SNAPSHOT

Event: 1539: Endocytotic lysosomal uptake

Short Name: endocytosis

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Endocytosis is an universal internalization route in eukaryotes (Dergai et al., 2016). In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway for taking up specific macromolecules from the extracellular fluid (Cooper, 2000) and it is also a mechanism for uptaking extracellular molecules in plant cells (Fan et al., 2015).

The experimental studies have been done on mice in vivo, primary human and rat cells, and human and animal (rat, mouse)-derived cell lines (Harusch Fraenkel et al., 2007; Ng et al., 2015; Nadanaciva et al., 2011; Lu et al., 2017; Xie et al., 2007; Verma and Stellacci, 2010; Zhang and Monteiro-Riviere, 2009).

Event: 898: Disruption, Lysosome

Short Name: Disruption, Lysosome

AOPs Including This Key Event

Biological Context

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Typically, human or murine cell lines are used to assess this event. Examples are

 murine (Reiners et al., 2002)

murine, human (Li et al., 2000)

murine, human (Ghosh et al., 2011)

human (Loos et al., 2014)

human, murine (Anguissola et al., 2014)

human (Hamacher-Brady et al., 2011)

 

Event: 177: N/A, Mitochondrial dysfunction 1

Short Name: N/A, Mitochondrial dysfunction 1

AOPs Including This Key Event

Biological Context

Cell term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).

Event: 55: N/A, Cell injury/death

Short Name: N/A, Cell injury/death

AOPs Including This Key Event

Biological Context

Cell term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).

Event: 1493: Increased Pro-inflammatory mediators

Short Name: Increased pro-inflammatory mediators

AOPs Including This Key Event

Biological Context

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

LIVER:

Human [Santibañez et al., 2011]

Rat [Luckey and Petersen, 2001]

Mouse [Nan et al., 2013]

Event: 1494: Leukocyte recruitment/activation

Short Name: Leukocyte recruitment/activation

AOPs Including This Key Event

Biological Context

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Event: 265: Activation, Stellate cells

Short Name: Activation, Stellate cells

AOPs Including This Key Event

Biological Context

Cell term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Human: Friedman, 2008

Rat: George et al.,1999

Mouse: Chang et al., 2014

Pig: Costa et al., 2001

Event: 68: Accumulation, Collagen

Short Name: Accumulation, Collagen

AOPs Including This Key Event

Biological Context

Organ term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Humans: Bataller and  Brenner, 2005; Decaris et al., 2015.  

Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013.

Rats: Hamdy and El-Demerdash, 2012; Li, Li et al., 2012; Natajaran et al., 2006; Luckey and Petersen, 2001.

 

Event: 344: N/A, Liver fibrosis

Short Name: N/A, Liver fibrosis

AOPs Including This Key Event

Biological Context

Organ term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Human: Bataller and Brenner, 2005;Merck Manual, 2015; Blachier et al., 2013.

Rat, mouse: Liedtke et al., 2013

Relationship: 1775: endocytosis leads to Disruption, Lysosome

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Mouse (Werneburg et al., 2002; Kagedal et al., 2001)

Rat (Jung et al., 2015)

Hamster (Hayashi et al., 2008)

Human (Wang et al., 2013)

Key Event Relationship Description

Different substances can be uptaken by endocytosis and localized in lysosomes, while some of them can cause lysosomal disruption. Lysosomotropic agents are mostly weak, lipophilic bases that diffuse across lysosomal membrane, get protonated in the acidic milieu of lysosome and therefore get trapped inside (de Duve et al., 1974). They accumulate and cause the destabilization of lysosomal membranes by acting as surfactants, incorporating its hydrophobic tail in the membrane and with the hydrophilic head facing the interior of the lysosome (de Duve et al.,1974; Firestone et al.,1979). Their accumulation increase the intralysosomal pH, which has many consequences, including the prevention of the further uptake of lysosomotropic compounds, an increase in size and number of lysosomes and the overloading of lysosomes with non-digestible materials. 

There are different mechanisms how lysosomotropic agents can disrupt lysosomal membrane. However, not all lysosomotropic agents disrupt lysosomes- for example ammonia salts, methylamine and related hydrophilic weak bases cause swelling of the lysosomes, but do not increase permeability of the membrane. Usually in order to do that, agent requires a certain degree of lipid solubility. The amine will accumulate in the lysosomes until its concentration is high enough to solubilize the lysosomal membrane (Dubowchik et al., 1995)

It has been demonstrated that as a result of protonated agents in lysosomes, there will be accumulation of non-permeable charged substances which will result in inflow of water by increased osmolarity (Bandyopadhyay et al., 2014). Inflow of water results in increase of size and can cause the rupture of lysosome.

Also, oxidative stress can cause destabilization of the lysosomal membrane and for this process, intra-lysosomal ferric ions are essential. They catalyse the formation of oxygen radicals from hydrogen peroxide (Zdoslek et al., 1993).

Evidence Supporting this KER

Biological Plausibility

Trapping of lysosomotropic agents accumulates substances inside of the lysosomes, increases volume of these organelles, and big lysosomes are more prone to rupture (Ono et al., 2003). However, there are many mechanisms for lysosomotropic substances to provoke lysosomal disruption, but their prior uptake by lysosomes is essential.

Empirical Evidence

Accumulation of different substances in lysosomes is causing severe dysfunction, increased permeability of lysosomal membrane and even their rupture. It was demonstrated that even accumulation of substances that are physiologically find in lysosomes can lead towards lysosomal dysfunction. Use of cathepsins' B and L inhibitors causes abnormal accumulation of pro-cathepsins, enlargement of lysosomes and their severe dysfunction (Jung et al., 2015)

Leu Leu OMe is one of the agents that accumulates in lysosomes, where it is converted to a membranolytic compound and increase permeability of lysosomal membrane (Uchimoto et al., 1999, Thiele and Lipsky, 1990)

Sphingosine is another lysosomotropic agent that accumulates within the lysosomes, where it permeabilizes the membrane via a detergent mechanism and provokes relocation of lysosomal enzymes to the cytosol (Kagedal et al., 2001). It induces lysosomal disruption in primary mouse hepatocytes as well as it permabilize isolated hepatic lysosomes in vitro (Werneburg et al., 2002) Also, exposure of J774 cells to ammonium chloride prior to sphingosine resulted in formation of NH₃, which entered into lysosomes and became protonated and increased the pH of the organelle. This prevented the accumulation of sphingosine in the lysosome and provided protection against its lysosomolytic and apoptosis-inducing effects (Kagedal et al., 2001).

Considering nanomaterials (NMs) as a trigger for lysosomal damage, recent studies underpinned the importance of lysosomal NM uptake for NM-induced toxicity. Once the material is taken up by a cell and transported to the lysosome, the acidic milieu herein can either enhance solubility of a NM, or the material remains in its initial nano form. Both can induce toxicity, causing lysosomal swelling, followed by lysosomal disruption and the release of pro-apoptotic proteins (Cho et al., 2011; Cho et al., 2012; Wang et al., 2013). 

H₂O₂ also changes the permeability of the lysosomal membrane. It reacts with redox-active iron in the lysosomes, and produces hydroxyl radicals in Fenton-type reactions (Kubota et al., 2010). These radicals can destabilize the membrane by lipid peroxidation and damage of lysosomal proteins. Presence of desferrioxamine, that binds iron, is protecting lysosomes from rupture (Brunk et al., 2001).

Hayashi et al. demonstrated the uptake of FITC-crotamine into endosomal compartment of cells, and increased accumulation in a time dependant manner. Only 15 minutes after the treatment it was possible to see morphological changes of the cell, disruption of the lipid bilayer of the lysosomal membrane and subsequent rupture of lysosomes. They presented that crotamine treatment triggered the release of cysteine cathepsins to the cell cytosol (Hayashi et al., 2008).

References

Bandyopadhyay D, Cyphersmith A, Zapata JA, Kim YJ, Payne CK. Lysosome transport as a function of lysosome diameter. PLOS ONE. (2014) 9:e86847.

Brunk UT, Neuzil J,  Eaton JW. Lysosomal involvement in apoptosis, Redox Report, (2001) 6 (2): 91-97.

Cho W-S, Duffin R, Howie SEM, Scotton CJ, Wallace WAH, Macnee W, Bradley M, Megson IL, Donaldson K. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Part Fibre Toxicol  (2011) 8:27.

Cho W-S, Duffin R, Thielbeer F, Bradley M, Megson IL, MacNee W, Poland CA, Tran CL, Donaldson K. Zeta potential and solubility to toxic ions as mechanisms of lung inflammation caused by metal/metal oxide nanoparticles. Toxicol Sci (2012) 126:469–477.

de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P,  Van Hoof  F. Commentary. Lysosomotropic agents. Biochem. Pharmacol. (1974) 23:2495- 2531.

Dubowchik GM, Gawlak SL, Firestone RA. The in vitro effects of three lysosomotropic detergents against three human tumor cell lines Bioorg. Med. Chem. Lett. (1995) 5:893-898.

Firestone RA, Pisano JM, Bonney, RJ. Lysosomotropic agents. 1. Synthesis and cytotoxic action of lysosomotropic detergents. J. Med. Chem. (1979) 22: 1130- 1133.

Hayashi MAF, Nascimento FD, Kerkis A, Oliveira V, Oliveira EB, Pereira A, Rádis-Baptista G, Nader HB, Yamane T, Kerkis I, Tersariol ILS. Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization. Toxicon. (2008) 52(3): 508–517.

Jung M, Lee J, Seo H-Y, Lim JS, Kim E-K. Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose. PLoS ONE. (2015) 10(1):e011697.

Kagedal K, Zhao M, Svensson I, Brunk UT. Sphingosine-induced apoptosis is dependent on lysosomal proteases. The Biochemical journal.  (2001) 359: 335-43.

Kubota C, Torii S, Hou N, Saito N, Yoshimoto Y, Imai H, Takeuchi T. Constitutive reactive oxygen species generation from autophagosome/lysosome in neuronal oxidative toxicity. J Biol Chem. (2010) 285(1):667-74.

