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Sotatercept analog suppresses inflammation to reverse experimental pulmonary arterial hypertension

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.

www.nature.com/scientificreports/ (BMPRII) mediates signaling by multiple BMPs, notably including circulating BMP9 and BMP10, and signals through SMAD1/5/8 intracellularly. This receptor is a particularly important regulator of vascular homeostasis and serves a critical gate-keeping function in PAH 10 . Consistent with the anti-inflammatory role of BMPRII in pulmonary endothelial cells [11][12][13] , inflammation has been implicated as a likely second hit required to induce severe vascular pathology in the context of reduced BMPRII signaling 14 .
In contrast to the protective function afforded by vascular SMAD1/5/8 signaling, multiple lines of evidence indicate that excessive activation of the SMAD2/3 branch is pathogenic in PAH 15,16 as well as in vascular disease broadly [17][18][19][20] . TGF-β is considered a prototypical SMAD2/3 pathway-activating ligand and a master homeostatic regulator of the respiratory system, with roles in inflammatory and immune regulation 21 as well as PAH pathogenesis 15,16 . Functional antagonism between SMAD1/5/8 and SMAD2/3 signaling pathways at multiple levels and under diverse pathologic conditions 22,23 supports the hypothesis that imbalance between these two superfamily branches is central to PAH pathology 15,16,24 . However, despite compelling evidence of SMAD2/3 involvement in PAH, few studies have implicated SMAD2/3-pathway ligands other than TGF-β in this disease [25][26][27][28][29] . These insights raise the possibility that multiple SMAD2/3 pathway-activating ligands drive pathologic vascular remodeling in PAH, but their identities, respective contributions, and cellular sites of action remain incompletely characterized.
Sotatercept and its rodent analog, RAP-011, are recombinant ActRIIA-Fc fusion proteins capable of sequestering multiple activin-class ligands-including activin A, activin B, growth differentiation factor 8 (GDF8) and GDF11-that preferentially activate the SMAD2/3 pathway 23,28 . In a phase 2 trial, sotatercept significantly improved pulmonary vascular resistance in patients with PAH receiving background therapy (NCT03496207) 30 , and this agent is the focus of ongoing clinical investigation (NCT03738150, NCT04576988, NCT04811092, NCT04896008). We recently described elevated expression of activin A, GDF8, and GDF11 in lung lesions from PAH patients and rodent models of PH, together with robust anti-proliferative and pro-apoptotic activity of ActRIIA-Fc in cellular and preclinical animal models of PAH 28 . Activin-class ligands, particularly activin A, promote inflammatory processes in some disease contexts [31][32][33] but have not been linked with pulmonary vascular inflammation in PAH 28,29 .
To better understand the mechanism by which ActRIIA-Fc exerts vascular anti-remodeling effects, in the present study we used RNA-seq and pathway analyses to determine the impact of ActRIIA-Fc therapy on the pulmonary gene signature in severe experimental PAH and to determine the degree to which this model of severe angio-obliterative PAH resembles the gene signature in PAH patients. Additionally, we explored the potential activities and immune effects of ActRIIA-Fc treatment in a model of heritable PAH arising from Bmpr2 haploinsufficiency. We also investigated the contribution of individual activin-class ligands to macrophage activation in vitro and the importance of multi-ligand sequestration for cardiopulmonary effects of ActRIIA-Fc in vivo. Finally, we sought to determine whether ActRIIA-Fc maintains its beneficial activity in severe experimental PAH when used in therapeutic combination with a vasodilator and whether therapeutic effects of ActRIIA-Fc in severe experimental PAH persist after treatment cessation as an indication of potential disease modification.

Results
Expression profiling and pathway analysis in a rat model of severe angio-obliterative PAH. To investigate the mechanistic bases for effects of RAP-011 reported previously in experimental PH 28 , we conducted RNA expression profiling and pathway analysis of lung tissue in a Sugen-hypoxia-normoxia (SuHxNx) rat model of severe angio-obliterative PAH (Fig. 1A, Supplemental Fig. 1). This model mimics important features of human PAH, including pathologic pulmonary vascular remodeling, perivascular pulmonary inflammation, marked RV dysfunction, and a progressive course culminating in severe occlusive arteriopathy 34,35 . Moreover, therapeutic effects observed in this preclinical model-with the normoxic progression phase included-are considered broadly predictive of therapeutic efficacy in patients 36 .
To set the stage for expression profiling in lung, we confirmed that hemodynamic parameters including RV systolic pressure (RVSP) and total pulmonary resistance index (TPRI) were significantly elevated in untreated SuHxNx rats at the onset of therapeutic treatment (week 5) and continued through week 9 (Supplemental Fig. 1). Delayed treatment with RAP-011 starting at week 5 markedly improved (reversed) hemodynamic deficits by week 9 compared to vehicle-treated SuHxNx rats (Supplemental Fig. 1). We confirmed that RAP-011 treatment produced significantly greater improvement than sildenafil (Supplemental Fig. 1), a conventional vasodilatory agent commonly used as first or second line therapy in PAH. Pulmonary histology in untreated week 5 and vehicle-treated week 9 SuHxNx rats revealed significantly elevated abundance of remodeled and occluded arteries, consistent with present hemodynamic results (Supplemental Fig. 1), and confirmed our previous observations that RAP-011 treatment causes regression of vascular remodeling more effectively than therapy with a standard vasodilator 28 .
