Heparan sulfate promotes recovery from acute liver injury: Inhibition of progressive cell death or enhanced regeneration?
Authors: Udayan Apte, Neil Kaplowitz
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article on Page 1601 Heparan sulfate (HS) proteoglycans (HSPGs), syndecans, and glycosyl‐phosphoinositide–linked glypicans are present in all adherent cells. HS, attached to these proteins, is a strongly anionic linear polysaccharide, which allows the cell surface to bind a variety of effectors and matrix components. Syndecan ectodomains can be proteolytically cleaved, releasing HS peptides that exert a variety of biological effects. HSPGs have roles in morphogenesis, tissue repair, host defense, and energy metabolism and act as coreceptors, potentiating the action of low concentrations of ligands. Shed ectodomains play a critical role in wound repair by binding cytokines and growth factors, protecting proteases from their inhibitors, and coordinating the repair process. Among the four‐member syndecan gene family, syndecan‐1 (Sdc1) is mainly expressed in epithelia including hepatocytes. Park et al. suggested that ectodomain shedding protects against dysregulated inflammation.1 Sdc1 shedding facilitates the removal of CXC chemokines, leading to the resolution of neutrophilic infiltration. Sdc1–/– mice developed normally but were more susceptible to lipopolysaccharide exposure, due to increased levels of chemokines and neutrophils. Delayed inhibition of metalloproteinases in wild‐type (WT) mice caused a similar phenotype as that of Sdc1–/– mice. Delayed administration of HS, the linear polysaccharide ligand binding moiety of Sdc1, protected the knockout mice from cell death.1 Nam et al., in this issue of hepatology, extend their work on the role of Sdc1 in acute liver injury and repair using the acetaminophen (APAP) mouse model, which closely resembles the human disease.2 APAP hepatotoxicity can be broadly divided into three phases.3 During initiation or the APAP metabolism phase, APAP is metabolized to its reactive metabolite N‐acetyl‐p‐quinone imine, which is quenched by glutathione (GSH), leading to GSH depletion and covalent protein binding, forming APAP adducts. This leads to the progression phase, which can be divided into two categories, the first involving intracellular events that include mitochondrial dysfunction and mitochondrial reactive oxygen species mediated by self‐sustaining mitogen‐activated protein kinase signaling through c‐Jun N‐terminal kinase.5 After a few hours during which antioxidants, GSH repletion, and c‐Jun N‐terminal kinase inhibitors are still effective at inhibiting cell death, a point of no return is reached when sufficient oxidative stress causes mitochondrial permeability transition, adenosine triphosphate depletion, and release of mitochondrial proteins, which induce DNA damage. The hallmark of injury at this stage is necrosis, which peaks in the mouse model at 12‐24 hours. This necrosis occurs mainly in zone 3, but there is a penumbra, i.e., an area surrounding the necrosis where hepatocytes appear to be reversibly stressed and where proliferation occurs. Though some controversy exists, most studies indicate that apoptosis does not occur, presumably because GSH depletion, reactive oxygen species, and adenosine triphosphate depletion incapacitate caspases. Following necrosis, the extracellular part of the progression phase starts, where damage‐associated molecular patterns are released from dying cells. Damage‐associated molecular patterns are implicated in propagation of injury, triggering inflammation, and initiating repair.3 The third and final stage is a recovery phase, where initially the hepatocytes surrounding necrotic zones divide and regenerate the damaged liver. Later hepatocyte proliferation spreads well beyond the penumbra.6 Nam et al. now demonstrate that global Sdc1 deletion unmasks enhanced liver injury from APAP, resulting in apparent delayed apoptosis usually not seen in WT mice. The early phase of APAP toxicity up to 12 hours was not affected either in vivo or in vitro (though a very high APAP concentration [50 mM] was used). After 12 hours, injury began to resolve in the WT but worsened in the knockout mice, suggesting that Sdc1 normally halts the progression of liver injury and/or is required to facilitate repair. Surprisingly, caspase 3 was activated in Sdc1–/– beginning at 12 hours. This was seen in the