Shadi Abu-Hayyeh

Intrahepatic cholestasis of pregnancy levels of sulfated progesterone metabolites inhibit farnesoid X receptor resulting in a cholestatic phenotype

Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent pregnancy‐specific liver disease and is associated with an increased risk of adverse fetal outcomes, including preterm labor and intrauterine death. The endocrine signals that cause cholestasis are not known but 3α‐sulfated progesterone metabolites have been shown to be elevated in ICP, leading us to study the impact of sulfated progesterone metabolites on farnesoid X receptor (FXR)‐mediated bile acid homeostasis pathways. Here we report that the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate is supraphysiologically raised in the serum of ICP patients. Mice challenged with cholic acid developed hypercholanemia and a hepatic gene expression profile indicative of FXR activation. However, coadministration of epiallopregnanolone sulfate with cholic acid exacerbated the hypercholanemia and resulted in aberrant gene expression profiles for hepatic bile acid‐responsive genes consistent with cholestasis. We demonstrate that levels of epiallopregnanolone sulfate found in ICP can function as a partial agonist for FXR, resulting in the aberrant expression of bile acid homeostasis genes in hepatoma cell lines and primary human hepatocytes. Furthermore, epiallopregnanolone sulfate inhibition of FXR results in reduced FXR‐mediated bile acid efflux and secreted FGF19. Using cofactor recruitment assays, we show that epiallopregnanolone sulfate competitively inhibits bile acid‐mediated recruitment of cofactor motifs to the FXR‐ligand binding domain. Conclusion: Our results reveal a novel molecular interaction between ICP‐associated levels of the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate and FXR that couples the endocrine component of pregnancy in ICP to abnormal bile acid homeostasis. (HEPATOLOGY 2013;)

Cadmium Accumulation in Aortas of Smokers

Abstract—Abdominal aortic aneurysm is a smoking-related disorder. Cadmium, inhaled from cigarettes, may accumulate in the aorta and facilitate weakening of the aorta through adverse effects on smooth muscle cell metabolism. Cadmium was measured by atomic absorption spectrometry in infrarenal aortas from 13 patients with abdominal aortic aneurysm and from 17 age- and sex-matched patients with normal-diameter abdominal aorta. Total cadmium content was associated with smoking, assessed as pack-years (r =0.54, P =0.004), but was similar in aneurysmal and undilated aortas. The cadmium content (mean±SE) was higher in the media (3.25±0.53 ng/mg dry wt, 7±1.2 &mgr;mol/L) than in the intima or adventitia (1.14±0.24 and 1.87±0.38 ng/mg dry wt, respectively; ANOVA, P <0.005). There was a strong correlation between medial cadmium content and pack-years of smoking (r =0.87, P <0.001). In aortic smooth muscle cells cultured on fibrillar collagen, cadmium inhibited DNA synthesis and collagen synthesis and diminished cell numbers (IC50 2 &mgr;mol/L, 6 &mgr;mol/L, and 6 &mgr;mol/L, respectively), but higher concentrations of cadmium were required for upregulation of metallothionein (EC50 23 &mgr;mol/L). The cadmium content of the aorta increases in direct proportion to the pack-years of cigarettes smoked, with selective accumulation in the medial layer. However, the cadmium content of aneurysmal aortas was not higher than that of nondilated aortas for patients with matched smoking history. In smokers, the level of cadmium accumulation is probably sufficient to impair the viability of cultured smooth muscle cells. Similar mechanisms could underlie the development of degenerative aortic disease in smokers.

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart. Methods and Results Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M2 receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M2 receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters. Conclusion We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M2 receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

Maternal cholestasis during pregnancy programs metabolic disease in offspring

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a ‘master’ protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na+-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [3H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na+-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [3H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na+-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.

Astroglia up-regulate transcription and secretion of 'readthrough' acetylcholinesterase following oxidative stress.

Novel and diverse functions of glial cells are currently the focus of much attention [A. Volterra and J. Meldolesi (2005)Nature Rev. 6, 626–640]. Here we present evidence that rat astroglia release acetylcholinesterase (AChE) as part of their response to hypoxic damage. Exposure of astroglia to tert‐butyl hydroperoxide, and hence oxidative stress, subsequently leads to a switching in mRNA from the classical membrane‐bound T‐AChE to a preferential increase in the splice variant for a soluble form, R‐AChE, This change in expression is reflected in increased perinuclear and reduced cytoplasmic AChE staining of the insulted glial cells, with a concomitant and marked increase in extracellular secretion that peaks at 1 h post‐treatment. An analogous increase in R‐AChE, over a similar time scale, occurs in response to psychological stress [D. Kaufer et al. (1998)Nature 93, 373–377], as well as to head injury and stroke [E. Shohami et al. (1999)J. Neurotrauma 6, 365–76]. The data presented here suggest that glial cells may be key chemical intermediaries in such situations and, perhaps more generally in pathological conditions involving oxidative stress, such as neurodegeneration.

Prognostic and mechanistic potential of progesterone sulfates in intrahepatic cholestasis of pregnancy and pruritus gravidarum

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9‐15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first‐trimester samples all progesterone sulfates were significantly elevated in serum from low‐risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5‐dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high‐risk population. Delineation of a progesterone sulfate‐TGR5 pruritus axis identifies a therapeutic target for itch management in ICP. (Hepatology 2016;63:1287–1298)

What a difference an egg makes

Institute of Reproductive and Developmental Biology, Imperial College London, London, UK (R A Fisher FRCPath, S Abu-Hayyeh PhD); IVF Hammersmith, Imperial College London, Hammersmith Hospital, London, UK (S A Lavery MRCOG, A Carby MBBS); Department of Obstetrics and Gynaecology, Gloucestershire Royal Hospital, Gloucester, UK (R Swingler MRCOG); and Trophoblastic Tumour Screening and Treatment Centre, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK (R A Fisher, Prof N J Sebire FRCPath, Prof M J Seckl FRCP)

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions. Methodology/Principal Findings We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration. Conclusion/Significance These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.