Fig. 4. ACTRIIA-Fc ameliorates PH and vascular remodeling in SU-Hx–exposed rats.

(A) Treatment timing for SU-Hx PH. Male adult rats were injected with vehicle or VEGFR1/2 inhibitor SUGEN5416 (200 mg/kg, sc), subjected to normoxia (Nx) or normobaric hypoxia (FiO₂ = 0.10) for 4 weeks, and received ACTRIIA-Fc (10 mg/kg, ip, twice weekly), sildenafil (60 mg/kg, twice daily, orally), or vehicle (10 mM TBS) for 4 weeks starting 1 day after SUGEN5416. (B) mPAP and (C) RVH in Nx-exposed and SU-Hx–exposed rats treated with ACTRIIA-Fc or sildenafil as compared to TBS vehicle–treated rats (n = 5 to 10 per group). Values are shown as means ± SEM. **P < 0.01 and #P < 0.0001, as indicated, one-way ANOVA with Dunnett’s multiple comparisons test. (D) Medial wall thickness and (E) the percentage of muscularized vessels (diameter, 10 to 50 μm) in rats treated with sildenafil or ACTRIIA-Fc as in (B). Values are shown as means ± SEM. n = 4 to 6 per group (30 to 50 vessels counted per sample). *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 as indicated, one-way or two-way ANOVA with Tukey’s or Dunnett’s test for multiple comparisons. (F) Immunofluorescence images of medial hypertrophy of pulmonary arterioles in Nx-exposed and SU-Hx–exposed rats treated with vehicle, ACTRIIA-Fc, or sildenafil. (G) Treatment timing for established PH in SU-Hx model. Male adult rats received a single injection of SUGEN5416 (20 mg/kg, sc) and were subjected to normobaric hypoxia (FiO₂ = 0.10) for 3 weeks, followed by 3 weeks of normoxia during which rats received varying doses of ACTRIIA-Fc (1, 3, or 10 mg/kg, ip, twice weekly) or isotype control (mIgG2a; 10 mg/kg, ip, twice weekly). (H) RVSP and (I) RVH in rats treated as in (G) (n = 5 to 8 per group). *P < 0.05, one-way ANOVA with Holm- Sidak’s test for multiple comparisons. (J and K) The highest dose of ACTRIIA-Fc attenuated medial wall thickening and (K) the percent-* age of muscularized vessels in rats treated as in (G). *P < 0.05 and **P < 0.01 as indicated, one-way and two-way ANOVA with Tukey’s and Dunnett’s tests for multiple comparisons. Values are shown as means ± SEM. n = 4 to 8 per group (30 to 50 vessels counted per sample). (L) Immunofluorescence images of medial hypertrophy and neointimal lesion formation in pulmonary arterioles from rats treated as in (G). (M) Quantification of proliferation index in small vessels and perivascular tissues of SU-Hx rats treated with isotype control or high dose of ACTRIIA-Fc. **P < 0.01, one-way ANOVA with Tukey’s test for multiple comparisons. Values are shown as means ± SEM. n = 3 to 4 per group (20 random high-powered fields of view quantified per sample). (N) Pai-1 mRNA expression via quantitative RT-PCR in lung tissues from rats with SU-Hx treated with ACTRIIA-Fc or control. *P < 0.05, one-way ANOVA with Tukey’s test for multiple comparisons. Values are shown as means ± SEM. n = 3 to 4 per group. (O) Treatment timing for rats with severe obliterative PH. SU-Hx rats were allowed to progress for 2 weeks under normoxia after 3 weeks of SU-Hx (20 mg/kg, sc; FiO₂ = 0.10), followed by 4 weeks of treatment with vehicle, ACTRIIA-Fc, or sildenafil. (P) RVSP and (Q) RVH in untreated rats after 5 and 9 weeks or rats treated with ACTRIIA-Fc or sildenafil from weeks 6 to 9 (n = 3 to 8). *P < 0.05 and **P < 0.01, one-way ANOVA with Tukey’s test for multiple comparisons. (R) Proportion of occluded vessels (n = 3 to 4) and (S) vascular wall thickness index (n = 3 to 4 and 30 to 50 vessels counted per sample) in rats as in (P). (T) Immunofluorescence images of intimal and medial remodeling in pulmonary arterioles in SU-Hx rats with or without ACTRIIA-Fc treatment. (U) Proliferative index in small vessels and perivascular tissues in SU-Hx rats with or without ACTRIIA-Fc (P = 0.19 ACTRIIA-Fc compared to week 9, n = 4 to 6, 20 random high-powered fields of view quantified per sample). (V) Percentage of apoptotic vascular cells (TUNEL⁺ vessel-associated cells) in ACTRIIA-Fc–treated rats as compared to untreated rats at 5 and 9 weeks after SU-Hx treatment (n = 4 to 6). (W) Percentage of p-SMAD2/3⁺ cells in the luminal wall and media of remodeled vessels in ACTRIIA-Fc–, sildenafil-, or vehicle-treated SU-Hx rats at 9 weeks (n = 4 to 5). Values for (P) to (W) are shown as means ± SEM. *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 as indicated, one-way ANOVA with Tukey’s test for multiple comparisons. Scale bars, 50 μm.