** 5 P , 0.001; NS 5 not significant.

PAH (42–44). Therefore, to investigate the association between hemoglobin levels and PAP values, we measured IL-6 levels by ELISA in 90 SLE patients whose serum samples were obtained at the time of echocardiogram; there was no significant difference in clinical and laboratory data between the 90 patients with IL-6 measured and those without (data not shown). As expected, SLE patients with increased PAP had significantly higher serum IL-6 levels than SLE patients with normal PAP (median 137 [IQR 57–585] versus median 14 [IQR 7–31] pg/ml; P , 0.001). Unlike other forms of inflammatory arthritis, serum IL-6 levels were not correlated with CRP levels; this finding is consistent with the previous reports (45,46). When the patients were divided into 3 groups according to their PAP (groups 1, 2, and 3 as above), serum IL-6 levels (log-transformed value) increased significantly with higher PAP group (P 5 0.001 for trend) (Figure 4A). However, serum IL-6 levels (log-transformed value) decreased across tertiles of hemoglobin level (P 5 0.008 for trend) (Figure 4B). The association of IL-6 with PAP remained significant after adjustment for confounding factors (SLEDAI, uric acid, and prednisolone dose; P 5 0.001 for trend), while association with hemoglobin disappeared because of collinearity between hemoglobin and SLEDAI (g 5 20.354, P , 0.001).

DISCUSSION In the present study, we found that SLE patients with increased PAP had a significantly higher frequency of anemia and lower hemoglobin levels than patients with normal PAP. Besides the well-known consequence of sickle cell anemia (22), our finding is consistent with the result of Coral-Alvarado et al (25), who demonstrated that ane-

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Figure 4. Association of serum interleukin-6 (IL-6) with pulmonary artery pressure (PAP) values and hemoglobin levels. When systemic lupus erythematosus patients were divided into 3 groups either by A, PAP (group 1, ,30 mm Hg [n 5 65]; group 2, 30–40 mm Hg [n 5 14]; and group 3, .40 mm Hg [n 5 11]), or B, tertiles of hemoglobin levels, serum IL-6 levels (log-transformed value) increased with higher groups of PAP (P 5 0.001 for trend), but decreased across tertiles of hemoglobin (P 5 0.008 for trend).

mia is one of the independent predictors for PAH in patients with systemic sclerosis. Furthermore, anemia was known to be associated with high mortality in patients with PAH (47). The mechanisms by which anemia participates in the pathogenesis of PAH associated with various disease conditions are still unclear. In the setting of hemolytic conditions from sickle cell or autoimmune hemolytic anemia, cell-free hemoglobin limits nitric oxide bioavailability by acting as a nitric oxide scavenger (48). Depletion of nitric oxide results in platelet activation, enhancing the endothelin 1 response and oxidative stress, all of which ultimately cause pulmonary vascular remodeling leading to pulmonary hypertension. However, the decreased hemoglobin levels in our patients with increased PAP did not appear to be related to either hemolytic anemia or renal insufficiency, as judged by serum lactate dehydrogenase levels and eGFR (data not shown). Further, the association between anemia and increased PAP was still significant after adjustment for eGFR (P , 0.001). Given the inverse correlation between hemoglobin level and SLEDAI score in our study (g 5 20.354, P , 0.001), as reported previously (49), anemia might be attributed to a chronic inflammatory process (i.e., ACD), being the most common form of anemia in SLE patients (27). Indeed, the percentage of ACD in SLE patients with anemia was 78.5%, and it was increased to 82.8% in those with severe anemia. Hypoxic pulmonary vasoconstriction can be an inciting and perpetuating factor for increased PAP (17). Hypoxia occurs when DO2 is unable to meet the metabolic need for oxygen; this point is called critical DO2. Earlier studies in animals and humans have shown that the critical DO2 is in the range of 8–10 ml/minute/kg (36,37). In our study,

