The extracellular matrix (ECM) consists of diverse components that work bidirectionally with surrounding cells to create a responsive microenvironment. In some contexts (e.g., hepatic fibrosis), changes to the ECM are well recognized and understood. However, it is becoming increasingly accepted that the hepatic ECM proteome (i.e., matrisome) responds dynamically to stress well before fibrosis. The term “transitional tissue remodeling” describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these changes in ECM may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM in liver injury are poorly understood. The goals of this work were to validate analysis of the ECM proteome and compare the impact of 6 weeks of ethanol diet and/or acute lipopolysaccharide (LPS). Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by solubility. Extracted proteins were identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Both ethanol and LPS dramatically increased the number of matrisome proteins ~25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes. Conclusion An extraction method to enrich the hepatic ECM was characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more-dramatic fibrotic changes to the liver occur. The changes to the mastrisome may contribute, at least in part, to the pathological responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. Nevertheless, there were responses that were unique to the individual and combined exposures.

Background It is well known that liver and lung injury can occur simultaneously during severe inflammation (e.g. multiple organ failure). However, whether these are parallel or interdependent (i.e. liver:lung axis) mechanisms is unclear. Previous studies have shown that chronic ethanol consumption greatly increases mortality in the setting of sepsis-induced acute lung injury (ALI). The potential contribution of subclinical liver disease in driving this effect of Ethanol on the lung remains unknown. Therefore, the purpose of this study was to characterize the impact of chronic Ethanol exposure on concomitant liver and lung injury. Methods Male mice were exposed to ethanol-containing Lieber-DeCarli diet or pair-fed control diet for 6 weeks. Some animals were administered lipopolysaccharide (LPS) 4 or 24 hours prior to sacrifice to mimic sepsis-induced ALI. Some animals received the TNFα blocking drug, etanercept, for the duration of alcohol exposure. The expression of cytokine mRNA in lung and liver tissue was determined by qPCR. Cytokine levels in the bronchoalveolar lavage fluid (BALF) and plasma were determined by Luminex assay. Results As expected, the combination of Ethanol and LPS caused liver injury, as indicated by significantly increased levels of the transaminases ALT/AST in the plasma and by changes in liver histology. In the lung, Ethanol preexposure enhanced pulmonary inflammation and alveolar hemorrhage caused by LPS. These changes corresponded with unique alterations in the expression of pro-inflammatory cytokines in the liver (i.e TNFα) and lung (i.e. MIP-2, KC). Systemic depletion of TNFα (etanercept) blunted injury and the increase in MIP-2 and KC caused by the combination of ethanol and LPS in the lung. Conclusions Chronic Ethanol preexposure enhanced both liver and lung injury caused by LPS. Enhanced organ injury corresponded with unique changes in the pro-inflammatory cytokine expression profiles in the liver and the lung.

Alcohol consumption is a common custom worldwide, and the toxic effects of alcohol on several target organs are well understood. The liver is the primary site of alcohol metabolism and is therefore the major target of alcohol toxicity. Alcoholic liver disease is a spectrum of disease states, ranging from simple steatosis (fat accumulation), to inflammation, and eventually to fibrosis and cirrhosis if untreated. The fibrotic stage of ALD is primarily characterized by robust accumulation of extracellular matrix (ECM) proteins (collagens) which ultimately impairs the function of the organ. The role of the ECM in early stages of ALD is poorly understood, but recent research has demonstrated that a number of changes in the hepatic ECM in prefibrotic ALD not only are present, but may also contribute to disease progression. The purpose of this review is to summarize the established and proposed changes to the hepatic extracellular matrix (ECM) that may contribute to earlier stages of ALD development and to discuss potential mechanisms by which these changes may mediate the progression of the disease.

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA−LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2-and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNAmediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.

Alcohol consumption is a common custom worldwide, and the toxic effects of alcohol on several target organs are well-understood. Given the poor prognosis of treating clinically-relevant alcoholic liver disease (ALD) (i.e., alcoholic hepatitis (AH) and cirrhosis), additional research is required to develop more effective therapies. While the stages of ALD have been well-characterized, targeted therapies to prevent or reverse this process in humans are still needed. Better understanding of risk factors and mechanisms underlying disease progression can lead to the development of rational therapies to prevent or reverse ALD in the clinic. A potential area of targeted therapy for ALD may be organ–organ communication in the early stages of the disease. In contrast to AH and end-stage liver diseases, the involvement of multiple organs in the development of ALD is less understood. The impact of these changes on pathology to the liver and other organs may not only influence disease progression during the development of the disease, but also outcomes of end stages diseases. The purpose of this review is to summarize the established and proposed communication between the liver and other organ systems that may contribute to the development and progression of liver disease, as well as to other organs. Potential mechanisms of this organ–organ communication are also discussed.

Introduction: Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods: Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin-mediated proteolysis (i.e., locked in the monomeric form), termed FibAEK mice, and Factor XIII A2 subunit-deficient mice (FXIII−/−). Female wild-type mice, FXIII−/− mice, and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4), twice weekly for 4 or 6 weeks (1 ml/kg, ip). Results: Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin-catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of cross-linked fibrin(ogen) was not reduced in CCl4-challenged FXIII−/− mice or FibAEK mice compared to wild-type mice. Rather, deposition of cross-linked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase-deficient mice (TGM2−/− mice). However, the reduction in cross-linked fibrin(ogen) in TGM2−/− mice did not affect CCl4-induced liver fibrosis. Conclusions: These results indicate that neither traditional fibrin clots, formed by the thrombin:FXIIIa pathway, nor atypical TGM2-cross-linked fibrin(ogen) contribute to experimental CCl4-induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.

The composition of the extracellular matrix (ECM) proteins and the expression of their cognate receptors dictate cell behavior and dynamics. In particular, the interactions of ECM proteins with integrin receptors are key mediators of these cellular processes, playing a crucial role in the progression of several diseases of the liver, including inflammation, fibrosis/cirrhosis and cancer. This study establishes a modeling approach combining computation and experiments to evaluate the kinetics of integrin receptor binding to hepatic ECM proteins. ECM ligand concentration was derived from LC-MS/MS quantification of the hepatic ECM from mice exposed to chronic carbon tetrachloride (CCl4); receptor density was derived from published literature. Mathematical models for ECM-integrin binding kinetics that were developed incorporate receptor divalence and an aggregation scheme to represent clustering. The computer simulations reproduced positive cooperativity in the receptor aggregation model when the aggregation equilibrium constant (Ka) was positive and greater than Keq for divalent complex formation. Importantly, the modeling projected an increase in integrin binding for several receptors for which signaling is known to be increased after CCl4 exposure in the liver. The proposed modeling approach may be of use to elucidate the kinetics of integrin receptor binding to ECM proteins for homeostatic and diseased livers.

Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII-/-), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII-/- mice yet cross-linking of hepatic fibrin(ogen) was dramatically reduced (>90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (e.g., peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.