Transport Properties of Statins by OATP1A2 and Regulation by Transforming Growth Factor-  (TGF-  ) Signaling in Human

Our in vivo rodent studies have shown that Organic Anion Transporting Polypeptide 1a4 (Oatp1a4) is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, Transforming Growth Factor-  (TGF-  ) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human orthologue of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-  /ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated, in vitro, using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-  /ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. Statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP)). Absorptive flux (i.e., apical-to-basolateral) for all statins was higher than secretory flux (i.e., basolateral-to-apical) and was decreased by an OATP inhibitor (i.e., estrone-3-sulfate). OATP1A2 protein expression, statin uptake, and cellular monolayer permeability were increased by BMP-9 treatment. This effect was attenuated in the presence of LDN193189. Apical-to-basolateral statin transport across human endothelial cellular monolayers requires functional expression of OATP1A2, which can be controlled by therapeutically targeting TGF-  /ALK1 signaling. Taken together with our previous work, the present data show that OATP-mediated drug transport is a critical mechanism in facilitating neuroprotective drug disposition across physiologically relevant multi-transporter environment. We demonstrate that all three statins are OATP1A2 substrates and that their uptake and monolayer permeability are also determined by P-gp and/or BCRP. We also show, for the first time, that activation of TGF-  /ALK1 signaling with the ALK1 agonist bone morphogenetic protein-9 (BMP-9) can increase HUVEC uptake and monolayer permeability of atorvastatin, pravastatin, and (1:40,000) anti-mouse (1:50,0000) secondary antibody (Jackson ImmunoResearch, West Grove, PA). Control western blot experiments to confirm antibody specificity were performed in the presence of secondary antibody only (i.e., absence of primary antibody) and using non-transfected HEK293 cells that do not express detectable levels of OATP transporters. Membranes were developed using enhanced chemiluminescence (Super Signal West Pico, Thermo-Fisher). Bands were quantitated using ImageJ software (Wayne Rasband, Research Services Branch, National Institute of (RSV) (D) were determined in the apical-to-basolateral and basolateral-to-apical direction. Experiments were conducted in the presence and absence of the OATP transport inhibitor E3S (50  M). E: BMP-9 (1 nM) is applied to the basolateral chamber of Transwell inserts. For inhibition experiments, LDN193189 (10  M) is added to the basolateral chamber 1 h prior to addition of BMP-9 to the same compartment. Following BMP-9 treatment, cellular monolayer permeability of atorvastatin (ATV) (B) , pravastatin (PRV) (C) , and rosuvastatin (RSV) (D) were determined in the apical-to-basolateral and basolateral-to-apical direction. Similar to apical compartment treatments, experiments were conducted in the presence and absence of the OATP transport inhibitor E3S (50  M). Data are expressed as mean  S.D. from three independent experiments with each data point in an individual experiment representing quadruplicate measurements. Asterisks indicate data points that are statistically significant from control (* p < 0.05; ** p < 0.01;  p < 0.01 comparing P App, A-B between