Fig 2: Treatment with a selective TLR inhibitor on hemodynamic parameters and RV hypertrophy in rats with SU5416/hypoxia/normoxia–induced PH rats. A, Experimental protocol. Rats received a single subcutaneous injection of SU5416 (20 mg/kg weight), and were housed in hypoxic condition (10% O2) for 3 weeks and then normoxic condition for 5 weeks. Some SU5416/hypoxia/normoxia–exposed rats were treated with E6446 (10 mg/kg per day, oral) from week 3 through week 8 after SU5416 injection according to a protocol similar to the reversal protocol (protocol 3) used for the MCT-exposed rats. Normal: normal controls. SU5416/hypoxia/normoxia: SuHxNx. B, RVSP. C, CI. D, TPRI. E, Right ventricular hypertrophy (RV/LV+S). Data are expressed as mean±SEM. N=5 to 8. **P<0.01 vs normal, based on a 1-way ANOVA followed by Bonferroni post hoc test. CI indicates cardiac index; LV, left ventricle; MCT, monocrotaline; PH, pulmonary hypertension; RV, right ventricle; RV/LV+S, weight ratio of RV free wall to the sum of LV free wall and septum; RVSP, right ventricular systolic pressure; TLR9, toll-like receptor 9; and TPRI, total pulmonary vascular resistance index.

Fig 3: Quantification of mitochondrial DNA genes, TLR9 activation, Il-6 and macrophage accumulation in normal control and MCT-exposed rats. A, NADH dehydrogenase subunit 4 (ND4, left), ATP synthase 6 (ATP6, middle) and cytochrome C oxidase subunit II (COX2, right) in plasma of normal and MCT-exposed rats at days 1, 7, and 14 after MCT injection, as measured by real-time PCR. 1/CT denotes the reciprocal of the count where the sequence is detected, and is a direct function of gene concentration. B, Protein expression level of cleaved TLR9 in whole lung measured by Western blotting. C, Il-6 mRNA expression level in whole lung measured by RT–PCR. D, Representative photomicrographs (left) and counts (right) of CD68-positive macrophages (arrowheads) in the lung. Scale bars indicate 50 µm. Con indicates normal rats injected with PBS; and MCT, monocrotaline. N=5–7. *P<0.05 and **P<0.01 vs Con., according to a 1-way ANOVA followed by Bonferroni post hoc test. HPF, high-power field; RT-PCR, reverse transcriptase polymerase chain reaction; and TLR9, toll-like receptor 9.

Fig 4: Preventive effects of long-term treatment with TLR9 inhibitors on pulmonary artery remodeling in MCT-exposed rats.Rats were given a subcutaneous injection of MCT on day 0. Some MCT-injected rats were treated with CLQ or E6446 from 3 days before to 21 days after MCT injection. Normal: normal controls. A, Representative photomicrographs of Verhoeff-van Gieson stained pulmonary arteries with outer diameters of 50 to 100 µm, and comparison of medial wall thickness in 4 groups. B, Representative photomicrographs of pulmonary arterioles with outer diameters of 50 µm or less immunostained for ß-smooth muscle actin showing NM, PM, and TM patterns, and comparison in 4 groups. C, Representative photomicrographs of Ki67-positive cells (arrowheads) in medial layer of pulmonary arteries with outer diameters of 50 µm or less, and comparison of counts in 4 groups. Bars indicate 50 µm. Data are expressed as mean±SEM. N=6 to 7. **P<0.01 vs Normal. ‡ P<0.01 vs MCT, according to a 1-way ANOVA followed by Bonferroni post hoc test. CLQ indicates chloroquine; FM, fully muscular; HPF, high-power field; MCT, monocrotaline; NM, nonmuscular; PM, partially muscular; TM, totally muscular; and TLR9, toll-like receptor 9.

Fig 5: Preventive effects of long-term treatment with TLR9 inhibitors on perivascular inflammation in MCT-exposed rats.Rats were given a subcutaneous injection of MCT on day 0. Some MCT-injected rats were treated with CLQ or E6446 from 3 days before to 21 days after MCT injection. Normal: normal controls. A and B, Representative photomicrographs of CD68-positive macrophages and NF-?B?positive cells (arrowheads) in medial layer of pulmonary arteries with outer diameters of 50 µm or less, and comparisons of counts in 4 groups. Scale bars indicate 50 µm. C, The mRNA expression levels of Il-6 in lung measured by RT-PCR. D, Representative photographs of Western blots of phospho-STAT3, STAT3, and ß-actin in lung (upper) and summary of the protein levels of phospho-STAT3/STAT3 in lung (lower). Data are expressed as mean±SEM. N=6 to 7. **P<0.01 vs Normal. ‡ P<0.01 vs MCT, according to a 1-way ANOVA followed by Bonferroni post hoc test. CLQ indicates chloroquine; HPF, high-power field; MCT, monocrotaline; NF-?B, nuclear factor ?B; RT-PCR, reverse transcriptase polymerase chain reaction; and TLR9, toll-like receptor 9.