Ono K, Kim SO, Han J. Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death. Mol Cell Biol. (2003) 23: 665-76.

Thiele DL, Lipsky PE. Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells. Proc Natl Acad Sci U S A. (1990) 87(1): 83–87.

Uchimoto T, Nohara H, Kamehara R, Iwamura M, Watanabe N, Kobayashi Y. Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester. Apoptosis. (1999) 4(5): 357–362.

Wang F, Bexiga MG, Anguissola S, Boya P, Simpson JC, Salvati A, Dawson KA: Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles. Nanoscale (2013) 5:10868–76.

Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ. Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent. Am J Physiol Gastrointest Liver Physiol. (2002) 283:G947–G956.

Zdolsek J, Zhang H, Roberg K, Brunk U, Sies H. H2O2-Mediated Damage to Lysosomal Membranes of J-774 Cells, Free Radical Research Communications, 1993, 18:2, 71-85

Relationship: 993: Disruption, Lysosome leads to N/A, Mitochondrial dysfunction 1

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Murine (Stoka et al., 2001; Zhang et al., 2009; Lindsten et al., 2000)

Human (Boya et al., 2003; Cirman et al., 2004)

Key Event Relationship Description

Disrupted lysosomal membrane release the content of lysosomes including cathepsins. Cathepsins take part in activation of BH3-only proteins, which directly or indirectly activate pro-apoptic Bax and Bak proteins. Once activated Bax and Bak form dimers and higher order oligomers, in order to form pores in outer mitochondrial membrane and cause mitochondrial injury.

Many evidences suggest that lysosomal disruption usually precedes mitochondrial injury (Guicciardi et al., 2000; Brunk et al., 2001; Zhao et al. 2003; Droga-Mazovec et al., 2008), with lysosomal proteases inducing mitochondrial dysfunction.

Zhao and colleagues have also proposed the existence of a positive feed-back mechanism between lysosomal damage and mitochondrial damage, in which early lysosomal rupture causes mitochondrial rupture and leakage of mitochondrial proteins that increase lysosomal damage and consequent apoptosis (Zhao et al., 2000).

The pathway between lysosomal membrane permeabilization (LMP) and mitochondrial membrane permeabilization (MMP) is regulated principally by Bcl-2 family of proteins. The family is subdivided into anti-apoptotic multidomain proteins (such as Bcl-2, Bcl-xl, Bcl-W, Mcl-1 and A1), pro-apoptotic multidomain proteins (Bax and Bak) and pro-apoptotic BH3-only proteins (such as Bid, Puma, Noxa, Bim, Bad, and Bik) (Fletcher and Huang, 2006; Youle and Strasser, 2008).

Cathepsins, released after lysosomal damage, have a role in the cell death through the cleavage of BH3-only proteins, such as Bid, to generate active tBid (truncated Bid) (Blomgran et al., 2007; Cirman et al., 2004; Droga-Mazovec et al., 2008; Houseweart et al., 2003; Stoka et al., 2001) and by degradation of the anti-apoptotic Bcl-2 molecules Bcl-2, Bcl-xl and Mcl-1 (Blomgran et al., 2007; Droga-Mazovec et al., 2008). However it was shown that though Bid is not the only substrate of lysosomal enzymes that induce cytochrome c release, it is the major one (Stoka et al., 2001). Droga-Mazovec and colleagues showed that Bid is cleaved by cathepsins in human liver carcinoma cells (HepG2) (Droga-Mazovec et al., 2008), while other study showed that particularly cathepsin B is active in hepatocytes (Guicciardi et al., 2000).

Bid is also cleaved by caspase 8, which represents a link between extrinsic and intrinsic (mitochondrial) pathway (Li et al., 1998).

Activated BH3-only proteins continue to activate pro-apoptic proteins Bax and Bak. Sarosiek et al. observed that Bid preferentially activates Bak, while Bim activates Bax (Sarosiek et al., 2013). The activation of Bax and Bak occurs after LMP, but before mitochondrial release of cytochrome c and caspase-3 activation (Boya et al., 2003). Currently there are two models describing activation of Bax and Bak proteins and the role of anti-apoptic and pro-apoptic multidomain proteins in it. In the indirect model, Bax and Bak are sequestered and inactivated by anti-apoptotic Bcl-2 proteins. The binding of pro-apoptotic BH3-only proteins to these Bcl-2 proteins triggers the release of Bax and Bak. The direct model proposes that Bax and Bak are activated by direct binding of pro-apoptotic BH3-only proteins, called the activators (Bid, Bim or Puma). However, these activators are normally sequestered by anti-apoptotic Bcl-2 proteins. In order to release the activators, other BH3-only proteins, called senzitizers, neutralize anti-apoptotic Bcl-2 proteins (Brenner and Mak, 2009; Willis et al., 2007).

Bak and Bax go under major conformational changes after binding of BH3 only proteins (as reviewed by Westphal et al., 2014). Once activated Bak or Bax molecules bind reciprocally to form symmetric homodimers. It is thought that homodimers of Bak or Bax must then associate to higher order oligomers to porate the mitochondrial outer membrane (Uren et al., 2017). Heterodimers form only a minor population compared with homodimers (Dewson et al., 2012; Mikhailov et al., 2003).

Bak and Bax shallow insertion into the outer leaflet of mitochondrial membrane (Westphal et al., 2014; Oh KJ et al., 2010) may destabilize the lamellar structure of the bilayer to induce lipidic pores in mitochondrial membrane. This induces release of proteins from the space between inner and outer mitochondrial membrane (Newmeyer et al., 2003).

Evidence Supporting this KER

Biological Plausibility

In the last decade there is a growing body of evidences about the strong functional link between lysosomes and mitochondria that play an important role in physiology and pathology. The evidences also showed link between lysosomal and mitochondrial damage, and that lysosomal damage precedes mitochondrial injury.

Empirical Evidence

LMP after exposure to ciprofloxacine, norfloxacine and hydroxychloroquine is detected couple of hourse earlier that MMP (Boya et al., 2003). The same study proves that the cells with signs of MMP are sub-ensemple of the group of the cells with signs of LMP, not vice-versa. Also inhibition of LMP with Baf A1 – that inhibits lysosomal vacuolar H⁺ ATP-ase, prevented MMP, while inhibition of MMP in Bax/Bak double knocks out cells didn't prevent LMP. However, inhibition of MMP prevented LMP to cause manifestations of the cell death. All the evidences from this and other studies (such as Droga-Mazovec et al., 2008; Ghosh et al., 2011) prove that LMP lies upstream from MMP and causes it.

When isolated mitochondria are incubated with purified cathepsin B in the presence of cytolic extracts, a release of cytochrome c from mitochondria is detected (Guicciardi et al., 2000). The microinjection of cathepsin D to the cell causes cytochrome c release, capsases activation and apoptosis (Roberg et al., 2002).

It was shown that mice deficient of stefin B (major intracellular cathepsins inhibitor) developed spontaneous cerebellar apoptosis (Houseweart et al., 2003). Pepstatin A, an inhibitor of cathepsin D, was found to inhibit caspase-3-like proteolytic activity and to prevent apoptosis (Roberg et al., 1999). The treatment of the cells with cathepsin B and cathepsin D inhibitors, pepstatin A and E-64-d, decreased MMP and activation of caspases (Kagedal et al., 2001).

Cathepsin B directly cuts Bid and produces active tBid, while cathepsin B inhibitors z-FA-fmk and E-64-d block Bid activation in cells (Zhang et al., 2009; Blomgran et al., 2007). When cathepsin B silenced HeLa cells were treated with granulysin Bid degradation was blocked, same as cytochrome c and apoptosis inducing factor (AIF) release (Zhang et al., 2009).

Stoka et al. showed that incubation of rat mitochondria to uncleaved Bid resulted only in insignificant levels of cytochrome c release, while exposure to both uncleaved Bid and lysosomal extracts resulted in cytochrome c release (Stoka et al., 2001). Other studies confirmed necessity of tBid for cytochrome c release (Gross et al., 1999; Luo et al., 1998).

Bid knockout mouse embryonic fibroblasts (MEFs) and Bax/Bak deficient MEFs were more resistant to granulysin induced death compared to wild type, and they released less cytochrome c and AIF (Zhang et al., 2009; Lindsten et al., 2000). Bcl-2-overexpressed HeLa cells almost completely blocked the release of cytochrome c and AIF after granulysin treatment (Zhang et al., 2009). Overexpression of Bcl-2 is also suppressing oxidative stress induced apoptosis (Zhao et al., 2000).

Some studies stated that certain cell types such as hepatocytes appear to require a Bid in order to disrupt mitochondrial membrane, release cytochrome c and following steps to execute apoptosis (Korsmeyer et al., 2000). They also proved that BH3 domain of tBid was not required for targeting mitochondrial membrane but it is required for cytochrome c release.

Using specific inhibitors it was demonstrated that cytosolic cathepsin D triggers Bax activation and translocation to mitochondria, resulting release of AIF from mitochondria, and the apoptosis (Bidere et al., 2003).

Incubation of cleaved Bid with mitochondria promotes oligomerization of membrane bound Bak and cytochrome c release (Wei et al., 2000).

Bax/Bak double knockout MEFs didn't show MMP induced by ciprofloxacin, norfloxacin and hydroxychloroquine, while increased LMP was detected (Boya et al., 2003).

Incubation of Bax with isolated mitochondria resulted in cytochrome c release while Bcl-xl inhibits this release (Jurgensmeier et al., 1998)

The length of the core dimer of Bax or Bak is the approximate width of the mitochondrial outer membrane, and the bend observed in the structures may be accommodated by the curved edge of the pore (Uren et al., 2017).

Uncertainties and Inconsistencies

Repnik and colleagues showed that inhibition of cysteine cathepsins by E-64-d had little effect on LDH (cytosolic enzyme lactate dehydrogenase) release in medium in LLOMe treated cells (Repnik et al., 2017). They also detected that after exposure to LLOMe, cathepsins remain in lysosomes and are being degraded there which is in contradiction with most of the previous studies.

