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Abstract
Rationale: Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis.
Objectives: We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis, and tested their functional and biomarker relevance.
Methods: Anti-AT1R and -ETAR Abs were detected by ELISA in different cohorts of patients and tested in vitro and in an animal model for their pathophysiological effects.
Measurements and Main Results: The Abs were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease–associated PAH (n = 110) compared with other forms of PAH/pulmonary hypertension (n = 106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and SSc-PAH–related mortality in a prospective analysis. Both Abs increased endothelial cytosolic Ca2+ concentrations in isolated perfused rat lungs, which could be blocked by respective specific receptor antagonists. Ab-mediated stimulation of intralobar pulmonary rat artery ring segments increased vasoconstrictive responses to Ang II and ET-1, and implicated cross-talk between both pathways demonstrated by reciprocal blockade with respective antagonists. Transfer of SSc-IgG containing both autoantibodies into healthy C57BL/6J mice led to more abundant vascular and airway α-smooth muscle actin expression and inflammatory pulmonary vasculopathy.
Conclusions: Anti-AT1R and -ETAR Abs are more frequent in SSc-PAH/connective tissue disease–PAH compared with other forms of pulmonary hypertension, and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy.
Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis.
Anti-AT1R and anti-ETAR antibodies are more frequent in SSc-PAH/connective tissue disease–PAH compared to other forms of pulmonary hypertension and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy.
Pulmonary arterial hypertension (PAH) is a rare yet frequently fatal clinical condition (1). Patients with connective tissue diseases (CTDs) account for 20–30% of patients with PAH in clinical trials. The majority suffers from systemic sclerosis (SSc; e.g., Ref. 2). Despite substantial therapeutic improvements, patients with CTD-PAH and patients with SSc-PAH have a reduced survival compared with patients with idiopathic PAH (IPAH) (3). The complex pathogenesis of PAH includes sustained vasoconstriction and vascular remodeling of small pulmonary arteries (4). Endothelin (ET) 1 or angiotensin II (Ang II) and their receptors have been implicated in disease pathogenesis. Pharmacologic targeting of ET-1 actions, mainly via activation of ET-1 receptor type A (ETAR), has become a mainstay of PAH therapy (5, 6). Increased Ang II type-1 receptor (AT1R) activation was linked to disease progression and mortality in patients with IPAH (7, 8). Pharmacologic targeting of AT1R with losartan alleviated experimental PAH (9).
In addition, dysregulated immunity and inflammation are also common in CTD-PAH and other PAH entities, as suggested by inflammatory infiltration, growth factor expression in the remodeled pulmonary vasculature, and increased cytokine and chemokine levels in the circulation (10). However, heterogeneity in therapeutic responses asks for a more precise definition of pathophysiological differences among PAH entities. Therefore new diagnostic and prognostic biomarkers will be critical to identify patient subsets that are likely to benefit from targeted therapies.
Recently, we have identified simultaneous presence of functional autoantibodies (Abs) against AT1R and ETAR in patients with SSc (11) and linked them to increased prevalence of SSc-related vascular and fibrotic complications and higher risk for SSc-related mortality. Both antibodies induce phosphorylation of extracellular signal–regulated kinase 1/2 and transforming growth factor-β expression in endothelial cells, and hence activate endothelial cells to secrete cytokines (11, 12). In addition, the antibodies mediate decreased wound repair by endothelial cells and act on immune cells (12, 13). Based on these data and inducible renal obliterative lesions after transfer of anti-AT1R Abs into animals (14), we hypothesized an involvement of anti-AT1R Abs and anti-ETAR Abs in the pathogenesis of CTD-associated PAH, especially SSc-related PAH. After investigating their presence in different PAH entities, we studied their potential relevance as predictive and prognostic biomarkers for SSc-PAH, and their contribution to its pathogenesis.
Some of the results of these studies have been previously reported in the form of abstracts (15, 16).
