Abstract 342: Analysis of Mechanism of a Novel Drug Candidate using an Organ-level Functional Microdevice that Reconstitutes Human Pulmonary Thrombosis
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Pulmonary microvascular thrombosis is a catastrophic medical condition and yet, it is very difficult to predict response and study mechanism of action of potential drug candidates to humans. This is partly so because currently available in vitro assays do not recapitulate physiologically-relevant forces and animal models can also be very complex, making it impossible to analyze intercellular signaling within the lung that occurs under coagulation or drug administration. We designed a model of lung thrombosis in which human primary alveolar and endothelial cells are co-cultured and maintained up to 2 weeks. The device consists of a top chamber seeded with human alveolar epithelial cells (AE) and a lower chamber seeded with endothelial cells, separated by a porous matrix-coated membrane. Whole blood was perfused at a physiological shear stress through the vascular channel and clots were visualized in real-time. When healthy cells were cultured, no intravascular blood clotting was observed, even when lipopolysaccharide (LPS) endotoxin was administered. In contrast, when LPS was added to the AE channel, it caused a significant increase in platelet adhesion at the endothelium, demonstrating that the presence of alveolar epithelium is critical to LPS-induced intravascular thrombosis in vitro . We evaluated this device by analyzing a novel protease activator receptor-1 (PAR1) antithrombotic compound, termed parmodulin 2 (PM2). When the endothelium was cultured along with PM2 under the condition of LPS stimulated AE, we found inhibition of clotting, demonstrating the therapeutic effect of PM2 in the presence of epithelial-endothelial-blood cell signaling. Finally, to test if PM2 performs the therapeutic function of Activated Protein C (APC) that has been reported to stimulate its cytosolic effects via the -arrestin pathway, we knocked down -arrestin in the endothelium and analyzed clot formation again. We found that clotting reoccurred in vascular channel, thus showing that PM2 acts like an APC-like drug candidate. In conclusion, the lung alveolus-on-a-chip reconstitutes organ-level responses to blood clotting and may offer a valuable platform for drug development by allowing to dissect contributions of various cells in their mechanism of action.
