Sotatercept analog suppresses inflammation to reverse experimental pulmonary arterial hypertension

Abstract

Sotatercept is an activin receptor type IIA-Fc (ActRIIA-Fc) fusion protein that improves cardiopulmonary function in patients with pulmonary arterial hypertension (PAH) by selectively trapping activins and growth differentiation factors. However, the cellular and molecular mechanisms of ActRIIA-Fc action are incompletely understood. Here, we determined through genome-wide expression profiling that inflammatory and immune responses are prominently upregulated in the lungs of a Sugen-hypoxia rat model of severe angio-obliterative PAH, concordant with profiles observed in PAH patients. Therapeutic treatment with ActRIIA-Fc-but not with a vasodilator-strikingly reversed proinflammatory and proliferative gene expression profiles and normalized macrophage infiltration in diseased rodent lungs. Furthermore, ActRIIA-Fc normalized pulmonary macrophage infiltration and corrected cardiopulmonary structure and function in Bmpr2 haploinsufficient mice subjected to hypoxia, a model of heritable PAH. Three high-affinity ligands of ActRIIA-Fc each induced macrophage activation in vitro, and their combined immunoneutralization in PAH rats produced cardiopulmonary benefits comparable to those elicited by ActRIIA-Fc. Our results in complementary experimental and genetic models of PAH reveal therapeutic anti-inflammatory activities of ActRIIA-Fc that, together with its known anti-proliferative effects on vascular cell types, could underlie clinical activity of sotatercept as either monotherapy or add-on to current PAH therapies.

Figure 1

Therapeutic treatment with ActRIIA-Fc broadly normalizes pulmonary gene expression in severe experimental PAH. (A) Experimental approach used to evaluate therapeutic effects of RAP-011 in a Sugen-hypoxia-normoxia (SuHxNx) rat model of severe PAH. Rats were treated on day 0 with a single dose of SU5416 (20 mg/kg) and exposed to normobaric hypoxia (10% O₂) for 3 weeks followed by 6 weeks of normoxia to allow disease progression. Rats were additionally treated with RAP-011 (2.5 mg/kg, s.c., twice weekly), sildenafil (30 mg/kg, p.o., twice daily), combination therapy with RAP-011 and sildenafil, or vehicle (PBS) for 4 weeks starting on week 5 post SU5416. (B) Heat map of differentially expressed genes (DEGs) in lung from untreated SuHxNx rats at week 5 (Wk 5) and vehicle-treated SuHxNx rats at week 9 (Wk 9 Veh), each compared to normal (Norm). Genes were clustered using the Ward method. (C) Heat map of DEGs at week 9 in lung from SuHxNx rats treated with RAP-011 or sildenafil (Sild), each compared to a normalized average from vehicle-treated SuHxNx rats at week 9 (right column). (D) IPA-based classification of selected genes exhibiting significant differential expression at week 9 in lung from SuHxNx rats treated with vehicle, RAP-011, or sildenafil.

Figure 2

Therapeutic treatment with ActRIIA-Fc suppresses pulmonary inflammation and aberrant immune responses in severe experimental PAH. (A) Levels of Il6, Ifng, Nfatc2, Havcr2, Ccl2, C6, Vcam1, and Sele mRNA in lung of normal rats (Norm) or SuHxNx rats treated with vehicle (Veh), RAP-011, sildenafil (Sild), or a combination of sildenafil and RAP-011. Data are means ± SEM (n = 6–9 rats per group). (B) Representative images of lung sections immunostained for macrophage marker CD11b revealing prominent clusters of labeled perivascular cells in severe experimental PAH after treatment with vehicle or sildenafil but not RAP-011. (C) Quantification of CD11b-positive cells in lung based on assessment of 40 high-magnification fields per rat. Scale bar, 50 µm. Data are means ± SEM (n = 6–9 rats per group). Analysis by one-way ANOVA and Tukey post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3