We next identified genes differentially expressed in SuHxNx rat lung compared with normal rat lung. Using hierarchical cluster analysis of RNA-seq data, we identified 345 differentially expressed genes (DEGs) with a foldchange ≥ 1.5 and adjusted p-value ≤ 0.05 at both week 5 and week 9. Of these 345 DEGs, 248 were upregulated and 97 were downregulated in diseased rat lung compared with normal tissue (Fig. 1B). Therapeutic treatment of SuHxNx rats with RAP-011 from week 5 to week 9 exerted a robust normalizing effect on this pathologic gene-expression profile (Fig. 1C). By week 9, RAP-011 treatment normalized expression of 207 out of 248 (84%) upregulated DEGs and 69 out of 97 (71%) downregulated DEGs. In contrast, therapeutic treatment of SuHxNx rats with sildenafil altered expression of only 27 of 345 (8%) total DEGs (Fig. 1C). Principal component analysis revealed that lung tissue from RAP-011-treated SuHxNx rats exhibited a gene expression profile globally resembling normal tissue, whereas the profile for sildenafil-treated SuHxNx rat lungs more closely resembled that of untreated SuHxNx rat lungs at week 5 (Supplemental Fig. 2). These results indicate that therapeutic treatment   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% O 2 ) 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. We then used Ingenuity Pathway Analysis (IPA) to identify dysregulated pathways and potential upstream regulators associated with all DEGs (defined by adjusted p-value < 0.001) based on a comparison of lung tissue in untreated SuHxNx rats at both week 5 and week 9 with lung tissue in normal rats. This analysis identified 58 pathways significantly dysregulated in diseased lungs at both week 5 and week 9 compared with normal tissue. As ranked by Fisher's method, the top canonical pathways include those mediating endothelial and vessel injury responses (coagulation, prothrombin activation, and glycoprotein VI pathways); inflammation and immune response (complement, dendritic cell maturation, pattern recognition, interleukin-10, and innate and adaptive immune response pathways); and TGF-β signaling ( Table 1), all of which have been implicated in PAH progression in either patients or preclinical models, or both 2,4 . Interestingly, the top-ranked upstream regulator identified by this analysis was tumor necrosis factor (TNF), a key regulator of inflammatory and immune responses that also inhibits expression of BMPRII 37 . Other top-ranked upstream regulators, such as VCAN, EPHB1, EGLN1, and TSC2, are known to regulate cell proliferation and migration. Additional candidate upstream regulators www.nature.com/scientificreports/ identified by IPA include TGFBR2 and genes involved in the mitogen-activated protein kinase pathway (ERK, p38MAPK, and MAP2k1), well-characterized regulators of inflammation and cell proliferation (Table 1). Overall, the pathways and upstream regulators implicated by this analysis in SuHxNx pulmonary pathology depict a phenotype of highly activated inflammation and proliferation.

Scientific Reports
Concordance in aberrant gene expression profiles between a rat model of severe angio-obliterative PAH and PAH patients. We next investigated the potential relevance of these dysregulated pathways to PAH pathogenesis in patients. Publicly available, human transcriptome data collected from 58 PAH lungs and 25 control lungs (https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE11 7261) were subjected to IPA to identify significantly dysregulated pathways, which were then compared with the pathways identified in SuHxNx rat lung (Table 1). Consistent with published human data, the top-ranked pathways identified here in lung tissue from PAH patients included those involving G protein-coupled receptors and several pathways associated with inflammation and immune responses. Importantly, IPA identified 18 pathways related to inflammatory and immune responses that are significantly dysregulated in lung tissue from both PAH patients and the SuHxNx rat model of severe PAH (Table 2). Among other such pathways are those mediating TGF-β and BMP signaling. These results obtained by IPA indicate concordance in dysregulated gene expression between the SuHxNx rat model and PAH patient lung tissues and provide corroborating evidence that the SuHxNx rat used here is a robust model of human PAH, particularly regarding its exuberant inflammatory gene expression signature.

ActRIIA-Fc targets pulmonary inflammatory markers and macrophage infiltration in diverse models of PH.
