LAUSR

Perivascular inflammation plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Recent studies have demonstrated that damaged mitochondrial DNA induces sterile inflammation by activating toll-like receptor (TLR)9 in spontaneous hypertensive rats. However, it remains unclear whether TLR9 is involved in perivascular inflammation and subsequent development of PAH. The purpose of the present study is to investigate whether chronic inhibition of TLR9 can ameliorate monocrotaline (MCT)-induced PAH in rats. Male Sprague-Dawley rats were injected with MCT (60 mg/kg). First, we conducted immunohistochemistory to examine which cell types express TLR9 in lungs of normal rats and MCT-exposed rats. Second, we extracted cell-free DNA from plasma of rats and amplified genes of COX2 by real-time PCR to detect circulating cell-free mitochondrial DNA, a ligand of TLR9. Third, the administration of a selective TLR9 inhibitor (E6446, 10mg/kg/day, drinking water) or non-selective TLR9 inhibitor (chloroquine: 50mg/kg/day, ip) started three days before MCT injection and sacrificed on day 21. We assessed hemodynamic data and histopathological analysis (EVG stain for medial wall thickness (MWT) in pulmonary arteries (outer diameter: 50 ∼ 100 μm) and CD68 for macrophage accumulation around pulmonary arteries (outer diameter: <50 μm)), and measured the levels of interleukin-6 (IL-6) in lungs by real time PCR. Finally, we investigated survival rate in the reversal protocol, where we started the administration of E6446 on day 14. TLR9 was expressed dominantly in pulmonary endothelial cells and macrophages in the lungs of both normal rats and MCT-exposed rats. Compared with normal rats, MCT-exposed rats showed increased gene expression of COX2 (0.048±0.001 vs. 0.052±0.001 expressed by 1/Ct) in plasma on day 14. MCT-exposed rats also had increased right ventricular systolic pressure (RVSP: 21±1 vs. 60±2 mmHg), total pulmonary vascular resistance index (TPRI: 0.07±0.01 vs. 0.43±0.02 mmHg/min/mL/kg), MWT (0.07±0.01 vs. 0.26±0,02) and accumulation of macrophages (1.6±0.3 vs. 20.0±1.7 cells/HPF) on day 21. In the prevention protocol, either E6446 or chloroquine significantly prevented the elevations of RVSP (49±4 or 48±3 mmHg) and TPRI (0.29±0.04 or 0.27±0.03 mmHg/min/mL/kg) with reducing MWT (0.18±0.01 or 0.18±0.01) and macrophage accumulation (9.7±1.3 or 9.8±2.5 cell/HPF) on day 21. In addition, these drugs significantly reduced the levels of IL-6 mRNA compared with MCT group (4.4±1.0 or 4.8±1.4 vs. 11.9±1.0). In the reversal protocol, the treatment of E6446 had significantly increased the survival rate (50 vs. 10%). TLR9 largely contributes to the development of PAH by reducing perivascular inflammation. Inhibition of TLR9 could be a novel therapeutic target for PAH.