Multiple ligands contribute to macrophage activation in vitro and cardiopulmonary remodeling in a SuHx rat model of PH. (A) Expression of markers of macrophage activation in THP-1 monocytes in vitro without treatment (Control) or after treatment with activin A (5 ng/ml), activin B (50 ng/ml), or GDF11 (5 ng/ml). Analysis by unpaired t-test (*P < 0.05, **P < 0.01 vs. control). (B) Experimental approach used to test effects of multi-ligand inhibition in a SuHx rat model of PH. Rats were treated with a single dose of SU5416 (20 mg/kg, s.c.), exposed to normobaric hypoxia (13% O₂), and treated s.c. twice weekly with separate antibodies against activin A and activin B (anti-Act, 10 mg/kg each), an antibody with dual specificity for GDF8 and GDF11 (anti-GDF, 10 mg/kg), combined anti-Act and anti-GDF, or vehicle (PBS) for 4 weeks starting one day post SU5416. (C) sPAP, (D) mPAP, and (E) Fulton index. Data are means ± SEM (n = 5–9 rats per group). Analysis by one-way ANOVA and Tukey post hoc test (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 4

ActRIIA-Fc reverses cardiac remodeling and expression of key cardiac genes in severe experimental PAH. (A) Experimental approach used to evaluate therapeutic effects of RAP-011 in the SuHxNx rat model of severe PAH. See Fig. 1 for details. (B) Pairs of representative echocardiographic images obtained at the end of diastole from the same SuHxNx rats before and after therapy. (C) RV fractional area change (RV FAC). Data are means ± SEM (n = 7–11 rats per group). (D) Ratio of myosin heavy-chain isoform expression (Myh7:Myh6) and levels of Inhba and Inhbb in the RV of normal or SuHxNx rats. Data are means ± SEM (n = 6–11 rats per group). Analysis by one-way ANOVA and Tukey post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ### P < 0.001 vs. wk 5, #### P < 0.0001 vs. wk 5).

Figure 5

ActRIIA-Fc reduces pulmonary macrophage infiltration and prevents PH in a mouse model of Bmpr2 haploinsufficiency. (A) Experimental approach used to evaluate cardiopulmonary effects of RAP-011 in mice with Bmpr2 haploinsufficiency. Bmpr2^+/R899X mice were housed under normoxic conditions (Nx) as controls or exposed to normobaric hypoxia (10% O₂) and treated twice-weekly with either RAP-011 (10 mg/kg, s.c.) or vehicle (PBS) for 5 weeks. (B) RVSP, (C) Fulton index, (D) PAAT, (E) TAPSE, and (F) RVWT. (G) Representative images of lung sections immunostained for macrophage marker F4/80. (H) Quantification of F4/80-positive cells in lung based on assessment of 30 high-magnification fields per mouse. Scale bar, 50 µm. Data are means ± SEM (n = 7–10 per group). Analysis by one-way ANOVA and Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Disease-reversing effects of ActRIIA-Fc in severe experimental PAH persist after treatment cessation. (A) Experimental approach used to evaluate the persistence of therapeutic effects of RAP-011 in a SuHxNx rat model of severe PAH. Rats were treated on day 0 with a single dose of SU5416 (20 mg/kg, s.c.) and exposed to 3 weeks of normobaric hypoxia (10% O₂) followed by 10 weeks of normoxia to allow disease progression. Rats were additionally treated twice weekly with RAP-011 (2.5 mg/kg, s.c.) or vehicle (PBS) from week 5 to week 9 post SU5416, at which time treatment was discontinued for the remaining 4 weeks. (B) RVSP, (C) TPRI, (D) Fulton index, (E) cardiac index (CI), (F) PAAT, and (G) TAPSE. Data are means ± SEM (n = 7–13 rats per group). Analysis by one-way ANOVA and Tukey post hoc test (*P < 0.05, **P < 0.01, ****P < 0.0001).