To assess the effects of therapeutic treatment with either RAP-011 or sildenafil on dysregulated pathways in SuHxNx rat lung, we determined relative expression levels of DEGs associated with these pathways. Therapeutic treatment with RAP-011 reversed the aberrant expression of many genes in lung that were otherwise activated in the SuHxNx disease state (Fig. 1D). Genes differentially expressed as a result of RAP-011 treatment prominently included those in pathways associated with inflammatory and aberrant immune responses (Fig. 1D). Other DEGs are associated with the TGF-β superfamily pathway, most notably Grem1 (gremlin-1), an endogenous BMP antagonist implicated in endothelial cell proliferation and PAH 38 . Changes in expression associated with RAP-011 treatment were especially pronounced in the cases of Col2A1, C6, and Grem1. In contrast with the striking corrective effects of RAP-011 treatment, therapeutic treatment with sildenafil produced limited changes in the expression of disease-associated genes (Fig. 1D). These results provide a pulmonary gene expression signature corresponding to the potent therapeutic anti-remodeling effects of RAP-011 in this model of severe PAH. We then examined the effects of RAP-011 therapy on expression of selected inflammatory and immune molecular markers in SuHxNx rat lung. PAH progression in SuHxNx rats was associated at week 9 with significantly elevated expression of eight key markers, including Il6 and Ccl2, and in each case therapeutic treatment with RAP-011-but not vehicle or sildenafil-fully normalized their mRNA levels ( Fig. 2A). Importantly, RAP-011 Table 2. Top common pathways in lungs from SuHxNx rats and PAH patients.  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). www.nature.com/scientificreports/ and sildenafil in combination normalized expression of these markers as effectively as RAP-011 monotherapy ( Fig. 2A), indicating that RAP-011 provides robust benefit in this model even when used in combination with a standard vasodilator. We investigated the effects of therapeutic treatment with RAP-011 on expression of CD11b, a marker for activated alveolar macrophages-the predominant immune cells within alveolar airspaces 39 . As determined by immunostaining, perivascular CD11b + cell abundance was significantly elevated in the lungs of SuHxNx rats at week 9 compared with levels in normal rat lungs (Fig. 2B,C). As with other immune markers, therapeutic treatment with RAP-011 fully blocked pulmonary infiltration of CD11b + cells, whereas treatment with either vehicle or sildenafil had no effect (Fig. 2B,C). RAP-011 fully normalized the number of pulmonary CD11b + cells when administered in combination with sildenafil ( Fig. 2B,C), thus demonstrating the anti-inflammatory efficacy of RAP-011 as either monotherapy or add-on therapy in this setting. As determined by fluorescence-activated cell sorting, preventive treatment with RAP-011 similarly blocked pulmonary infiltration of CD11b + cells in monocrotaline-treated rats, another established model of inducible PH 40 , while also preventing development of elevated RVSP and RV hypertrophy (Supplemental Fig. 3). These results demonstrate that therapeutic treatment with ActRIIA-Fc-unlike standard PAH therapy with sildenafil-robustly inhibits inflammation and perivascular monocytic infiltration as important components of its tissue-level anti-remodeling activity in PH models.

− Log 10 (P value) Z-Score − Log 10 (P value) Z-Score
Activin-class ligands contribute to macrophage activation and cardiopulmonary remodeling. We next examined effects of activin-class ligands on expression of molecular markers of inflammatory macrophage activation in vitro. THP1 cells, a human monocytic cell line, exhibited differential patterns of gene induction on exposure to activin A, activin B, or GDF11 (Fig. 3A). Specifically, activin A increased expression of Ccl2, Il6, Il1b, and Tnf; activin B selectively increased Il6; and GDF11 increased Ccl2, Il6, and Il1b. As expected, treatment of THP1 cells with a triple combination of activin A, activin B, and GDF11 also caused upregulation of www.nature.com/scientificreports/ inflammatory molecular markers, and co-treatment with a human ActRIIA-Fc analog (ACE-011) prevented this effect (Supplemental Fig. 4). These results indicate that activin-class ligands can individually exert differential activating effects on monocytes to promote a proinflammatory macrophage phenotype in vitro. We used neutralizing antibodies directed against either activins or GDFs to investigate the respective contribution of these ligands to in vivo cardiopulmonary effects of RAP-011, which binds activins and GDFs with high affinity and exhibits slow off-rates advantageous for ligand sequestration 23 . In a preventive SuHx rat model (Fig. 3B), elevated hemodynamic parameters such as systolic pulmonary artery pressure (sPAP) and mean pulmonary artery pressure (mPAP) were normalized more effectively by dual combination treatment with antibodies directed against activin A/B and GDF8/GDF11 than by separate antibody treatments (Fig. 3C,D). Dual antibody treatment also normalized RV hypertrophy more effectively than separate antibody treatments (Fig. 3E). Although sequestration of either activins or GDFs conveyed partial protection in these experiments, our results imply that sequestration of multiple SMAD2/3-pathway ligands-likely activins, GDF8, and GDF11 in combination-provides a greater spectrum of therapeutic benefit and underlies RAP-011-induced reversal of cardiopulmonary impairments in experimental PH.
ActRIIA-Fc reverses cardiac remodeling in severe experimental PAH. We further investigated the cardioprotective effects of RAP-011 monotherapy and compared them with sildenafil in the SuHxNx model, which exhibits RV hypertrophy and an impaired cardiac index at the onset of therapeutic treatment 28 (Supplemental Fig. 5). Therapeutic treatment with RAP-011 starting 5 weeks after disease initiation improved these parameters significantly by week 9 and yielded greater improvement than treatment with sildenafil (Supplemental Fig. 5). Serial echocardiography revealed that RAP-011 therapy reversed RV dilatation, septal wall flattening, and RV fractional area change (RVFAC), whereas sildenafil did not (Fig. 4B,C). We confirmed that RAP-011 therapy alleviated abnormalities in pulmonary artery acceleration time (PAAT) and RV free-wall thickness (RVFWT) more effectively than sildenafil (Supplemental Fig. 5).
