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Gap junctions in liver disease: Implications for pathogenesis and therapy

Published:December 29, 2018DOI:https://doi.org/10.1016/j.jhep.2018.12.023

      Summary

      In the normal liver, cells interact closely through gap junctions. By providing a pathway for the trafficking of low molecular mass molecules, these channels contribute to tissue homeostasis and maintenance of hepatic function. Thus, dysfunction of gap junctions affects a wide variety of liver processes, such as differentiation, cell death, inflammation and fibrosis. In fact, dysfunctional gap junctions have been implicated, for more than a decade, in cholestatic disease, hepatic cancer and cirrhosis. Additionally, in recent years there is an increasing body of evidence that these channels are also involved in other relevant and prevalent liver pathological processes, such as non-alcoholic fatty liver disease, acute liver injury and portal hypertension. In parallel to these new clinical implications the available data include controversial observations. Thus, a comprehensive overview is required to better understand the functional complexity of these pores. This paper will review the most recent knowledge concerning gap junction dysfunction, with a special focus on the role of these channels in the pathogenesis of relevant clinical entities and on potential therapeutic targets that are amenable to modification by drugs.

      Keywords

      Gap junctions, hemichannels and connexins: Molecular characteristics and function

      Cell-to-cell communication is of extreme importance in tissue homeostasis, which is maintained by transmission of regulatory signals
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      (Fig. 1). Intercellular communication via gap junctions (GJs) represents one of the most important routes of rapid signalling between cells. GJ channels span 2 plasma membranes and consist of 2 hemichannels (connexons), one belonging to each cell. Each hemichannel is formed by 6 connexin (Cx) subunits and is permeable to small molecules up to 1–1.5kD.
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      Figure thumbnail gr1
      Fig. 1Representation of hepatocytes and gap junction functions, structure, trafficking messengers and strategies to evaluate gap junction functions. a) Gap junctions participate in different functions. b) Individual connexins assemble intracellularly into hexamers, called connexons (hemichannels), which dock with other connexons in adjacent cells, assembling an axial channel spanning 2 plasma membranes and a narrow extracellular gap. c) Different molecules pass through the gap junctions. d) With different approaches the function of different connexins has been evaluated. 2-APB, 2-aminoethoxydiphenyl-borate.
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      Therefore, this paper will review recent knowledge concerning the role of GJs in the pathogenesis of liver diseases.

      Modulators of GJ function and targeting in diseases outside the liver

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      • Ghatnekar G.S.
      Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: a multicenter, randomized trial.
      In addition to the direct beneficial effects of simply potentiating or blocking the channel, the Cx targeting drugs may be used as adjuvants potentiating the effects of other known therapeutic agents. This is of particular interest in hepatocellular carcinoma (HCC) where GJs may favour the delivery of cytotoxic drugs to tumour cells. In this regard, studies have shown that GJ mimetics facilitate the spread of cisplatin, conferring a better therapeutic effect.
      • Peterson-Roth E.
      • Brdlik C.M.
      • Glazer P.M.
      Src-Induced cisplatin resistance mediated by cell-to-cell communication.
      Quinolone, a GJ opener was recently shown to enhance cisplatin-induced cytotoxicity,
      • Ding Y.
      • Nguyen T.A.
      Gap junction enhancer potentiates cytotoxicity of cisplatin in breast cancer cells.
      supporting the rationale for combination therapies that include GJ openers in the treatment of various cancers such as colon,
      • Nakamura Y.
      • Chang C.C.
      • Mori T.
      • Sato K.
      • Ohtsuki K.
      • Upham B.L.
      • et al.
      Augmentation of differentiation and gap junction function by kaempferol in partially differentiated colon cancer cells.
      prostate
      • Fukushima M.
      • Hattori Y.
      • Yoshizawa T.
      • Maitani Y.
      Combination of non-viral connexin 43 gene therapy and docetaxel inhibits the growth of human prostate cancer in mice.
      and breast.
      • Grek C.L.
      • Rhett J.M.
      • Bruce J.S.
      • Abt M.A.
      • Ghatnekar G.S.
      • Yeh E.S.
      Targeting connexin 43 with alpha-connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1.
      In addition, inhibition of GJ may reduce the toxic effects of drugs by preventing the propagation of inflammatory or death stimuli to neighbouring cells.
      • Grek C.L.
      • Rhett J.M.
      • Ghatnekar G.S.
      Cardiac to cancer: connecting connexins to clinical opportunity.
      Given the potentially opposing effects of modulating GJ function, clinical application in a given disease needs to be carefully considered.

      Acute liver injury and inflammation

      GJs and Cxs are involved in settings where homeostatic regulation is crucial, such as during inflammation and cell death. Available data indicate that Cx26, Cx32 and Cx43 can contribute to acute liver injury and inflammation related to drugs, lipopolysaccharide (LPS) and ischaemia-reperfusion injury. Given that several immune cells including monocytes, macrophages and Kupffer cells express Cx43
      • Branes M.C.
      • Contreras J.E.
      • Saez J.C.
      Activation of human polymorphonuclear cells induces formation of functional gap junctions and expression of connexins.
      and are known to be involved in autoimmune liver diseases, the role of GJs in specific autoimmune liver diseases should be explored.
      • Valdebenito S.
      • Barreto A.
      • Eugenin E.A.
      The role of connexin and pannexin containing channels in the innate and acquired immune response.

