Mark A. Ross

A Whole-Organ Regenerative Medicine Approach for Liver Replacement

BACKGROUND & AIMS The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue. METHODS A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion. RESULTS The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction. CONCLUSIONS Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.

Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice.

Sterile inflammatory insults are known to activate innate immunity and propagate organ damage through the recognition of extracellular damage‐associated molecular pattern (DAMP) molecules. Although DAMPs such as endogenous DNA and nuclear high‐mobility group box 1 have been shown to be critical in sterile inflammation, the role of nuclear histone proteins has not yet been investigated. We report that endogenous histones function as DAMPs after ischemic injury through the pattern recognition receptor Toll‐like receptor (TLR) 9 to initiate inflammation. Using an in vivo model of hepatic ischemia/reperfusion (I/R) injury, we show that levels of circulating histones are significantly higher after I/R, and that histone neutralization significantly protects against injury. Injection of exogenous histones exacerbates I/R injury through cytotoxic effects mediated by TLR9 and MyD88. In addition, histone administration increases TLR9 activation, whereas neither TLR9 nor MyD88 mutant mice respond to exogenous histones. Furthermore, we demonstrate in vitro that extracellular histones enhance DNA‐mediated TLR9 activation in immune cells through a direct interaction. Conclusion: These novel findings reveal that histones represent a new class of DAMP molecules and serve as a crucial link between initial damage and activation of innate immunity during sterile inflammation. (HEPATOLOGY 2011; 54:999–1008)

In vivo immune modulatory activity of hepatic stellate cells in mice

Accumulating data suggest that hepatic tolerance, initially demonstrated by spontaneous acceptance of liver allografts in many species, results from an immune regulatory activity occurring in the liver. However, the responsible cellular and molecular components have not been completely understood. We have recently described profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro. In this study, we demonstrate in vivo evidence of immune modulatory activity of HSCs in mice using an islet transplantation model. Co‐transplanted HSCs effectively protected islet allografts from rejection, forming a multi‐layered capsule, which reduced allograft immunocyte infiltrates by enhancement of apoptotic death. The immune modulation by HSCs appeared to be a local effect, and regulated by inducible expression of B7‐H1, an inhibitory molecule of B7 family. This may reflect an intrinsic mechanism of immune inhibition mediated by liver‐derived tissue cells. In conclusion, these results may lead to better understanding of liver immunobiology and development of new strategies for treatment of liver diseases. (HEPATOLOGY 2006;44:1171–1181.)

Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase–generated superoxide

Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase-generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cell-permeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures.

Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.

Trivalent arsenic [As(III)] is a well‐known environmental toxicant that causes a wide range of organ‐specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)‐induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)‐exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM‐1 and laminin‐1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin‐1 expression and SEC caveolae, as well as increased membrane‐bound Rac1‐GTPase. Conclusion: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver. (HEPATOLOGY 2007; 45:205–212.)

A mouse model of accelerated liver aging caused by a defect in DNA repair.

The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging‐related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1−/Δ mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1−/Δ mice, we systematically compared liver from 5‐month‐old progeroid Ercc1−/Δ mice to old (24‐36‐month‐old) wild‐type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1−/Δ mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1−/Δ mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1−/Δ liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1−/Δ and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome‐wide transcriptional changes between old WT and 16‐week‐old, but not 5‐week‐old, Ercc1−/Δ mice, emphasizing that the Ercc1−/Δ mice acquire an aging profile in early adulthood. Conclusion: There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging‐related hepatic changes. (HEPATOLOGY 2012)

Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury†

The nuclear protein high mobility group box 1 (HMGB1) is an important inflammatory mediator involved in the pathogenesis of liver ischemia/reperfusion (I/R) injury. Strategies aimed at preventing its release from stressed or damaged cells may be beneficial in preventing inflammation after I/R. Cisplatin is a member of the platinating chemotherapeutic agents and can induce DNA lesions that are capable of retaining high mobility group proteins inside the nucleus of cells. In vitro studies in primary cultured rat hepatocytes show that nontoxic concentrations of cisplatin can sequester HMGB1 inside the nucleus of hypoxic cells. Similarly, the in vivo administration of nontoxic doses of cisplatin prevents liver damage associated with a well‐established murine model of hepatic I/R as measured by lower circulating serum aminotransferase levels, lower hepatic inflammatory cytokine levels including tumor necrosis factor α and interleukin‐6, lower inducible NO synthase expression, and fewer I/R‐associated histopathologic changes. The mechanism of action in vivo appears to involve the capacity of cisplatin to prevent the I/R‐induced release of HMGB1 as well as to alter cell survival and stress signaling in the form of autophagy and mitogen‐activated protein kinase activation, respectively. Conclusion: Low, nontoxic doses of cisplatin can sequester HMGB1 inside the nucleus of redox‐stressed hepatocytes in vitro and prevent its release in vivo in a murine model of hepatic I/R. Furthermore, cell survival and stress signaling pathways are altered by low‐dose cisplatin. Therefore, platinating agents may provide a novel approach to mitigating the deleterious effects of I/R‐mediated disease processes. (HEPATOLOGY 2009.)

