Kevin Moore

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development.

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.

The lack of a simple assay for the quantification of S-nitrosothiols in complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of approximately 4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28+/-7 nM.

Sepsis is a common complication of cirrhosis with a high mortality. In this study, we have investigated some of the pathways that may be involved in tissue injury and death. Bile duct-ligated (BDL) cirrhotic and control rats were challenged with lipopolysaccharide (LPS). Sensitivity to LPS was markedly enhanced in the BDL group, and was associated with increased liver injury and mortality. There was a 5-fold constitutive activation of nuclear factor kappa B (NFkappaB) in the liver of BDL rat controls (P <.001), and this was activated further, but to a similar extent, in the liver of both sham and BDL rats after injection of LPS. Plasma tumor necrosis factor alpha (TNF-alpha) increased more markedly in the BDL cirrhotic rats (2,463 +/- 697 pg/mL in BDL rats versus 401 +/- 160 pg/mL in the controls at 3 hours; P <.01). Plasma nitrite/nitrate concentrations were increased in the BDL controls at baseline, and increased further after LPS (P <.05), but did not differ from sham controls at 6 hours. Plasma F(2)-isoprostanes increased 6-fold in the cirrhotic rats and 2-fold in the controls (P <.01) indicative of lipid peroxidation. Esterified F(2)-isoprostanes in the liver increased 2- to 3-fold at 1 hour in control and BDL rats, but returned to baseline levels by 3 hours. Esterified F(2)-isoprostanes in the kidney increased by 2-fold in the BDL rats after LPS administration, but remained unchanged in sham controls. We conclude that there is a marked increase in sensitivity to LPS in BDL cirrhotic rats. This is associated with an enhanced TNF-alpha response and increased lipid peroxidation. These may be directly and causally related to mortality.