Anti-AT1R/ETAR Ab levels were first measured in serum samples from 62 consecutive patients with SSc-PAH admitted to the Rheumatology Department of the Charité University Hospital Berlin (Berlin, Germany). Additional serum samples were obtained from 182 patients with different pulmonary hypertension (PH)/PAH entities, followed at the Department of Respiratory Medicine at the University Hannover (Hannover, Germany; n = 115), and at the Justus-Liebig University (Giessen, Germany; n = 67). Of these 182 patients, 62 had IPAH, 31 were classified as having chronic thromboembolic PH (CTEPH), 14 had PH due to congenital heart disease (CHD), 19 had SSc-PAH, and 56 were patients with PAH associated with CTD, which was not further specified. Combining all cases from the three centers, the cohort included 110 patients with CTD-PAH, of which 81 had SSc-PAH. Patients with SSc fulfilled the new European League against Rheumatism/American College of Rheumatology criteria for SSc (17). In addition, 253 consecutive patients with SSc entering our out-patient clinic between 2006 and 2011, without right heart catheter (RHC)-proven PAH and naive to the use of ETAR or AT1R blockers, were tested for anti-AT1R and anti-ETAR Ab levels, and were prospectively observed. The epidemiologic data of the patients are shown in Table 1. Under clinical routine conditions, patients were screened for PAH at least in 1-year intervals by assessment of World Health Organization functional class, echocardiography, lung function with detection of predicted single-breath diffusion capacity for carbon monoxide (DlCO-SB), and, during the last years, also by detection of N-terminal pro-brain natriuretic peptide (NT-proBNP) levels. Only if PAH was suspected was RHC performed, using a mean pulmonary arterial pressure (mPAP) of 25 mm Hg or greater and a pulmonary capillary wedge pressure of 15 mm Hg or less at rest for PAH diagnosis (18). All patients were examined in experienced PAH centers. Pulmonary vascular resistance (PVR) and cardiac index (CaI) were determined according to the local guidelines, yet not reported for all patients. The study was approved by the local ethics committee of each contributing center, and included written consent of each patient.
| SSc-PAH (n = 67) | SSc Non-PAH (n = 217) | IPAH (n = 62) | CTEPH (n = 31) | CHD (n = 14) | CTD-PAH (n = 110) | |
|---|---|---|---|---|---|---|
| Female sex, % | 71.6 | 83.5 | 56.5 | 51.6 | 57.1 | 81.8 |
| Age, yr | 61.9 (37.7–81.9) | 54.2 (29.0–71.0) | 51 (46.8–55.2) | 61.6 (57–66.1) | 59.4 (50.4–68.4) | 58.9 (56.4–61.3) |
| Duration of PAH, yr | 3.5 (1.30.6–8.7) | n.a. | 3.6 (2.8–4.5) | 3.1 (2–4.1) | 2.2 (0.4–4.1) | 2.9 (1.9–4.0) |
| Duration of SSc, yr | 8.81 (0.85–25.2)* | 6.0 (0–20.0)* | n.a. | n.a. | n.a. | n.a. |
| mPAP, mm Hg | 40.5 (36.6–44.4) | n.a. | 53.4 (48.7–58.4) | 43.6 (40.8–47.2) | 36.9 (22.3–51.5) | 40.8 (37.7–44) |
| PVR, dyn ⋅ s/cm5† | 557.6 (457.1–658.0) | n.a. | 874 (747–1001) | 747 (621–737) | 555 (117–993.6) | 608.7 (512–706) |
| Cardiac index†, L/min/m2 | 2.6 (2.4–2.8) | n.a. | 2.3 (2.1–2.6) | 2.2 (2.1–2.4) | 2.5 (2.1–2.9) | 2.8 (2.5–3.0) |
| Anti-AT1R Ab levels, U | 17.9 (14.4–21.4) | 19.5 (17.7–21.2) | 8.1 (5.5–10.7) | 5.5 (4.3–6.7) | 6 (3.1–8.8) | 16.9 (14.3–19.6) |
| Anti-ETAR Ab levels, U | 17.2 (13.7–20.8) | 21.1 (19.2–22.9) | 6.4 (3.5–9.3) | 3.5 (2.8–4.1) | 4.9 (1.5–8.2) | 15.8 (13.2–18.4) |
Serum antibody levels were measured by an ELISA (CellTrend GmbH, Luckenwalde, Germany [since 2012, One Lambda, Inc., Canoga Park, CA) as previously described (11). Briefly, polystyrene plates were coated with extracts from cells overexpressing the human AT1R or the human ETAR in native conformation, respectively. Detection of the Ab levels was performed by staff unaware of patient data.