We examined effects of therapeutic treatment with RAP-011 on selected molecular markers of cardiac dysfunction in the SuHxNx model of severe PAH. Cardiac remodeling and heart failure are associated with increased activin-ActRIIA/B signaling and an expression shift from myosin heavy chain isoform α to isoform β (increased Myh7:Myh6 ratio) 41,42 . As compared with normal heart tissue, RV tissue from vehicle-treated SuHxNx rats at week 9 displayed increased expression of β-subunits for activin A (Inhba) and activin B (Inhbb) and an increased ratio of Myh7:Myh6 expression (Fig. 4D). In each case, therapeutic treatment with RAP-011 partially or fully normalized expression of these markers of cardiac dysfunction, whereas treatment with either vehicle or sildenafil did not. Moreover, co-treatment of SuHxNx rats with RAP-011 and sildenafil decreased levels of pSmad3 in the RV, and RAP-011 monotherapy was sufficient to increase levels of pSmad1/5/8 in the RV (Supplemental Fig. 6). Together, these results extend our previous findings in a SuHxNx model and confirm that inhibition of multiple activin-class ligands by ActRIIA-Fc reverses aberrant cardiac gene expression, reverses cardiac structural remodeling, and partially corrects an imbalance in RV Smad signaling in severe experimental PAH.
ActRIIA-Fc is effective when used in combination with a vasodilator in severe experimental PAH. As part of the experiments with SuHxNx rats described above, we investigated whether combined therapy with RAP-011 and sildenafil conferred greater therapeutic benefit for established disease than their respective monotherapies. In comparisons not reported previously, combined therapy with RAP-011 and sildenafil produced significantly greater improvement in cardiac endpoints than treatment with sildenafil alone (Fig. 4B-D; Supplemental Fig. 5). A similar pattern was observed for hemodynamic deficits and vascular occlusion (Supplemental Fig. 1). For some parameters, combination therapy seemed more effective than RAP-011 monotherapy ( Fig. 4; Supplemental Figs. 1, 5), although the majority of the benefit was provided by RAP-011. These results demonstrate greater effectiveness of ActRIIA-Fc monotherapy compared to sildenafil monotherapy, as well as ActRIIA-Fc effectiveness as an add-on therapy in this rat model of severe angio-obliterative PAH, consistent with the efficacy observed in PAH patients receiving background therapies 30 . These results further solidify the view that ActRIIA-Fc operates through mechanisms largely distinct from those of current PAH therapies.
ActRIIA-Fc alleviates cardiopulmonary remodeling and macrophage infiltration in a model of heritable PAH arising from Bmpr2 haploinsufficiency. Loss-of-function mutations in BMPR2 have been identified in heritable PAH, and even idiopathic forms of PAH are associated with either reduced BMPRII protein expression or diminished BMPRII signaling 3,43 . Therefore, we generated Bmpr2 haploinsufficient mice as reported by others 44 and evaluated RAP-011 activity in these mutant mice under hypoxic conditions (Fig. 5A). Analysis of genomic DNA confirmed that Bmpr2 +/R899X mice possess a heterozygous nucleotide substitution at the expected position, and immunoblotting confirmed reduced levels of BMPRII protein in lung lysates consistent with a truncated protein product and/or nonsense-mediated mRNA degradation (Supplemental Fig. 7).
Exposure of Bmpr2 +/R899X mice to hypoxia elevated RVSP, induced RV hypertrophy, and produced abnormalities in PAAT, TAPSE, and RVWT, whereas Bmpr2 +/R899X mice under normoxic conditions lacked these cardiovascular phenotypes (Fig. 5B-F). Preventive treatment with RAP-011 normalized each endpoint (Fig. 5B-F). In addition, exposure of Bmpr2 +/R899X mice to hypoxia for 5 weeks caused pulmonary macrophage infiltration (Fig. 5G,H), as determined by immunostaining for the macrophage marker F4/80. Similar to its therapeutic effects in the SuHxNx rat model, RAP-011 prevented infiltration of macrophages into the lungs of Bmpr2 +/R899X mice (Fig. 5G,H). However, in contrast to results in SuHxNx rats ( Fig. 2A), we did not find evidence that molecular markers of inflammation are upregulated in the lungs of Bmpr2 +/R899X mice subjected to hypoxia (Supplemental Fig. 8), suggesting that the inflammatory phenotype in the mouse model is less severe. Together, these results indicate that a mouse model of heritable PAH, like the SuHxNx rat model of induced PAH described above, is www.nature.com/scientificreports/ characterized by marked inflammatory infiltrates, and that treatment with ActRIIA-Fc in either case is associated with suppressed macrophage infiltration and restored cardiopulmonary structure and function.
