Fig 1.
Experimental timeline in days for the monocrotaline (MCT)-induced pulmonary hypertension protocol.
(a) MCT was injected at a dose of 60 mgkg-1 i.p. on day 1. Fourteen days (washout period) after MCT injection, rats were orally treated with sildenafil (S), LASSBio-1359 (L), or both substances for 14 days. On days 1, 14, and 28, echocardiographic examinations were performed to monitor cardiopulmonary system function and structure. On day 28, right ventricle (RV) catheterization was performed to measure RV systolic pressure (RVSP). Following this measurement, the animals were euthanized, the hearts were removed and weighed, and the RV was dissected. Pulmonary arteries (PAs) were rapidly removed for the vascular reactivity study. Representative images of the PA outflow profile are presented for (b) day 0, (c) day 14, and (d) day 28 after MCT injection in an animal from the vehicle (V) group. Note the progressive change in the shape of the PA waveform from day 14 to day 28 after MCT injection, which confirmed the development of PH.
Fig 2.
(a) Relaxation effect of sildenafil, LASSBio-1359, or a combination of these drugs on phenylephrine-induced contraction of isolated pulmonary arteries from male Wistar rats. Each point represents the mean ± standard error of the mean (SEM) of 6 experiments. (b) Isobologram showing the interaction of sildenafil with LASSBio-1359. Note that the experimental EC50 is located at the left and below the theoretical EC50. A synergistic interaction between sildenafil and LASSBio-1359 was detected using the Student’s t-test. *P < 0.05 compared with EC50add. EC50, concentration of each drug that produces 50% of the maximal relaxation; EC50mix, experimental EC50 of the drug combination; EC50add, theoretical additive combination EC50.
Table 1.
Comparative data on body, heart, and right ventricle weights.
Fig 3.
Effects of MCT injection and treatments with vehicle, monotherapy, or combination of sildenafil and LASSBio-1359 on the PA outflow profile.
(a) Representative images of the PA outflow profile in the group receiving MCT injection (after 28 days). (b) Ratio between PA acceleration time and TET. (c) PA peak velocity. Data represent the mean ± SEM (n = 6 rats per group). One-way analysis of variance was used to determine the significance of differences among groups. Significance of interactions between groups was determined by Tukey’s post-hoc tests. *P < 0.05 compared with C group rats; #P < 0.05 compared with V group rats; †P < 0.05 compared with S-34 group rats, and °P < 0.05 compared with L-34 group rats. C, control; V, MCT + vehicle; S-34, MCT + sildenafil (34 μmolkg-1day-1); S-170, MCT + sildenafil (170 μmolkg-1day-1); L-34, MCT + LASSBio-1359 (34 μmolkg-1day-1); L-170, MCT + LASSBio-1359 (170 μmolkg-1day-1); S + L, combination of sildenafil and LASSBio-1359 (34 μmolkg-1day-1 of each drug); PA, pulmonary artery; PAAT, pulmonary artery acceleration time; TET, total ejection time of the right ventricle; MCT, monocrotaline.
Fig 4.
Effects of MCT injection and treatments with vehicle, monotherapy, or combination of sildenafil and LASSBio-1359 on PA reactivity to ACh and RVSP on day 28.
(a) Linear regression between RVSP and ACh-induced maximal relaxation in PAs from experimental groups. (b) ACh-induced maximal relaxation. (c) RVSP. Data represent the mean ± SEM (n = 6 rats per group). One-way analysis of variance was used to determine the significance of differences among groups. Significance of interactions between groups was determined by Tukey’s post-hoc tests. *P < 0.05 compared with C group rats; #P < 0.05 compared with V group rats; †P < 0.05 compared with S-34 group rats, °P < 0.05 compared with L-34 group rats, ΦP < 0.05 compared with S-170 group rats, and ∞P < 0.05 compared with L-170 group rats. C, control; V, MCT + vehicle; S-34, MCT + sildenafil (34 μmolkg-1day-1); S-170, MCT + sildenafil (170 μmolkg-1day-1); L-34, MCT + LASSBio-1359 (34 μmolkg-1day-1); L-170, MCT + LASSBio-1359 (170 μmolkg-1day-1); S + L, combination of sildenafil and LASSBio-1359 (34 μmolkg-1day-1 of each drug); ACh, acetylcholine; RVSP, right ventricular systolic pressure; MCT, monocrotaline.
Fig 5.
Effects of MCT injection and treatments with vehicle, monotherapy, or combination of sildenafil and LASSBio-1359 on RV geometry and function 28 days after MCT administration.
(a) Representative images of RV free wall obtained by M-mode echocardiography. (b) RV free wall thickness. (c) RV stroke volume. (d) Heart rate. (f) RV cardiac output. Data represent the mean ± SEM (n = 6 rats per group). One-way analysis of variance was used to determine the significance of differences among groups. Significance of interactions between groups was determined by Tukey’s post-hoc tests. *P < 0.05 compared with C group rats; #P < 0.05 compared with V group rats; †P < 0.05 compared with S-34 group rats, °P < 0.05 compared with L-34 group rats, and ΦP < 0.05 compared with S-170 group rats. C, control; V, MCT + vehicle; S-34, MCT + sildenafil (34 μmolkg-1day-1); S-170, MCT + sildenafil (170 μmolkg-1day-1); L-34, MCT + LASSBio-1359 (34 μmolkg-1day-1); L-170, MCT + LASSBio-1359 (170 μmolkg-1day-1); S + L, combination of sildenafil and LASSBio-1359 (34 μmolkg-1day-1 of each drug); RV, right ventricle; MCT, monocrotaline.
Fig 6.
Kaplan—Meier survival curve of MCT-injected rats treated with vehicle (DMSO) or therapeutic regimens.
MCT, monocrotaline.