Recombinant factor XIII diminishes multiple organ dysfunction in rats caused by gut ischemia-reperfusion injury

Abstract

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Because FXIII has also been shown to modulate inflammation and endothelial permeability, we hypothesized that FXIII diminishes multiple organ dysfunction caused by gut I/R injury. A model of superior mesenteric artery occlusion (SMAO) was used to induce gut I/R injury. Rats were subjected to 45-min SMAO or sham SMAO and treated with recombinant human FXIII A2 subunit (rFXIII) or placebo at the beginning of the reperfusion period. Lung permeability, lung and gut myeloperoxidase activity, gut histology, neutrophil respiratory burst, and microvascular blood flow in the liver and muscles were measured after a 3-h reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells was tested in vitro. Superior mesenteric artery occlusion-induced lung permeability as well as lung and gut myeloperoxidase activity was significantly lower in rFXIII-treated versus untreated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. Rats treated with rFXIII also had higher liver microvascular blood flow compared with the placebo group. Superior mesenteric artery occlusion did not cause FXIII consumption during the study period. In vitro, activated rFXIII caused a dose-dependent increase in human umbilical vein endothelial cell monolayer resistance to thrombin-induced injury. Thus, administration of rFXIII diminishes SMAO-induced multiple organ dysfunction in rats, presumably by preservation of endothelial barrier function and the limitation of polymorphonuclear leukocyte activation.

Fig. 1. rFXIII diminishes lung permeability alterations after gut I/R injury

Lung permeability is evaluated by measuring concentration of EBD in BALF and expressed as the percentage of that present in the plasma. Data are expressed as means ± SD (n = 8 in each group). *P < 0.05 vs. all other groups.

Fig. 2. rFXIII reduces neutrophil sequestration in lung and gut after gut I/R injury

The level of neutrophil sequestration is evaluated by measuring MPO activity. Data are expressed as means ± SD (n = 8 in each group). *P < 0.05 vs. all other groups; ^#P < 0.05 vs. shams.

Fig. 3. rFXIII decreases the level of oxidative stress after gut I/R injury

The level of oxidative stress is assessed by neutrophil respiratory burst activity. Data are expressed as means ± SD (n = 8 in each group). *P < 0.05 vs. all other groups.

Fig. 4. rFXIII reduces the severity of ileal mucosal damage after gut I/R injury

Hematoxylineosin staining. Original magnification ×100. A, Rat is subjected to SMAO and placebo treatment. Villi tips are disrupted. Red blood cell congestion is seen. Gut injury level is classified as grade 3 mucosal damage score. B, Rat is subjected to SMAO and rFXIII treatment. Extension of the subepithelial space as well as villi edema is seen. Gut injury level is classified as grade 2 mucosal damage score. C, Rat is subjected to sham SMAO and placebo treatment. Almost normal villi are seen (grade 0–1 mucosal damage score). D, Rat is subjected to sham SMAO and rFXIII treatment. Almost normal villi are seen (grade 0–1 mucosal damage score).

Fig. 5. FXIII activity does not change after gut I/R injury

Factor XIII activity in rat plasma is determined using Berichrom assay before and 3 h after SMAO or sham SMAO. Data are expressed as means ± SD (n = 8 in each group). *P < 0.05 vs. before SMAO; ^#P < 0.05 vs. sham + placebo and SMAO + placebo.

Fig. 6. rFXIIIa protects from thrombin-induced HUVEC resistance alterations

Transendothelial electrical resistance of HUVECs after the following types of treatment is shown: 0.1 nM thrombin, 50 nM rFXIIIa, combination of thrombin and rFXIIIa, or albumin (control). Data are expressed as means ± SE (n = 8 in each group). Human umbilical vein endothelial cell resistance after thrombin administration (starting from 0.5 h after treatment) is lower than in all other groups (P < 0.05). Human umbilical vein endothelial cell resistance after thrombin + rFXIIIa administration (starting from 0.5 h and up to 1.2 h after treatment) is higher than after sole thrombin administration alone but lower than after sole rFXIIIa treatment or albumin administration (P < 0.05).

Fig. 7. Immunofluorescent staining of HUVECs treated with the following agents: thrombin, combination of thrombin and rFXIIIa, rFXIII, or albumin (control)

Cells are fixed in 4% paraformaldehyde and stained with anti–VE-cadherin antibody (Cell Signaling, Beverly, Mass), Alexa-Fluor488–conjugated secondary antibody (Invitrogen, Carlsbad, Calif), and DAPI (blue) at 60 min after treatment. Disruption of adherens junctions by thrombin is seen by the decrease in green signal intensity. Addition of rFXIIIa reduces HUVEC alterations.