27 (20.5%) of 132 SLE patients had DO2 values less than 8 ml/minute/kg, and DO2 values were significantly lower in SLE patients with increased PAP than in those with normal PAP (Figure 1A), suggesting that a considerable number of SLE patients are at risk of hypoxic injury, and hypoxia reflected by decreased DO2 could contribute to the development of PAH in SLE patients. The latter assertion is supported by observations that DO2 values were inversely correlated with values of PAP and plasma NTproBNP (Figures 1B and C), a validated biomarker of PAH (39). Furthermore, our data showing a positive correlation between DO2 values and hemoglobin levels in SLE patients indicate that anemic hypoxia is responsible for increased PAP in SLE patients. Anemia is often overlooked as an important risk factor for development of adverse cardiovascular outcomes in several disease conditions (50–52). A hematocrit level of less than 30% was shown to be associated with high damage accrual in the disease course of SLE patients (38). We therefore defined severe anemia as hemoglobin levels below 10 gm/dl and compared the cumulative duration of severe anemia among groups stratified by PAP. As shown in Figure 2, SLE patients with increased PAP (group 3) had significantly longer durations of severe anemia than patients with normal PAP (groups 1 and 2). The length of exposure to hypoxia that causes a remodeling of the pulmonary vascular bed is still unknown, and it may be different regarding the severity of hypoxia, hosts (animal versus human), and inciting insults. In healthy volunteers, exposure to hypobaric hypoxia over a 6-week period increased PAP (53). Repeated lung injury in mice with 10–14 weeks of endotoxin injection caused PAH (54). In our study, SLE patients with anemia of a longer duration

Anemic Hypoxia With PAH in SLE ($3 months) in the preceding 6 months had a higher risk of increased PAP compared to those with a shorter duration (,3 months; P , 0.001), whereas remote exposure to anemia did not confer risk of increased PAP. These observations agree with previous data demonstrating that chronic exposure to hypoxia results in the remodeling of the pulmonary vascular bed leading to PAH (55,56), while acute hypoxia causes a rise in pulmonary blood pressure by vasoconstriction, which is reversible by euoxia (57). Several lines of evidence have also shown that IL-6 is implicated in the development and progression of PAH (42,43). Elevated serum IL-6 levels are found in patients with idiopathic PAH and rheumatic diseases with PAH (44,58). Transgenic overexpression of IL-6 leads to severe PAH in mice (42), and IL-6 deficiency protected mice from hypoxia-driven experimental PAH (43). IL-6 also has been shown to play a role in the development of anemia. Serum IL-6 levels were inversely correlated with hemoglobin levels in SLE patients (40), and administration of recombinant human IL-6 in cancer patients and rhesus monkeys led to a rapid decrease in hemoglobin concentrations (59,60). Consistent with these reports, we found that serum IL-6 levels increased across the groups with higher PAP, but decreased across tertiles of hemoglobin (Figure 4). These findings, together with previous reports (40,42,43,58,61), corroborate a central role for IL-6 in the development of PAH associated with anemia in SLE patients. It is notable that a patient with mixed connective tissue disease who had refractory PAH with marked anemia dramatically responded to treatment with tocilizumab (a monoclonal antibody against IL-6 receptor) that aimed at treating coexisting multicentric Castleman’s disease (62). Contrary to IL-6, we did not find any significant difference in serum EPO levels between patients with normal and increased PAP (data not shown), although EPO is known to be associated with development of pulmonary hypertension as well as anemia (63). Taken together, chronic inflammation over a longer period contributes to the development of anemia and PAH, both of which are known to be mediated by the action of increased IL-6 (64). However, considering that anemia per se is responsible for the increase of PAP by decreasing DO2 value (Figure 1), correction of anemia might be beneficial for decreasing PAP in SLE patients with PAH or patients at risk for PAH. In conclusion, our study suggests that chronic exposure to anemic hypoxia is responsible for the increased PAP in SLE patients, and IL-6 could play an important role in the development of PAH associated with anemia. However, this study had several limitations to be addressed. First, selection bias has to be considered, since we pooled the data of SLE patients from affiliated hospitals to increase the precision of the results. Second, we did not confirm our findings of echocardiography with catheterization of the right side of the heart due to ethical issues. Third, because of the cross-sectional study design, we were not able to infer causality of the findings and assess the longitudinal changes of PAP after anemia correction. Finally, the small number of increased PAP cases precluded the impact of anemia on PAP with respect to severity and types of anemia. Further studies are required to clarify these issues.

1709 AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Dr. Cho had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. K-J Kim, Cho. Acquisition of data. K-J Kim, Baek, Yoon, W-U Kim. Analysis and interpretation of data. K-J Kim, Yoon, W-U Kim, Cho.

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