All ex vivo and in vivo experiments were conducted with endotoxin-free IgG isolated from serum samples as previously described (11, 12). For in vitro experiments, final concentration was 1 mg/ml if not stated otherwise. Purified pooled IgG from 14 patients with SSc with high levels of anti-AT1R Abs (mean concentration, 21.8 U) and of anti-ETAR Abs (mean concentration, 17.91 U) was used for animal experiments. IgG from 15 healthy donors served as control (mean concentration, 3.85 U for anti-AT1R Abs and 2.5 U for anti-ETAR Abs, respectively).
Isolated lungs from male Sprague-Dawley rats (350–450 g body weight) were prepared (n = 9 per group), perfused, and ventilated as described previously (19, 20). For imaging of endothelial cytosolic Ca2+ concentration ([Ca2+]i), lungs were positioned under an upright fluorescence microscope and superfused with normal saline at 37°C. Fura-2-acetoxymethylester (Fura-2 AM, 5 μmol/L), which is de-esterified intracellularly to the Ca2+-sensitive dye, fura-2, was loaded to lung endothelial cells for 20 minutes via a microcatheter. Fura-2 fluorescence in endothelial cells of subpleural lung single venular capillaries of 15–30 μm in diameter was excited as described previously (21). Endothelial [Ca2+]i was determined from the 340 nm:380 nm ratio based on a Kd of 224 nmol/L and appropriate calibration parameters (21). After baseline recordings, purified IgG from either patients with SSc or from healthy donor sera was directly infused in HEPES dextran buffer into the imaged microvessels via a microcatheter over 30 minutes. The AT1R blocker, valsartan (a gift of D. N. Müller, Max Delbrück Center for Molecular Medicine, Berlin, Germany), and the ETAR blocker, sitaxsentan (provided by Pfizer Deutschland GmbH, Berlin, Germany) were each administered 10 minutes before IgG infusion in respective experiments (1 μmol/L each). All animal studies were approved by the local government authorities.
Third- to fourth-generation intralobar pulmonary rat artery ring segments from Sprague-Dawley rats (external diameter, 100–300 μm; length, 2 mm) were freed from connective tissue and mounted in a small vessel myograph (DMTDanish Myotechnology, Aarhus, Denmark). Contractile responses were measured as described previously (22). As ET-1–induced contraction in rat small pulmonary arteries is insensitive to either BQ-123, a selective ETAR antagonist, or the selective ETBR antagonist, BQ-788 (22), we used the combined ETA/BR antagonist, bosentan (a gift from Actelion, Basel, Switzerland). The vessels were incubated with 10 μM bosentan for 20 minutes, after which IgG (0.1 mg/mL) was added for 30 minutes. Thereafter, vessels were exposed to cumulatively increasing concentrations of Ang II. After repeated washing, the vessels were treated following the same protocol substituting valsartan 10 μM for bosentan and ET-1 for Ang II. Contractile responses to Ang II and ET-1 were measured as a percentage of the response to 60 mM K+; the effects were reversible by the respective receptor blockers.
For short-term experiments, female 7-week-old C57BL/6J mice received a single intravenous injection of 800 μg IgG dissolved in 100 μl NaCl 0.9%. Lungs were harvested for histological and immunological analyses at Day 7. For long-term experiments, mice received repeated IgG injections of 200 μg IgG dissolved in 100 μl NaCl 0.9% at Days 1, 17, 30, and 93. Organs were harvested at Day 100. Body weight (Days 30, 58, 78, and at readout day) and rectal temperature control (at read out day; BAT-12 Microprobe; Physitemp Instruments, Clifton, NJ) were monitored.