      Acute liver injury

      To better understand the role of GJs in drug-induced liver injury, studies in cells and animal models have been conducted wherein Cx expression was modified by gene therapy or drugs (Table 1). The observation that HeLa cells transfected with herpes simplex virus induced the killing of a neighbouring cell through the diffusion of toxic phosphorylated ganciclovir molecules after enhancement of GJs,
      • Mesnil M.
      • Piccoli C.
      • Tiraby G.
      • Willecke K.
      • Yamasaki H.
      Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.
      provided the rationale to explore the role of GJs in acute liver injury. Acute administration of carbon tetrachloride and dimethynitrosamine, which induce acute liver injury, resulted in reduced expression of Cx32
      • Miyashita T.
      • Takeda A.
      • Iwai M.
      • Shimazu T.
      Single administration of hepatotoxic chemicals transiently decreases the gap-junction-protein levels of connexin 32 in rat liver.
      due to transcriptional downregulation.
      • Maes M.
      • McGill M.R.
      • Da Silva T.C.
      • Abels C.
      • Lebofsky M.
      • Monteiro Maria
      • et al.
      Involvement of connexin43 in acetaminophen-induced liver injury.
      Additionally, Cxs were mislocalized from the cell surface to the cytoplasm. Cx32 depleted animals exhibited less severe liver injury after acute administration of D-galactosamine, carbon tetrachloride, thioacetamide and acetaminophen.
      • Asamoto M.
      • Hokaiwado N.
      • Murasaki T.
      • Shirai T.
      Connexin 32 dominant-negative mutant transgenic rats are resistant to hepatic damage by chemicals.
      • Naiki-Ito A.
      • Asamoto M.
      • Naiki T.
      • Ogawa K.
      • Takahashi S.
      • Sato S.
      • et al.
      Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat.
      The severity of liver injury increased to that in wild-type animals following restoration of Cx32 by gene transfection.
      • Park W.J.
      • Park J.W.
      • Erez-Roman R.
      • Kogot-Levin A.
      • Bame J.R.
      • Tirosh B.
      • et al.
      Protection of a ceramide synthase 2 null mouse from drug-induced liver injury: role of gap junction dysfunction and connexin 32 mislocalization.
      The potential role of GJs in contributing to cell death is further supported by studies in cultured hepatocytes, where suppression of Cx26 and Cx32 reduced the synchronization of cell death after administration of acetaminophen.
      • Saito C.
      • Shinzawa K.
      • Tsujimoto Y.
      Synchronized necrotic death of attached hepatocytes mediated via gap junctions.
      Taken together, these data suggest that the reduction in Cx32 during acute liver injury is likely to be an adaptive response aimed at protecting healthy cells from the propagation of toxins or messengers associated with cell death. These data have been translated into potential novel therapeutics targeting blockade of Cx26 and Cx32 using 2-aminoethoxydiphenyl-borate. Administration of 2-aminoethoxydiphenyl-borate
      • Tao L.
      • Harris A.L.
      2-aminoethoxydiphenyl borate directly inhibits channels composed of connexin26 and/or connexin32.
      before, concurrently or after inducing acute liver injury was protective.
      • Patel S.J.
      • Milwid J.M.
      • King K.R.
      • Bohr S.
      • Iracheta-Velle A.
      • Li M.
      • et al.
      Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure.
      Table 1Experimental studies describing the role of connexins in acute liver injury.
      Studied CxAnimal modelType and dose of toxicEffectsReference
      Cx32Sprague-Dawley male transgenic ratSingle intraperitoneal injection of D-galactosamine (300 mg/kg body wt) and carbon tetrachloride (0.5 ml/kg body wt)Less evident necrosis and ballooning findings in Cx32 deficient ratsAsamoto et al.
      • Asamoto M.
      • Hokaiwado N.
      • Murasaki T.
      • Shirai T.
      Connexin 32 dominant-negative mutant transgenic rats are resistant to hepatic damage by chemicals.
      Cx32, Cx43Sprague-Dawley male transgenic ratSingle intraperitoneal injection of acetaminophen (250, 500, 1000 mg/kg body wt)Less inflammation after insult in rats lacking Cx32, and induction of Cx43 expressionNaiki-Ito et al.
      • Naiki-Ito A.
      • Asamoto M.
      • Naiki T.
      • Ogawa K.
      • Takahashi S.
      • Sato S.
      • et al.
      Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat.
      Cx32Sprague-Dawley male ratsSingle intraperitoneal injection of carbon tetrachloride (1.0 mL/kg body wt) and dimethylnitrosamine (6.3–25 mg/kg body wt)Decrease in hepatic Cx32 expression after injury and inverse correlation with the increase in plasmic alanine-aminotransferase activityMiyashita T et al.
      • Miyashita T.
      • Takeda A.
      • Iwai M.
      • Shimazu T.
      Single administration of hepatotoxic chemicals transiently decreases the gap-junction-protein levels of connexin 32 in rat liver.
      Cx32Ceramide synthase (CerS2) null mice (altered gap junction function)Intraperitoneal injection of acetaminophen (300 mg/kg body wt), D-galactosamine (800 mg/kg body wt), carbon tetrachloride (2 ml/kg body wt) and thioacetamide (200 mg/kg body wt)Less acetaminophen-induced hepatotoxicity after ablation of Cx32Park WJ et al.
      • Park W.J.
      • Park J.W.
      • Erez-Roman R.
      • Kogot-Levin A.
      • Bame J.R.
      • Tirosh B.
      • et al.
      Protection of a ceramide synthase 2 null mouse from drug-induced liver injury: role of gap junction dysfunction and connexin 32 mislocalization.
      Cx26, Cx32C57BL/6 Knock-out mice and wildtype mice treated with 2-aminoethoxydiphenyl-borate (Cx blocker)Intraperitoneal injection of thioacetamide (200, 500 or 1000 mg/kg body wt) and acetaminophen (500 or 750 mg/kg body wt)Mice deficient in Cx32 and wildtype mice (cotreated with 2APB) showed reduced inflammation and oxidative stressPatel SJ et al.
      • Patel S.J.
      • Milwid J.M.
      • King K.R.
      • Bohr S.
      • Iracheta-Velle A.
      • Li M.
      • et al.
      Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure.
      Cx32C57BL/6 mice treated with 2-aminoethoxydiphenyl-borateIntraperitoneal injection of acetaminophen (400 mg/kg body wt)Protection by attenuation of c-jun-N-terminal kinase but not related to a specific role for Cx32Du K et al.
      • Du K.
      • Williams C.D.
      • McGill M.R.
      • Xie Y.
      • Farhood A.
      • Vinken M.
      • et al.
      The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation.
      Cx32C57BL/6 Knock-out miceIntraperitoneal injection of acetaminophen (300 mg/kg body wt)No influence of Cx32 deletionMaes M et al.
      • Maes M.
      • McGill M.R.
      • Da-Silva T.C.
      • Lebofsky M.
      • Maria Monteiro de Araújo C.
      • Tiburcio T.
      • et al.
      Connexin32: a mediator of acetaminophen-induced liver injury?.
      Cx32C57BL/6 Knock-out miceIntraperitoneal injection of acetaminophen (100, 200, or 300 mg/kg body wt)More susceptible to liver damage 24 hours after the insult in Cx32 deficient miceIgarashi I et al.
      • Igarashi I.
      • Maejima T.
      • Kai K.
      • Arakawa S.
      • Teranishi M.
      • Sanbuissho A.
      Role of connexin 32 in acetaminophen toxicity in a knockout mice model.
      Cx43C57BL/6 Knock-out miceIntraperitoneal injection of acetaminophen (300 mg/kg body wt)Cx43-deficient animals tended to show increased liver cell death, inflammation and oxidative stress in comparison with wild type counterpartsMaes M et al.
      • Maes M.
      • McGill M.R.
      • Da Silva T.C.
      • Abels C.
      • Lebofsky M.
      • Monteiro Maria
      • et al.
      Involvement of connexin43 in acetaminophen-induced liver injury.
      Cx, connexin; wt, weight.
      Connexins form gap junctions and hemichannels, which have different expression patterns depending of the type of liver disease.
      In contrast to the decreased expression of Cx26 and Cx32 during acute liver injury, Cx43 expression increases.
      • Maes M.
      • McGill M.R.
      • Da Silva T.C.
      • Abels C.
      • Lebofsky M.
      • Monteiro Maria
      • et al.
      Involvement of connexin43 in acetaminophen-induced liver injury.
      • Naiki-Ito A.
      • Asamoto M.
      • Naiki T.
      • Ogawa K.
      • Takahashi S.
      • Sato S.
      • et al.
      Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat.
      This unexpected increase suggests that Cx43 may play a role in propagating death signals.
      • Krutovskikh V.A.
      • Piccoli C.
      • Yamasaki H.
      Gap junction intercellular communication propagates cell death in cancerous cells.
      Accordingly, in liver cell cultures, a progressive increase in Cx43 mRNA and protein expression was observed during apoptosis.
      • Vinken M.
      • Decrock E.
      • Vanhaecke T.
      • Leybaert L.
      • Rogiers V.
      Connexin43 signaling contributes to spontaneous apoptosis in cultures of primary hepatocytes.
      It is possible that Cx43 mediates propagation of cell death through caspase-3, a relevant factor in the apoptotic cascade, as they co-localize when apoptosis is induced. In support of this hypothesis, inhibition of Cx43 resulted in downregulation of caspase-3.
      • Naiki-Ito A.
      • Asamoto M.
      • Naiki T.
      • Ogawa K.
      • Takahashi S.
      • Sato S.
      • et al.
      Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat.
      Overall, these data suggest that blockade of Cx26 and Cx32, and counteracting Cx43 overexpression may represent potential therapeutic targets to reduce acute toxic liver injury. Despite this, there are data contradicting the protective role of Cx26 and Cx32 and the deleterious role of Cx43 in acute liver injury, suggesting that the situation may be more complex than first thought. Complete deletion of Cx32 was shown to worsen acute liver injury
      • Igarashi I.
      • Maejima T.
      • Kai K.
      • Arakawa S.
      • Teranishi M.
      • Sanbuissho A.
      Role of connexin 32 in acetaminophen toxicity in a knockout mice model.
      and another recent study has suggested that the increase in Cx43 may well be an adaptive response, as knocking out Cx43 was actually associated with worse liver injury.
      • Maes M.
      • McGill M.R.
      • Da Silva T.C.
      • Abels C.
      • Lebofsky M.
      • Monteiro Maria
      • et al.
      Involvement of connexin43 in acetaminophen-induced liver injury.
      It is possible that these radically different observations may be due to differences in the animal species used, type of blocker/deletion and the route and dose of administration of toxins (Table 1).
      Altogether, by means of targeted disruption of Cx genes or drug manipulation, these results argue for a crucial role of Cxs in the propagation of acute liver injury, irrespective of the type of hepatotoxin. However, the exact contribution of Cx remains unknown, as GJs may provoke a positive or negative effect on the severity of injury. The complexity lies in the fact that the cell death or survival response mediated by GJs may be determined by the transfer of molecules that can pass through them.
      • Decrock E.
      • Vinken M.
      • De V.E.
      • Krysko D.V.
      • D'Herde K.
      • Vanhaecke T.
      • et al.
      Connexin-related signaling in cell death: to live or let die?.