As stated earlier there are empirical evidences that incubation of cathepsin B with mitochondria and cytosolic factors increase mitochondrial permeabilization. However, in some studies pharmacological inhibition of cathepsin B, L and D didn't suppress Bid cleavage, suggesting that other lysosomal proteases might be responsible for Bid cleavage (Reiners et al., 2002).

The knockout of genes coding for cathepsins B, D, L and S failed to prevent induced MMP and cell death (Boya et al., 2003).

Housewert et al. showed that the amount of cerebellar granule cell apoptosis in cystatin B-deficient mice did not change when Bid was removed.  This indicates that cathepsins can use other mechanisms to initiate apoptosis. They concluded that another molecule may partially substitute for Bid when it is missing (Housewert et al., 2003). Willis and colleagues showed that neither Bim nor Bid are necessary for apoptosis, as their absences didn't stop apoptosis or prevented Bax activation (Willis et al., 2007).

Some  reports described Bax/Bak-independent mechanisms of cytochrome c release, involving either cyclosporine A sensitive mitochondrial membrane permeability (Wan et al., 2008) or a serine protease(s)-dependent mechanism (Mizuta et al., 2007).

References

Baixauli F, Acin-Perez R, Villarroya-Beltri C, Mazzeo C, Nunez-Andrade N, Gabande-Rodriguez E, Ledesma MD, Blázquez A, Martin MA, Falcón-Pérez JM, Redondo JM, Enríquez JA, Mittelbrunn M. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses. Cell Metab. (2015) 22, 485–498.

Bidere N, Lorenzo HK,  Carmona S, Laforge M, Harper F, Dumont C, Senik A. Cathepsin D triggers Bax activation, resulting in selective AIF relocation in T lymphocytes entering the early commitment phase to apoptosis. J. Biol. Chem. (2003) 278:31401–31411.

Blomgran R, Zheng L, Stendah O. Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization, J. Leukoc. Biol. (2007) 81 1213–1223.

Boya P, Andreau K, Poncet D, Zamzami N, Perfettini JL, Metivier D, Ojcius DM, Jäättelä M, Kroemer G. Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion. J. Exp. Med. (2003) 197:1323–1334.

Brenner D, Mak TW. Mitochondrial cell death effectors.  Curr Opin Cell Biol. (2009) 21(6): 871–877.

Brunk UT, Dalen H, Roberg K, Hellquist HB. Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radicals Biol. Med. (1997) 23, 61 -626.

Cirman T, Orešić K, Mazovec GD, Turk V, Reed JC, Myers RM, Salvesen GS, Turk B. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins, J. Biol. Chem. 279 (2004) 3578–3587.

Dewson G, Ma S, Frederick P, Hockings C, Tan I, Kratina T, Kluck RM. Bax dimerizes via a symmetric BH3:groove interface during apoptosis. Cell Death Differ. (2012) 19, 661–670.

Droga-Mazovec G, Bojič L, Petelin A, Ivanova S, Romih R, Repnik U, Salvesen GS, Stoka V, Turk V, Turk B. Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues, J. Biol. Chem. (2008)  283: 19140–19150.

Fernandez-Mosquera L, Diogo CV, Yambire KF, Santos GL, Luna Sanchez M, Benit P, Rustin P, Lopez LC, Milosevic I, Raimundo N. Acute and chronic mitochondrial respiratory chain deficiency differentially regulate lysosomal biogenesis. Sci. Rep. (2017) 7:45076.

Fletcher JI, Huang DC. BH3-only proteins: orchestrating cell death. Cell Death Differ. (2006) 13: 1268-1271.

Ghosh M, Carlsson F, Laskar A, Yuang XM, Li W. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages. FEBS Lett. (2011) 585(4): 623–629.

Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer SJ. Caspase cleaved BID targets mitochondrial and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. (1999) 274: 1156 ± 1163.

Guicciardi ME,  Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J. Clin. Invest. (2000) 106:1127–1137.

Houseweart MK, Vilaythong A, Yin XM, Turk B, Noebels JL, Myers RM. Apoptosis caused by cathepsins does not require Bid signaling in an in vivo model of progressive myoclonus epilepsy (EPM1). Cell Death and Differentiation, (2003): 10(12), 1329-1335.

Jürgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC.  Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A. (1998) 95(9): 4997–5002.

Kagedal K, Zhao M, Svensson I, Brunk UT. Sphingosine-induced apoptosis is dependent on lysosomal proteases. The Biochemical journal.  (2001) 359: 335-43.

Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c.  Cell Death Differ. (2000); 7(12): 1166–1173.

Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. (1998) 94(4): 491–501.

Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA, Ulrich E, Waymire KG, Mahar P, Frauwirth K, Chen Y, Wei M, Eng VM, Adelman DM, Simon MC, Ma A, Golden JA, Evan G, Korsmeyer SJ, MacGregor GR, Thompson  CB. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell. (2000) 6: 1389–1399.

Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, aBcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell (1998) 94: 481 - 490.

Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White E, Saikumar P. Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J. Biol. Chem. (2003) 278: 5367–5376.

Mizuta T, Shimizu S, Matsuoka Y, Nakagawa T, Tsujimoto Y. A Bax/Bak-independent mechanism of cytochrome c release. J. Biol. Chem. (2007) 282, 16623–16630.

Newmeyer DD, Ferguson-Miller S. Mitochondria: releasing power for life and unleashing the machineries of death. Cell (2003) 112: 481–490.

Oh KJ, Singh P, Lee K, Foss K, Lee S, Park M, Lee S, Aluvila S, Park M, Singh P, Kim RS, Symersky J, Walters DE. Conformational Changes in BAK, a Pore-forming Proapoptotic Bcl-2 Family Member, upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers. The Journal of Biological Chemistry. (2010) 285(37):28924-28937.

Palmieri M, Impey S, Kang H, di Ronza A, Pelz C, Sardiello M, Ballabio A. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Hum. Mol. Genet. (2011).  20: 3852–3866.

Reiners J, Caruso J, Mathieu P, Chelladurai B, Yin X-M, Kessel D. Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves bid cleavage. Cell death and differentiation. (2002) 9(9):934-944.

Repnik U, Borg Distefano M,  Speth MT, Ng MYW, Progida C, Hoflack B, Gruenberg J, Griffiths G. L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes. J. Cell Sci. (2017) 130: 3124–3140.

Roberg K, Johansson U, Ollinger K. Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress. Free Radic Biol Med. (1999) 27(11-12): 1228–1237.

Roberg K, Kagedal K, Ollinger K. Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts. Am. J. Pathol. (2002) 161:89–96.

Sarosiek KA, Chi X, Bachman JA, Sims JJ, Montero J, Patel L, Flanagan A, Andrews DW, Sorger P, Letai A. BID preferentially activates BAK while BIM preferentially activates BAX, affecting chemotherapy response Mol Cell. Mol Cell. (2013) 51(6): 751–765.

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Relationship: 363: N/A, Mitochondrial dysfunction 1 leads to N/A, Cell injury/death

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Neuronal necrosis has been noted in sea lions accidentally exposed to DomA (Silvagni et al., 2005) that correlated well with the histopathological findings previously reported in experimental studies (Tryphonas et al., 1990).

Key Event Relationship Description

ROS generation is known to activate different pathways leading to apoptosis, whereas depletion of energy production induces necrotic cell death.

Evidence Supporting this KER

Biological Plausibility

There is functional mechanistic understanding supporting this relationship between KE3 and KE4.

ROS are known to stimulate a number of events and pathways that lead to apoptosis, triggered by ROS-induced ER stress signalling pathway (Lu et al., 2014), caspase-dependent and -independent apoptosis (Zhou et al., 2015), mitogen-activated protein kinase (MAPK) signal transduction pathways (reviewed in Cuadrado and Nebreda, 2010, Harper and LoGrasso, 2001).

Depletion of cellular ATP is known to cause switching from apoptotic cell death triggered by a variety of stimuli to necrotic cell death (Leist et al., 1997) suggesting that the level of intracellular ATP determines whether the cell dies by apoptosis or necrosis (Nicotera et al., 1998). There is strong proof that apoptosis requires energy, as it is a highly regulated process involving a number of ATP-dependent steps such as caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, bleb formation and apoptotic body formation (Richter et al., 1996).

Empirical Evidence

Include consideration of temporal concordance here

In the case of DomA, in vitro studies have shown that oxidative stress and oxidative stress-induced activation of the stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK) pathway is implicated in DomA-mediated apoptosis (Giordano et al., 2007; 2008; 2009; Lu et al., 2010). In vivo findings also show that ROS-mediated cognitive deficits are associated with apoptosis induced by activation of the JNK pathway (Lu et al., 2010; 2011).

• Mice injected intraperitoneally (i.p.) with DomA at a dose of 2 mg/kg once a day for 4 weeks have shown increase (6 fold) of the TUNEL positive cells in the hippocampus . In the same study they have found that indicators of mitochondria function are markedly decreased (1.5-2 fold) and ROS levels are elevated (3.2 fold) (Lu et al., 2012). DomA treatment also significantly decreases the levels of bcl-2, procaspase-3 and procaspase-12 and increases the activation of caspase-3 and caspase-12 in the mouse hippocampus (Lu et al., 2012). The same research group using similar dose but longer exposure (4 weeks), has shown increase of ROS (3 fold) and NOX (2 fold) and elevated (8 fold) mean value of TUNEL-positive cells in the hippocampal CA1 sections as well as increase in the activation of caspase-8 and caspase-3 (Wu et al., 2012). These two in vivo studies (Lu et al., 2012; Wu et al., 2012) suggest that both KEs are affected in response to the same dose of DomA and exposure paradigm and that the incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction).

