Dual inhibition of TGFβ and PDGF improves RV remodeling and function in response to RV pressure or volume‐loading

Figures

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  FIGURE 1. PAB increases RVSPs and PR results in diastolic retrograde blood flow. (a, b) Representative images showing colour flow and pulsed wave (PW) Doppler echocardiography. (a) Depicts a lack of red colour Doppler signal during diastole in sham rats while (b) PR rats show diastolic retrograde blood flow in the main pulmonary artery. Retrograde flow originating from branch pulmonary arteries indicates severe PR. (c) Quantitative analysis of RV systolic pressures measured by conductance catheters in PR, PAB, and sham controls. Graphs depict that PAB induced elevated RV systolic pressures while no differences were found in PR rat groups compared to sham controls. Data are expressed as mean ±SD. PA, pulmonary artery; PAB, pulmonary artery band; PR, pulmonary regurgitation; PW, pulse wave; RV, right ventricle; TRN, Tranilast.

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  FIGURE 2. Tranilast reduced RV fibrosis and cardiomyocyte hypertrophy in male PAB rats. (a) PAB rats had increased RV fibrosis (i–iv) and cardiomyocyte hypertrophy (v–viii) compared to sham controls. (b) PR did not induce RV fibrosis (i–iv) or cardiomyocyte hypertrophy (v–viii). TRN treatment reduced RV fibrosis (ix) and cardiomyocyte hypertrophy (x) in PAB. Data are expressed as mean ± SD, CSA, cross‐sectional areas; PAB, pulmonary artery band; PR, pulmonary regurgitation; RV, right ventricle; TRN, Tranilast.

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  FIGURE 3. Tranilast reduced TGFβ1 and SMAD signaling associated with RV fibrosis and attenuated PDGFRβ expression in vivo. (a, b) Representative western blots and quantitative analysis of TGFβ1 canonical (a) and non‐canonical signaling (b) and (c) PDGFRβ expression in PAB and PR groups. TRN reduced TGFβ1 expression and SMAD2/3 activity in PAB rats and normalized ERK1/2 activity in PR rats. PDGFRβ expression was increased in vehicle‐treated PAB and PR rats and attenuated with TRN treatment. Data expressed as mean±SD. Sham versus PAB groups are expressed as relative fold‐change relative to sham + vehicle controls, n = 9–12/group; n = 3–6/group in 12W sham versus PR. 12W, 12 week; ERK, extracellular signal‐regulated kinase; PAB, pulmonary artery band; PDGFRβ, platelet‐derived growth factor receptor β; PR, pulmonary regurgitation; TGFβ1, transforming growth factor receptor β1; TRN, Tranilast.

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  FIGURE 4. PDGF inhibition reduced SMAD2/3 and JNK activity and β‐catenin phosphorylation to attenuate POSTN expression in TGFβ‐stimulated and reduced RV fibroblast proliferation in mechanically stimulated RV fibroblasts. (a–c) Western blots and quantitative analysis of (a) TGFβ canonical, (b, c) non‐canonical signaling (p38, ERK, JNK MAPKs, and Akt), (c) β‐catenin phosphorylation, and (d) POSTN expression in RV fibroblasts. (d) Representative fields of immunofluorescent stained mechanically stimulated RV fibroblasts to detect proliferating vimentin‐positive cardiac fibroblasts (green), by ki‐67 (red) positive nuclei. Cellular nuclei were stained blue (Hoescht stain). Images are in 20× magnification. Proliferating nuclei are depicted by the overlay of blue and red (purple). Microscopy images are in 20× magnification. Quantitative analysis of the proliferative index (PI), ratio of Ki‐67 positive nuclei to total nuclei. DMSO + stretch (62% ± 19%); AG 1295 + Stretch (32% ± 22%); TRN + Stretch (46% ± 19%). Data are expressed as mean ±SD, NS, non‐stretch; TRN, Tranilast.

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  FIGURE 5. TGFβ1 increases PDGFRβ expression but not PDGFRα. (a) Representative western blots of PDGFRα and PDGFRβ in RV fibroblasts. (b) Quantitative analysis of PDGFRα and PDGFRβ expression. TGFβ1 and AG1295 stimulated PDGFRβ expression, but no significant changes were found in PDGFRα expression. Data expressed as mean ± SD, n = 3/group. PDGFR, platelet‐derived growth factor receptor; TGFβ, transforming growth factor β.