Cationic Colloidal Silica Membrane Perturbation as a Means of Examining Changes at the Sinusoidal Surface during Liver Regeneration

By employing the cationic colloidal silica membrane density perturbation technique, we examined growth factor receptor and extracellular matrix (ECM) changes at the sinusoidal surface during rat liver regeneration 72 hours after 70% partial hepatectomy (PHx). At this time after PHx, hepatocyte division has mostly subsided, while sinusoidal endothelial cell (SEC) proliferation is initiating, resulting in avascular hepatocyte islands. Because of the discontinuous nature of the surface of liver SEC, ECM proteins underlying the SEC, as well as SEC luminal membrane proteins, are available to absorption to the charged silica beads when the liver is perfused with the colloid. Subsequent liver homogenization and density centrifugation yield two separate fractions, enriched in SECs as well as hepatocyte basolateral membrane-specific proteins up to 50-fold over whole liver lysates. This technique facilitates examination of changes in protein composition that influence or occur as a result of SEC mitogenesis and migration during regeneration of the liver. When ECM and receptor proteins from SEC-enriched fractions were examined by Western immunoblotting, urokinase plasminogen activator receptor, fibronectin, and plasmin increased at the SEC surface 72 hours after PHx. Epidermal growth factor receptor, plasminogen, SPARC (secreted protein, acidic and rich in cysteine, also called osteonectin or BM40), and collagen IV decreased, and fibrinogen subunits and c-Met expression remained constant 72 hours after PHx when compared to control liver. These results display the usefulness of the cationic colloidal silica membrane isolation protocol. They also show considerable modulation of surface components that may regulate angiogenic processes at the end stage of liver regeneration during the reformation of sinusoids.

Interferon regulatory factor 1 mediates acetylation and release of high mobility group box 1 from hepatocytes during murine liver ischemia-reperfusion injury.

Damage-associated molecular patterns (DAMPs) initiate inflammatory pathways that are common to both sterile and infectious processes. The DAMP, high-mobility group box 1 (HMGB1), and the transcription factor, interferon regulatory factor 1 (IRF-1), have been independently associated as key players in ischemia-reperfusion (I/R) injury. Our study demonstrates that IRF-1 contributes to hepatocellular release of HMGB1 and further that IRF-1 is a necessary component of HMGB1 release in response to hypoxia or after liver I/R. We also link the nuclear upregulation of IRF-1 to the presence of functional Toll-like receptor 4 (TLR4), a pattern recognition receptor also important in sterile and infectious processes. Using IRF-1 chimeric mice, we show that IRF-1 upregulation in hepatic parenchymal cells, and not in the bone marrow-derived immune cells, is responsible for HMGB1 release during ischemic liver injury. Finally, our study also demonstrates a role for IRF-1 in modulating the acetylation status and subsequent release of HMGB1 through histone acetyltransferases. We found that serum HMGB1 is acetylated after liver I/R and that this process was dependent on IRF-1. Additionally, liver I/R induced a direct association of IRF-1 and the nuclear histone acetyltransferase enzyme p300. Together, these findings suggest that I/R-induced release of acetylated HMGB1 is a process that is dependent on TLR4-mediated upregulation of IRF-1.ABBREVIATIONS-IRF-1-interferon regulatory factor 1; HMGB1-high-mobility group box 1; I/R-ischemia-reperfusion; PRR-pattern recognition receptor; DAMP-damage-associated molecular pattern; TLR4-Toll-like receptor-4; HAT-histone acetyltransferase; HDAC-histone deacetylase; WT-wild-type; MOI-multiplicity of infection

Sinusoidal endothelial cell repopulation following ischemia/reperfusion injury in rat liver transplantation

We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold‐stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re‐endothelialize within 48 hours of transplantation, and repopulation is coincident with up‐regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor‐2 expression on large vessel endothelial cells and repopulating LSECs. Although re‐endothelialization occurs rapidly, we show here, using several high‐resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow–derived cells into functioning LSECs is routinely between 1% and 5%. Conclusion: Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow–derived LSECs after cold ischemia and warm reperfusion. (HEPATOLOGY 2007.)