Cecilia L. Basiglio

Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

Complement activation and disease: protective effects of hyperbilirubinaemia.

Complement, an important effector mechanism of the immune system, is an enzymatic cascade of approx. 30 serum proteins leading to the amplification of a specific humoral response. It can be activated through the classical or alternative pathways, or through the mannose-binding lectin pathway. The activation of the classical pathway is initiated by the binding of the C1 component to antigen-bound antibodies, known as immunocomplexes. C1 is a complex of one molecule of C1q, two molecules of C1r and two molecules of C1s. C1q contains three copies of a Y-shaped fundamental unit with globular heads included in its structure, which play a major role in the interaction with the Fc portion of immunoglobulins. Deficient or exacerbated activation of the complement system leads to diseases of variable severity, and pharmacological inhibition of the complement system is considered as a therapeutic strategy to ameliorate the inflammatory effects of exacerbated complement activation. Bilirubin is a product of haem degradation by the concerted action of haem oxygenase, which converts haem into biliverdin, and biliverdin reductase, which reduces biliverdin to UCB (unconjugated bilirubin). UCB exerts both cytoprotective and cytotoxic effects in a variety of tissues and cells, acting either as an antioxidant at low concentrations or as an oxidant at high concentrations. In the present review, we describe in detail the anti-complement properties of bilirubin, occurring at levels above the UCB concentrations found in normal human serum, as a beneficial effect of potential clinical relevance. We provide evidence that UCB interferes with the interaction between C1q and immunoglobulins, thus inhibiting the initial step in the activation of complement through the classical pathway. A molecular model is proposed for the interaction between UCB and C1q.

Differential effects of silymarin and its active component silibinin on plasma membrane stability and hepatocellular lysis

Silymarin (SIL) is a natural extract with hepatoprotective properties composed mainly of flavonolignans, with silibinin (SB) being its principal constituent. SB is thought to be the main responsible for SIL hepatoprotective properties, although this has not been corroborated systematically. We analysed comparatively the effects of SIL and SB on hepatocellular plasma membrane stability. SIL (500 microM concentration in SB) protected significantly the plasma membrane disruption induced by Triton X-100 (TX-100) and taurochenodeoxycholate (TCDC), both in isolated plasma membrane (assessed by recording the plasma membrane transition from bilayer to micelle using the R18 self-quenching assay) and in isolated rat hepatocytes (assessed by the release into the incubation medium of the cytosolic enzymes lactate dehydrogenase and alanine aminotransferase). Contrarily, SB (500 microM) exacerbated plasma membrane disruption induced by TX-100 in both systems at detergent concentrations relevant to induce hepatocellular lysis, although it displayed some stabilizing properties at higher concentrations. SB showed a lower stabilizing effect against TCDC-induced plasma membrane disruption when assayed in both models. In addition, SB exposure made the plasma membrane more labile to disruption induced by osmotic lysis. These results show that SIL and SB have differential effects on membrane stability; whereas SIL shows consistently stabilizing effects, SB exacerbates hepatocellular lysis or exerts only minimal stabilizing effects. This differential behaviour should be taken into account when considering the pros and cons of using purified SB vs. the whole SIL extract in medicinal formulations for liver diseases.

G‐protein‐coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß‐d‐glucuronide‐induced cholestasis

Estradiol‐17ß‐d‐glucuronide (E17G) activates different signaling pathways (e.g., Ca2+‐dependent protein kinase C, phosphoinositide 3‐kinase/protein kinase B, mitogen‐activated protein kinases [MAPKs] p38 and extracellular signal‐related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance‐associated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G‐protein‐coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G‐induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G‐induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30‐AC‐PKA. PKA inhibition prevented E17G‐induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30‐AC‐PKA pathway totally prevented E17G‐induced alteration in Abcb11 and Abcc2 function and localization. Conclusion: Activation of GPR30‐AC‐PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation. (Hepatology 2014;59:1016–1029)

Tauroursodeoxycholate counteracts hepatocellular lysis induced by tensioactive bile salts by preventing plasma membrane-micelle transition

