Fibrinogen inhibits microRNA-19b, a novel mechanism for repair of haemorrhagic shock-induced endothelial cell dysfunction

Abstract

Background: The benefits of plasma as an adjunct to the treatment of haemorrhagic shock are well established; however, the mechanism by which plasma modulates the endotheliopathy of trauma remains unclear. Our recent data demonstrated a novel role of microRNA-19b in post-haemorrhagic shock endothelial dysfunction via targeting of syndecan-1. Additionally, fibrinogen, as a key component of plasma or an isolated haemostatic protein, protects the endothelium by stabilizing syndecan-1. We therefore hypothesized that fibrinogen would inhibit microRNA-19b to mitigate the endotheliopathy of trauma in a murine model of haemorrhagic shock.

Materials and methods: C57BL/6J mice were subjected to haemorrhagic shock (mean arterial pressure 35±5 mmHg for 90 minutes) followed by resuscitation with lactated Ringer's, fresh frozen plasma, fibrinogen or no resuscitation. MicroRNA-19b and syndecan-1 mRNA were measured in lung tissue by qRT-PCR. Lungs were stained for histopathologic injury, and broncheoalveolar lavage was collected for protein as a permeability indicator.

Results: Pulmonary microRNA-19b was increased after haemorrhagic shock and lactated Ringers, but reduced to sham levels by plasma and fibrinogen. Conversely, pulmonary syndecan-1 mRNA was downregulated by haemorrhagic shock and lactated Ringers, but returned to sham levels by plasma and fibrinogen. Plasma and fibrinogen-based resuscitation reduced lung injury compared to haemorrhagic shock and lactated Ringers while fibrinogen also reduced broncheoalveolar lavage protein.

Discussion: We have demonstrated a novel mechanism by which fibrinogen, a key component of plasma and haemostatic agent, inhibits miR-19b, possibly by mitigating the endotheliopathy of trauma. Complete demonstration of the mechanism of fibrinogen inhibition of endotheliopathy via microRNA, however, remains to be elucidated. These findings support the early and empiric use of fibrinogen in post-haemorrhagic shock resuscitation.

Figure 1

Mean arterial pressure over time Mice underwent 90 minutes of haemorrhagic shock followed by resuscitation with either fibrinogen, FFP, or LR, and were compared to mice undergoing haemorrhagic shock only and sham mice. Data is reported as mean ± SD with n=6–10/group; two-way ANOVA. Asterisk indicates resuscitation time points at which MAP of fibrinogen resuscitated mice was significantly higher than MAP of FFP, LR and HS only groups. fib: fibrinogen, FFP: fresh frozen plasma, LR: lactated ringer’s, HS: haemorrhagic shock.

Figure 2

Pulmonary miR-19b Mice underwent 90 minutes of haemorrhagic shock followed by resuscitation with either fibrinogen, FFP, or LR, and were compared to mice undergoing haemorrhagic shock only and sham mice. Pulmonary miR-19b was measured by qRT-PCR. Data is reported as mean ± SD with n=3/group; one-way ANOVA. Bars indicate relationships with p<0.05. fib: fibrinogen, FFP: fresh frozen plasma, LR: lactated ringer’s, HS: haemorrhagic shock.

Figure 3

Pulmonary Syndecan-1 Mice underwent 90 minutes of haemorrhagic shock followed by resuscitation with either fibrinogen, FFP, or LR, and were compared to mice undergoing haemorrhagic shock only and sham mice. Pulmonary syndecan-1 mRNA was measured by qRT-PCR. Data is reported as mean ± SD with n=3/group; one-way ANOVA. Bars indicate relationships with p<0.05. fib: fibrinogen, FFP: fresh frozen plasma, LR: lactated ringer’s, HS: haemorrhagic shock.

Figure 4

Lung histopathologic injury Mice underwent 90 minutes of haemorrhagic shock followed by resuscitation with either fibrinogen, FFP, or LR, and were compared to mice undergoing haemorrhagic shock only and sham mice. Shown are representative images and the corresponding lung injury scores (scale of 0–3). Data is reported as mean ± SD with n=4–6/group; one-way ANOVA. Bars indicate relationships with p<0.05. fib: fibrinogen, FFP: fresh frozen plasma, LR: lactated ringer’s, HS: haemorrhagic shock.

Figure 5

Lung permeability Mice underwent 90 minutes of haemorrhagic shock followed by resuscitation with either fibrinogen, FFP, or LR, and were compared to mice undergoing haemorrhagic shock only and sham mice. BAL protein concentration is shown as an indicator of lung permeability. Data is reported as mean ± SD with n=6/group; one-way ANOVA. Bars indicate relationships with p<0.05. fib: fibrinogen, FFP: fresh frozen plasma, LR: lactated ringer’s, HS: haemorrhagic shock.