For immunohistochemistry, lungs were removed and immediately snap frozen in O.C.T. medium (Tissue-Tek, Sakura, Europe). Sections of 3 μm were prepared and stained with a polyclonal anti–α-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, UK) and with an anti-human IgG F(ab′)2-fragment (mouse absorbed; Dianova, Hamburg, Germany) to detect human IgG deposition. 4′,6-diamidino-2-phenylindole (Sigma Aldrich, Seelze, Germany) was used for nuclear DNA staining. For histological assessment, lungs were fixed in 4% paraformaldehyde at room temperature for 24 hours and embedded in paraffin using standard procedures. Anti-CD31 antibody was used (clone SZ31; Dianova) to stain endothelial cells. Hematoxylin and eosin staining was performed to visualize cellular organization. Signals were detected by light microscopy (Axioplan; Carl Zeiss MicroImaging GmbH, Jena, Germany) as well as by immunofluorescence (LSM710; Carl Zeiss Microscopy GmbH) and analyzed by ZEN 2009 Light Edition software (Carl Zeiss Microscopy GmbH).
Comparisons between groups were analyzed by Mann-Whitney U test for continuous variables. Kaplan-Meier analyses were performed to investigate the onset of PAH development and mortality after antibody detection. Significance was determined by log-rank test. Receiver-operating characteristic (ROC) curves were plotted to determine area under the curve (AUC), sensitivity, and specificity using calculated distance to 0.1. Small-vessel myography data were analyzed by ANOVA. Results are expressed as means (±SEM); in myograph experiments, n stands for the number of the studied pulmonary artery ring segments. Differences were considered statistically significant at a P value of 0.05 or less. GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) as well as IBM SPSS 21.0 (Ehningen, Germany) were used for statistical analyses.
Epidemiologic and hemodynamic data of the cohorts studied are shown in Table 1. PH/PAH groups did not differ significantly among centers. Higher percentages of female patients and shorter duration of PAH were observed in CTD-PAH and SSc-PAH. mPAP, as detected by RHC in these two groups, was significantly lower as compared with IPAH (P < 0.001) and CTEPH (P = 0.029). Accordingly, PVR was significantly lower in patients with SSc-PAH compared with IPAH (P < 0.0001) and patients with CTEPH (P = 0.002). The CaI was significantly higher in patients with SSc-PAH compared with patients with IPAH or CTEPH, and similar to patients with CHD.
The levels of the anti-AT1R and anti-ETAR Abs were significantly higher in CTD-PAH and SSc-PAH as compared with IPAH (P < 0.0001), CTEPH, and CHD groups (P < 0.001; Figures 1A and 1B), with highest levels in patients with SSc-PAH. The majority of the patients with SSc-PAH (69.1%) and patients with CTD (62.7%) were positive for anti-AT1R Abs (cut-off, 9.2 units) as compared with patients with IPAH (21%), CTEPH (8%), and CHD (21.4%) (11). In addition, 65.4% of the patients with SSc-PAH and 54.5% of the patients with CTD, yet only 11.3% of the patients with IPAH, none of the patients with CTEPH, and 14.3% of the patients with CHD, were positive for anti-ETAR Abs (cut-off, 10.4 units) (11). The antibody levels were overall equally high or even slightly higher in patients with SSc that did not develop PAH (Table 1). The Abs did not show significant correlations (r2 > 0.2) with hemodynamic parameters (PVR, CaI, or mPAP) or NT-proBNP levels (data not shown). However, a strong correlation between anti-AT1R and anti-ETAR Abs was observed in patients with SSc-PAH (r2 = 0.81, Figure 1C). Results of ROC curve analyses show sensitivity, specificity, and AUC values (Table 2). All differences of the groups shown were highly significant (P < 0.001, respectively).
Figure 1. Detection of anti–angiotensin receptor type-1 (AT1R) (A) and anti–endothelin (ET) receptor type A (ETAR) autoantibodies (Abs) (B) in patients with pulmonary arterial hypertension (PAH) due to connective tissue disease (CTD-PAH), due to systemic sclerosis (SSc-PAH), in patients with idiopathic PAH (IPAH), chronic thromboembolic pulmonary hypertension (CTEPH), and pulmonary hypertension due to congenital heart disease (CHD) by ELISA. Linear regression analysis revealed strong correlations between the anti-AT1R and anti-ETAR Ab levels in patients with SSc (C). n.s. = not significant.