penumbra, the area surrounding the necrosis, the area where liver cell proliferation is seen in WT mice.6Sdc1–/– exhibited suppressed AKT activation and increased glycogen synthase kinase 3β (GSK3β) activation, the importance of which was confirmed by inhibition experiments. Caspase 3 and GSK3β inhibitors arrested the progression of injury, whereas AKT inhibition worsened it. Interestingly, in WT mice given a lethal dose of APAP, which is known to impair regeneration,6 the delayed administration of caspase 3 and GSK3β inhibitors protected against progressive injury. The authors interpret this as GSK3β‐mediated apoptosis. Whereas the authors do acknowledge the alternative, that GSK3β inactivation may promote liver repair (through β‐catenin activation) and the delayed apoptosis may be a default pathway due to failure to regenerate, these studies need further detailed analysis to determine the effect of GSK3β inhibition on various downstream targets. Among the derivatives of HS the authors tested heparin, which is similar to heparan but has less glucuronic acid in the disaccharide chain than iduronic acid and is highly sulfated. Heparin was also protective in the APAP model. Although not addressed here, the authors previously showed that the protective effect of HS could be disassociated from its anticoagulant property. Further analysis of heparin's protective role is needed in light of the recent report that coagulation pathways may stimulate liver regeneration after APAP overdose and thus could be actually beneficial.7 HS can modulate several growth factors and cytokines, and such interactions may be involved in its protective effect in acute liver injury. For example, heparin activates hepatocyte growth factor, one of the primary mitogens for hepatocytes,8 which may in turn drive liver repair following APAP overdose. The authors tested the therapeutic potential of the Sdc1 ectodomain, which contains HS as well as HS alone. Pretreatment protected Sdc1–/– mice but, even more remarkably, when the ectodomain or HS was administered 3 or 10 hours after APAP, beyond when N‐acetylcysteine is effective. This approach also was beneficial in WT mice given a lethal dose of APAP. Structural analysis revealed that 2‐0‐sulfated domains in Sdc1 HS were critical for inhibiting the progression of liver injury. Although this approach did not seem to affect liver injury in the concanavalin A model, it did show efficacy in furosemide‐induced hepatic necrosis, suggesting potential value beyond APAP. It is important to note that protection was seen in WT mice at the lethal dose, which impairs regeneration, highlighting the potential therapeutic value of HS in the treatment of human APAP toxicity. A major missed opportunity in this study is to test the role of HS and Sdc1 in liver regeneration after APAP. Although the authors briefly investigated mechanisms of liver repair, lack of an extended time course complicates data interpretation. Studies have shown that the final recovery phase starts around 24 hours after APAP exposure in mice, when cells in the necrotic penumbra enter the cell cycle by expressing cyclin D1.6 They undergo S‐phase DNA synthesis around 48 hours and mitosis around 72 hours after APAP exposure. The proliferation ceases by 96 hours, and the regenerative process is completed by 120 hours. The duration and extent of the liver regeneration are based on the dose and initial injury. High doses of APAP and/or higher initial liver injury as observed in Sdc–/– mice results in delayed liver regeneration. The role of proteoglycans in liver regeneration has long been suspected. HS is required for proper functioning of various growth factors involved in hepatocyte proliferation, and Sdc1 overexpression resulted in increased hepatocyte proliferation.9 Taken together with the promitogenic effects of heparin, it is very likely that Sdc1 and other HSPGs are involved in stimulation of liver regeneration after APAP overdose, which needs further investigation. In summary, these novel data demonstrate that HSPGs, and specifically Sdc1, are critically involved in APAP‐induced liver injury and liver repair through multiple mechanisms including preventing progression of liver injury and initiation of liver repair. These studies have highlighted HSPGs as yet another promising therapeutic target in the treatment of APAP‐induced acute liver failure. Author names in bold designate shared co‐first authorship.