Persistence of ActRIIA-Fc-induced cardiopulmonary benefits in severe experimental PAH. We investigated whether cardiopulmonary benefits of therapeutic RAP-011 treatment in severe experimental PAH are sustained after treatment cessation (Fig. 6A). In untreated SuHxNx rats, we confirmed that structural and functional abnormalities present by week 5, including altered RVSP, TPRI, RV hypertrophy, cardiac index, PAAT, and TAPSE, remain largely unchanged at week 13 ( Fig. 6B-G). Therapeutic treatment with RAP-011, starting at week 5, produced significant improvement in these parameters by week 9 (Fig. 6B-G). Importantly, in SuHxNx rats treated therapeutically with RAP-011 from weeks 5 to 9, improvements in each of these endpoints persisted for 4 weeks after treatment cessation, until week 13 ( Fig. 6B-G). Circulating levels of RAP-011 were undetectable 2 weeks after treatment was withdrawn. These results indicate that concurrent inhibition of activin-class 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.

Discussion
The complex, multifactorial etiology of PAH represents a daunting challenge for development of disease-modifying therapies. Low penetrance of mutant alleles such as BMPR2 3,4 indicates that factors in addition to BMPRII deficiency are required to induce PAH in most cases and underscores the importance of therapeutic approaches targeting multiple disease mediators in combination. We previously identified an unexpectedly prominent role of activin-class ligands as drivers of pulmonary vascular disease and established ActRIIA-Fc as a potential therapeutic approach for restoring the balance between pulmonary vascular SMAD1/5/8 and SMAD2/3 signaling in www.nature.com/scientificreports/ PAH 28 . Here, we identify activin-class ligands as key mediators of inflammatory and immune responses-either directly or indirectly-in severe experimental PAH and point to important regulatory effects of these ligands on macrophage activation and perivascular infiltration in diseased lung tissue. These results are the first to implicate activin-driven inflammation in pulmonary vascular remodeling in PAH and broaden the spectrum of known pathologic effects for these important TGF-β superfamily ligands. Our results demonstrate that inflammatory gene signatures and macrophage perivascular infiltrates in severe experimental PAH were normalized by therapeutic treatment with ActRIIA-Fc, and the concordance we observed between aberrant gene profiles in this rodent model and in PAH patients strongly supports the translatability of these findings to human PAH. As confirmed by principal component analysis, therapeutic treatment with ActRIIA-Fc reversed this inflammatory pathologic phenotype to an extent far exceeding that of a standard vasodilator. ActRIIA-Fc similarly prevented macrophage infiltration in lung tissue while exerting beneficial cardiopulmonary effects in two other models, most notably a mouse model of heritable PAH arising from Bmpr2 haploinsufficiency. Additionally, our results support a disease mechanism in which activins and GDFs with overlapping activity profiles act in a concerted manner to promote pulmonary inflammation and cardiopulmonary remodeling. Thus, whereas blockade of either activins or GDFs with their respective antibodies elicited structural and functional improvements, their combined neutralization conferred additive benefit. Finally, anti-remodeling effects of ActRIIA-Fc treatment in severe experimental PAH were undiminished by concurrent vasodilator therapy, indicating the potential of this agent as an effective add-on therapy as well as monotherapy. Beneficial effects were sustained for at least one month after treatment cessation, suggesting that ActRIIA-Fc, unlike standard vasodilators, could be disease modifying.
There is a growing consensus that early and persistent inflammation and altered immune responses underlie PAH pathophysiology. It has been proposed that advanced vascular remodeling might be reversible by approaches that address specific inflammatory and immune processes 2 . Consistent with the normal anti-inflammatory role of BMPRII in pulmonary endothelial cells [11][12][13] , inflammation has been implicated as a likely second hit required to induce severe vascular pathology in the context of reduced BMPRII signaling 14 . In one study, Tian and co-workers found that an acute inflammatory insult caused mesenchymal transdifferentiation by pulmonary endothelial cells (EndMT) through activation of canonical SMAD2/3 signaling, an effect which was reversible in vitro by knockdown of Tgfbr1 (ALK5) or Smad3 and reversible in Bmpr2 mutant rats by a small-molecule inhibitor of ALK5. Their findings further implicate SMAD1/5/8-and SMAD2/3-pathway interactions as an important point for convergence of early pathogenic factors in PAH 10 ; however, it is noteworthy that interventions targeting ALK5 or SMAD3 could also potentially inhibit signaling by activins, GDF8, and GDF11, which share downstream effectors with TGF-β.
Our results identify perivascular macrophages as an important cell type by which ActRIIA-Fc reverses pulmonary vascular remodeling in severe experimental PAH. Although their roles require further investigation, monocytes-macrophages are heavily implicated in PAH [45][46][47][48][49] , potentially orchestrating both the initiation and resolution of pulmonary inflammation. Activin A in particular is involved broadly in macrophage activation, inflammation, and fibrosis 33,[50][51][52] . In PAH patients, alveolar macrophages produce activin A, and elevated levels of circulating activin A are predictive of patient mortality 25 . Our results indicate that activin-class ligands can exert distinct but overlapping effects on monocytes-macrophages, with one shared effect of activin A and GDF11 being increased expression of Ccl2, which encodes an important chemokine that promotes monocyte-macrophage chemotaxis and endothelial permeability 53 . In contrast with the CCL2-promoting effect of SMAD2/3 pathway activators, BMP9 and BMP10 inhibit release of CCL2 by pulmonary endothelial cells to promote vascular quiescence 54 . Reciprocal regulation of CCL2 by activin-class ligands and BMPs suggests that CCL2 could be a key mediator through which activin-class ligands disrupt vascular quiescence and promote pathologic remodeling-an elegant example of bidirectional homeostatic regulation by SMAD2/3 and SMAD1/5/8 pathways. In addition, the joint regulation of CCL2 by both activin A and GDF11 underscores the need to target multiple ligands concurrently to achieve a robust therapeutic outcome.
