Shock Induces Endothelial Permeability After Trauma Through Increased Activation of Rhoa Gtpase

Abstract

Introduction: Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivo plasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results: Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitro trauma-induced permeability (−0.05, P > 0.05), whereas base excess (BE) does correlate with permeability (−0.47, P = 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < −9) compared with the injury only (ISS > 15, BE > −9; P = 0.04) or minimally injured (ISS < 15, BE > −9; P = 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation (P = 0.002) and a trend toward decreased Rac1 activation (P = 0.07) compared with minimally injured control. Conclusions: Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitro using plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro. Our ECIS model of trauma-induced permeability using ex vivo plasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation

“…Importantly, we also here provide evidence for the first time that 3K3A-aPC, which retains its cytoprotective function but has only approximately 5% of its anticoagulant function, abrogated the endotheliopathy/permeability after trauma. Furthermore, it has been previously shown that trauma-induced endothelial dysfunction is driven by a decreased Rac1/RhoA activation and associated loss of junctional integrity 19 . Building upon this ex vivo mechanistic data, we show that pre-incubation with aPC prior to treatment with ex vivo trauma plasma dramatically increases the Rac1/RhoA activation ratio, which corroborates our ECIS results.…”

“…Furthermore, it has been previously shown that trauma-induced endothelial dysfunction is driven by a decreased Rac1/RhoA activation and associated loss of junctional integrity. 19 Building upon this ex vivo mechanistic data, we show that pre-incubation with aPC prior to treatment with ex vivo trauma plasma dramatically increases the Rac1/RhoA activation ratio, which corroborates our ECIS results. This suggests that aPC's barrier protective effects, which are driven through PAR-1 signaling, involve the Rac1 and RhoA GTPase signaling pathways.…”

“…Exposure to ex vivo plasma was 5 minutes and 10 minutes in duration. Cells were then washed with ice cold PBS, and lysate was harvested prior to performing a protein concentration quantification and GLISA assays for Rac1 and RhoA activity as previously described 19 . GTPase activity levels were expressed as a ratio to levels in lysate from untreated control.…”

“…Then, they were treated with a 2 μg/mL concentration 3K3A-aPC in 180 μL of un-supplemented VascuLife media 30 minutes prior to stimulation with 20 μL of ex vivo plasma obtained from patients with severe injury and shock (ISS >15, BE < −6), resulting in a concentration of ex vivo trauma plasma of 10% as previously described. 19 A separate group of cells were treated with thrombin to be used as our positive control while a group of untreated cells were used as our negative control. We then traced the normalized resistance, and thus permeability changes, in real time via ECIS for 30 minutes after treatment with ex vivo plasma.…”

Injury-induced endotheliopathy: What you need to know

“…58 Wu et al 59 demonstrated that this binding activates an intracellular PAK-1 signaling pathway, which disassembles stress fibers to reduce endothelial permeability and maintain barrier integrity. DeBot et al 60 demonstrated that RhoA GTPase is activated after trauma and reduces endothelial permeability. RhoA GTPAse is upstream of PAK-1and likely is involved in the same pathway of endothelial protection.…”