[More] [Minimize]| Question Addressed | Anti-AT1R Abs Sensitivity/Specificity in % (AUC) | Anti-ETAR Abs Sensitivity/Specificity in % (AUC) |
|---|---|---|
| SSc-PAH vs. IPAH | 68.8/85.5 (0.772) | 72.5/85.5 (0.786) |
| Non–SSc-PAH vs. SSc-PAH | 68.8/78.0 (0.735) | 70.0/82.4 (0.754) |
| CTD-PAH vs. non–CTD-PAH | 62.4/82.5 (0.760) | 72.5/78.1 (0.786) |
During prospective follow-up of 253 consecutive patients with SSc at the department of Rheumatology, 36 patients were diagnosed with PAH (mean observation period: 73 mo). Epidemiologic and hemodynamic data of the prospective PAH-SSc cohort were not different from the cross-sectional SSc-PAH cohorts (data not shown). By comparison of various clinical parameters for the ability to predict PAH, AUC was best for systolic pulmonary arterial pressure (sPAP) and predicted values of DlCO-SB. The predictive capacity of anti-AT1R and anti-ETAR Ab levels was comparable to NT-proBNP values (Figure 2A). The best calculated cut-off from the ROC analysis was then used for a Kaplan-Meier analysis, shown in Figure 2B. Both anti-AT1R (cut-off, 19.0 units; P < 0.0001), as well as anti-ETAR Abs (cut-off, 23.0 units; P = 0.0056), predicted development of PAH in patients with SSc. The autoantibodies were equally predictive as a FVC:DlCO ratio greater than 1.6 (P = 0.046; Figure 2C) or DlCO less than 55% (P < 0.0001; Figure 2D), that are known risk factors for PAH (23). For anti-AT1R– and anti-ETAR–positive patients with SSc, there was an increased risk for PAH with a hazard ratio of 4.3 (95% confidence interval [CI] = 2.2–8.4) and of 3.5 (CI = 1.51–5.60), respectively. However, in a multivariate analysis, sPAP, as detected by echocardiography, was the only independent predictor for PAH proven by RHC (P < 0.001; odds ratio = 1.102; CI = 1.055–1.152).
Figure 2. Capacity of the autoantibodies (Abs) to predict pulmonary arterial hypertension (PAH) in patients with systemic sclerosis (SSc). (A) Comparison between the Abs and hemodynamic parameters for the prediction of PAH in SSc as detected by receiver-operating characteristic (ROC) analyses. (B) Kaplan-Meier analysis on cumulative PAH-free survival with number of patients at risk followed up after anti–angiotensin receptor type-1 (AT1R) and anti–endothelin (ET) receptor type A (ETAR) autoantibody testing in 253 patients with SSc. Vertical spikes represent censored patients. The cut-off used to discriminate patients with PAH-SSc from those without PAH was determined by ROC analysis (A). Given was also the hazard ratio (HR). The table shows the number of subjects at risk. (C and D) Kaplan-Meier analysis on cumulative PAH-free survival in the same cohort as (B) according to an FVC/lung diffusing capacity for carbon monoxide (DlCO) ratio greater than 1.6 (C) and DlCO less than 55% expected (D). NT-proBNP = N-terminal pro-brain natriuretic peptide; sPAP = systolic pulmonary arterial pressure.
[More] [Minimize]We analyzed the autoantibody levels in those 36 patients from our own cohort for their capacity to discriminate between patients that died and those who did not and the ROC AUC was 0.766 for the anti-AT1R Ab and 0.662 for the anti-ETAR Ab (data not shown). In addition, we had follow-up data from 34 patients with SSc-PAH from the Hannover cohort (70 patients with SSc-PAH in total). By using an ROC analysis and cut-off calculation, anti-AT1R Ab levels above 15.8 units predicted mortality with a sensitivity of 68.2% and a specificity of 62.2% (AUC = 0.669; P = 0.03). For the anti-ETAR antibodies, a cut-off of 18.3 units was associated with mortality, with a sensitivity of 68.2% and a specificity of 71.1% (AUC = 0.672; P = 0.02). As shown in Figure 3A, autoantibody levels against ETAR and AT1R predicted mortality better than the hemodynamic parameters mPAP, CaI, or PVR (each at PAH diagnosis). Kaplan-Meier survival analysis with respective cut-off levels (Figure 3A) showed a significantly different survival for anti-ETAR Ab–positive and –negative patients (P = 0.027; hazard ratio = 2.7; CI = 1.2–6.1; Figure 3B). This was not statistically significant for the anti-AT1R Abs in this cohort (P = 0.13). However, when the hemodynamic data (mPAP, CaI, and PVR) were compared with the anti-AT1R and anti-ETAR Ab levels in a multivariate analysis, anti-AT1R Abs were the only independent predictors of death (P = 0.03; odds ratio = 1.053; CI = 1.004–1.104). Autoantibody status in other PAH/PH entities (patients with CTD in general, patients without CTD, all patients with PH/PAH) did not reveal differences in survival.
