540 - The Effect of LRRC8A Modulation on Inflammatory Signaling and Barrier Function in Endothelial Cells Exposed to Lipopolysaccharide

Publication Number: 1519.540

Tristan Loveday, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States; Ryan Stark, Vanderbilt University Medical Center, Nashville, TN, United States; Fred S. Lamb, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States

Background: Sepsis, a dysregulated response to infection, causes significant pediatric morbidity and mortality. The vascular endothelium plays a key role by regulating vascular tone, leukocyte infiltration, and in particular, tissue edema. Endothelial dysfunction in sepsis is often linked to excessive reactive oxygen species (ROS), production of which requires voltage-regulated anion channels (VRACs), especially the LRRC8A subunit. VRAC inhibitors like DCPIB and tamoxifen might improve endothelial barrier function by inhibiting LRRC8A and reducing LPS-induced inflammation.

Objective: To characterize the impact of LRRC8A genetic and pharmacologic modification on endothelial cell signaling and barrier function.

Design/Methods: Human microvascular endothelial cells (HMVECs) were plated on 0.4 µM transwell inserts in a CellZScope to measure transendothelial electrical resistance (TEER). Cells were treated with scrambled siRNA or siLRRC8A with or without LPS (100 ng/ml) given at 24 hours for genetic manipulation. Pharmacological blockade occurred via tamoxifen (10 µM) or DCPIB (30 µM) either 30 minutes prior to or 90 minutes after LPS exposure with proper vehicle controls. TEER data was recorded for 24 hours after LPS. ELISA was performed on culture supernatants 6 hours after LPS treatment. TEER data were quantified by net area under curve (AUC) compared to baseline. Data were compared via one-way ANOVA. A p-value of < 0.05 was considered significant.

Results: Knockdown of LRRC8A via siRNA significantly increased resting TEER (mean AUC of -311 and 174.3 for siControl and siLRRC8A respectively, p < 0.0001) and resistance after LPS (mean AUC of -469.3 and 295.4 respectively, p = 0.0005). Tamoxifen improved resting TEER (mean AUC of 220.2 for control and 426.1 for tamoxifen, p = 0.0279), and appeared to increase resistance after LPS, though not significant by AUC. DCPIB pre-treatment significantly preserved monolayer integrity (mean AUC of -766.7 for LPS and -320.3 for DCPIB 30' prior to LPS, p = 0.0011). However, DCPIB post-treatment significantly reduced endothelial resistance beyond that of LPS alone (mean AUC of -1,131.2 for LPS 90' prior to DCPIB, p = 0.0036). DCPIB pre-treatment significantly reduced IL-8 concentration (p < 0.0001). Tamoxifen did not reduce LPS-mediated IL-8.

Conclusion(s): Modulation of LRRC8A may improve endothelial barrier integrity when challenged with gram-negative LPS. However, despite both DCPIB and tamoxifen affecting the LRRC8A channel, they have different effects that are time and outcome-based, suggesting a nuanced role of VRAC-mediated ROS in regulating the endothelial response to sepsis.

Figure 1
TEER of HMVECs normalized as indicated; A) siControl vs siLRRC8A exposed to LPS (100 ng/ml) N = 4, B) Control vs tamoxifen (10 µM) applied alone, 30 min prior to LPS, or 90 min after LPS, N = 4, error bars = mean + SEM. Data normalization indicated by y-axis labels.

Figure 2
A) TEER of HMVECs, DCPIB (30 µM) applied either 30 minutes prior to or 90 minutes after exposure to LPS (100 ng/ml), N = 4 (5 for LPS group) vs control and LPS-exposed groups. Normalized to TEER measurement prior to LPS exposure, data normalization indicated by y-axis label.

Figure 3
IL-8 supernatant concentrations for cells treated with Tamoxifen 10 µM (A) or DCPIB 30 µM (B).