The soluble guanylate cyclase stimulator riociguat ameliorates pulmonary hypertension induced by hypoxia and SU5416 in rats

Michaela Lang, Baktybek Kojonazarov, Xia Tian, Anuar Kalymbetov, Norbert Weissmann, Friedrich Grimminger, Axel Kretschmer, Johannes-Peter Stasch, Werner Seeger, Hossein Ardeschir Ghofrani, Ralph Theo Schermuly, Michaela Lang, Baktybek Kojonazarov, Xia Tian, Anuar Kalymbetov, Norbert Weissmann, Friedrich Grimminger, Axel Kretschmer, Johannes-Peter Stasch, Werner Seeger, Hossein Ardeschir Ghofrani, Ralph Theo Schermuly

Abstract

Background: The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63-2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO-sGC-cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.

Methods and results: Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55 ± 0.02, p<0.05), increased cardiac output (60.8 ± .8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03 ± 0.3 mmHg min(-1) ml(-1) 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).

Conclusion: Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.

Conflict of interest statement

Competing Interests: JPS is employee of Bayer HealthCare, who also provided an unrestricted research grant. RTS declares he has been a consultant for Bayer Healthcare and has received speakers bureau (honoraria) from Bayer Healthcare. RTS also declares he has received grant/research support from Bayer HealthCare, Excellence Cluster Cardiopulmonary System, German Research Foundation, and the University of Giessen and Marburg Lung Center. HAG declares he has been a consultant for Bayer Healthcare and has received speakers bureau (honoraria) from Bayer Healthcare. HAG also declares he has received unrestricted research grant from Bayer Healthcare (related to this study), supports/grants Excellence Cluster Cardiopulmonary System, German Research Foundation, and the University of Giessen and Marburg Lung Center. FG declares he has been a consultant for Bayer Healthcare and has received grant/research support from Excellence Cluster Cardiopulmonary System, German Research Foundation, and the University of Giessen and Marburg Lung Center. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. ML, BK, NW and WS have declared that no competing interests exist.

Competing Interests: ML, BK, NW and WS have declared that no competing interests exist.

Figures

Figure 1. Effects of riociguat and sildenafil…
Figure 1. Effects of riociguat and sildenafil on hemodynamics in SUHx rats.
(A) RVSP, (B) SAP, (C) CO and (D) TPR of different treatment groups. *p<0.05. Note. CO, cardiac output; RVSP, right ventricular systolic pressure; SAP, systemic arterial pressure; TPR, total pulmonary resistance.
Figure 2. Effects of riociguat and sildenafil…
Figure 2. Effects of riociguat and sildenafil on RVH and fibrosis in SUHx rats.
(A) Ratio of the RV to LV+S and (B) RV collagen contents of different treatment groups. *p<0.05. Note. LV, left ventricle; RV, right ventricle; RVH, right ventricular hypertrophy; S, interventricular septum.
Figure 3. Effects of riociguat and sildenafil…
Figure 3. Effects of riociguat and sildenafil on pulmonary vascular remodeling in SUHx rats.
(A) Proportion of non-, partially or fully muscularized pulmonary arteries as a percentage of the total pulmonary artery cross section (p<0.05. Note. c, closed; F, fully muscularized; N, non-muscularized; o, opened; P, partially muscularized; pc, partly closed.
Figure 4. Localization of sGCα1, sGCβ1, eNOS…
Figure 4. Localization of sGCα1, sGCβ1, eNOS and PDE-5 in pulmonary vessels of SUHx rats.
Localization of sGCα1, sGCβ1, eNOS and PDE-5 are presented in healthy controls, SUHx 3-week control animals and vehicle-treated animals. Note. eNOS, endothelial nitric oxide synthase; PDE-5, phosphodiesterase type 5; sGCα1, soluble guanylate cyclase α1; sGCβ1, soluble guanylate cyclase β1.
Figure 5. Effects of riociguat and sildenafil…
Figure 5. Effects of riociguat and sildenafil on apoptosis and cell proliferation in the lung tissue of SUHx rats.
(A) Western blotting of caspase-3 and cleaved caspase-3 with densitometric analysis and (B) PCNA immunostaining with representative examples of different treatment groups. *p<0.05. Note. PCNA, proliferating cell nuclear antigen.
Figure 6. Effects of riociguat and sildenafil…
Figure 6. Effects of riociguat and sildenafil on sGC and eNOS protein levels, and cGMP concentration in the lung tissue of SUHx rats.
(A) Western blotting of eNOS, sGCα1 and sGCβ1, (B) densitometric analysis of sGCα1/β-actin, (C) densitometric analysis of sGCβ1/β-actin, (D) densitometric analysis of eNOS/β-actin and (E) concentration of cGMP, in lung tissue of different treatment groups. *p<0.05. Note. eNOS, endothelial nitric oxide synthase; PDE-5, phosphodiesterase type 5; sGCα1, soluble guanylate cyclase α1; sGC, soluble guanylate cyclase; sGCβ1, soluble guanylate cyclase β1; cGMP, cyclic guanosine monophosphate.

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