Figure 3. Capacity of the autoantibodies (Abs) to predict death in patients with systemic sclerosis (SSc)-pulmonary arterial hypertension (PAH). (A) Comparison between the Abs and hemodynamic parameters for the prediction of mortality in patients with SSc-PAH as detected by receiver-operating characteristic (ROC) analyses. ROC analyses show the capability of the Abs to distinguish patients that survived with PAH from those with a fatal disease course. (B) Anti–angiotensin receptor type-1 (AT1R)/endothelin (ET) receptor type A (ETAR) Abs and survival. In a Kaplan-Meier analysis of 67 patients with SSc-PAH, anti-ETAR Abs predicted death in patients with SSc-PAH, and a similar tendency was also found for anti-AT1R Abs. The cut-off used to discriminate patients with PAH-SSc from those without PAH was determined by ROC analysis (A). Given was also the hazard ratio (HR). The table below shows the number of subjects at risk. mPAP = mean pulmonary arterial pressure; PVR = pulmonary vascular resistance.
[More] [Minimize]To evaluate the potential of these autoantibodies to elicit functional agonist effects in the intact pulmonary vasculature, we imaged endothelial [Ca2+]I as an established and assessable downstream signal of AT1R or ETAR activation during infusion of SSc and control IgG. SSc-IgG induced a marked increase in endothelial [Ca2+]i that was not detected after infusion of control IgG (Figures 4A–4C) and could be blocked by both valsartan and sitaxsentan (Figure 4D).
Figure 4. Functional effects of the autoantibodies (Abs) on endothelial cytosolic Ca2+ concentration ([Ca2+]i) and on contraction of pulmonary resistance vessels. Representative images show fura-2–loaded endothelial cells color coded for [Ca2+]i in a venular capillary of the isolated perfused rat lung (A and B). Images were obtained at baseline and 20 minutes after infusion of purified systemic sclerosis (SSc)-IgG (1 mg/ml); vessel margins are depicted by dotted lines; arrows indicate the direction of perfusate flow. Group data show mean endothelial [Ca2+]i at baseline and during infusion of purified SSc-IgG or control IgG from healthy donors (1 mg/ml starting at t = 0 min, arrow) in the absence (C) or presence of the endothelin (ET) receptor type A (ETAR) blocker, sitaxsentan, or the angiotensin receptor type-1 (AT1R) blocker, valsartan (1 μMol/L each), respectively (D). IgG from patients with SSc containing anti-AT1R and anti-ETAR Abs increase Ang II– (E) and endothelin-1 (F) –mediated contraction in rat pulmonary resistance vessels that was blocked by the noncorresponding receptor blocker to a level of control IgG (n ≥ 6 each group). Data are expressed as means ± SEM.
[More] [Minimize]In small-vessel myography of intralobar pulmonary rat artery ring segments, anti-AT1R and anti-ETAR Abs amplified the vasoconstrictive responsiveness to ET-1 and Ang II (Figures 4E and 4F). Pretreatment of the vessels with bosentan abolished the response to Ang II (Figure 4E), whereas AT1R antagonist valsartan (Figure 4E) reduced responses to ET-1 (Figure 4F).
For assessment of the pathophysiologic relevance of anti-AT1R and anti-ETAR Abs in vivo, SSc-IgG or healthy control (HC) IgG were injected into 7-week-old C57BL/6 mice (Figure 5). Human IgG was detected in frozen lung sections 7 days after a single injection of SSc-IgG, but not with HC-IgG (Figure 5A, shown in red), implicating Ab binding in murine lung tissue. In parallel, increased α-SMA expression was observed around the walls of small pulmonary vessels adjacent to airways in the SSc-IgG–treated group as compared with HC-IgG–treated mice (Figure 5B).