      Lipopolysaccharide-induced liver injury

      There is compelling evidence in experimental animal models that administration of LPS, which induces an inflammatory response, results in decreased expression of Cx26 and Cx32
      • Correa P.R.
      • Guerra M.T.
      • Leite M.F.
      • Spray D.C.
      • Nathanson M.H.
      Endotoxin unmasks the role of gap junctions in the liver.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      • De M.A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      • Lidington D.
      • Tyml K.
      • Ouellette Y.
      Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43.
      (Table 2). This reduction in levels of Cx26 and Cx32 protein expression in hepatocytes was related to inflammation.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Temme A.
      • Traub O.
      • Willecke K.
      Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.
      However, a downregulation of these Cxs at the level of gene expression by a post-transcriptional mechanism has also been postulated.
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      In the setting of experimental cirrhosis, the administration of LPS resulted in a further reduction in both Cx26 and Cx32.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      This argues in favour of a protective role of these Cxs, which shut down intercellular communication and propagation of inflammation.
      Table 2Experimental studies describing the role of connexins in inflammation induced by LPS.
      Studied CxAnimal modelType and dose of toxicEffectsReference
      Cx32C57BL/6 Knock-out miceIntravenous injection of LPSHypoglycemia was slightly prolonged, and cholestasis was much worse in Cx32-deficient miceCorrea PR et al.
      • Correa P.R.
      • Guerra M.T.
      • Leite M.F.
      • Spray D.C.
      • Nathanson M.H.
      Endotoxin unmasks the role of gap junctions in the liver.
      Cx26, Cx32, Cx43Sprague-Dawley male ratsIntravenous injection of LPS 2 mg/kg body wtCx26 and Cx32 were reduced after LPS whereas Cx43 increased associated with prominent inflammationGonzalez HE et al.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      Cx32Sprague-Dawley male ratsIntravenous injection of LPS 1 mg/kg body wtA decrease in the level of Cx32 mRNA in rat liver occurred at the posttranscriptional levelGingalewski C et al.
      • Gingalewski C.
      • Wang K.
      • Clemens M.G.
      • De Maio A.
      Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.
      Cx26,d Cx32Sprague-Dawley male ratsIntravenous injection of LPS 1 mg/kg body wtDecreased communication was observed associated to Cx mislocalization and decreased Cx32 mRNADe MA et al.
      • De M.A.
      • Gingalewski C.
      • Theodorakis N.G.
      • Clemens M.G.
      Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide.
      Cx43Cell cultureLPS in culture mediumCx43 is tyrosine phosphorylated showing intercellular resistance following exposure to LPSLidington D et al.
      • Lidington D.
      • Tyml K.
      • Ouellette Y.
      Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43.
      Cx26, Cx32, Cx43Sprague-Dawley male rats induced to bile-duct ligationIntraperitoneal injection of LPS 1 mg/kg body wtCx26/32 expression inversely correlates with Cx43 expression after LPS. However, inhibiting Cx43 produced hepatocellular necrosisBalasubramaniyan V et al.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      Cx26, Cx32, Cx43Cell culture from Wistar male ratsLPS 1 µg/ml in culture mediumLPS up-regulate Cx43 protein and messenger RNA expression, and enhance intercellular communication in hepatic stellate cellsFischer R et al.
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      Cx43Sprague-Dawley male ratsIntraperitoneal injection of LPS 6 mg/kg body wtKuppfer cells exposed to LPS showed Cx43 at cell-cell contacts associated with higher dye couplingEugenin EA et al.
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      Cx, connexin; LPS, lipopolysaccharide; wt, weight.
      Meanwhile, increased expression of Cx43 has been shown in stellate cells, macrophages, endothelial cells and also in leukocytes in response to LPS.
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      • Jara P.I.
      • Boric M.P.
      • Saez J.C.
      Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.
      This increase in Cx43 expression was also associated with increased activity of Cx43, indicated by a higher dye coupling, suggesting that Cx43 may play a role in liver inflammation.
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      Interestingly, inhibiting Cx43 in rats treated with LPS using mimetic peptides was associated with increased hepatocellular necrosis, suggesting that the increased hepatic Cx43 expression is most likely an adaptive protective response.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.

