Mitochondrial Transplantation from Bone Marrow Mesenchymal Stromal Cells Combined with Sildenafil Attenuated Vascular Remodeling and Improved Right Ventricular Dysfunction in Experimental Pulmonary Arterial Hypertension

Figures

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  Figure 1. Biodistribution of mitochondria derived from bone marrow mesenchymal stromal cells (BM-MSCs) after intravenous administration. BM-MSC–derived mitochondria were labeled with MitoTracker Deep Red and administered intravenously via the jugular vein. (A) Representative ex vivo fluorescence images of the heart, liver, kidneys, and lungs obtained 3 h and 24 h after injection. (B) Quantification of the fluorescence signal (radiant efficiency) in each organ. Data are shown as means ± standard deviation (SD); n = 3 per time point. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test.

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  Figure 2. Echocardiographic assessment of right ventricular outflow and pulmonary artery flow on day 28. (A) Representative echocardiographic images showing pulmonary artery flow (yellow contour) and right ventricular (RV) outflow tract diameter (yellow calipers). (B) Pulmonary artery acceleration time to ejection time ratio (PAT/PET). (C) RV outflow tract diameter. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 8 per group. Data are shown as means ± SD. One-way ANOVA with Tukey’s post hoc test.

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  Figure 3. Right ventricular systolic pressure (RVSP). (A) Representative right ventricular pressure tracings. (B) Quantification of RVSP measured on day 28. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 8 per group. Data are shown as means ± SD. One-way ANOVA with Tukey’s post hoc test.

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  Figure 4. Right ventricular hypertrophy assessed by the Fulton index. The Fulton index was calculated as right ventricle weight divided by left ventricle plus septum weight and normalized to body weight. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 8 per group. Data are shown as means ± SD. One-way ANOVA with Tukey’s post hoc test.

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  Figure 5. Perivascular collagen deposition in lung tissue. (A) Representative lung sections stained with Masson’s trichrome showing perivascular collagen (blue). (B) Quantification of perivascular collagen deposition (%). Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 6 per group. Data are presented as means ± SD. One-way ANOVA with Tukey’s post hoc test (p < 0.05).

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  Figure 6. Pulmonary arterial muscularization assessed by α-smooth muscle actin (α-SMA) immunostaining. (A) Representative images showing α-SMA staining (brown) in the pulmonary artery wall. (B) Quantification of α-SMA expression (%) in the vascular wall. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. Arrows represent intense pulmonary arterial muscularization in PAH and PAH-sil groups. n = 6 per group. Data are expressed as means ± SD. One-way ANOVA with Tukey’s post hoc test (p < 0.05).

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  Figure 7. Endothelial-to-mesenchymal transition markers in lung tissue. (A) Vimentin (mesenchymal marker) and (B) VE-cadherin (endothelial marker) gene expression were analyzed by reverse transcriptase quantitative polymerase chain reaction (qPCR). Data were normalized to GAPDH and expressed as fold change relative to the CTRL group. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 6 per group. Data are presented as medians and interquartile ranges. Kruskal–Wallis followed by Dunn’s test (p < 0.05).

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  Figure 8. Expression of genes related to mitochondrial homeostasis in lung tissue. Reverse transcriptase quantitative polymerase chain reaction analysis of (A) Succinate dehydrogenase complex, subunit A (Sdha), (B) mitochondrial transcription factor A (TFAM), (C) optic atrophy protein 1 (OPA1), and (D) mitofusin-1 (Mfn-1). Data were normalized to GAPDH and are expressed as fold change relative to the CTRL group. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 6 per group. Data are presented as medians and interquartile ranges. Kruskal–Wallis followed by Dunn’s test (p < 0.05).

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  Figure 9. Plasma levels of inflammatory markers, IL-6 (A) and IL-1β (B). Data were normalized to total protein content using the Bradford assay. Groups: CTRL, control; PAH, pulmonary arterial hypertension; PAH-sil, PAH treated with sildenafil (20 mg/kg/day); PAH-imt, PAH treated with BM-MSC–derived mitochondrial fraction (100 µg); PAH-sil & imt, combination therapy. n = 6 per group. Data are presented as medians with interquartile ranges. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s post hoc test (p < 0.05).