One prominent proinflammatory cytokine identified in our study is IL-6, whose gene expression is elevated in severe experimental PAH and normalized by ActRIIA-Fc therapy. In addition, we found that activin A, activin B, and GDF11 each increase Il6 expression levels as one component of their proinflammatory effects on monocytes-macrophages in vitro. IL-6 mediates pulmonary macrophage activation by adventitial fibroblasts, is implicated in human PAH, and when overexpressed causes spontaneous development of PH in mice 47,55-59 . Furthermore, BMPRII-and IL-6-associated pathways display reciprocal regulation in pulmonary smooth muscle cells 60 , providing yet another possible link between BMPRII signaling and inflammation in PAH pathogenesis. The extensive evidence for a pathogenic role of IL-6 suggested that this cytokine could be targeted to reduce inflammation and thereby attenuate other PAH disease components. However, a clinical trial in patients with PAH failed to demonstrate robust hemodynamic benefits of tocilizumab, a monoclonal antibody directed against the IL-6 receptor 61 . Although follow-up study will be required in larger patient populations, these results suggest that targeting inflammation alone might be insufficient for treatment of PAH and underscore the complex, multifactorial mechanisms of PAH disease progression.
It is particularly noteworthy that ActRIIA-Fc treatment in severe experimental PAH reverses elevated pulmonary expression of Grem1, which encodes an endogenous BMP antagonist (gremlin-1) regarded as an important promoter of vascular remodeling in PAH 38 . Hypoxia stimulates gremlin secretion by pulmonary microvascular endothelial cells, and Grem1 haploinsufficiency reduces pulmonary vascular remodeling in mice exposed to chronic hypoxia 38 . Other cell types, including arterial smooth muscle cells and macrophages, could also be sources of gremlin-1, and the former type exhibits increased gremlin-1 expression in response to mechanical stretch in vitro 62,63 . Best known for its pro-proliferative actions, gremlin-1 has also been linked to inflammation in the kidney and the lung through effects on Notch signaling and macrophage migration, respectively 62,64 . Gremlin-1 was found to play vital roles in PAH associated with congenital heart disease (systemic-to-pulmonary www.nature.com/scientificreports/ shunts), which does not typically arise from BMPR2 mutation; in this case, gremlin-1 could help to explain reduced BMPRII-SMAD1/5/8 pathway activity in the presence of intact BMPR2 63 . Importantly, therapeutic immunoneutralization of gremlin-1 reduces pulmonary vascular remodeling in experimental PAH 65 . Thus, reversal of Grem1 overexpression could be a key mechanism by which ActRIIA-Fc rebalances SMAD1/5/8 signaling with SMAD2/3 signaling in the pulmonary vasculature 28 . Because gremlin-1 acts as a mediator of BMPRII pathway inhibition by endothelin 66 , gremlin-1 modulation could also be a potential point of mechanistic convergence between therapeutic effects of ActRIIA-Fc and those of endothelin receptor antagonists in patients with PAH. Vascular remodeling in PAH is understood in broad terms to arise from physiologic cellular responses to stress or injury that eventually become dysregulated and persistent. Inflammation, hypoxia, or biomechanical stress in individuals with impaired BMPRII pathway activity may promote pathologic remodeling of the extracellular matrix, abnormal cellular proliferation, and potentially EndMT 67 . Recent findings indicate that BMPRII plays a protective role in endothelial cell homeostasis, with loss of BMPRII favoring EndMT and driving cells toward a primed biomechanical state in which changes in stiffness or shear stress provide a second hit and initiate a self-sustaining cycle of excess TGF-β signaling 10 . Chronic TGF-β1 signaling in PAH also induces sustained SMAD3 activation in pulmonary artery smooth muscle cells, which correlates with the hemodynamic and morphologic PAH phenotype in rodents 68 . Extensive involvement of TGF-β in these important disease processes raises the possibility that other SMAD2/3-pathway ligands may play under-appreciated pathologic roles that reinforce or overlap with those of TGF-β. Indeed, recent findings indicate that activin A produced by pulmonary microvascular endothelial cells can promote PAH through increased internalization and degradation of BMPRII 29 , in yet another example of crosstalk between SMAD2/3 and SMAD1/5/8 pathway branches.