      Liver ischaemia and reperfusion

      GJ channels and Cxs have a role in ischaemia-reperfusion injury of the heart,
      • Garcia-Dorado D.
      • Inserte J.
      • Ruiz-Meana M.
      • Gonzalez M.A.
      • Solares J.
      • Julia M.
      • et al.
      Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion.
      brain
      • Lin J.H.
      • Weigel H.
      • Cotrina M.L.
      • Liu S.
      • Bueno E.
      • Hansen A.J.
      • et al.
      Gap-junction-mediated propagation and amplification of cell injury.
      and vascular tissues.
      • Rodriguez-Sinovas A.
      • Garcia-Dorado D.
      • Ruiz-Meana M.
      • Soler-Soler J.
      Protective effect of gap junction uncouplers given during hypoxia against reoxygenation injury in isolated rat hearts.
      • Rawanduzy A.
      • Hansen A.
      • Hansen T.W.
      • Nedergaard M.
      Effective reduction of infarct volume by gap junction blockade in a rodent model of stroke.
      • Schwanke U.
      • Konietzka I.
      • Duschin A.
      • Li X.
      • Schulz R.
      • Heusch G.
      No ischemic preconditioning in heterozygous connexin43-deficient mice.
      • Schwanke U.
      • Li X.
      • Schulz R.
      • Heusch G.
      No ischemic preconditioning in heterozygous connexin 43-deficient mice–a further in vivo study.
      The proposed mechanism is the initiation of an injury-signalling cascade that is propagated through GJs, affecting cellular metabolism.
      • Aggarwal S.
      • Randhawa P.K.
      • Singh N.
      • Jaggi A.S.
      Preconditioning at a distance: Involvement of endothelial vasoactive substances in cardioprotection against ischemia-reperfusion injury.
      In addition to the exchange of signals, ions and messengers between adjacent cells, functions independent of intercellular communication and related to the presence of Cx in the mitochondria have also been shown. In this case, Cx43 has important functions including modulation of mitochondrial respiration and production of reactive oxygen species.
      • Rodriguez-Sinovas A.
      • Ruiz-Meana M.
      • Denuc A.
      • Garcia-Dorado D.
      Mitochondrial Cx43, an important component of cardiac preconditioning.
      Hepatic ischaemia-reperfusion injury is commonly observed during partial hepatectomy and liver transplantation, and Cxs have been studied in this setting. In animal models of hepatic ischaemia-reperfusion, an early decrease in Cx26 and Cx32 mRNA and protein expression was observed.
      • Gingalewski C.
      • De M.A.
      Differential decrease in connexin 32 expression in ischemic and nonischemic regions of rat liver during ischemia/reperfusion.
      • Nakashima Y.
      • Kohno H.
      • El-Assal O.N.
      • Dhar D.K.
      • Ono T.
      • Yamanoi A.
      • et al.
      Sequential changes of connexin32 and connexin26 in ischemia-reperfusion of the liver in rats.
      Partial prevention of this effect was obtained with actinomycin D, which prevents the degradation of Cx32 mRNA, although protein expression of Cxs remained low, suggesting that its regulation occurs by different post-transcriptional and post-translational mechanisms.
      • Nakashima Y.
      • Ono T.
      • Yamanoi A.
      • El-Assal O.N.
      • Kohno H.
      • Nagasue N.
      Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.
      This alteration is likely to represent an adaptive response aimed at restricting the spread of noxious signals to healthy areas. In keeping with this hypothesis, in vitro experiments using cell cultures, targeting the Cx32 gene increased cell survival, which was associated with decreased molecular permeability of GJs.
      • Wang R.
      • Huang F.
      • Chen Z.
      • Li S.
      Downregulation of connexin 32 attenuates hypoxia/reoxygenation injury in liver cells.
      An alternative explanation is that a reduction of cell-to-cell communication prevents disruption of cellular metabolism.
      • De M.A.
      • Vega V.L.
      • Contreras J.E.
      Gap junctions, homeostasis, and injury.
      Ischaemic preconditioning, which attenuates and protects against ischaemia-reperfusion damage, is NO dependent.
      • Bolli R.
      • Bhatti Z.A.
      • Tang X.L.
      • Qiu Y.
      • Zhang Q.
      • Guo Y.
      • et al.
      Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide.
      • Bolli R.
      • Manchikalapudi S.
      • Tang X.L.
      • Takano H.
      • Qiu Y.
      • Guo Y.
      • et al.
      The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning.
      In this condition, cell-to-cell coupling appears to be necessary for the protective effect of preconditioning, as uncoupling by chemical inhibitors significantly reduced the protection provided by hypoxic preconditioning. In addition, preconditioning led to an increase in Cx43 expression, which was associated with increased GJ permeability.
      • Rath G.
      • Saliez J.
      • Behets G.
      • Romero-Perez M.
      • Leon-Gomez E.
      • Bouzin C.
      • et al.
      Vascular hypoxic preconditioning relies on TRPV4-dependent calcium influx and proper intercellular gap junctions communication.
      Clearly, more research is needed to understand the pathophysiological alterations in ischaemia-reperfusion injury that are related to Cxs. This research may identify potential therapeutic approaches to reverse the effects of ischaemia-reperfusion injury.