• The cell viability has been measured by the MTT reduction assay in mouse cerebellar granule neurons (CGNs) and showed that the IC50 values for DomA are 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons (Giordano et al., 2006). This reduction in cell viability has been demonstrated to be concentration dependent after studying a range of concentrations of DomA (0.01 and 10 µM). Giordano et al. 2007 have shown that 100 nM DomA induce apoptotic cell death in mouse CGNs. In a follow-up study, the same research group has performed a dose response evaluation and showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs derived from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione are more sensitive as 10 nM DomA induces a significant increase in apoptotic cell number (Giordano et al., 2009). The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes has been caused by 100 nM DomA. Interestingly, 1 and 10 µM DomA still cause significant apoptosis in both cell types but to a lesser extent compared to 100 nM DomA. ROS have been measured only at the dose of 100 nM DomA, 30 min after treatment and showed 2.5 fold increase compared to controls in CGNs from Gclm (+/+) mice (Giordano et al., 2009). Caspase 3 activity has also been measured after 12 h with prior 1 h exposure to 100 nM DomA and found to be increased (2.2 fold). In the same study, DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h exposure. Again these in vitro studies (Giordano et al., 2007; 2009) suggest that both KEs are affected by the same dose of DomA and that the incidence of KE down (cell death) is higher than the incidence of KE up (mitochondrial dysfunction). Furthermore, KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that takes place 12-24 h later.

• Mixed cortical cultures have been treated with 3, 5, 10, or 50 μM DomA for a variety of exposure durations (10 min, 30 min, 1 h, or 2 h), after which DomA is washed out and the culture medium is replaced with conditioned medium from unexposed sister cultures (Qiu et al., 2006). In all cases neuronal death has been measured 24 h following the beginning of exposure. The results show that DomA-induced neuronal death is determined by both concentration and duration of exposure. After a 10-min exposure, 50 μM DomA produces marked neuronal death of 47.4 %, whereas by 1 h of treatment, the same concentration produces near maximal neuronal death but longer exposures do not increase neuronal death further (Qiu et al., 2006). Regarding time dependence, this study shows that low concentrations of DomA produces more neuronal death if this is measured 22 h after the washout than if measured immediately after DomA treatment, while higher concentrations of DomA (20–100 μM) produces equivalent degrees of neuronal death when measured at these two time points (Qiu et al., 2006). Based on these findings, three EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration of exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) (Qiu et al., 2006).

• The mean concentration of DomA in rat brain samples obtained at 30 min after intraperitoneal (i.p.) administration of 1 mg/kg DA is 7.2 ng/g (Tsunekawa et al., 2013). These animals have been examined and revealed after histopathological analysis neuronal shrinkage and cell death, including an increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and after 5 days (19.0 %) compared to the controls (1.7 %) (Tsunekawa et al., 2013). In the same study, indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone (Tsunekawa et al., 2013).

• Brain slices from 8-day-old pups have been treated after 2 weeks with 10 μM DomA and assessed with propidium iodine (PI) stain to determine cellular damage (Erin and Billingsley, 2004). A time course has been carried out and viable cultures have been visualized 12, 24, 48 and 92 h after DomA treatment. Changes in PI uptake has been detected after 24 h post-treatment and at 4h the average fold-increase of PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively (Erin and Billingsley, 2004). In the same study, incubation of brain slices with DomA induces degradation of α-spectrin to the 120-kDa product after 18 h of treatment but no change has been noted after 12 h incubation, whereas caspase 3 activity results have not been conclusive (Erin and Billingsley, 2004).

• Using observations of neuronal viability and morphology, exposure of cultured murine cortical neurones to DomA for 24 h have shown to induce concentration-dependent neuronal cell death and the EC50 determined to be 75 µM (Larm et al., 1997).

Stressor	Experimental Model	Tested concentrations	Exposure route	Exposure duration	Mitochondrial dysfunction (KE up) (measurements, quantitative if available)	Cell death (KE down) (measurements, quantitative if available)	References	Temporal Relationship	Dose-response relationship	Incidence	Comments
DomA	16-month-old male ICR mice	2 mg/kg	Intraperitoneally (i.p.)	Once a day for 4 weeks	Indicators of mitochondrial function were markedly decreased (1.5-2 fold) and ROS levels were elevated (3.2 fold).	The mean of TUNEL positive cells in the hippocampus was increased (6 fold). The levels of bcl-2, procaspase-3 and procaspase-12 were significantly decreased and the activation of caspase-3 and caspase-12 in the mouse hippocampus were increased.	Lu et al., 2012		Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	16-month-old male ICR mice	2 mg/kg	i.p.	Once a day for 4 weeks	ROS levels were increased (3 fold) and NOX (2 fold).	The mean value of TUNEL-positive cells in the hippocampal CA1 sections was elevated (8 fold) and the activation of caspase-8 and caspase-3 was increased.	Wu et al., 2012		Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		Time course (15 to 120 min)	DomA caused a significant time- and concentration-dependent increase in ROS production. The higher ROS production (2.5 fold increase) was recorded after 1 h of exposure.	IC50 values for DomA were 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons based on MTT assay after 24 h of exposure.	Giordano et al., 2006	KE up (mitochondrial dysfunction) happens earlier than KE down (cell death)	Same doses
DomA	CGNs from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		Time course (0 to 180 min)	DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.	0.1μM DomA was maximally effective in inducing apoptosis, while a concentration causing high toxicity (10μM) induced very limited apoptosis, 24 h after exposure.	Giordano et al., 2007	KE up (mitochondrial dysfunction) happens earlier (1-2 h) than KE down (cell death) that occurs after 24 h	Same doses
DomA	CGNs from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		For ROS: 30min, Apoptosis: 12-24 h.	ROS levels were measured only at the dose of 100 nM DomA 30 min after treatment in CGNs from Gclm (+/+) mice and showed 2.5 fold increase compared to controls .	A dose response study that showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione were more sensitive as 10 nM DomA induced a significant increase in apoptotic cells number .The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes was caused by 100 nM DomA. 1 and 10 µM DA caused significant apoptosis in both cell types but to less extend compared to 100 nM DomA. Caspase 3 activity after 12 h with prior 1 h exposure to 100 nM DomA found to be increased (2.2 fold). DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h from exposure.	Giordano et al., 2009	KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that take place 12-24 h later	Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	Mixed cortical cultures obtained from pregnant Holtzman rats on embryonic day (ED) 16–18	3, 5, 10, or 50 μM		10 min, 30 min, 1 h or 2 h, after which DomA was washed out and the culture medium replaced with conditioned medium from unexposed sister cultures .		EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) .	Qiu et al., 2006
DomA	Rat	1 mg/kg DA	i.p.		Indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone .	Neuronal shrinkage and cell drop out as well as increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and 5 days (19.0 %) has been found compared with that of controls (1.7 %) .	Tsunekawa et al., 2013
DomA	Rat rain slices from 8-day-old pups	10 μM		Time course (12, 24, 48 and 92 h) after DomA treatment.		PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively . Degradation of α-spectrin to the 120-kDa product after 18 h of DomA treatment was noted but no change was noted after 12 h incubation, whereas caspase 3 activity results were not conclusive.	Erin and Billingsley, 2004
DomA	Cultured murine cortical neurones					DomA induces concentration-dependent neuronal cell death and the EC50 determined to be 75 µM .	Larm et al., 1997

Gap of knowledge: there are no studies showing that GLF induces neuronal cell death through mitochondrial dysfunction.

Uncertainties and Inconsistencies

Rats have been administered with DA at the dose of 1.0 mg/kg for 15 days. The histochemical analysis of hippocampus from these animals has revealed no presence of apoptotic bodies and no Fluoro-Jade B positive cells (Schwarz et al., 2014).

References

Cuadrado A, Nebreda AR., Mechanisms and functions of p38 MAPK signalling. Biochem J., 2010, 429(3): 403–417.

Erin N, Billingsley ML., Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices. Brain Res., 2004, 1014: 45-52.

Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70: 2116-2126.

Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.

Giordano G, Klintworth HM, Kavanagh TJ, Costa LG., Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases. Neurochem Int., 2008, 52: 1100-1105.

Giordano G, Li L, White CC, Farin FM, Wilkerson HW, Kavanagh TJ, Costa LG., Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells. J Neurochem., 2009, 109: 525-538.

Harper SJ, LoGrasso P., Signalling for survival and death in neurones: the role of stress-activated kinases. JNK and p38. Cell Signal., 2001, 13(5): 299–310.

Larm JA, Beart PM, Cheung NS., Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones. Neurochem Int., 1997, 31: 677-682.

Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P., Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med., 1997, 185: 1481−1486.

Lu J, Wu DM, Zheng YL, Hu B, Zhang ZF., Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol., 2010, 20: 598-612.

Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B, Zhang ZF., Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain., 2011, 134: 783-797.

Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52(3): 646-59.

Lu TH, Su CC, Tang FC, Chen CH, Yen CC, Fang KM, Lee KL, Hung DZ, Chen YW., Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway. Chem Biol Interact., 2014, 225: 1-12.

Nicotera P, Leist M, Ferrando-May E., Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicol Lett., 1998, 102-103: 139-142.

Qiu S, Pak CW, Currás-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical cultures: effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006, 89: 243-256.

Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett., 1996, 378: 107-110.

Schwarz M, Jandová K, Struk I, Marešová D, Pokorný J, Riljak V. Low dose domoic acid influences spontaneous behavior in adult rats. Physiol Res., 2014, 63: 369-76.

Silvagni PA, Lowenstine LJ, Spraker T, Lipscomb TP, Gulland FMD., Pathology of Domoic Acid Toxicity in California Sea Lions (Zalophus californianus). Vet Path., 2005, 42: 184-191.

Tryphonas L, Truelove J, Iverson F, Todd EC, Nera EA. Neuropathology of experimental domoic acid poisoning in non-human primates and rats. Can Dis Wkly Rep. 1990 Sep;16 Suppl 1E:75-81.

Tsunekawa K, Kondo F, Okada T, Feng GG, Huang L, Ishikawa N, Okada S., Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation. BMC Neurosci., 2013, 14: 4-16.

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Zhou Q, Liu C, Liu W, Zhang H, Zhang R, Liu J, Zhang J, Xu C, Liu L, Huang S, Chen L., Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis. Toxicol Sci., 2015, 143: 81-96.

