Endothelin B Receptor Immunodynamics in Pulmonary Arterial Hypertension

Introduction Inflammation is a major pathological feature of pulmonary arterial hypertension (PAH), particularly in the context of inflammatory conditions such as systemic sclerosis (SSc). The endothelin system and anti-endothelin A receptor (ETA) autoantibodies have been implicated in the pathogenesis of PAH, and endothelin receptor antagonists are routinely used treatments for PAH. However, immunological functions of the endothelin B receptor (ETB) remain obscure. Methods Serum levels of anti-ETB receptor autoantibodies were quantified in healthy donors and SSc patients with or without PAH. Age-dependent effects of overexpression of prepro-endothelin-1 or ETB deficiency on pulmonary inflammation and the cardiovascular system were studied in mice. Rescued ETB-deficient mice (ETB -/-) were used to prevent congenital Hirschsprung disease. The effects of pulmonary T-helper type 2 (Th2) inflammation on PAH-associated pathologies were analyzed in ETB -/- mice. Pulmonary vascular hemodynamics were investigated in isolated perfused mouse lungs. Hearts were assessed for right ventricular hypertrophy. Pulmonary inflammation and collagen deposition were assessed via lung microscopy and bronchoalveolar lavage fluid analyses. Results Anti-ETB autoantibody levels were elevated in patients with PAH secondary to SSc. Both overexpression of prepro-endothelin-1 and rescued ETB deficiency led to pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy with accompanying lymphocytic alveolitis. Marked perivascular lymphocytic infiltrates were exclusively found in ETB -/- mice. Following induction of pulmonary Th2 inflammation, PAH-associated pathologies and perivascular collagen deposition were aggravated in ETB -/- mice. Conclusion This study provides evidence for an anti-inflammatory role of ETB. ETB seems to have protective effects on Th2-evoked pathologies of the cardiovascular system. Anti-ETB autoantibodies may modulate ETB-mediated immune homeostasis.


Supplementary Figure 2: ETB deficiency was associated with reduced dynamic lung compliance.
Lungs of 8-12 weeks (wks) and 6 months (mo) old rescued endothelin B receptor-deficient (ETB -/-) and corresponding wild-type (WT) mice were prepared and livers were removed. (A) Liver weight related to body weight was similar in all groups. (B) In isolated perfused and ventilated lungs, dynamic lung compliance (Cdyn) was decreased in ETB -/compared to WT mice, independent of age. Data are represented as box plots depicting median, quartiles, and ranges excluding outliers (open circles); N=7-28. **p<0.01, ***p<0.001 (Mann-Whitney U test).

Supplementary Figure 3: Dynamic lung compliance in prepro-endothelin-1 overexpressing mice.
Lungs of 2-6 months (mo) or 16-18 mo old prepro-endothelin-1 overexpressing (preET tg ) mice and corresponding wild-type (WT) mice were prepared. In isolated perfused and ventilated lungs, dynamic lung compliance was comparable in both groups of the same age. Data are represented as box plots depicting median, quartiles, and ranges excluding outliers (open circles); N=7-12; # indicates significant difference between 16-18 mo vs. 2-6 mo old groups. ### p<0.001 (Mann-Whitney U test). Figure 4: ETB deficiency was associated with perivascular lymphocytic infiltrates in the lung. Lungs of rescued endothelin B receptor-deficient (ETB -/-) and corresponding wild-type (WT) mice were harvested and lung tissue sections stained with hematoxylin and eosin. Representative images (N=20-25 per group) of ≥12 mo old mice are shown (identical lungs as shown in Figure 4, here with lower magnification for a broader overview). Scale bars represent 100 µm.

Supplementary Figure 5: The perivascular space of small pulmonary arteries was unaltered in preproendothelin-1 overexpressing mice compared to wild-type mice.
Lungs of prepro-endothelin-1 overexpressing (preET tg ) mice and corresponding wild-type (WT) mice were harvested and lung tissue sections stained with hematoxylin and eosin. Representative images (N=23-32 per group) of ≥12 mo old mice are shown. Scale bars represent 20 µm. Figure 6: ETB deficiency did not affect Th2-mediated pulmonary vascular hyperresponsiveness to serotonin. Rescued endothelin B receptor-deficient (ETB -/-) and corresponding wild-type (WT) mice were systemically sensitized with OVA (or PBS as control) and repeatedly exposed to aerosolized OVA (OVA/OVA) or PBS (PBS/PBS). Forty-eight hours after the last challenge, lungs were prepared and livers were removed. (A) In isolated perfused and ventilated lungs, pulmonary vascular responsiveness to increasing concentrations of serotonin was determined. Data ( Ppa) represent the difference between the highest pressure response to serotonin and the basal Ppa. (B) Liver weight was determined and related to body weight. In (A), values are given as mean and SEM, and analyzed using two-way repeated measures ANOVA (N=5-7). In (B), data are represented as box plots depicting median, quartiles, and ranges excluding outliers (open circles), and analyzed using Mann-Whitney U test (N=13-14).

Supplementary Figure 7: ETB deficiency aggravated Th2-induced perivascular inflammation in the lung.
Rescued endothelin B receptor-deficient (ETB -/-) and corresponding wild-type (WT) mice were systemically sensitized with OVA (or PBS as control) and repeatedly exposed to aerosolized OVA (OVA/OVA) or PBS (PBS/PBS). Forty-eight hours after the last challenge, lungs were removed and fixed. Lung tissue sections were stained with hematoxylin and eosin. Representative images (n=7) per group are shown.

Supplementary Figure 8: Quantification of plasma levels of vasoactive intestinal peptide (VIP).
Rescued endothelin B receptor-deficient (ETB -/-) and corresponding wild-type (WT) mice were systemically sensitized with OVA (or PBS as control) and repeatedly exposed to aerosolized OVA (OVA/OVA) or PBS (PBS/PBS). Forty-eight hours after the last challenge, plasma samples were collected and stored at -80°C. VIP levels in plasma were quantified by EIA. The detection limit was 0.05 ng/mL (dotted line). Data are represented as box plots depicting median, quartiles, and ranges excluding outliers (open circles), and analyzed using Mann-Whitney U test (N=7-8 per group).