Resuscitative Strategies to Modulate the Endotheliopathy of Trauma: From Cell to Patient

Abstract

Clinical data has supported the early use of plasma in high ratios of plasma to red cells to patients in hemorrhagic shock. The benefit from plasma seems to extend beyond its hemostatic effects to include protection to the post-shock dysfunctional endothelium. Resuscitation of the endothelium by plasma and one of its major constituents, fibrinogen, involves cell surface stabilization of syndecan-1, a transmembrane proteoglycan and the protein backbone of the endothelial glycocalyx. The pathogenic role of miRNA-19b to the endothelium is explored along with the PAK-1-mediated intracellular pathway that may link syndecan-1 to cytoskeletal protection. Additionally, clinical studies using fibrinogen and cyroprecipitate to aid in hemostasis of the bleeding patient are reviewed and new data to suggest a role for plasma and its byproducts to treat the dysfunctional endothelium associated with nonbleeding diseases is presented.

Figure 1.. Summary of proposed pathway for fibrinogen’s protection of the endothelium.

Following hemorrhagic shock, shedding of the endothelial cell (EC) syndecan-1 ectodomain is accompanied by an increase in EC miRNA-19b and a decrease in EC syndecan-1 mRNA. This leads to inhibition of the PAK1 mediated intra-cellular signaling pathway with a subsequent increase in stress fiber formation and breakdown of endothelial barrier integrity. With resuscitation, fibrinogen binds to syndecan-1, leading to activation of the PAK1 pathway, inhibition of miRNA19b, decreased stress fiber formation and restitution of the endothelial barrier integrity.

Figure 2.. Endothelial cell permeability is reduced by cryoprecipitate.

Human lung microvascular endothelial cells were serum deprived then incubated in either 10% lactated Ringers (LR), conventional cryoprecipiate (CC), or pathogen-reduced cryoprecipitate (PRC) for 6 hours. The cryoprecipitate products were diluted to a final fibrinogen concentration of approximately 2 mg/ml in media. Permeability was assessed by FITC-dextran. Experiments were performed in triplicate; mean ±SEM, ANOVA with Bonferroni.

Figure 3.. Endothelial cell resistance after treatment with cryoprecipitate.

(A) Cell barrier function was assessed by measuring transendothelial electrical resistance (TEER) real time using electric cell-substrate impedance sensing (ECIS) tracing of human umbilical vein endothelial cells (HUVECs) treated with conventional cryoprecipitate (CC), pathogen reduced cryoprecipitate (PRC), or fresh frozen plasma (FFP) and compared to media alone. (B) Area under the curve (AUC) histogram represents quantitation of changes in barrier resistance. (C) TEER ECIS tracing of HUVECs pretreated with CC, PRC, or FFP then challenged with thrombin and compared to control media. (D) AUC histogram represents quantitation of changes in barrier resistance. The cryoprecipitate products were diluted to a final fibrinogen concentration of approximately 2 mg/ml in media. FFP was treated at 10% of total volume. Experiments were performed in triplicate; mean ±STD, one-way ANOVA. NS indicates no significant difference between groups.

Figure 4.. Endothelial cell permeability is reduced by pathogen-reduced plasma (PRP) and pathogen-reduced plasma cryoprecipitate reduced (PRPCR).

Human lung microvascular endothelial cells were serum deprived then incubated in either 10% Lactate Ringers (LR), pathogen-reduced plasma (PRP), or pathogen-reduced plasma cryoprecipitate reduced (PRPCR) for 6 hours. Permeability was assessed by FITC-dextran. Experiments were performed in triplicate; mean ±SEM, ANOVA with Bonferroni’s correction.