Relationship: 1776: N/A, Cell injury/death leads to Increased pro-inflammatory mediators

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Human (Andersson et al., 2000; Scaffidi et al., 2002; Bell et al., 2006; Clarke et al., 2010)

Mouse (Faouzi et al., 2001; Chen et al., 2007)

Key Event Relationship Description

Cell death, including both necrosis and apoptosis can lead toward inflammation. Faouzi and colleagues showed that apoptosis can induce hepatic inflammation equally as necrosis (Faouzi et al., 2001). Some studies indicate that phagocytes can produce inflammatory cytokines upon ingestion of apoptotic bodies (Uchimura et al., 1997).

When cells undergo necrosis they lose the integrity of their plasma membrane and release their intracellular contents, into the extracellular space. The same process can occur when apoptotic cells aren't cleared fast enough and their membrane becomes permeable to macromolecules, which presents secondary necrosis (Majno et al., 1995). There is evidence that the immune system has evolved the capacity to detect the release of intracellular molecules which stimulates the generation of adaptive immune responses to dying cells.

Intracellular content of dying cells that triggers immune response when excreted contains molecules named danger associated molecular patterns (DAMPs). DAMPs include for example HMGB-1, IL-1α, uric acid, DNA fragments, mitochondrial content, and ATP (Eigenbrod et al., 2008; Kono et al., 2010a; Sauter et al., 2000). DAMPs can be molecules that have non-inflammatory functions in living cells (such as HMGB-1, ATP) and acquire immunomodulatory properties when released (Rock and Kono, 2008), or alarmins, molecules that have cytokine-like properties (such as IL-1α, IL-6), which are stored in cells and released after cell lysis and contribute to the inflammatory response (Oppenheim and Yang, 2005; Vanden Berghe et al., 2006).

One of the most investigated DAMPs is HMGB-1 (Lotze et al., 2005). HMGB-1 is a nuclear protein that binds to chromatin and has a role in bending DNA and regulating gene transcription (Landsman et al., 1993). HMGB-1 is released by both necrotic and apoptotic cells (Scaffidi et al., 2002; Bell et al., 2006), but also apoptotic cells activate macrophages that engulf them to secrete HMGB-1 (Qin et al., 2006). This protein induces inflammation, dendritic cells maturation, migration, and T-cell activation (Scaffidi et al., 2002; Messmer et al., 2004; Rovere –Querini et al., 2004; Dumitriu et al., 2005; Yang et al., 2007).

HMGB-1 is a stimulus for tumour necrosis factor (TNF) synthesis and release, but it also significantly activates the synthesis of IL-1 α, IL-1 β, IL-1RA, IL-6, IL-8, MIP-1 a, and MIP-1 (Andersson et al., 2000). It was shown that HMGB-1 released from late apoptotic cells remains bound to nucleosomes and that HMGB1-nucleosome complexes activate antigen-presenting cells (APC) and induce secretion of cytokines by macrophages and expression of co-stimulatory molecules in DCs (Urbonaviciute et al., 2008).

HMGB-1 is not the only pro-inflammatory DAMP released from dying cells. Other DAMPs, S100A8/A9 and S100A12 proteins induce pro-inflammatory cytokine production by macrophages (Hofmann et al., 1999; Yang et al., 2001; Viemann et al., 2004; Ehlerman et al., 2006; Pouliot et al., 2008).

The adjuvant activity of cells was reduced by enzymatic depletion of uric acid, indicating that it is a major DAMP, at least in some cells (Shi et al., 2003). Uric acid is a mediator released from necrotic or apoptotic cells that has immunostimulatory properties in vivo (Gordon et al., 1985; Shi et al., 2003). 

Insufficient autophagy of deteriorated mitochondria could lead to massive release of DAMPs such as mtDNA and possibly other mitochondrial proteins (Oka et al., 2012).

Receptors on host cells sense when DAMPs are released and that triggers the inflammatory process. These receptors are pattern-recognition receptors (PRRs) (Chen and Nunez, 2010). PRRs represent proteins by which cells recognize microbial entities, but also some of the host's own molecules and direct an immune response (Piccinini et al., 2010). PRRs can be broadly divided in five subfamilies: Toll-like receptors (TLRs), RIG-1-like receptors (RLRs), NOD like receptors (NLRs), AIM2-like receptors (ALRs) and C-type lectin receptors (CLRs) (Takeuchi and Akira, 2010; Wang et al., 2014). For example, HMGB-1 was reported to stimulate TLR2 and TLR4 (Park et al. 2004) and receptor for advanced glycation end products (RAGE) (Dumitriu et al., 2005), while NLRP3 has been involved in the inflammatory response to mono-sodium urate (MSU) (Martinon et al., 2006). Cellular nucleic acids can stimulate TLR7 and TLR9 on B cells to promote antibody responses (Green and Marshak-Rothstein, 2011; Leadbetter et al., 2002).

TLRs are placed either at the cell surface (TLR1, TLR2, TLR4, TLR5, and TLR6) or in the endolysosomal compartment (TLR3, TLR7, and TLR9) (Barton and Kagan, 2009). Upon binding with the ligand, they undergo a conformational change and initiate a signalling cascade via signal adaptor molecules: myeloid differentiation primary response gene 88 (MyD88), MyD88 adaptor-like protein (MAL, also known as TIR-domain-containing adaptor protein; TIRAP), TIR domain-containing adaptor protein inducing interferon-β (TRIF), and TRIF-related adaptor molecule (TRAM). MyD88 was essential for the inflammatory response to injected dead cells (Chen et al., 2007).

All TLRs, except TLR3, associate with MyD88, and this stimulates a kinase cascade resulting in the activation of mitogen activated protein kinases (MAPKs), c-Jun N-terminal kinases, p38, and extracellular signal–regulated kinases, and nuclear factor NF-kB (Akira and Takeda, 2004; Lee and Kim, 2007). NF-kB is an important transcription factor for IL -1β and NLRP3 (Wang et al., 2004; Bauernfeind et al., 2009).

NF-kB is a central mediator of pro-inflammatory gene induction and functions in both types of immune cells. NF-kB pathway is responsible for transcriptional induction of pro-inflammatory cytokines, chemokines and additional inflammatory mediators, such as NLRP3, pro-IL-1β and pro-IL-18 (Sun et al., 2013; Ghosh and Karin, 2002; Hayden and Ghosh, 2013).

Macrophages must first be ‘primed’ with a stimulus that induces the synthesis of pro-IL -1β and also upregulates the expression of NLRP3 (Bauernfeid et al., 2009; Franchi et al., 2009). The stimuli that can prime macrophages include TLR agonists and cytokines like TNF. When macrophages producing pro-IL -1β are stimulated with ATP or irritant particles, inactive pro-caspase 1 assembles into a molecular complex called the inflammasome and is cleaved into active form (Stutz et al., 2009; Schroder and Tschopp, 2010). Inflammasomes consist of caspase 1, apoptosis-associated speck-like protein containing CARD (ASC) and an NLRP (Schroder and Tschopp, 2010). The catalytically active caspase 1 then cleaves pro-IL-1β to its mature and active form (Stutz et al., 2009).  Macrophages lacking any of the inflammasome components don't make mature IL-1 when stimulated in culture with sterile particles (Hornung et al., 2008; Halle et al., 2008). NF-κB signaling pathway is also involved in the regulation of inflammasome (Guo et al., 2015).

Sometimes substantial sterile inflammatory response can be seen in caspase 1-deficient mice (eg. Chen et al., 2007). This contrasts with the much more marked reduction of these responses that is consistently observed in IL-1β -deficient mice. These data imply that there must be a caspase 1-independent pathway for generating mature IL-1β in vivo (Dinarello, 2009).

In the sterile inflammatory response to cell death, the contribution of TNF appears to be more modest than IL-1 (Rock et al., 2011). A possible explanation might be that the IL-1 is being released from the dying cells themselves (Eigenbrod et al., 2008).

After engulfment of apoptotic bodies, Kupffer cells in liver express TNF, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL) (Canbay et al. 2003), which can induce apoptosis in hepatocytes and further aggravate liver inflammation. Engulfment of apoptotic bodies by macrophages also induces FasL expression (Kiener et al., 1997), which is known to exert a pro-inflammatory activity (Chen et al., 1998).

Evidence Supporting this KER

Biological Plausibility

The severity of cell death activation determines the outcome for the cell: inflammation is part of the tissue regeneration process, and intermediate apoptotic stimuli are able to trigger this response. Recruitment of inflammatory cells such as neutrophils is meant as a beneficial process, as for example apoptotic bodies of bacteria-infected cells can be removed. Thus the apoptotic cells can secrete soluble "find-me" factors that trigger infiltration of immune cells. However, if this becomes chronic it has the potential to enhance tissue damage and ultimately induce fibrosis (Jaeschke, 2002; Cullen et al., 2013).

Empirical Evidence

During the apoptosis of Jurkat cells treated with various agents, HMGB-1 was released into the media as assessed by Western blotting. This release was blocked by an inhibitor of apoptosis (Bell et al., 2006).

Some studies reported that purified HMGB-1 activate leukocytes and stimulate the production of pro-inflammatory mediators in vitro (Li et al., 2004; Zimmermann et al., 2004).

Injecting a neutralizing HMGB-1 antibody into animals treated with a hepatotoxic drug, reduced inflammation in the damaged liver (Scaffidi et al., 2002). Other studies showed that anti-HMGB-1 antibodies could also reduce inflammation in livers that had suffered from ischemia-reperfusion injury (Tsung et al., 2005).

In uric acid-depleted mice, the inflammatory responses to cell death were significantly reduced (Kono et al., 2010a).

ATP can stimulate the production of pro-inflammatory cytokines from macrophages (Ferrari et al., 1997; Ferrari et al., 2006). Depletion of ATP or elimination of its receptor inhibited inflammation in vivo in reaction to thermal injury of the liver (McDonald et al., 2010).

Injection of an agonistic anti-FAS antibody into mice causes hepatocytes to undergo apoptosis which stimulates inflammation. If apoptosis is blocked, then this inflammatory response is inhibited (Faouzi et al., 2001).

