Rationale: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) contribute to significant intensive-care unit (ICU) morbidity and mortality. Acute chest syndrome (ACS), a feared complication of sickle cell disease, may also lead to ARDS. Rap1 is a small GTPase that stimulates adhesion of endothelial cells (EC) to the extracellular matrix and enhances cell-cell junction complexes, leading to decreased endothelial permeability. RASA3, a GTPase-activating protein (GAP), is able to function as a GAP for Rap1. We propose that RASA3 is a potential novel target in ALI and ACS pathobiology. Methods: Whole lung lysates were collected from 6-8 week old male transgenic HbSS sickle cell mice and C57BL/6 mice and RASA3 expression was measured. Human lung microvascular endothelial cells (hLMVECs) were cultured to confluency in 6 well plates. Cells were treated with increasing concentrations of hemin (10μM, 20 μM or 40 μM) for 24 hours, or LPS at 1 μg/mL and collected at various time points (1, 6, 12 and 24 hours). Cells were harvested using RIPA buffer, Pierce protein assay was performed and Western blotting was performed. RASA3 and Rap1 antibodies were used to assay protein content and normalized to β-actin. Results: We found that RASA3 expression was slightly lower in SCD mice compared to wild type animals. Treatment of hLMVECs with LPS significantly increased RASA3 expression, peaking at 12 hours, with a trend towards Rap1 expression decreasing 24 hours after treatment. Treatment of hLMVECs with hemin also demonstrated increased expression of RASA3 and decreased Rap1 expression in a dose-dependent manner. Conclusions: Our findings suggest that RASA3 plays a potential role in endothelial cell barrier function in vitro. Further work is ongoing to elucidate the role of RASA3 in ALI models. A schematic of our working model for the role of RASA3 in ALI is shown in Figure 1.

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