      Role of connexins in hepatic fibrosis

      Fibrosis is a consequence of pro-inflammatory cytokine release, oxidative stress and necrosis/apoptosis. Fibrosis is associated with the involvement of stellate cells, which transform from a quiescent state into a proliferative and contractile myofibroblast-like phenotype. Other neighbouring non-parenchymal cells including cholangiocytes, Kupffer cells and infiltrating monocytes interact and contribute to further activation of stellate cells.
      In the normal liver, fenestrated liver sinusoidal endothelial cells induce senescence of hepatic stellate cells. Capillarization of sinusoids reduces the ability of endothelial cells to suppress stellate cell activity.
      • DeLeve L.D.
      • Wang X.
      • Guo Y.
      Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence.
      • Xie G.
      • Wang X.
      • Wang L.
      • Wang L.
      • Atkinson R.D.
      • Kanel G.C.
      • et al.
      Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats.
      Although GJs may provide a direct pathway of intercellular communication, functional communication between endothelial cells and stellate cells has yet to be consistently identified.
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      • Rojkind M.
      • Novikoff P.M.
      • Greenwel P.
      • Rubin J.
      • Rojas-Valencia L.
      • de Carvalho A.C.
      • et al.
      Characterization and functional studies on rat liver fat-storing cell line and freshly isolated hepatocyte coculture system.
      However, as previously discussed, Cxs may contribute to intercellular transfer of angiocrine signals after injury
      • Ding B.S.
      • Cao Z.
      • Lis R.
      • Nolan D.J.
      • Guo P.
      • Simons M.
      • et al.
      Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.
      or may be incorporated into microvesicles involved in promoting fibrogenesis.
      • Lemoinne S.
      • Thabut D.
      • Housset C.
      • Moreau R.
      • Valla D.
      • Boulanger C.M.
      • et al.
      The emerging roles of microvesicles in liver diseases.
      In this regard, Cxs, in particular Cx43, have been shown to contribute to the composition of membrane vesicles, making this an important target for future research.
      • Soares A.R.
      • Martins-Marques T.
      • Ribeiro-Rodrigues T.
      • Ferreira J.V.
      • Catarino S.
      • Pinho M.J.
      • et al.
      Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells.
      The role of GJs in liver fibrogenesis was studied recently.
      • Cogliati B.
      • Crespo Y.S.
      • Da Silva T.C.
      • Aloia T.P.
      • Nogueira M.S.
      • Real-Lima M.A.
      • et al.
      Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice.
      Studies showed a significant decrease in liver fibrosis in Cx32 knockout mice compared to wild-type mice. Although the mechanism underlying this protective effect of Cx32 deletion is not clear, reduced oxidative stress was suggested as a possible explanation. In experimental models of cirrhosis induced by carbon tetrachloride, a downregulation of Cx32 was observed.
      • Nakata Y.
      • Iwai M.
      • Kimura S.
      • Shimazu T.
      Prolonged decrease in hepatic connexin32 in chronic liver injury induced by carbon tetrachloride in rats.
      In humans, reduced expression, as well as a re-localization from the membrane to the cytoplasm were also observed.
      • Nakashima Y.
      • Ono T.
      • Yamanoi A.
      • El-Assal O.N.
      • Kohno H.
      • Nagasue N.
      Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.
      This evidence argues for a protective role of Cx32.
      In general, connexins aim to protect the liver from injury in response to several insults.
      In models of fibrosis such as after Schistosoma mansoni inoculation
      • Oloris S.C.
      • Mesnil M.
      • Reis V.N.
      • Sakai M.
      • Matsuzaki P.
      • Fonseca E.S.
      • et al.
      Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.
      or common bile duct ligation,
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      Cx43 expression was increased while the expression of Cx26 and Cx32 decreased.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      • Kojima T.
      • Yamamoto T.
      • Murata M.
      • Lan M.
      • Takano K.
      • Go M.
      • et al.
      Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver.
      By contrast, others have observed decreased expression,
      • Nakata Y.
      • Iwai M.
      • Kimura S.
      • Shimazu T.
      Prolonged decrease in hepatic connexin32 in chronic liver injury induced by carbon tetrachloride in rats.
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      or aberrant Cx43 positioned within the cytoplasm of cells,
      • Cogliati B.
      • Da Silva T.C.
      • Aloia T.P.
      • Chaible L.M.
      • Real-Lima M.A.
      • Sanches D.S.
      • et al.
      Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.
      after chronic carbon tetrachloride administration. Phenobarbital, which itself decreases GJs,
      • Mesnil M.
      • Fitzgerald D.J.
      • Yamasaki H.
      Phenobarbital specifically reduces gap junction protein mRNA level in rat liver.
      is usually co-administrated to promote fibrosis,
      • Garner R.C.
      • McLean A.E.
      Increased susceptibility to carbon tetrachloride poisoning in the rat after pretreatment with oral phenobarbitone.
      possibly explaining the observed discrepancy.
      Current evidence points towards a role of Cx43 in collagen matrix deposition. Administration of chronic carbon tetrachloride to Cx43 deficient mice resulted in a similar grade of fibrosis as observed in wild-type animals. Nevertheless, an intensification of collagen deposition and nodule formation with retraction of the liver capsule was more evident in Cx43 deficient animals.
      • Cogliati B.
      • Da Silva T.C.
      • Aloia T.P.
      • Chaible L.M.
      • Real-Lima M.A.
      • Sanches D.S.
      • et al.
      Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.
      In apparent contradiction, another study evaluating the role of Cx43 in fibrosis, aimed at discriminating between GJs and hemichannels. In both cases when Cx43 was inhibited, mice treated chronically with thioacetamide exhibited less fibrosis. Additionally, the authors concluded that hemichannel blockade mediated reduced stellate cell activation and reduced deposition of collagen.
      • Crespo Y.S.
      • Da Silva T.C.
      • Pereira I.V.A.
      • Willebrords J.
      • Maes M.
      • Sayuri N.M.
      • et al.
      TAT-Gap19 and carbenoxolone alleviate liver fibrosis in mice.
      To add more complexity, pannexins are involved in the transport of ATP to the extracellular space where it is converted to adenosine, which acts on its receptors to stimulate fibrosis. In another recent study, tenofovir, acting as a pannexin hemichannel blocker had a direct antifibrotic effect.
      • Feig J.L.
      • Mediero A.
      • Corciulo C.
      • Liu H.
      • Zhang J.
      • Perez-Aso M.
      • et al.
      The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin.