Chen and colleagues injected dead cells into mice that genetically lacked various TLR. Inflammation was reduced in mice that were doubly-deficient in TLR2 and TLR4, confirming that these receptors play a role in the sterile inflammatory response. However reduction in the response was mild, indicating that there must be other receptors involved in this process (Chen et al., 2007).

IL-1 receptor antagonist (IL-1RA) administration resulted in a significant decrease in IL-6, and a borderline decrease in TNF production. This antagonist does not reduce the number of apoptotic bodies present, indicating that its effect on IL-6 and TNF- levels were due to neutralization of IL-1 (Clarke et al., 2010). Knock out of IL-1 receptor antagonist results in lethal artery inflammation (Nicklin et al., 2000).

The IL-1-dependent inflammatory response to cell death in vivo is significantly reduced in NLRP3-deficient mice (Imaeda et al., 2009; Iyer et al., 2009).

Death-induced neutrophilic inflammation was markedly decreased in mice lacking MyD88. However, this effect was primarily due to a key role for the IL-1 receptor in the recruitment of neutrophils (Chen et al., 2007).

Neutralization of IL-1α or genetic deficiency of IL-1 inhibited inflammation responses to injected dead cells (Kono et al., 2010b; Chen et al., 2007).

Injection into mice of a variety of other dead cell types that genetically lack both IL-1α and IL- 1β stimulated an inflammatory response that was equivalent to that of wildtype necrotic cells (Kono et al., 2010b). This implicates that IL-1 that is driving the sterile inflammatory response in many cases is not coming directly from the dead cell but is produced by cells in the host upon recognition of cell death. That this is the case was formally shown by the loss of inflammatory response to dead cells in mice that genetically lack either IL-1α or IL-1β (Kono et al., 2010b).

Uncertainties and Inconsistencies

The inflammatory role of HMGB-1 is still not completely clear. There are many studies that confirm its pro-inflammatory activity. However, in some experiments highly purified HMGB-1 had little pro-inflammatory activity (Rouhiainen et al., 2007), while in another injection of recombinant HMGB-1 into infarcted heart muscle in vivo stimulated regeneration and repair (Limana et al., 2005).

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Relationship: 1777: Increased pro-inflammatory mediators leads to Leukocyte recruitment/activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Human (Bleul et al., 1996; Miyamoto et al., 2000; Yamada et al., 2001; Sun et al., 2015)

Sheep (Nikiforou et al., 2016)

Mouse (Narumi et al., 1992; Fahy et al., 2001; Lee et al., 2009)

Key Event Relationship Description

Circulating blood leukocytes are required to migrate to sites of injury and infection with the aim to eliminate the primary inflammatory trigger and contribute to tissue repair. In this process are involved selectins (expressed both on leukocytes and endothelium) and integrins (expressed on leukocytes) (von Andrian et al., 1991), with the essential role of the vascular endothelium.

Fast activation of the endothelium with inflammatory stimuli such as histamine and PAF (type I) or slow activation with tumor necrosis factor (TNF) or cytokine interleukin-1 β (IL-1β) (type II), makes the surface of endothelium adhesive (Bevilacqua and Gimbrone, 1987; Pober and Sessa, 2007). This transformation is mediated by a transcriptionally regulated program involving the nuclear factor NF-kB dependent pathway triggered by pro-inflammatory cytokines or bacterial endotoxins (reviewed by Collins et al., 1995).

Integrins mediate attachment between cells or to basement membrane. The β2 integrin family is exclusively expressed on leukocytes and is essential for leukocyte arrest on the endothelium and for migration across the endothelium (Ley et al., 2007). In unstimulated leukocytes integrins are usually in a conformation with low binding affinity, until they receive signals from other receptors, such as chemokine receptors (G-protein-coupled receptors), when they change their conformation and display high affinity for ligands (Luo et al., 2007). Chemokines activate β1 or β2 integrins on monocytes, neutrophils, and lymphocytes and as such serve as chemoattractant for these cells during inflammation (Huber et al., 1991; Tanaka et al., 1993; Gunn et al., 1998).

The chemokines are a family of structurally related cytokines that can act as pro-inflammatory agents (Baggiolini et al., 1994; Vaddi et al., 1997). They have the ability to attract leukocyte subsets to specific sites. They recruit neutrophils, monocytes, natural killer cells (NK) and natural killer T (NKT) cells, all of which express inflammatory chemokine receptors and immature dendritic cells (DCs) that provide the link between innate and adaptive immunity (Oo et al., 2010). After antigen-specific activation of lymphocytes by activated DCs, inflammatory chemokines then attract antigen-specific effector T cells to the inflammatory site (Heydtmann and Adams, 2002).

During diapedesis, leukocytes migrate across the endothelium and basement membrane to enter tissue (Ley et al., 2007; Yadav et al., 2003). Once in tissue, the leukocyte follows chemokine gradients to sites of inflammation, using chemokine-mediated changes in the actin cytoskeleton to propel migration. For example, it was demonstrated that chemokines CXCL9, CXCL10 and CXCL11 are important not only in adhesion, but also in transmigration of effectors T lymphocytes through hepatic endothelium (Curbishley et al., 2005; Eksteen et al., 2004). Intracellular actin reorganization is a prerequisite for cell movement, and it has been shown that chemokines such as SDF-1 induce and increase intracellular filamentous actin in lymphocytes (Bleul et al., 1996).

There is essential role of interleukins, but also other factors such as tumor necrosis factor (TNF), interferon (IFN) in leukocyte recruitment and production of chemokines.

Normally, IL-1β binds to IL-1R1 receptor on the surface of target cells. Following ligand binding the adaptor molecule, myeloid differentiation factor-88 (MyD88), interacts with IL-1R1 via its toll interleukin receptor (TIR) domain (O'Neill, 2008). Signal transduction leads to activation of both mitogen-activated protein kinases (MAPKs) and the transcription factor NF-kB, and resulting in pro-inflammatory cytokine expression. For example, chemokine RANTES production requires the transcription factor NF-kB and the activation of mitogen-activated protein kinases (MAPKs) (Genin et al., 2000; Miyamoto et al., 2000; Kujime et al., 2000, Maruoka et al., 2000; Yang et al., 2000).

TNF-α cleavage produces an intracellular domain that translocates to the nucleus and induces pro-inflammatory cytokine signalling, particularly the expression of IL-12 (Friedman et al., 2006). IL-18 induces natural killer and natural killer T cells to produce IFN-γ (Okamura et al., 1998), but it requires IL-12 to induce IFN-γ production by Th1 cells (Nakanishi et al., 2001). There is an essential role of IFN-I in promoting the chronic recruitment of Ly6Chi monocytes. IFN-I production is elicited via a toll like receptor-7 (TLR-7) and MyD88-dependent pathway (Lee et al., 2008).

While CXC-chemokines, e.g. IL-8, act mostly on neutrophils (Springer, 1995), members of the CC-chemokines, e.g. RANTES and macrophage inflammatory protein have been shown to exert function on monocytes, eosinophils and lymphocytes (Baggiolini et al., 1994; Carr et al., 1994). This depends on the receptors that are expressed on leukocytes. Th1 express preferentially CCR5 and CXCR3, while Th2 cells have CCR3, CCR4 and CCR8 on their surface (Syrbe et al., 1999). Monocytes and macrophages express CCR5 and other receptors for RANTES (Weber et al., 2000).

RANTES chemokine is produced by many cells in the extravascular compartment, including fibroblasts, epithelial cells, and tissue-infiltrating lymphocytes and monocytes (MacEwan, 2002; Hehlgans and Männel, 2002; Black et al., 1997). It acts as a potent chemoattractant for monocytes, memory T cells, eosinophils, and basophils (Schall et al., 1988, 1990; Baggiolini and Dahinden, 1994). Elevated levels of RANTES transcripts are detected within hours of exposure to pro-inflammatory stimuli, including IL-1β, TNF-α, IFN-γ, viruses and LPS (Barnes et al., 1996).

Evidence Supporting this KER

Biological Plausibility

There is much evidence that application of chemokines attract leukocytes to specific site in different species (Beck et al., 1997; Lee et al., 2000; Fahy et al., 2001; Nikiforou et al., 2016).

Empirical Evidence

It was shown that a number of chemokines destabilize the rolling of lymphocytes on L-selectin ligands, suggesting that chemokines are capable of regulating the rolling process (Grabovsky et al., 2002).

Jorgensen and colleagues showed that exposure of mice to FliC^ind strain S. Typhimurium triggered a significant neutrophil influx in the spleen of wild-type mice, but not Il1b⁻/⁻Il18⁻/⁻ mice (Jorgensen et al., 2016).

The expression of chemokines CCL2, CCL7, and CCL12 was reduced dramatically in MyD88-/- mice (Lee et al., 2009).

Miyamoto and colleagues showed that exposure of cells to IL-1β, TNF-α, and IFN-γ resulted in the induction of RANTES mRNA and protein (Ortiz et al., 1996; Miyamoto et al., 2000). The levels of RANTES production by the fibroblasts in the presence of IL-1β or TNF-α were significantly elevated compared with those in the absence of these factors (Yamada et al., 2001).

In the mouse, IFN-γ administration induces high levels of IP-10 expression in liver and kidneys, with lower levels in spleen (Narumi et al., 1992).

CAPE inhibitor of NF-kB blocked partially IL-1β induced expression of chemokines MIP-1a and MIP-1b (Guo et al., 2003).

CCR2 and CCR5 receptors on the CD8 T cells are enriched in the inflamed human liver, and CCR1 is important in the regulation of hepatic inflammation in murine models (Shields et al., 1999; Boisvert et al., 2003).  

Intradermal injection of RANTES induces a potent T-lymphocyte and eosinophils recruitment (Fahy et al., 2001; Beck et al., 1997).  Intradermal administration of MIP-1a resulted in accumulation of monocytes, lymphocytes, eosinophils and recruitment of neutrophils (Lee et al., 2000).