Ursodeoxycholic acid is widely used as a therapeutic agent for the treatment of cholestatic liver diseases. In these hepatopathies, the bile secretory failure produces accumulation of endogenous, tensioactive bile salts, leading to plasma membrane damage and, eventually, hepatocellular lysis. In the present study, we analyzed the capacity of the ursodeoxycholic acid endogenous metabolite, tauroursodeoxycholate (TUDC), to stabilize the hepatocellular plasma membrane against its transition to the micellar phase induced by the tensioactive bile salt taurochenodeoxycholate (TCDC), the main endogenous bile salt accumulated in cholestasis. The disruption of the plasma membrane was evaluated (i) in isolated hepatocytes, through the release of the enzyme lactate dehydrogenase to the incubation medium and (ii) in isolated plasma membranes, through the self-quenching assay of the membranotropic probe octadecylrhodamine B; this assay allows for detergent-induced transition from membrane bilayer to micelle to be monitored. Our results showed that isolated hepatocytes treated with TUDC are more resistant to TCDC-induced cell lysis. When this effect was evaluated in isolated plasma membranes, the TCDC concentration necessary to reach half of the transition from bilayer to micelle was increased by 22% (p<0.05). This difference remained even when TUDC was removed from the incubation medium before adding TCDC, thus indicating that TUDC exerted its effect directly on the plasma membrane. When the same experiments were carried out using the non-ionic detergent TX-100 or the cholesterol-complexing detergent digitonin, no protective effect was observed. In conclusion, TUDC prevents selectively the bilayer to micelle transition of the hepatocellular plasma membrane induced by hydrophobic bile salts that typically build up and accumulate in cholestatic processes. Our results suggest that formation of a complex between negatively charged TUDC and cholesterol in the membrane favours repulsion of negatively charged detergent bile salts, thus providing a basis for the understanding of the TUDC protective effects.

Unconjugated bilirubin inhibits C1 esterase activity

OBJECTIVE To evaluate if unconjugated bilirubin (UB) inhibits C1 esterase activity. DESIGN AND METHODS Esterase activity was evaluated by C1-mediated hydrolysis of N-acetyl-L-tyrosine ethyl ester, and binding of UB to C1r and C1s was assessed by dot-blot analysis. RESULTS UB inhibited C1 enzymatic activity. C1r, C1s and human serum albumin bound [(14)C]-UB to a similar extent. CONCLUSIONS UB inhibits C1 esterase activity, apparently due to a direct pigment-protein interaction. This could explain the inhibitory action of UB on complement activation.

Antiphospholipid and antioangiogenic activity in females with recurrent miscarriage and antiphospholipid syndrome

Background Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis, fetal losses and thrombocytopenia associated to antiphospholipid antibodies. They are directed to phospholipids, such as cardiolipins (anticardiolipin) and lupus anticoagulant or to complexes formed by phospholipids and protein cofactors, such as β2 glycoprotein 1 (a-β2GP1) and annexin V (a-annexin V). These auto-antibodies may be considered as a family of antibodies involved in thrombotic events and antiphospholipid activity. On the other hand, some proangiogenic factors are involved in the normal development of placental vasculature, such as the vascular endothelial growth factor. Overexpression of vascular endothelial growth factor receptor in its soluble form (sVEGFR-1) has been associated to a higher antiangiogenic activity. Our aim was to analyse the association between anticardiolipin, lupus anticoagulant, a-β2GP1, a-annexin V and sVEGFR-1 with recurrent miscarriage before week 10 of gestation in females with antiphospholipid syndrome. Methods We studied 24 females (primary or secondary antiphospholipid syndrome), who were divided into two groups: females with recurrent miscarriage before week 10 of gestation (M; n = 12) and females with no history of fetal loss (NM; n = 12). Anticardiolipin, a-β2GP1, a-annexin V and sVEGF-R1 concentrations were assessed by ELISA, while lupus anticoagulant was assessed by screening and confirmatory tests. Results A significant association was observed between the number of positive biomarkers and the belonging group (P < 0.05). Besides, a positive result for lupus anticoagulant and a-β2GP1 was found to be significantly associated to the M group (P < 0.05). Conclusions Lupus anticoagulant and a-β2GP1 may be implicated in pregnancies complicated by recurrent miscarriage in females with antiphospholipid syndrome.