      Cirrhosis and its complications

      Portal hypertension

      In liver disease, increased intrahepatic vascular resistance contributes to the severity of portal hypertension.
      • Hernandez-Guerra M.
      • Garcia-Pagan J.C.
      • Bosch J.
      Increased hepatic resistance: a new target in the pharmacologic therapy of portal hypertension.
      In addition to the structural component of portal hypertension caused by fibrosis, a more dynamic component is also present.
      • Bathal P.S.
      • Grossmann H.J.
      Reduction of the increased portal vascular resistance of the isolated perfused cirrhotic rat liver by vasodilators.
      In cirrhosis, intrahepatic vascular tone is increased due to dysfunction of sinusoidal cells and decreased NO, resulting in impaired vasorelaxation in response to acetylcholine.
      • Gupta T.K.
      • Toruner M.
      • Chung M.K.
      • Groszmann R.J.
      Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.
      GJs connect endothelial cells and allow for propagation of vasodilation.
      • Chaytor A.T.
      • Evans W.H.
      • Griffith T.M.
      Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries.
      • Tallini Y.N.
      • Brekke J.F.
      • Shui B.
      • Doran R.
      • Hwang S.M.
      • Nakai J.
      • et al.
      Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC GCaMP2 transgenic mice.
      Indeed, binding of acetylcholine stimulates calcium-activated potassium channels in the plasma membrane evoking hyperpolarization, which is conducted from cell to cell through GJs.
      Cx37, Cx40, Cx43 and Cx45 regulate vascular tone.
      • Begandt D.
      • Good M.E.
      • Keller A.S.
      • DeLalio L.J.
      • Rowley C.
      • Isakson B.E.
      • et al.
      Pannexin channel and connexin hemichannel expression in vascular function and inflammation.
      Cx40 and Cx43 are involved in regulation of hepatic blood flow and are expressed in sinusoidal and endothelial cells of hepatic arteries and portal veins
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      • Shiojiri N.
      • Niwa T.
      • Sugiyama Y.
      • Koike T.
      Preferential expression of connexin37 and connexin40 in the endothelium of the portal veins during mouse liver development.
      (Fig. 4). This is consistent with the observation that Cx43 expression is likely to be absent during the resting state but induced during endothelial dysfunction.
      • Gabriels J.E.
      • Paul D.L.
      Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed.
      It is noteworthy that the expression of Cx43 in stellate cells is increased in parallel with its activation, while its blockade inhibits propagated contraction in response to calcium.
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      • Toyofuku T.
      • Yabuki M.
      • Otsu K.
      • Kuzuya T.
      • Hori M.
      • Tada M.
      Intercellular calcium signaling via gap junction in connexin-43-transfected cells.
      Experiments conducted in our laboratory showed that blocking GJs increases portal perfusion pressure and reduces vasodilatory response to acetylcholine.
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      The mechanism underlying this observation is not clear but the data suggests this may be modulated by Cx-mediated NO release.
      • Poisson J.
      • Lemoinne S.
      • Boulanger C.
      • Durand F.
      • Moreau R.
      • Valla D.
      • et al.
      Liver sinusoidal endothelial cells: physiology and role in liver diseases.
      Figure thumbnail gr4
      Fig. 4Role of connexins in portal hypertension in cirrhosis. Different cells in the liver participate in fibrosis and vascular tone, contributing to increased intrahepatic resistance. Cxs participate in arterial vasodilation by conducting hyperpolarization directly from endothelium to vascular smooth muscular cell in the arteries. Cx, connexin. (
      • Fischer R.
      • Reinehr R.
      • Lu T.P.
      • Schonicke A.
      • Warskulat U.
      • Dienes H.P.
      • et al.
      Intercellular communication via gap junctions in activated rat hepatic stellate cells.
      ,
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      ,
      • Branes M.C.
      • Contreras J.E.
      • Saez J.C.
      Activation of human polymorphonuclear cells induces formation of functional gap junctions and expression of connexins.
      ,
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      ,
      • Eugenin E.A.
      • Gonzalez H.E.
      • Sanchez H.A.
      • Branes M.C.
      • Saez J.C.
      Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro.
      ,
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      ,
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      ,
      • Shiojiri N.
      • Niwa T.
      • Sugiyama Y.
      • Koike T.
      Preferential expression of connexin37 and connexin40 in the endothelium of the portal veins during mouse liver development.
      ,
      • Barriere E.
      • Tazi K.A.
      • Rona J.P.
      • Pessione F.
      • Heller J.
      • Lebrec D.
      • et al.
      Evidence for an endothelium-derived hyperpolarizing factor in the superior mesenteric artery from rats with cirrhosis.
      ,
      • Griffith T.M.
      • Chaytor A.T.
      • Edwards D.H.
      The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization.
      ,
      • Mather S.
      • Dora K.A.
      • Sandow S.L.
      • Winter P.
      • Garland C.J.
      Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries.
      .)
      Decreased endothelial NO synthase activity in the liver may also be due to upregulation of caveolin-1.
      • Shah V.
      • Toruner M.
      • Haddad F.
      • Cadelina G.
      • Papapetropoulos A.
      • Choo K.
      • et al.
      Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the Rat.
      Interestingly, a strong association of Cx40 and Cx43 with caveolin-1 has been identified in endothelial and epidermal cells.
      • Schubert A.L.
      • Schubert W.
      • Spray D.C.
      • Lisanti M.P.
      Connexin family members target to lipid raft domains and interact with caveolin-1.
      It is possible that Cx43 expression is implicated in caveolin-1 overexpression in cirrhotic livers. Shear stress is a potent inducer of NO production and its relationship with GJs has been evaluated. Shear stress promoted Cx43 expression in endothelial cells.
      • Inai T.
      • Mancuso M.R.
      • McDonald D.M.
      • Kobayashi J.
      • Nakamura K.
      • Shibata Y.
      Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves.
      Although increased expression of Cx43 seems not to be limited to the sinusoidal liver cells, it is possible that the induction of Cx43 expression seen during cirrhosis is a compensatory mechanism to favour the transfer of molecules in response to shear stress. By contrast, changes in the Cx37 expression pattern caused by shear stress are less clear.
      • Pfenniger A.
      • Wong C.
      • Sutter E.
      • Cuhlmann S.
      • Dunoyer-Geindre S.
      • Mach F.
      • et al.
      Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells.
      Interestingly, Kruppel-like factor 2 (KLF2), which is activated after induction of shear stress and upregulates eNOS, has been suggested to regulate Cx37 expression. Indeed, shear stress induced Cx37 expression was abrogated following KLF2 suppression, suggesting that KLF2 acts as a transcription factor for Cx37. Here again, the association of a relevant NO promoter such as KLF2 with Cxs suggests a role for GJs in the regulation of vascular tone.
      In cirrhosis, following an increase in intrahepatic resistance, a progressive cascade of events leads to splanchnic and peripheral vasodilation. Sodium retention and volume expansion increases cardiac output that in turn contributes to the development of ascites, circulatory dysfunction and renal failure.
      • EASL
      clinical practice guidelines on the management of ascites, spontaneous bacterial peritonitis, and hepatorenal syndrome in cirrhosis.
      In opposition to the hepatic circulation, systemic NO is elevated. In addition to NO, other factors have also been hypothesized to participate in arterial vasodilation, such as the endothelium-derived hyperpolarizing factors
      • Barriere E.
      • Tazi K.A.
      • Rona J.P.
      • Pessione F.
      • Heller J.
      • Lebrec D.
      • et al.
      Evidence for an endothelium-derived hyperpolarizing factor in the superior mesenteric artery from rats with cirrhosis.
      and more specifically epoxyeicosatrienoic acids.
      • Griffith T.M.
      • Chaytor A.T.
      • Edwards D.H.
      The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization.
      GJs have been described as being fundamental in conducting hyperpolarization directly from the endothelium to vascular smooth muscle cells in the arteries.
      • Griffith T.M.
      • Chaytor A.T.
      • Edwards D.H.
      The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization.
      • Mather S.
      • Dora K.A.
      • Sandow S.L.
      • Winter P.
      • Garland C.J.
      Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries.
      In small resistance mesenteric arteries of cirrhotic rats, inhibiting epoxyeicosatrienoic acids promoted vasoconstriction, an effect that was shown to be independent of NO and prostaglandin, as it was still observed after their inhibition.
      • Bolognesi M.
      • Zampieri F.
      • Di P.M.
      • Verardo A.
      • Turato C.
      • Calabrese F.
      • et al.
      Increased myoendothelial gap junctions mediate the enhanced response to epoxyeicosatrienoic acid and acetylcholine in mesenteric arterial vessels of cirrhotic rats.
      However, the effect of epoxyeicosatrienoic acids was blunted following pretreatment with a GJ blocker, suggesting that epoxyeicosatrienoic acids may initiate a hyperpolarizing response that is conducted to vascular smooth muscle cells by myoendothelial GJs with consequent vasorelaxation. NO is also responsible for improving Cx43 communication between endothelial and myoendothelial cells. This is because of its ability to nitrosylate proteins, thus modifying protein function.
      • Lima B.
      • Forrester M.T.
      • Hess D.T.
      • Stamler J.S.
      S-nitrosylation in cardiovascular signaling.
      Indeed, NO has been shown to s-nitrosylate Cx43 channels.
      • Straub A.C.
      • Billaud M.
      • Johnstone S.R.
      • Best A.K.
      • Yemen S.
      • Dwyer S.T.
      • et al.
      Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.

      Hepatic encephalopathy

      Hepatic encephalopathy (HE) is an important neuropsychiatric complication that is associated with end-stage liver disease and has a multifactorial pathogenesis. Work from our laboratory recently demonstrated that Cx-hemichannel functionality, and consequently lactate transport, was impaired in the cerebral cortices of bile duct ligated rats with mild HE.
      • Hadjihambi A.
      • De C.F.
      • Hosford P.S.
      • Habtetion A.
      • Karagiannis A.
      • Davies N.
      • et al.
      Ammonia mediates cortical hemichannel dysfunction in rodent models of chronic liver disease.
      While the expression of the main astrocytic and neuronal Cxs was unaffected, the results of this study suggest that HE is associated with impairment of hemichannel functionality in the central nervous system, with ammonia playing a key role. The data supporting Cx-hemichannel dysfunction provide evidence of a possible mechanism underlying the pathogenesis of HE, involving a potential neuronal energy deficit due to impaired hemichannel-mediated lactate transport between astrocytes and neurons.