Direct IL-1α exposure to the gut resulted in increased numbers of CD3+ cells in the fetal sheep ileum when compared with control animals on the first day after the exposure. The number of white blood cells, monocytes, and neutrophils was increased in cord blood after 6 days of IL-1α exposure to the lung and chorioamnion/skin. The number of lymphocytes on the day 6 was increased for the lung. Compared with controls, gut mRNA levels of TNF-α and IL-1 was significantly increased at 6 days after IL-1α exposure to the GI tract (Nikiforou et al., 2016).

IL-1 β induced up-regulation of CXCR4 in certain cancer cells, but in order to do so necessary is that these cells have IL-1R1. Presence of IL-1R antagonist significantly inhibited the up-regulation of CXCR4 induced by IL-1β at both mRNA and protein level (Sun et al., 2015).

SDF-1 is an efficacious chemoattractant and showed a similar dose response for murine lymphocytes and human monocytes, but was not active on human or murine neutrophils. SDF-1 is a highly effective transendothelial chemoattractant (Bleul et al., 1996).

Uncertainties and Inconsistencies

Lloyd and colleagues found that several chemokines can stimulate the adherence of peripheral blood lymphocytes to ICAM-1 coated slides (Loyd et al., 1996). However, by using a parallel plate flow chamber, other study failed to observe such an effect (Carr et al., 1996).

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Relationship: 1778: Leukocyte recruitment/activation leads to Activation, Stellate cells

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Human (Zimmermann et al., 2010; Liaskou et al., 2013)

Mouse (Seki et al., 2007; Gäbele et al., 2009; Pradere et al., 2013; McHedlidze et al., 2014)

Rat (Reeves et al., 2000;  Duffield et al., 2005; Imamura et al., 2005)

Key Event Relationship Description

During hepatic injury quiescent hepatic stellate cells (HSCs) undergo activation which is associated with proliferation, increased contractile activity, fibrogenesis, changes in matrix protease activity, loss of intracellular retinoid storage, production of cytokines, and phenotypic transformation (Friedmann, 2000).

Different inflammatory cells activate HSCs to secrete collagen (Casini et al., 1997). Factors that promote activation of HSC include the cytokines, TNF-α and TGF-β (Bachem et al., 1993), endothelin-l (ET-1) (Rockey and Chung, 1996) and oxidative stress (Lee et al., 1995).

TGF-β is considered the most powerful mediator of HSC activation in vitro and in vivo (Friedmann, 2000; Bataller and Brenner, 2005). TGF-β triggers phenotypical HSC activation by paracrine and autocrine action, and induces collagen I expression and α-smooth muscle actin (α-SMA) stress fiber organization (Gressner et al., 2002; Dooley et al., 2003). TGF-β binds to heteromeric transmembrane receptors, including TβRI and TβRII (Heldin et al., 1997). Binding to TβRII triggers heteromerization with and transphosphorylation of TβRI. The signal is then propagated through phosphorylation of receptor associated Smad2 and Smad3 and their oligomerization with the common mediator Smad4. Complexes of phosphorylated Smad2 or 3 and Smad4 translocate into the nucleus, where they modulate the transcription of target genes, including those encoding extracellular matrix components (ECM) components (Piek et al., 1999; Miyazono et al., 2000; Moustakas et al., 2001). IFN-γ suppresses TGF-β and PDGF-dependent signalling pathways (Fujita et al., 2006).

IL-33 is released by stressed hepatocytes and attracts type 2 innate lymphoid cells (ILC2), which trigger the profibrogenic activation of HSCs via mediators such as IL-13 (McHedlidze et al., 2013; Heymann and Tacke, 2016). The binding of IL-33 to ST2 receptor activates NF-kB and mitogen activated protein kinases (MAPKs) and drives the production of pro-inflammatory and Th2 associated cytokines (Schmitz et al., 2005), which resulted in the stimulation of α-SMA and collagen expression in HSCs (Tan et al., 2018).

IL-13 indirectly activates TGF-β by upregulating the expression of matrix metalloproteinases (MMPs) that cleave the LAP–TGF-β1 complex (Lee et al., 2001; Lanone et al., 2002). Also, IL-13 has been reported to directly induce production of TGF-β1 in HSCs during liver fibrosis (Shimamura et al., 2008). When treating HSCs with IL-13 and performing a time-course analysis, a time-dependent activation of Smad proteins was observed (Liu et al., 2011).

TNF-α stimulated both p38 MAPK and JNK activity in a time-dependent manner, same as ET-1. However, TGF-β had no significant stimulatory effect on either of these MAPKs. The addition of p38 MAPK inhibitor pyridinyl imidazole derivative SB202190 resulted in reduction of α-SMA, indicator of activated HSC (Reeves et al., 2000)

Oxidative stress enhanced activation of HSCs and collagen synthesis in them, whereas antioxidants stopped the stimulatory effect of free radicals (Lee et al., 1995; Svegliati-Baroni et al., 2001). Oxidative stress molecules, such as superoxide, hydrogen peroxide, hydroxyl radicals, may be derived from hepatocytes, activated KCs, other inflammatory cells and HSCs (Natarajan et al., 2006; Kisseleva and Brenner, 2007; Lee and Friedman, 2011).

Liver-infiltrating CD14+ CD16+ monocytes secrete high levels of chemokines (such as CCL1, CCL2, CCL3, CCL5), cytokines (IL-1α, IL-1β, IL-6, IL-13, IL-16, TNF-α and macrophage migration inhibitory factor), growth factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor) and can efficiently activate primary HSC in vitro (Liaskou et al., 2013; Zimmermann et al., 2010).

Neutrophils may activate HSCs through matrix degradation, secreting compounds like elastase, which then degrades laminin, an extracellular matrix protein in normal liver that is critical for keeping stellate cells in a quiescent state (Friedman et al., 1989). Neutrophil-derived reactive oxygen species (ROS) significantly stimulated procollagen type I accumulation in the HSC culture medium, while the addition of vitamin E or SOD impaired the ROS stimulated stimulation of procollagen I (Casini et al., 1997).

Macrophages are primary source of TGF-β1 in the fibrotic liver (Bataller and Brenner, 2009). Macrophages release several pro-inflammatory cytokines like TNF-α or IL-1 (Tacke and Zimmermann, 2014), which activate the transcription factor NF-kB in HSC and promote the survival of activated HSC (Pradere et al., 2013).

Kupffer cells, liver resident macrophages, after their activation, activate HSC via mechanisms that involve the potent profibrotic cytokines like TGF-β and platelet derived growth factor (PDGF), and ROS (Karlmark et al., 2008; Bataller and Brenner, 2009; Bataller and Lemon, 2012). Apart from directly stimulating matrix-secreting HSC, hepatic macrophages may aggravate scarring by promoting HSC survival via IL-1 and TNF-α induced NF-kB activation (Pradere et al., 2013).  Beside resident macrophages, infiltration of macrophages from blood is essential for liver fibrogenesis (Duffield et al., 2005; Imamura et al., 2005).

Th2 cell– derived cytokines, IL-4, IL-5, IL-13, can enhance fibrosis progression by stimulating TGF-β production in macrophages and by direct effects on HSCs (Wynn, 2004).

DCs have a minor contribution to NF-kB activation (Pradere et al., 2013).

NKT cells were also found to promote liver fibrogenesis in vivo, likely by releasing pro-inflammatory cytokines and activating HSCs (Wehr et al., 2013; Syn et al., 2012). However, there are also studies demonstrating that NKT cells may exert antifibrotic actions, because they can, under certain conditions, also kill HSC and produce IFN-γ, like NK cells (Gao et al., 2013; Park et al., 2009).

Signalling pathways for HSC activation include, for example, NF-kB that is involved in HSC activation upon lipopolysaccharide (LPS) or TLR4 stimulation or ATP induced cytosolic Ca²⁺ influx via purinergic signalling receptors (Dranoff et al., 2004). The activation of TLR4 receptor in HSC downregulates TGF-β pseudoreceptor BAMBI and sensitizes these cells for TGF-β, resulting in increased ECM production by HSCs and fibrosis (Seki et al., 2007).

Activated HSCs secrete inflammatory chemokines, express cell adhesion molecules, and modulate the activation of lymphocytes (Vinas et al., 2003). Therefore, a vicious circle in which inflammatory and fibrogenic cells stimulate each other is likely to occur (Maher, 2001).

Evidence Supporting this KER

Biological Plausibility

The recruitment of immune cells from the circulation into the injured tissue is the key mechanism during fibrogenesis in the liver (Heymann and Tacke, 2016).

Empirical Evidence

Upon stimulation, Th1 cells produce interferon-gamma, which is antifibrogenic in liver. C57BL/6 mice contain primarily Th1 cells. Thus, the weak response to CCl₄ in these mice is consistent with an interferon effect. In contrast, BALB/c mice have primarily Th2 lymphocytes. Th2 cells produce IL-4, which induces TGF-β. Both of these cytokines are profibrogenic in liver, which explains why BALB/c mice display enhanced fibrosis in response to CCl₄ in comparison with C57BL/6 mice (Maher, 1999).

In vivo data from Karlmark et al., 2009 suggested that intrahepatic CD11bF4/80 monocyte-derived cells, same as liver resident macrophages, produce TGF-β1 and thereby directly activate HSCs. This was confirmed in an in vitro experiment, in which only intrahepatic recruited monocytes could readily activate HSCs in a TGF-β–dependent manner.

There is strong evidence that the blockade of TGF-β alone is sufficient to completely block experimental fibrogenesis in liver (reviwed in Gressner et al., 2002).

Overexpression of Smad7, a natural antagonist of TGF-β signalling, prevents activation of HSCs and liver fibrosis in rats (Dooley et al., 2003).  

The activation of HSC by CD14 ⁺ CD16⁺ monocytes could be partially blocked by anti- TGF-β antibodies (Zimmermann et al., 2010).

In rats receiving CCl₄, HSC activation correlates temporally and spatially with superoxide production (Montosi et al., 1998).

Macrophage depletion by administration of diphtheria toxin intraperitoneally or intravenous led to a significant reduction in the number of HSCs (Duffield et al., 2005).