In this report, we compared the effects of ActRIIA-Fc with those of the phosphodiesterase type 5 inhibitor sildenafil in PAH models and found far greater anti-inflammatory efficacy for ActRIIA-Fc. However, vasodilator therapies approved for use in PAH can act through one of several distinct mechanisms, also including stimulation of soluble guanylate cyclase and inhibition of endothelin receptors. Our present results should therefore be interpreted conservatively, as further studies will be required to compare anti-inflammatory effects of ActRIIA-Fc with those of other vasodilator classes, both individually and in combination. In addition, clinical data will be required to determine whether the anti-inflammatory effects seen with ActRIIA-Fc treatment in rodent models also contribute to efficacy in patients with PAH.
Complete reversal of PAH in rare cases 69 offers hope that cardiopulmonary remodeling in PAH patients can be reversed more generally with a sufficiently robust therapeutic approach. The ability of ActRIIA-Fc to reverse established pulmonary inflammation and cardiopulmonary remodeling in severe experimental PAH indicates that the SMAD2/3-pathway ligands targeted by this agent mediate key interactions between cell types implicated in this disease, potentially including perivascular immune cells, endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts. We speculate that the breadth and robustness of ActRIIA-Fc activity revealed here in experimental PAH, attained through mutually reinforcing effects on pathogenic components of inflammation and cardiopulmonary remodeling, could potentially translate to disease-modifying activity of sotatercept in patients with PAH, as either a monotherapy or add-on to currently available therapies for PAH.

Materials and methods
All experiments were performed in accordance with the relevant guidelines and regulations approved by Acceleron Pharma Inc., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.
Animal studies. Animal studies were approved by the Institutional Animal Care and Use Committee at Acceleron Pharma Inc., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, in accordance with ARRIVE guidelines. Adult male Sprague-Dawley (SD) and Wistar (WI) rats (150-180 gm) (Envigo, Indianapolis, IN) were used as SuHxNx and MCT rat models, respectively. The SuHxNx model was established by a single subcutaneous injection of vascular endothelial growth factor receptor antagonist semaxanib (SU5416, 20 mg/kg; Cayman Chemical) with immediate onset of exposure to normobaric hypoxia (10% O 2 ) for 3 weeks followed by normoxia (21% O 2 ) for 6 weeks as described 34,35,70 . Sildenafil was obtained from Cayman Chemical. The MCT model was established by a single subcutaneous injection of MCT (60 mg/kg, Torris) followed by 4 week exposure to normoxia. Bmpr2 +/R899X mice were generated as a model of BMPR2 haploinsufficiency as described 44 . Fusion protein and neutralizing antibodies. RAP-011 was constructed and purified essentially as described 71 . Anti-activin A antibody (A2) 72 and an antibody with dual specificity for myostatin and GDF11 (RK35) 73 were modified internally for use in mice by substitution of murine IgG2a Fc. Anti-activin B antibody was generated using the Adimab platform and validated internally.
Hemodynamic and RV measurements. Animals were anesthetized with 3-4% isoflurane and placed on controlled heating pads. RV systolic pressure (RVSP) was measured by advancing a curved-tip pressure transducer catheter, 2F (SPR-513, Millar Instruments) for rats and 1F (SPR-1000, Millar Instruments) for mice, into the RV via the right jugular vein under 1-2% isofluorane anesthesia. In rats, cardiac output was assessed by advancing a 2F microtipped PV catheter (SPR 838, Millar Instruments) into the left ventricle through the right carotid artery under 1.5-2% isofluorane anesthesia. Cardiac index (CI) was calculated by dividing cardiac output by body weight. Total pulmonary vascular resistance index (TPRI) was estimated by dividing RVSP by CI 35 . Heart and lungs were collected en bloc and lungs were perfused with physiological saline via the RV outflow tract to flush the blood cells from the pulmonary circulation. RV hypertrophy was determined by calculating the weight ratio of the RV free wall to the combined left ventricle and septum (Fulton index Histopathology and immunohistochemistry. After perfusion, the right pulmonary lobe was separated and snap frozen for biochemical analysis while the left pulmonary lobe was collected and preserved in neutral buffered formalin as described 34,35

Morphological analyses.
Lung sections prepared with modified Verhoeff-Van Gieson stain and anti-αSMA immunostaining were used to assign grades for vascular occlusion and analyzed with HALO software (Indica Labs) to determine wall thickness of pulmonary arteries. Wall thickness was expressed as [(OD -ID)/ OD] × 100%, where OD is vessel outer diameter and ID is inner diameter. Briefly, each lung section was subdivided by rectangular grid, and outer and inner perimeters of vessels in randomly selected rectangles were measured to calculate OD and ID, respectively. Wall thickness was stratified on the basis of OD into three groups, < 50 μm, 50-100 μm, and > 100 μm. The degree of vascular occlusion was classified as grade 0 (no luminal occlusion), grade 1 (< 50% luminal occlusion), or grade 2 (> 50% luminal occlusion) as described 35 . Approximately 100 arteries per lung section were analyzed in each of four animals per treatment group. All measurements and scoring of occlusive lesions were performed by an investigator blinded to treatment grouping.

Echocardiography.