      Cholestatic disease

      GJs are involved in bile secretion and regulation of bile flow,
      • Nathanson M.H.
      • Rios-Velez L.
      • Burgstahler A.D.
      • Mennone A.
      Communication via gap junctions modulates bile secretion in the isolated perfused rat liver.
      • Serriere V.
      • Berthon B.
      • Boucherie S.
      • Jacquemin E.
      • Guillon G.
      • Claret M.
      • et al.
      Vasopressin receptor distribution in the liver controls calcium wave propagation and bile flow.
      • Temme A.
      • Stumpel F.
      • Sohl G.
      • Rieber E.P.
      • Jungermann K.
      • Willecke K.
      • et al.
      Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.
      • Yang J.
      • Ichikawa A.
      • Tsuchiya T.
      A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line.
      and any alteration in intercellular transmission of secondary messengers might be expected to result in cholestasis. After bile duct ligation, GJ expression was decreased.
      • Metz J.
      • Aoki A.
      • Merlo M.
      • Forssmann W.G.
      Morphological alterations and functional changes of interhepatocellular junctions induced by bile duct ligation.
      • Traub O.
      • Druge P.M.
      • Willecke K.
      Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis.
      This was associated with a marked reduction in protein levels of Cx26 and Cx32,
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      • Kojima T.
      • Yamamoto T.
      • Murata M.
      • Lan M.
      • Takano K.
      • Go M.
      • et al.
      Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver.
      which seems to be related to the associated inflammatory response.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      In addition, an increase in cholestatic bile acids such as taurolithocholate, taurolithocholate-sulfate and taurochenodeoxycholate promotes the closed state of GJs and worsens intercellular communication, making cholestasis worse.
      • Boucherie S.
      • Koukoui O.
      • Nicolas V.
      • Combettes L.
      Cholestatic bile acids inhibit gap junction permeability in rat hepatocyte couplets and normal rat cholangiocytes.
      However, the expression of Cx43 increases following bile duct ligation
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.
      • Fallon M.B.
      • Nathanson M.H.
      • Mennone A.
      • Saez J.C.
      • Burgstahler A.D.
      • Anderson J.M.
      Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.
      and after the development of cirrhosis.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      • Hernandez-Guerra M.
      • Gonzalez-Mendez Y.
      • de Ganzo Z.A.
      • Salido E.
      • Garcia-Pagan J.C.
      • Abrante B.
      • et al.
      Role of gap junctions modulating hepatic vascular tone in cirrhosis.
      The protein expression of Cx43 was further increased following LPS challenge and reduced following treatment with anti-TNF drugs.
      • Balasubramaniyan V.
      • Dhar D.K.
      • Warner A.E.
      • Vivien Li W.Y.
      • Amiri A.F.
      • Bright B.
      • et al.
      Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.
      These data suggest that the activation and infiltration of macrophages contribute to this adaptive response, which involves the in the synthesis and recycling of Cx43.
      • Gonzalez H.E.
      • Eugenin E.A.
      • Garces G.
      • Solis N.
      • Pizarro M.
      • Accatino L.
      • et al.
      Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.

      Non-alcoholic fatty liver disease

      Non-alcoholic fatty liver disease comprises a complex disease spectrum, including hepatic steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis and eventually HCC. Intracellular signalling cascades favour the deposition of fat in hepatocytes and induce inflammation.
      • Marra F.
      • Svegliati-Baroni G.
      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      Since GJs can modulate the transfer of molecules, Cxs potentially have an important role in NASH. Cx32 knockout rats with diet induced non-alcoholic fatty liver disease developed more pronounced oxidative stress, inflammation and liver injury than wild-type controls,
      • Sagawa H.
      • Naiki-Ito A.
      • Kato H.
      • Naiki T.
      • Yamashita Y.
      • Suzuki S.
      • et al.
      Connexin 32 and luteolin play protective roles in non-alcoholic steatohepatitis development and its related hepatocarcinogenesis in rats.
      • Tiburcio T.C.
      • Willebrords J.
      • Da Silva T.C.
      • Pereira I.V.
      • Nogueira M.S.
      • Crespo Y.S.
      • et al.
      Connexin32 deficiency is associated with liver injury, inflammation and oxidative stress in experimental non-alcoholic steatohepatitis.
      suggesting that GJ plays a protective role by maintaining homeostasis through cell-to-cell communication. However, using specific peptides to block Cx hemichannels decreased triglycerides, cholesterol, and inflammatory markers compared to controls, in animals fed a high-fat diet.
      • Willebrords J.
      • Cogliati B.
      • Pereira I.V.A.
      • Da Silva T.C.
      • Crespo Y.S.
      • Maes M.
      • et al.
      Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice.
      This apparently paradoxical observation may be explained by the fact that hemichannels are constitutively closed and open after a pathological stimulus contrary to Cx forming GJs. During injury different deleterious molecules are exchanged between the extracellular and intracellular environment of cells, so blocking hemichannels may be responsible for the beneficial phenotype reported in these studies. In keeping with this study, genetically modified obese rats treated with carbenoxolone, a non-specific blocker of Cx, had decreased liver steatosis, along with a significantly decreased body fat percentage, hypertriglyceridemia, hypercholesterolemia and insulin resistance.
      • Prasad Sakamuri S.S.
      • Sukapaka M.
      • Prathipati V.K.
      • Nemani H.
      • Putcha U.K.
      • Pothana S.
      • et al.
      Carbenoxolone treatment ameliorated metabolic syndrome in WNIN/Ob obese rats, but induced severe fat loss and glucose intolerance in lean rats.
      The functions of gap junctions and hemichannels are amenable to modification with drugs, making them attractive therapeutic targets.
      Pannexins, which form channels connecting cells with the extracellular environment, have also been studied in the setting of NASH.
      • Willebrords J.
      • Maes M.
      • Pereira I.V.A.
      • Da Silva T.C.
      • Govoni V.M.
      • Lopes V.V.
      • et al.
      Protective effect of genetic deletion of pannexin1 in experimental mouse models of acute and chronic liver disease.
      When open these channels participate in inflammatory processes.
      • Crespo Y.S.
      • Willebrords J.
      • Johnstone S.R.
      • Maes M.
      • Decrock E.
      • De B.M.
      • et al.
      Pannexin1 as mediator of inflammation and cell death.
      A decrease in lobular inflammation and oxidative stress was observed in mice with pannexin deletion. However, in this study a different gene expression profile was observed in pannexin deficient animals, particularly affecting lipid metabolism and genes involved in the inflammatory and oxidative stress response, suggesting that more experiments focussed on specifically blocking pannexins without modifying gene expression need to be performed. Interestingly, at a cellular level, pannexins contribute to ATP release, which functions as a pro-inflammatory signal for recruitment and activation of inflammatory cells in lipoapoptosis.
      • Xiao F.
      • Waldrop S.L.
      • Khimji A.K.
      • Kilic G.
      Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells.
      Overall, these studies suggest that improving GJ permeability, or blocking hemichannels either constituted by Cxs or pannexins may represent relevant therapeutic targets.

