ONLINE DATA SUPPLEMENT
Nebivolol for improving endothelial dysfunction, pulmonary vascular remodeling,
and right heart function in pulmonary hypertension
Frédéric Perros, PhD,*†‡ Benoît Ranchoux, PhD,*†‡ Mohamed Izikki, PhD,** Sana Bentebbal, PhD,** Chris
Happé, PhD,¶ Fabrice Antigny, PhD,*†‡ Philippe Jourdon, PhD,*†‡ Peter Dorfmüller, MD, PhD,*†‡∥ Florence
Lecerf,*†‡ Elie Fadel, MD,§ Gerald Simonneau, MD,*†‡ Marc Humbert, MD, PhD,*†‡ Harm Jan Bogaard, MD,
PhD,¶ Saadia Eddahibi, PhD**
Short title: Nebivolol in pulmonary hypertension
Legends of supplemental figures
Supplemental figure 1: In vitro effects of nebivolol and metoprolol on human pulmonary endothelial cells
proliferation. Pulmonary endothelial cells (P-EC) from control and SPH lungs were either cultured with basal
medium, fetal calf serum (FCS) or FCS+nebivolol (Nebi) or FCS+metoprolol (Meto) at 10-6M and 10-5M.
Results are expressed in europium counts (Eu count)
Comparison between control and PAH: * P<0.05, ** P<0.01
Comparison to cells in the same group without treatment: # P<0.05, ## P<0.01
N=6 in each condition
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Supplemental figure 2: In vitro effects of nebivolol and metoprolol on the human pulmonary artery smooth cell
(PASMC) proliferation induced by P-EC (P-EC to PASMC crosstalk). PASMC were either cultured with basal
medium, or human P-ECs-conditioned medium from control and SPH patients, with or without nebivolol (Nebi)
or metoprolol (Meto) at 10-6M, 10-5M and 10-4M.
Results are expressed in europium counts (Eu count)
Comparison between control and PAH: * P<0.05, ** P<0.01
Comparison to cells in the same group without treatment: # P<0.01, ## P <0.001
N=6 in each condition
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Supplemental figure 3: In vitro effects of nebivolol and metoprolol on human P-EC production of
proinflammatory cytokines/chemokines (IL6, IL1β, and MCP1), growth factors (PDGF, EGF, and FGF2), and
vasoconstrictive factor (ET1). The P-ECs from control and SPH lungs were serum starved in MCDB131 medium
with or without increasing concentrations of nebivolol or metoprolol (10 −6M to 10−4M). After incubation for 24
hours, the medium was collected for ELISAs.
Comparison between control and PAH: * P <0.01, ** P <0.001, *** P < 0.0001
Comparison to cells in the same group without treatment: # P <0.05, ## P <0.01, ### P <0.001
N=6 in each condition
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Supplemental figure 4: In vivo effects of nebivolol and metoprolol on hemodynamics and right heart
hypertrophy in the MCT-induced PH in rats. Three groups were compared: 1) saline-treated control group
(Control); 2) MCT-exposed group (MCT); 3) MCT-exposed and 10 mg.kg-1.day-1 (day 14-28) nebivolol-treated
group (MCT+N10). In vivo effects of nebivolol and metoprolol on: A: right ventricular systolic pressures (RVSP,
mmHg), B: mean pulmonary artery pressure (mPAP, mmHg), C: systolic carotid artery pressure (sCAP),
D: Heart Rate (HR, bpm), E: cardiac output (CO, ml.min-1), F: total pulmonary vascular resistance (PVR,
mmHg.min/ml-1), G: Fulton’s index of right ventricular hypertrophy, calculated as the ratio of the right
ventricular weight to left ventricular plus septal weight (RV/LV+S), H: Right ventricular BNP expression relative
to Gapdh expression.
Control, n=5
MCT, n=6
MCT+N10, n=5
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Supplemental figure 5: In vivo effects of nebivolol and metoprolol on pulmonary vascular remodeling and
inflammation in the MCT-induced PH in rats. Three groups were compared: 1) saline-treated control group
(Control); 2) MCT-exposed group (MCT); 3) MCT-exposed and 10 mg.kg-1.day-1 (day 14-28) nebivolol-treated
group (MCT+N10). The analysis of the neomuscularization of normally non-muscularized small distal PA
(≤50µm) is a common and robust way to quantify the degree of remodelling of the rat pulmonary vasculature.
A: % of non muscularized (NM) PA. B: % of partially muscularized (PM) PA. C: % of fully muscularized (FM) PA.
D: Fully occluded distal PA.
Control, n=5
MCT, n=6
MCT+N10, n=5
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