Figure 5. Human IgG staining in cryo lung sections of C57BL/6 mice (n = 7/group) treated with systemic sclerosis (SSc)-IgG show a high signal of red immunofluorescence 7 days after injection (white arrows), and this is not present in healthy control (HC)-IgG–treated mice (A). 4′,6-Diamidino-2-phenylindole (DAPI; shown in blue) was used to stain nuclear DNA. α-smooth muscle actin (α-SMA) was increased in lungs of SSc-IgG–treated mice in small vessels accompanying airways (black arrow) compared with healthy donor controls (B). After repetitive IgG transfers (n = 7–8 per group), H&E staining (C) and endothelial staining with anti-CD31 (D) revealed obliterative vasculopathy (black arrows) with increased perivascular inflammation (C), and confirmed increased α-smooth muscle actin expression in small vessels around airways (black arrows, E). Representative pictures are shown. H&E = hematoxylin and eosin.
[More] [Minimize]Repetitive injections of SSc-IgG (n = 8), HC-IgG (n = 7), and vehicle (n = 7) were administered in long-term pilot experiments over a period of 100 days. Obliterative pulmonary arteriopathy of various extent and increased cellular infiltrates were observed in mice injected with SSc-IgG, and were either not observed or observed to a lesser degree in controls (Figures 5C–5E). Fulton index (right ventricular weight over left ventricular plus interventricular septum weight ratio, RV/LV + S) and PVR were not different compared with controls.
We identified elevated autoantibodies against AT1R and ETAR in patients with SSc-PAH and CTD-PAH as compared with patients with IPAH or other PH entities. Simultaneously present anti-AT1R and anti-ETAR antibodies in patients with SSc predicted development of PAH and its associated mortality. Both receptor autoantibodies act as agonists by means of endothelial responses in isolated rat lungs and augmented vasoconstriction in response to natural ligands in rat pulmonary resistance vessels. Passive transfer of anti-AT1R and anti-ETAR antibody–positive human SSc-IgG increased α-SMA expression in healthy mice in concordance with human IgG detection within lung tissue 7 days after injection. Repetitive injections of SSc-IgG induced obliterative vasculopathy with perivascular lymphocyte infiltration. Our data unify autoimmune receptor activation processes with increased vascular reactivity and vascular remodeling. These data expand on our previous findings concerning the molecular and cellular effects of anti-AT1R and anti-ETAR autoantibodies on immune cells, endothelial cells, and fibroblasts, as well as their pathogenic role in mice after antibody transfer (12, 13).
Recently, IgG from both IPAH and patients with SSc-PAH was found to recognize antigens in vascular smooth muscle cells and fibroblasts (24, 25). The protein extraction and two-dimensional electrophoresis precluded isolation of membrane antigens, such as AT1R and ETAR (24, 25). Unlike intracellular antigens, functional consequences of autoimmune-mediated AT1R and ETAR activation are pleiotropic, and range from increase in endothelial [Ca2+]i influx, and enhanced vasoconstrictive responses to natural ligands, to the enhancement of chronic remodeling process. Vascular hyperreactivity, obliterative microvascular disease, and interstitial fibrosis are prominent features of SSc pulmonary involvement, which could all be influenced by anti-AT1R and anti-ETAR Abs. Autoantibody-mediated receptor cross-talk, together with enhanced reactivity to natural ligands, could represent an additional pathophysiological aspect of vascular dysregulation (22). The simultaneous occurrence of anti-AT1R and anti-ETAR Abs implies the importance of possible receptor heterodimerization, which could affect the influence of agonists and antagonists on signal transduction (26).
