Microcirculatory disturbance in acute liver injury

Abstract

Microcirculatory disturbance is thought to be involved in the pathogenesis of acute liver injury (ALI). The current study examined the pathophysiologic role of hepatic microcirculatory disturbance in patients with ALI and in mouse models of ALI. Using serum aminotransferase (ALT)/lactate dehydrogenase (LDH) ratio as a hypoxic marker, 279 patients with ALI were classified into the low ALT/LDH ratio (ALT/LDH ≤1.5) and high ALT/LDH ratio group (ALT/LDH >1.5). In the low ALT/LDH ratio group, serum ALT, LDH, fibrinogen degradation products and prothrombin time-international normalized ratio were increased relative to the high ALT/LDH ratio group. Histologically, hepatic expression of tissue factor (TF) and hypoxia-related proteins was enhanced in the low ALT/LDH ratio group, and this was accompanied by sinusoidal fibrin deposition. Sinusoidal hypercoagulation and intrahepatic hypoxia was also analyzed in two different mouse models of ALI; Concanavalin A (ConA) mice and Galactosamine/tumor necrosis factor (TNF)-α (G/T) mice. Serum ALT/LDH ratio in ConA mice was significantly lower compared with G/T mice. Pimonidazole staining revealed the upregulation of hypoxia-related proteins in ConA mice. Recombinant human soluble thrombomodulin improved liver damage in ConA mice in association with reduced sinusoidal hypercoagulation and intrahepatic hypoxia. The present study provides evidence that serum ALT/LDH ratio aids in the identification of patients with ALI and intrahepatic hypoxia as a result of microcirculatory disturbance. The results facilitate the improved understanding of the pathogenesis of ALI, thereby offering a novel therapeutic strategy against ALI, which arises from sinusoidal hypercoagulation.

Keywords: acute liver failure; intrahepatic hypoxia; microcirculatory disturbance; serum aminotransferase/lactate dehydrogenase ratio; sinusoidal hypercoagulation.

Figure 1

Serum ALT/LDH4 ratio in every etiology. ALT/LDH, aminotransferase/lactate dehydrogenase ratio.

Figure 2

Histological evaluation of intrahepatic hyper-coagulation and hypoxia related proteins of 17 patients with ALI. (A) TF staining (magnification, x100) and phosphotungstic acid-hematoxylin staining (magnification, x400) of liver sections in a patient with low ALT/LDH ratio and a patient with high ALT/LDH ratio were performed to evaluate sinusoidal coagulopathy. The arrows indicate fibrin depositions in sinusoids. Black Scale bar=100 µm. Red Scale bar=25 µm. (B) HIF-1α, HIF-2α, LDH-V and VEGFA staining of liver sections in a patient with low ALT/LDH ratio and a patient with high ALT/LDH ratio were performed to evaluate intrahepatic hypoxia (magnification, x100). Black Scale bar=100 µm. (C) The percentage of positive area in HIF-1α, HIF-2α, LDH-V and VEGFA staining of liver sections. ^*P<0.05. ALI, acute liver injury; ALT/LDH, aminotransferase/lactate dehydrogenase ratio; HIF, hypoxia-inducible factor; LDH-V, lactate dehydrogenase-V; TF, tissue factor.

Figure 3

Serum ALT/LDH ratio and FDP in ALI model mice. (A) Hematoxylin and eosin staining of liver sections. Scale bar=100 µm. (B) Serum ALT, LDH, ALT/LDH ratio and FDP levels. Data are expressed as the mean ± SD. ^*P<0.05, ns, non-significant. ALT/LDH, aminotransferase/lactate dehydrogenase ratio; FDP, fibrin degradation products; ALI, acute liver injury; ConA, Concanavalin A.

Figure 4

Intrahepatic hyper-coagulation and hypoxia related proteins expressions of ALI model mice. (A) Hepatic mRNA expression of TF and PAI-1 were quantified by RT-q PCR. Data are expressed as the mean ± SE (control mice, n=5; G/T induced ALI model mice, n=5; ConA induced ALI model mice, n=5). (B) TF staining (magnification, x100) and PTAH staining (magnification, x400) of GT and ConA induced ALI model mice liver. The arrows indicate fibrin depositions in sinusoids. Black Scale bar=100 µm. Red Scale bar=5 µm. (C) Hepatic mRNA expression of HIF-1α, HIF-2α, LDHA and VEGFA were quantified by RT-qPCR. Data are expressed as the mean ± SE (control mice, n=5; G/T induced ALI model mice, n=5; ConA induced ALI model mice, n=5). (D) HIF-1α, HIF-2α, LDH-V, VEGFA and pimonidazole staining of GT and ConA induced ALI model mice liver were performed to evaluate intrahepatic hypoxia (magnification, x100). Black Scale bar=100 µm. (E) Western blot analysis showing the levels of expression of HIF-1α and HIF-2α in GT and ConA induced ALI model mice liver. ^*P<0.05, ^**P<0.01. ns, non-significant. ALI, acute liver injury, RT-q, reverse-transcription quantitative; PAI-1, plasminogen activation inhibitor-1; ConA, Concanavalin A; PTAH, phosphotungstic acid-hematoxylin; HIF, hypoxia-inducible factor; LDH, lactate dehydrogenase; TF, tissue factor.

Figure 5

rhTM suppresses liver damage in ConA-induced ALF model mice. (A) Serum ALT and LDH. Data are expressed as the mean ± SD. (B) Hematoxylin and eosin staining of liver sections (magnification, x100). Scale bar=100 µm. (C) Hepatic mRNA expression levels of TF and PAI-1 were quantified by RT-qPCR. Data are expressed as the mean ± SE. (D) TF staining (magnification, x100) and phosphotungstic acid-hematoxylin staining (magnification, x400) of liver. The arrows indicate fibrin depositions in sinusoids. Black Scale bar=100 µm. Red Scale bar=5 µm. (E) Hepatic mRNA expression levels of HIF-1α, HIF2α, LDHA and VEGFA were quantified by RT-qPCR. Data are expressed as the mean ± SE. (F) HIF-1α, HIF-2α, LDH-V, VEGFA and pimonidazole staining of liver were performed to evaluate intrahepatic hypoxia (magnification, x100). Black Scale bar=100 µm. ^*P<0.05, ^**P<0.01. ns, non-significant. rhTM, human soluble thrombomodulin; ConA, Concanavalin A; ALF, Acute liver failure; ALT, aminotransferase; LDH, lactate dehydrogenase ratio; PAI-1, plasminogen activation inhibitor-1; RT-q, reverse-transcription quantitative; HIF, hypoxia-inducible factor; TF, tissue factor.