      Hepatocellular carcinoma

      The ability of GJs to regulate cellular proliferation
      • Loewenstein W.R.
      • Kanno Y.
      Intercellular communication and the control of tissue growth: lack of communication between cancer cells.
      • Loewenstein W.R.
      • Rose B.
      The cell-cell channel in the control of growth.
      supports the idea that these channels could be involved in cancer pathophysiology. In addition, there is evidence based on experimental studies suggesting a possible role of GJs in liver carcinogenesis. Targeted disruption of the Cx32 gene was associated with an increase in hepatic tumours, possibly because of a reduction in the propagation of apoptotic signals to adjacent cells.
      • Moennikes O.
      • Buchmann A.
      • Ott T.
      • Willecke K.
      • Schwarz M.
      The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.
      • Temme A.
      • Buchmann A.
      • Gabriel H.D.
      • Nelles E.
      • Schwarz M.
      • Willecke K.
      High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.
      • Kato H.
      • Naiki-Ito A.
      • Naiki T.
      • Suzuki S.
      • Yamashita Y.
      • Sato S.
      • et al.
      Connexin 32 dysfunction promotes ethanol-related hepatocarcinogenesis via activation of Dusp1-Erk axis.
      In keeping with this and further supporting the idea that Cxs may show tumour suppressive properties, both Cx26 and Cx32 expression are decreased in HCC while a mislocalization (and dysfunction) of the Cxs from the cell membrane to the cytoplasm has also been observed.
      • Shimizu K.
      • Onishi M.
      • Sugata E.
      • Sokuza Y.
      • Mori C.
      • Nishikawa T.
      • et al.
      Disturbance of DNA methylation patterns in the early phase of hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined diet in rats.
      • Tsujiuchi T.
      • Shimizu K.
      • Itsuzaki Y.
      • Onishi M.
      • Sugata E.
      • Fujii H.
      • et al.
      CpG site hypermethylation of E-cadherin and Connexin26 genes in hepatocellular carcinomas induced by a choline-deficient L-Amino Acid-defined diet in rats.
      • Dagli M.L.
      • Yamasaki H.
      • Krutovskikh V.
      • Omori Y.
      Delayed liver regeneration and increased susceptibility to chemical hepatocarcinogenesis in transgenic mice expressing a dominant-negative mutant of connexin32 only in the liver.
      • Igarashi I.
      • Makino T.
      • Suzuki Y.
      • Kai K.
      • Teranishi M.
      • Takasaki W.
      • et al.
      Background lesions during a 24-month observation period in connexin 32-deficient mice.
      • Yang Y.
      • Zhu J.
      • Zhang N.
      • Zhao Y.
      • Li W.Y.
      • Zhao F.Y.
      • et al.
      Impaired gap junctions in human hepatocellular carcinoma limit intrinsic oxaliplatin chemosensitivity: a key role of connexin 26.
      In HCC tissues, a reduction in the expression of Cx32 was associated with larger more aggressive tumours, vascular invasion, and poorer survival. Thus, experiments exploring the potential benefit of GJ opening drugs should be explored. Concerning this observation, doxorubicin resistant HCC cell lines showed reduced expression of Cx32. By contrast, overexpression of Cx32 resulted in increased sensitivity of HCC cells to the chemotherapy drug, supporting the hypothesis that Cx32 could be also an important target for counteracting drug resistance of HCC.
      • Yu M.
      • Zou Q.
      • Wu X.
      • Han G.
      • Tong X.
      Connexin 32 affects doxorubicin resistance in hepatocellular carcinoma cells mediated by Src/FAK signaling pathway.
      • Yang Y.
      • Qin S.K.
      • Wu Q.
      • Wang Z.S.
      • Zheng R.S.
      • Tong X.H.
      • et al.
      Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition.
      More recently, sorafenib, an oral multikinase inhibitor approved for advanced HCC, was shown to be more efficacious after increasing GJ intercellular communication with all-trans retinoic acid. This effect was abolished after co-incubation with GJ inhibitor 18-alpha glycyrrhetinic acid and oleamide.
      • Mesnil M.
      • Piccoli C.
      • Tiraby G.
      • Willecke K.
      • Yamasaki H.
      Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.
      • Yang Y.
      • Qin S.K.
      • Wu Q.
      • Wang Z.S.
      • Zheng R.S.
      • Tong X.H.
      • et al.
      Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition.
      In an apparent contradiction, the observation that Cx43 expression is increased in HCC cells suggests that Cx43 may possibly have oncogenic properties instead of suppressing tumorigenesis.
      • Krutovskikh V.
      • Mazzoleni G.
      • Mironov N.
      • Omori Y.
      • Aguelon A.M.
      • Mesnil M.
      • et al.
      Altered homologous and heterologous gap-junctional intercellular communication in primary human liver tumors associated with aberrant protein localization but not gene mutation of connexin 32.
      • Oyamada M.
      • Krutovskikh V.A.
      • Mesnil M.
      • Partensky C.
      • Berger F.
      • Yamasaki H.
      Aberrant expression of gap junction gene in primary human hepatocellular carcinomas: increased expression of cardiac-type gap junction gene connexin 43.
      • Wang Z.S.
      • Wu L.Q.
      • Yi X.
      • Geng C.
      • Li Y.J.
      • Yao R.Y.
      Connexin-43 can delay early recurrence and metastasis in patients with hepatitis B-related hepatocellular carcinoma and low serum alpha-fetoprotein after radical hepatectomy.
      • Zhang D.
      • Kaneda M.
      • Nakahama K.
      • Arii S.
      • Morita I.
      Connexin 43 expression promotes malignancy of HuH7 hepatocellular carcinoma cells via the inhibition of cell-cell communication.
      In fact, the magnitude of expression of Cx43 and its localization correlated with the malignant potential,
      • Kawasaki Y.
      • Kubomoto A.
      • Yamasaki H.
      Control of intracellular localization and function of Cx43 by SEMA3F.
      migration, invasive capacity and metastatic ability of HCC.
      • Ogawa K.
      • Pitchakarn P.
      • Suzuki S.
      • Chewonarin T.
      • Tang M.
      • Takahashi S.
      • et al.
      Silencing of connexin 43 suppresses invasion, migration and lung metastasis of rat hepatocellular carcinoma cells.
      However, an alternative explanation could be that the increased expression of Cx43 is a compensatory response to mislocalization of Cx43 as has been postulated to occur in breast cancer.
      • Jamieson S.
      • Going J.J.
      • D'Arcy R.
      • George W.D.
      Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours.
      Additional studies are needed to elucidate the exact role of Cx43 in hepatocarcinogenesis.

      Conclusions

      In conclusion, there is accumulating evidence that GJs have important functions related to cell-to-cell communication and that they contribute to tissue homeostasis. These functions have relevant consequences for the liver’s tolerance to acute injury as well as chronic insult, such as that observed in cirrhosis. It is clear that Cxs are expressed in multiple cell types and have distinct or even opposing roles depending on the liver cell type studied and type of constituted channel. Different Cx subtypes are both downregulated and upregulated in many liver disease conditions. This may form the basis for new therapeutic strategies focussed on specifically limiting or improving the traffic of messengers. However, more research is needed to elucidate the exact molecular mechanisms involved in order to exploit this pathway for the treatment of liver diseases.

      Financial support

      Dr. M. Hernández-Guerra is the recipient of a Grant from Instituto de Salud Carlos III ( PI14/01243 ). This work was supported by a grant for MHG from the Asociación Española para el Estudio del Hígado (AEEH) and Fundación Canaria Doctor Manuel Morales .

      Conflict of interest

      Rajiv Jalan has research collaborations with Takeda, Ocera, and Yaqrit, and consults with Yaqrit. Rajiv Jalan is the founder of Yaqrit Limited, which is developing UCL inventions for treatment of patients with cirrhosis. Rajiv Jalan is an inventor of ornithine phenylacetate, which was licensed by UCL to Mallinkrodt Pharma. He is also the inventor of Yaq-001, DIALIVE and Yaq-005, the patents for which have been licensed by UCL into a spinout company, Yaqrit Ltd. The other authors declare that they have no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      MHG and RJ contributed in the concept and design of the manuscript; MHG and AH written the article.

      Acknowledgements

      MHG would like to thank Prof. Alexander V Gourine Laboratory and Liver Failure Group at UCL for excellent support.

      Supplementary data

      The following are the Supplementary data to this article:

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