We are aware that our experimental data derived from mice injected with anti-AT1R and anti-ETAR containing IgG isolated from representative patients pose several limitations. In these preliminary experiments, we were not able to document increases in mPAP or PVR, or induction of right ventricular hypertrophy. However, endothelial cell activation, enhanced vasoconstriction, small-vessel obliteration, with increased α-SMA expression and perivascular infiltrates, indicate a vasculopathy that partly resembles changes seen in hypoxia and monocrotaline PAH models (27). Altered hemodynamics and right ventricular hypertrophy are also not commonly observed in early stages of other pulmonary hypertension models (27, 28). The utility and relevance of the Abs against AT1R and ETAR as predictive biomarkers of SSc-PAH development was underscored by similar sensitivity values as documented for NT-proBNP values, FVC:DlCO ratios, and DlCO values less than 55%, established predictors for SSc-PAH (29–32). Concordant with this published work, DlCO and sPAP values were best predictors for PAH in our study. We suggest that the measurement of autoantibody levels could provide an additional important prognostic tool, together with echocardiography, with the calculation of sPAP and a detailed lung function analysis (33). We are aware of the putative limitation that our study focused on very specific patient cohorts treated at tertiary care centers. However; our SSc-PAH cohorts were representative of other cohorts, as reflected by lower mPAP and PVR values and higher CaIs in patients with SSc-PAH as compared with patients with IPAH (30–32).
The clinical importance of the Abs against AT1R and ETAR in patients with SSc-PAH is further underscored by the fact that they were predictive of death. As hemodynamic parameters failed to predict death in other cohorts as well (30), our data indicate that the anti-AT1R and anti-ETAR Abs may have a role in risk stratification of SSc-PAH. In the era of personalized medicine, algorithms based on multiple parameters are increasingly valued for the identification of patients at risk, as recently demonstrated (33).
Although therapeutic implications of our findings remain to be established, the current study implies a contribution of functional autoimmunity to the development of PAH-associated vasculopathy. Immunosuppressive treatment with cyclophosphamide alone did not improve hemodynamic parameters in SSc-PAH (35). In the setting of heart and kidney transplantation, where AT1R autoantibodies are well established as diagnostic and prognostic biomarkers, intensified triple immunosuppression and AT1R blockade seem to be effective for the majority (36, 37), although some patients with fulminant phenotypes also required antibody removal by plasmapheresis (14).
In conclusion, our study suggests an involvement of functional autoantibody-mediated vascular receptor activation in SSc-PAH, and emphasizes anti-AT1R and anti-ETAR Abs as novel prognostic markers for CTD-PAH and SSc-PAH.
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* These authors equally contributed to the manuscript.
Supported by University Hospital Charité, Deutsche Forschungsgemeinschaft grant SFB-TR84 C3 and C6 (M.W.), European Community’s Seventh Framework Programme under grant agreement 241544–Systems Biology towards Novel Chronic Kidney Disease Diagnosis and Treatment (D.D.), Stiftung Sklerodermie e.V., Deutsches Netzwerk für systemische Sklerodermie (both G.R.), Heart & Stroke Foundation of Canada (W.M.K.), ARTICULUM Fellowship, Ministry of Industry grant KF2441006AJ3, CellTrend GmbH, and Actelion Pharmaceuticals Germany GmbH. The assay for measuring N-terminal pro-brain natriuretic peptide was kindly provided by Roche Germany.
Author Contributions: Conception and design—M.O.B., M.W., H.H., I.L., M.G., W.M.K., G.R.B., D.D., and G.R.; data acquisition—M.O.B., A.K., M.K., J.G., A.R., C.T., M.W., A.A.K., H.H., H.A.G., H.T., N.N., M.M.H., I.L., M.G., W.M.K., S.B., D.D., and G.R.; data analysis and interpretation—M.O.B., A.K., M.K., J.G., A.R., C.T., M.W., A.A.K., H.H., H.A.G., H.T., R.T.S., N.N., M.M.H., I.L., M.G., W.M.K., S.B., G.R.B., D.D., and G.R.; drafting or critically revising of the manuscript for important intellectual content—M.O.B., A.K., M.K., J.G., C.T., M.W., A.A.K., R.T.S., N.N., M.M.H., W.M.K., S.B., G.R.B., D.D., and G.R.
Originally Published in Press as DOI: 10.1164/rccm.201403-0442OC on September 2, 2014
Author disclosures are available with the text of this article at www.atsjournals.org.
