Endothelial cell-derived MMP19 promotes pulmonary fibrosis by inducing E(nd)MT and monocyte infiltration.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in remodeling the extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). MMP19, which is an MMP, was significantly upregulated in hyperplastic alveolar epithelial cells in IPF lung tissues and promoted epithelial-mesenchymal transition (EMT). Recent studies have demonstrated that endothelial-to-mesenchymal transition (E(nd)MT) contributes to pulmonary fibrosis. However, the role of MMP19 in pulmonary vascular injury and repair and E(nd)MT remains unclear. METHODS: To determine the role of MMP19 in E(nd)MT and pulmonary fibrosis. MMP19 expressions were determined in the lung endothelial cells of IPF patients and bleomycin (BLM)-induced mice. The roles of MMP19 in E(nd)MT and endothelial barrier permeability were studied in the MMP19 cDNA-transfected primary human pulmonary microvascular endothelial cells (HPMECs) and MMP19 adenoassociated virus (MMP19-AAV)-infected mice. The regulatory mechanism of MMP19 in pulmonary fibrosis was elucidated by blocking its interacting proteins SDF1 and ET1 with AMD3100 and Bosentan, respectively. RESULTS: In this study, we found that MMP19 expression was significantly increased in the lung endothelial cells of IPF patients and BLM-induced mice compared to the control groups. MMP19 promoted E(nd)MT and the migration and permeability of HPMECs in vitro, stimulated monocyte infiltration into the alveolus, and aggravated BLM-induced pulmonary fibrosis in vivo. SDF1 and Endothelin-1 (ET1) were physically associated with MMP19 in HPMECs and colocalized with MMP19 in endothelial cells in IPF patient lung tissues. AMD3100 and bosentan alleviated the fibrosis induced by MMP19 in the BLM mouse model. CONCLUSION: MMP19 promoted E(nd)MT by interacting with ET1 and stimulated monocyte infiltration into lung tissues via the SDF1/CXCR4 axis, thus aggravating BLM-induced pulmonary fibrosis. Vascular integrity regulated by MMP19 could be a promising therapeutic target for suppressing pulmonary fibrosis. Video abstract.

Bosentan Alters Endo- and Exogenous Bile Salt Disposition in Sandwich-Cultured Human Hepatocytes.

Bosentan, a well-known cholestatic agent, was not identified as cholestatic at concentrations up to 200 µM based on the drug-induced cholestasis (DIC) index value, determined in a sandwich-cultured human hepatocyte (SCHH)-based DIC assay. To obtain further quantitative insights into the effects of bosentan on cellular bile salt handling by human hepatocytes, the present study determined the effect of 2.5-25 µM bosentan on endogenous bile salt levels and on the disposition of 10 µM chenodeoxycholic acid (CDCA) added to the medium in SCHHs. Bosentan reduced intracellular as well as extracellular concentrations of both endogenous glycochenodeoxycholic acid (GCDCA) and glycocholic acid in a concentration-dependent manner. When exposed to 10 µM CDCA, bosentan caused a shift from canalicular efflux to sinusoidal efflux of GCDCA. CDCA levels were not affected. Our mechanistic model confirmed the inhibitory effect of bosentan on canalicular GCDCA clearance. Moreover, our results in SCHHs also indicated reduced GCDCA formation. We confirmed the direct inhibitory effect of bosentan on CDCA conjugation with glycine in incubations with liver S9 fraction. SIGNIFICANCE STATEMENT: Bosentan was evaluated at therapeutically relevant concentrations (2.5-25 µM) in sandwich-cultured human hepatocytes. It altered bile salt disposition and inhibited canalicular secretion of glycochenodeoxycholic acid (GCDCA). Within 24 hours, bosentan caused a shift from canalicular to sinusoidal efflux of GCDCA. These results also indicated reduced GCDCA formation. This study confirmed a direct effect of bosentan on chenodeoxycholic acid conjugation with glycine in liver S9 fraction.

Comparison of the inhibitory effect of ketoconazole, voriconazole, fluconazole, and itraconazole on the pharmacokinetics of bosentan and its corresponding active metabolite hydroxy bosentan in rats.

1. This study aimed to investigate the inhibitory effect of azole antifungal agents, including ketoconazole, voriconazole, fluconazole, and itraconazole, on the pharmacokinetics of bosentan (BOS) and its active metabolite hydroxy bosentan (OHBOS) in Sprague-Dawley (SD) rats.2. A total of 25 healthy male SD rats were divided into five groups and treated with various azole antifungal agents by gavage, followed by a single dose of BOS after 30 min.3. The study found that ketoconazole led to a significant increase (5.1-fold) in the AUC(0-t) of BOS, associated with a 5.8-fold elevation in the Cmax, which was greater than that for fluconazole (2.6- and 2.9-fold) and voriconazole (1.1- and 1.7-fold). Accordingly, the Vz/F and CLz/F of BOS reduced by 89.2% and 83.7%, respectively, on administering ketoconazole concomitantly. However, fluconazole caused a decrease in Vz/F and CLz/F by 77.4% and 72.2%, respectively, compared with voriconazole that exhibited a decrease in CLz/F by 51.7% with a negligible change in Vz/F. Also, obvious differences were observed in the pharmacokinetic parameters of OHBOS between the control and treated groups.4. Collectively, treatment with ketoconazole resulted in a prominent inhibitory effect on the metabolism of BOS, followed by treatment with fluconazole, voriconazole, and itraconazole. Therefore, these details of animal studies may help draw more attention to the safety of BOS while combining it with ketoconazole, voriconazole, fluconazole, or itraconazole clinically.