Intratracheally Administered Peptide-Modified Lipid Admixture Containing Fasudil and/or DETA NONOate Ameliorates Various Pathologies of Pulmonary Arterial Hypertension

Figures

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  Figure 1. Experimental design plan: On Day 0, the rats were given a subcutaneous injection of 20 mg/kg of Sugen. This was followed by a 3-week stay in a hypoxic chamber maintained at 10% oxygen. Subsequently, the rats were housed in a normoxic environment for an additional 3 weeks. Post this period, the rats received one of four treatments via intratracheal administration: saline, CAR-modified lipid mixture containing fasudil, CAR-modified lipid mixture containing DN, or a CAR-modified lipid mixture containing both fasudil and DN. These treatments were administered every 48 h for a span of 3 weeks.

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  Figure 2. MALDI-TOF spectra of (A) the targeting peptide CAR, (B) DSPE-PEG 2000, and (C) the CAR-DSPEG conjugate. The molecular weights of CAR and DSPE-PEG were <1290 Da and ~3000 Da, respectively. The mass of the conjugate (3400–4200) was the additive masses of CAR and DSPE-PEG.

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  Figure 3. (A) Mean pulmonary arterial pressure (mPAP), (B) right ventricular systolic pressure (RVSP) and (C) Fulton index, in the Sugen/Hypoxia rat model upon treatment with the CAR-modified lipid mixture of fasudil, DN and fasudil plus DN. Data are presented as mean ± SEM, n = 3–5, ** p < 0.01, *** p < 0.001.

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  Figure 4. (A) Pulmonary acceleration time (PAT), (B) ratio of PAT/ejection time (PET), (C) tricuspid annual plane systolic excursion (TAPSE), and (D) right ventricular free wall thickness (RVFWT) of PAH rats treated with saline, CAR-modified lipid mixture of fasudil, DN and fasudil plus DN. Data are presented as mean ± SEM, n = 3–5, * p < 0.05, ** p < 0.

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  Figure 5. Representative images of pulmonary acceleration time (PAT), right ventricular free wall thickness (RVFWT), and tricuspid annular plane systolic excursion (TAPSE) of PAH rats treated with saline, the CAR-modified lipid mixture of fasudil, DN and fasudil plus DN.

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  Figure 6. Representative images of pulmonary arteries/arterioles of PAH rat lungs treated with (A) saline, he CAR-modified lipid mixture of (B) fasudil, (C) DN and (D) fasudil plus DN.

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  Figure 7. Pulmonary arteries/arterioles with an external diameter of (A) <50 µm, (B) 50 µm–100 µm, and (C) >100 µm. For each rat lung sample, 20 pulmonary arteries/arterioles were identified and analyzed to determine the ratio of wall area to total vessel area upon treatment with (1) saline, the CAR-modified lipid mixture containing (2) fasudil alone, (3) DN alone, and (3) a combination of fasudil and DN. Data are presented as mean ± SEM, n = 3–5, * p < 0.05, ** p < 0.01.

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  Figure 8. (A) Calculation of area of collagen deposition using the following formula: % area of collagen deposition = (collagen area/(background area − transparent area)) × 100. (B) Percentage of collagen deposition. For each rat lung sample, 20 pulmonary arteries/arterioles were identified and analyzed for collagen deposition upon treatment with the CAR-modified lipid mixture of fasudil, DN or a combination of fasudil and DN. Data are presented as mean ± SEM, n = 3–5, *** p < 0.001.

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  Figure 9. Representative images of collagen deposition in pulmonary arteries/arterioles of PAH rat lungs treated with (A) saline, the CAR-modified lipid mixture containing (B) fasudil alone, (C) DN alone, and (D) a combination of fasudil and DN. Arrows indicate blue color collagen deposition.