Echocardiography was performed with a Vevo 3100 imaging system with MX201 scanhead (VisualSonics, Toronto, ON, Canada) on rats anesthetized with 3-4% isoflurane and maintained with 1.5-2% isoflurane. B-Mode, M-Mode and pulse-wave Doppler flow imaging were performed in each rat at the end of weeks 5 and 9. Briefly, rats were placed supine on a heated platform and allowed to breathe spontaneously. The RV outflow tract was visualized using a modified parasternal long axis view. Pulmonary artery acceleration time (PAAT) was measured as the time from start to peak velocity of blood flow in the lumen of the main pulmonary artery distal to the pulmonary valve as obtained from the pulse-wave doppler recording. B-Mode parasternal short-axis view of a mid-ventricular cross section of the heart was visualized at the level of the papillary muscles. RV wall thickness (RVWT) was measured using M-mode in a modified parasternal long-axis view through the aortic valve. RV fractional area change (RVFAC) was measured using a B-mode apical fourchamber view. Tricuspid annular plane systolic excursion (TAPSE) was obtained from the apical four-chamber view directing the M-mode doppler beam through the lateral annulus of the tricuspid valve plane. For each parameter, measurements from three individual heartbeats per animal were taken and averaged. Values shown for echocardiographic parameters PAAT, RVWT, and TAPSE from rats treated with sildenafil monotherapy are historical data from our laboratory.
Quantitative PCR. Frozen tissues were pulverized using a stainless steel mortar and pestle (Cellcrusher) chilled in liquid nitrogen and homogenized in 700 μl QIAzol (79306, QIAGEN) with a Precellys CK28-R hard tissue homogenizing kit (P000916-LYSK0A, Bertin). Total RNA was extracted from homogenized tissues by using the miRNeasy Mini Kit (217994, QIAGEN), and the concentration and quality of RNA was determined by absorbance at 260/280 nm with a NanoDrop One Spectrophotometer (Thermo Scientific, USA). cDNA was prepared using RNA to cDNA EcoDry™ Premix-Random Hexamers (639546, Takara Bio, USA). Quantitative PCR was performed on reverse-transcribed cDNA using TaqMan™ Universal PCR Master mix (4304437, Applied Biosystem) to analyze mRNA expression levels. Relative expression of mRNA was determined by the ΔΔCt method. www.nature.com/scientificreports/ penicillin-streptomycin-amphotericin B solution (PCS-999-002, ATCC) at a dilution of 1:1000. To assess macrophage activation, 1.5 × 10 6 THP-1 cells were treated with activn A (5 ng/mL) or GDF11 (5 ng/mL) for 24 h, or activin B (50 ng/mL) for 6 h, in serum-free media. Prior to treatment, cells were growth-arrested overnight in serum free media. After treatment, cells were washed twice with ice-cold PBS and lysed with Buffer RLT plus (1,053,393, Qiagen). Total RNA was extracted with RNAeasy plus kit (74,134,Qiagen) according to the manufacturer's instructions. cDNA was prepared using RNA-to-cDNA EcoDry™ Premix-Random Hexamers (639,546, Takara Bio, USA). Quantitative PCR was performed on reverse-transcribed cDNA using TaqMan™ Universal PCR Master mix (4,304,437, Applied Biosystem) to analyze mRNA expression levels. Relative expression of mRNA was determined by the ΔΔCt method.
Pulmonary cell suspensions and fluorescence-activated cell sorting (FACS). Single-cell suspensions were prepared from lung as described 74 . Briefly, heart and lungs were collected en bloc, and lungs were perfused with phosphate buffered saline (PBS) via the RV outflow tract to flush blood cells from the pulmonary circulation. After perfusion, lung lobes were diced for 2 min in 20 ml ice-cold digestion buffer (HBSS), containing 1.5 mg/ml collagenase A (Roche), 0.4 mg/ml DNase I (Roche), 5% fetal bovine serum, and 10 mM HEPES, pH 7.4. This tissue suspension was incubated at 37 °C for 30 min with continuous gentle shaking. After digestion, 20 ml of ice-cold PBS was added and gently mixed. The resulting cell suspension was strained through a 70 μm cell strainer, centrifuged at 12,000 rpm at 4 °C for 5 min, and treated with red blood cell lysis buffer on ice for 10 min. Following red blood cell lysis, remaining cells were washed twice with PBS at 12,000 rpm at 4 °C for 5 min. For FACS, cells were incubated in 100 µl of flow mix solution composed of stain buffer (BD Biosciences) with 2% Fc block containing antibodies at 1:33 fold dilution (except for stained controls) for 30 min at 4 °C. After staining, cells were washed with 1 ml of stain buffer twice at 12,000 rpm at 4 °C for 5 min. The cell pellet was resuspended in 500 µl stain buffer, and propidium iodide was added to allow exclusion of dead cells. Data was acquired with an LSRII flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences). Compensation was performed on the flow cytometer at the beginning of each experiment. Data were analyzed using Flowjo v10.
Statistical analysis. Statistical analysis was performed using GraphPad Prism 8. Data are reported as means ± SEM. Differences between groups were analyzed using Student's t-test or ANOVA with Tukey post hoc test for multiple comparisons. Differences were considered significant at P < 0.05.