Abstract Rationale The acute respiratory distress syndrome (ARDS) results in alveolar edema, respiratory failure and high mortality. A dysfunctional pulmonary endothelial cell (EC) barrier is a key pathologic hallmark of ARDS. Protein regulation of the actin cytoskeleton is key to maintaining this barrier. We have previously identified a role for the cytoskeletal regulator capping protein Arp2/3 complex myosin-I linker (CARMIL1). In the present study, we link endothelial CARMIL1 expression to alterations in cell junctional proteins and markers of inflammation. Methods EC barrier function was assessed by a FITC-dextran transwell assay. CARMIL 1 expression was augmented in human pulmonary artery ECs via transduction with lentiviral particles encoding WT CARMIL1 or treatment with CARMIL1 siRNA (Santa Cruz) x 48 hrs. ECs were treated with sphingosine-1-phosphate (S1P) x 10 minutes, thrombin (1U/ml) or LPS x 18 hrs. Inflammatory markers (IL-6 and IL-8) were measured by ELISA. Cell fractions (membrane vs cytosol) were separated by differential centrifugation. CARMIL1 and junctional protein expression levels were quantified by western blot. Results CARMIL1 silenced cells exhibited reduced barrier integrity at baseline (884.1 ±34v 1409±26.8 (A.U) and following thrombin (1305.1 ±33.07vs 1771.5±62.8 (A.U) as measured by the FITC-dextran permeability assay. Reduced CARMIL1 expression results in decreased expression of the tight junction proteins ZO-1, claudin-5 and occludin. After LPS treatment, cells transduced with CARMIL1 had significantly decreased levels of IL-6 compared to vector (1216.2±227.1 vs 829.16± 141.01pg/ml, P 0.05) and a trend in reduced IL-8 (11277.2±1767.1 vs9488±1186.254pg/mL). We observed increased CARMIL1 translocation to the cell membrane following S1P treatment. Conclusion Endothelial CARMIL1 expression is required for baseline barrier integrity and preserves barrier function after inflammatory stimuli. CARMIL1 overexpression reduces EC inflammatory markers. Alterations in junctional proteins offer a mechanistic explanation for the role of endothelial CARMIL1 in pulmonary vascular leak and ARDS outcomes. This abstract is funded by: NIH 1R01HL170118
Chen et al. (Fri,) studied this question.