Search

Background and Aim: We previously reported that hypothermic machine perfusion (HMP) for liver preservation is feasible, but hepatic microcirculatory dysfunction and significant liver damage remain major obstacles in its application when the preservation is extended to 24 h. The underlying injury mechanism is not well understood. The present study sought to investigate the role of thromboxane A2 (TXA2) in the pathogenesis of liver injury after prolonged HMP. Methods: Livers isolated from Sprague–Dawley rats were subjected to continuous machine perfusion with University of Wisconsin (UW) solution at a flow rate of 0.4 mL/min/g liver at 4°C for 24 h. A specific TXA2 synthase inhibitor, OKY-046 (OKY), was added to UW solution during the preservation period and to the Krebs–Henseleit buffer during reperfusion. The performance of the livers after preservation was evaluated using an isolated liver perfusion system with Krebs–Henseleit buffer at a flow rate of 15 mL/min at 37°C for 30 min. Results: Prolonged HMP induced a significant release of TXA2 into the portal circulation as indicated by markedly increased levels of TXB2 in the perfusate during reperfusion (at 30 min, 1447.4 ± 163.6 pg/mL vs 50.91 ± 6.7 pg/mL for control). Inhibition of TXA2 synthesis with OKY significantly decreased releases of TXA2 (69.8 ± 13.4 pg/mL) concomitant with reduced lactate dehydrogenase (LDH) releases (at 30 min, HMP + OKY: 144.9 ± 27.9 U/L; HMP: 369.3 ± 68.5 U/L; simple cold storage or SCS: 884.4 ± 80.3 U/L), decreased liver wet/dry weight ratio (HMP + OKY vs SCS and HMP: 3.6 ± 0.3 vs 4.4 ± 0.1 and 3.9 ± 0.2, respectively) and increased hyaluronic acid uptake (at 30 min, HMP + OKY vs SCS, HMP: 33.1 ± 2.9% vs 13.9 ± 3.6%, 18.6 ± 2.4%, respectively). Liver histology also showed significant improvement in tissue edema and hepatocellular necrosis with OKY compared with HMP without OKY. Conclusion: The results demonstrate that TXA2 is involved in the development of hepatocellular injury induced by HMP, and inhibition of TXA2 synthesis during preservation and reperfusion protects liver hepatocytes and sinusoidal endothelial cells from injuries caused by prolonged HMP. [ABSTRACT FROM AUTHOR]

We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml·min-1·kg body wt-1). ET-1 (10 pmol/ min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 ± 0.4 vs. 3.6 ± 0.5 vs. 2.6 ± 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 ± 0.4 mmHg vs. 2.9 ± 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 ± 73.4 vs. 110.8 ± 0.8 vs. 114.8 ± 54.7 vs. 135.2 ± 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release. [ABSTRACT FROM AUTHOR]

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A[sub 2] (TXA[sub 2]) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA[sub 2] in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After I wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 rnin of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B[sub 2] (TXB[sub 2]), the stable metabolite of TXA[sub 2]. Although recirculation of the buffer caused no significant change in shamoperated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly-with a significant increase of TXB[sub 2] in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 ± 803 vs. 10,210 ± 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB[sub 2] release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA[sub 2] release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA[sub 2] in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA[sub 2], which is associated with upregulation of the COX-2 enzyme. [ABSTRACT FROM AUTHOR]

Background/Aims: Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance. Methods: One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment. Results: The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2. Conclusions: The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction. [Copyright &y& Elsevier]