After bile duct ligation, Kupffer cell–depleted mice showed almost complete suppression of HSC activation and fibrosis (Seki et al., 2007).

Liver injury caused by ischemia and reperfusion, along with TNF-α, CXCL1, and endothelin-A receptor expression, was significantly decreased in HSC-depleted mice compared with controls, with decreased neutrophil infiltration and parenchymal cell death. This suggests that HSCs are involved in hepatic production of CXCL1 and contribute to neutrophil recruitment (Stewart et al., 2014).

Important evidence that HSCs produce chemoattractant is that MCP-1 mRNA was clearly co-distributed with cells expressing α-SMA, a marker of activated HSCs (Marra et al., 1998).

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Relationship: 295: Activation, Stellate cells leads to Accumulation, Collagen

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Human: Safadi and Friedman, 2002; Bataller and Brenner, 2005; Lee und Friedman 2011.

Rat: Li, Li et al., 2012; Luckey and Petersen, 2001;  Rockey et al., 1992

 

Key Event Relationship Description

Up-regulation of collagen synthesis following hepatic stellate cell (HSC) activation is among the most striking molecular responses of HSCs to injury and is mediated by both transcriptional and post-transcriptional mechanisms. Activated HSCs do not only proliferate and increase cell number, but also increase collagen production per cell. Synthesis of type I collagen is initiated by expression of the col1a1 and col1a2 genes, giving rise to α 1(I) and α 2(I) procollagen mRNAs in a 2:1 ratio. Upon activation of HSCs and other myofibroblast precursors, there is a > 50-fold increase in α 1(I) procollagen mRNA levels. The half-life of collagen α1(I) mRNA increases 20-fold in activated HSCs compared with quiescent HSCs. Monocytes and macrophages are involved in inflammatory actions by producing large amounts of Nitric oxide (NO) and inflammatory cytokines such as TNF-α which have a direct stimulatory effect on HSC collagen synthesis. Synthesis of TGF-α and TGF-β promotes activation of neighbouring quiescent HSCs, whereas the release of HGF (hepatocyte growth factor) stimulates regeneration of adjacent hepatocytes.

The basement membrane-like matrix is normally comprised of collagens IV and VI, which is progressively replaced by collagens I and III and cellular fibronectin during fibrogenesis. Although multiple extracellular matrix (ECM) components are up-regulated, type I collagen is the most abundant protein. These changes in ECM composition initiate several positive feedback pathways that further amplify collagen production. Increasing matrix stiffness is a stimulus for HSC activation and matrix-provoked signals link to other growth factor receptors through integrin-linked kinase and transduce via membrane-bound guanosine triphosphate binding proteins, in particular Rho67 and Rac, signals to the actin cytoskeleton that promote migration and contraction.

The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Down-regulated expression of degrading Matrix metalloproteinases (MMPs) and up-regulation of tissue inhibitors of metalloproteinases (TIMPs), MMP- inhibitors, lead to a net decrease in protease activity, and therefore, matrix accumulation. Chronic inflammation, hypoxia and oxidative stress reactivate epithelial-mesenchymal transition (EMT) developmental programmes that converge in the activation of NF-kB. Cells that may transdifferentiate into fibrogenic myofibroblasts are hepatocytes and cholangiocytes. Additional sources of ECM include bone marrow (which probably gives rise to circulating fibrocytes) and portal fibroblasts (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008;  Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009; Friedman, 2010; 2008; Dalton et al., 2009; Leung, et al., 2008; Nan et al., 2013;  Hamdy and El-Demerdash, 2012;Li, Li et al., 2012; Natajaran et al., 2006; Luckey and Petersen, 2001;  Chen and Raghunath, 2009;Thompson et al., 2011; Henderson and Iredale, 2007).

 

 

Evidence Supporting this KER

Biological Plausibility

There is general acceptance that HSCs are collagen producing cells and key actors in fibrogenesis. The functional relationship between these KEs is consistent with biological knowledge (Benyon and Arthur; 2001; Milani et al., 1994; Safadi and Friedman, 2002; Kolios et al.,2006; Bataller and Brenner, 2005; Lee und Friedman 2011; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008;  Kershenobich Stalnikowitz and Weisssbrod , 2003; López-Novoa and Nieto, 2009).

Empirical Evidence

 

It is difficult to stimulate sufficient collagen production and its subsequent incorporation into a pericellular matrix in vitro; therefore analytical methods have focused on measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver begin to express α-SMA coincident with culture-induced activation ( Chen and Raghunath, 2009; Rockey et al.,1992).

Uncertainties and Inconsistencies

no inconsistencies

References

• Benyon, R.C. and M.J. Arthur (2001), Extracellular matrix degradation and the role of stellate cells, Semin Liver Dis, vol. 21, no. 3, pp. 373-384.
• Milani, S. et al. (1994), Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver, Am J Pathol, vol. 144, no. 3, pp. 528-537.
• ↑Safadi, R. and S.L. Friedman (2002), Hepatic fibrosis--role of hepatic stellate cell activation, MedGenMed, vol 4, no. 3, p. 27.
• Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.
• Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.
• Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.
• Guo, J. and S. L. Friedman (2007), Hepatic fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.
• Brenner, D.A. (2009), Molecular Pathogenesis of Liver Fibrosis, Trans Am Clin Climatol Assoc, vol. 120, pp. 361–368.
• Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.
• Kershenobich Stalnikowitz, D. and A.B. Weisssbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.
• López-Novoa, J.M. and M.A. Nieto (2009), Inflammation and EMT: an alliance towards organ fibrosis and cancer progression, EMBO Mol Med, vol. 1. no. 6-7, pp. 303–314.
• Friedman, S.L (2010), Evolving challenges in hepatic fibrosis, Nat. Rev. Gastroenterol. Hepatol, vol. 7, no. 8, pp. 425–436.
• Friedman, S.L. (2008), Mechanisms of Hepatic Fibrogenesis, Gastroenterology, vol. 134, no. 6, pp. 1655–1669.
• Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.
• Leung, T.M. et al. (2008), Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis, Int J Exp Pathol, vol. 89, no. 4, pp. 241-250.
• Nan, Y.M. et al. (2013), Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice, Lipids in Health and Disease, vol. 12, p. 11.
• Hamdy, N. and E. El-Demerdash. (2012), New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage, Toxicol Appl Pharmacol, vol. 261, no. 3, pp. 292-299.
• Li, Li et al. (2012), Establishment of a standardized liver fibrosis model with different pathological stages in rats, Gastroenterol Res Pract; vol. 2012, Article ID 560345.
• Natajaran, S.K. et al. (2006), Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J Gastroenterol Hepatol, vol. 21, no. 6, pp. 947-957.
• Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.
• Chen, C. and M. Raghunath (2009), Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art, Fibrogenesis Tissue Repair, vol. 15, no. 2, p. 7.
• Thompson, K.J., I.H. McKillop and L.W. Schrum (2011), Targeting collagen expression in alcoholic liver disease, World J Gastroenterol, vol. 17, no. 20, pp. 2473-2481.
• Henderson, N.C. and J.P. Iredale (2007), Liver fibrosis: cellular mechanisms of progression and resolution, Clin Sci (Lond), vol. 112, no. 5, pp. 265-280.
• Rockey, D.C. et al. (1992), Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture, J Submicrosc Cytol Pathol, vol. 24, no. 2, pp. 193-203.

Relationship: 82: Accumulation, Collagen leads to N/A, Liver fibrosis

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Human: Lee and Friedman, 2011; Bataller and Brenner, 2005; Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997.  

Rat :Liedtke et al., 2013.

Key Event Relationship Description

Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) proteins including collagen. Liver fibrosis results from an imbalance between the deposition and degradation of ECM and a change of ECM composition; the latter initiates several positive feedback pathways that further amplify fibrosis. With chronic injury, there is progressive substitution of the liver parenchyma by scar tissue. Deposition of collagen in the liver progressively disrupts the normal hepatic architecture so that the normal relationship between vascular inflow and outflow is destroyed and the normal collagen content around hepatic sinusoids in regenerating nodules becomes modified.Advanced liver fibrosis results in cirrhosis (Lee and Friedman, 2011; Bataller and Brenner, 2005; Pellicoro et al., 2014;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). 

 

Evidence Supporting this KER

Biological Plausibility

By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between this KE and the AO is undisputed (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). 

 

Empirical Evidence

 

There is a smooth transition from ECM accumulation to liver fibrosis without a definite threshold and plenty in vivo evidence exists that ECM accumulation is a pre-stage of liver fibrosis (Lee and Friedman, 2011; Bataller and Brenner, 2005;Brancatelli et al., 2009; Rockey and Friedman, 2006; Poynard et al., 1997). 

 

Uncertainties and Inconsistencies

no inconsistencies

References

• Lee, U.E. and S.L. Friedman (2011), Mechanisms of Hepatic Fibrogenesis, Best Pract Res Clin Gastroenterol, vol. 25, no. 2, pp. 195-206.
• Bataller, R. and D.A. Brenner (2005), Liver Fibrosis, J.Clin. Invest, vol. 115, no. 2, pp. 209-218.
• Pellicoro, A. et al. (2014), Liver fibrosis and repair: immune regulation of wound healing in a solid organ, Nat Rev Immunol, vol. 14, no. 3, pp. 181-194.
• Brancatelli, G. et al. (2009), Focal confluent fibrosis in cirrhotic liver: natural history studied with serial CT, AJR Am J Roentgenol, vol. 192, no. 5, pp. 1341-1347.
• Rockey, D.C. and S.L. Friedman (2006), Hepatic fibrosis and cirrhosis, Zakim and Boyer's Hepatology, 5th edition, section 1, chapter 6, pp. 87-109.
• Poynard, T., P. Bedossa and P. Opolon (1997), Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups, Lancet, vol. 349, no. 9055, pp. 825-832.
• Liedtke, C. et al. (2013), Experimental liver fibrosis research: update on animal models, legal issues and translational aspects, Fibrogenesis Tissue Repair, vol. 6, no. 1, p. 19.