Lofthouse EM et al. (2019), Ursodeoxycholic acid inhibits uptake and vasoco...

XB-ART-55813

FASEB J 2019 Jul 01;337:8211-8220. doi: 10.1096/fj.201900015RR.

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Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Lofthouse EM , Torrens C , Manousopoulou A , Nahar M , Cleal JK , O'Kelly IM , Sengers BG , Garbis SD , Lewis RM .

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Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion-transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.-Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O'Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

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Species referenced: Xenopus laevis
Genes referenced: ibsp slco1a2 slco4a1

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	Figure 1. The uptake of OATP substrates, ES and TC, are inhibited by UDCA in placental villous fragments. A) 3H-ES uptake by villous fragments was shown to be linear up to 5 min (n = 2 placentas). B) 3H-TC uptake by villous fragments was shown to be linear up to 2.5 min (n = 3 placentas). C) 3H-ES uptake was inhibited by 1 mM ES (*P < 0.001, n = 5), 1 mM TC (P < 0.001, n = 5), and 1 mM UDCA (P < 0.01, n = 5) but not by the negative control glycine (n = 5). D) 3H-TC uptake was inhibited by 100 μM UDCA (P < 0.01, n = 5), 100 μM TC (P < 0.01, n = 5), and 100 μM ES (***P < 0.01, n = 5), but not by the negative control glycine (n = 5). Data are presented as means and sem and were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-ES and 3H-TC alone).
	Figure 2. UDCA inhibits TC uptake in placental villous fragments in a dose-dependent manner. Compared with 3H-TC alone, 100–200 μM UDCA inhibited TC uptake (*P < 0.05, n = 3 placentas, triplicate conditions, 3 fragments/replicate). Data are presented as means and sem and were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-TC alone).
	Figure 3. UDCA inhibits OATP4A1 but does not cause transstimulation, suggesting it is not transported. A) 3H-ES uptake into oocytes by OATP4A1 was shown to be linear up to 10 min, and this was inhibited by cold 2.5 mM ES (n = 5 ovaries, 5 oocytes per condition). B) 3H-ES uptake (under sodium-free conditions) in OATP4A1-injected oocytes was inhibited by 500 μM ES, TC, and UDCA (P < 0.05, n = 5 ovaries, 5 oocytes/condition), but not by negative control glycine. Data are presented as means and sem and are analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-ES alone). C) 3H-ES efflux in OATP4A1-injected oocytes is transstimulated by ES (P < 0.05, n = 3) and TC (**P < 0.01), but not by UDCA or negative control glycine. This suggests that UDCA is not a substrate of OATP4A1. Data are adjusted for background water-injected responses and presented as means and sem. Data were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with buffer alone); n > 3 individual ovaries, 5 oocytes per condition for uptake, 15 oocytes/condition for efflux.
	Figure 4. TC induces vasoconstriction in human placental vasculature. A) In placental perfusions, infusion of a 1 μM TC bolus into the fetal circulation (P < 0.01, n = 4 perfusions) and subsequently 20 nM angiotensin II (P < 0.011, n = 4 perfusions) significantly increased fetoplacental pressure from baseline. Data are presented as means and sem and were analyzed using an unpaired Student’s t test in which data were compared with a hypothetical value of 0. B) In chorionic plate arteries from human term placenta, TC (n = 5) produced a modest constriction that was significantly less potent than the thromboxane mimetic U46619 (1 μM) (n = 3, P < 0.01). Although this was a modest response, it was abolished by both nifedipine (1 μM, = 2) and ES (100 μM, n = 3, P < 0.01).
	Figure 5. TC-induced concentration-dependent vasoconstriction in rat aortas is inhibited by OATP substrates, UDCA, and the calcium channel blocker nifedipine. A) Vasoconstriction is induced by phenylephrine and TC (n = 13). B) TC responses were abolished in the presence of 1 μM nifedipine, a calcium channel blocker (n = 3, *P < 0.005). C) TC constriction is abolished in the presence of UDCA (n = 5, P < 0.01). D) TC constriction is abolished in the presence of OAT inhibitor BSP (n = 7, *P < 0.005) and ES (n = 4, *P < 0.005).
	Figure 6. A) GO analysis of the differentially expressed proteins following 8 h exposure of placental villous fragments to TC vs. controls showed significant enrichment for GO terms related to metabolism, cell death, oxidative stress, and vesicle-mediated transport. The dotted line indicates a value of P = 0.05. B) Differentially expressed proteins that map to significant pathways (cellular response to oxidative stress, cell death, and vesicle-mediated transport) presented in heatmap format representing the ratio of expression in treated vs. control samples for each placenta.
	Figure 7. A) In cholestasis, high maternal bile acid concentrations lead to reversal of the normal direction of bile acid transfer being reversed. Placental OATP4A1 activity inhibition via UDCA prevents toxic bile acid uptake across the microvillous membrane of the placental syncytiotrophoblast. B) We propose that vasoconstriction of placental smooth muscle results from altered calcium signaling induced by a rise in intracellular calcium from M2 receptors and l-type calcium channels.

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	Figure 1. The uptake of OATP substrates, ES and TC, are inhibited by UDCA in placental villous fragments. A) 3H-ES uptake by villous fragments was shown to be linear up to 5 min (n = 2 placentas). B) 3H-TC uptake by villous fragments was shown to be linear up to 2.5 min (n = 3 placentas). C) 3H-ES uptake was inhibited by 1 mM ES (*P < 0.001, n = 5), 1 mM TC (P < 0.001, n = 5), and 1 mM UDCA (P < 0.01, n = 5) but not by the negative control glycine (n = 5). D) 3H-TC uptake was inhibited by 100 μM UDCA (P < 0.01, n = 5), 100 μM TC (P < 0.01, n = 5), and 100 μM ES (***P < 0.01, n = 5), but not by the negative control glycine (n = 5). Data are presented as means and sem and were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-ES and 3H-TC alone).
	Figure 2. UDCA inhibits TC uptake in placental villous fragments in a dose-dependent manner. Compared with 3H-TC alone, 100–200 μM UDCA inhibited TC uptake (*P < 0.05, n = 3 placentas, triplicate conditions, 3 fragments/replicate). Data are presented as means and sem and were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-TC alone).
	Figure 3. UDCA inhibits OATP4A1 but does not cause transstimulation, suggesting it is not transported. A) 3H-ES uptake into oocytes by OATP4A1 was shown to be linear up to 10 min, and this was inhibited by cold 2.5 mM ES (n = 5 ovaries, 5 oocytes per condition). B) 3H-ES uptake (under sodium-free conditions) in OATP4A1-injected oocytes was inhibited by 500 μM ES, TC, and UDCA (P < 0.05, n = 5 ovaries, 5 oocytes/condition), but not by negative control glycine. Data are presented as means and sem and are analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with 3H-ES alone). C) 3H-ES efflux in OATP4A1-injected oocytes is transstimulated by ES (P < 0.05, n = 3) and TC (**P < 0.01), but not by UDCA or negative control glycine. This suggests that UDCA is not a substrate of OATP4A1. Data are adjusted for background water-injected responses and presented as means and sem. Data were analyzed by a 1-way ANOVA with Dunnett’s post hoc (compared with buffer alone); n > 3 individual ovaries, 5 oocytes per condition for uptake, 15 oocytes/condition for efflux.
	Figure 4. TC induces vasoconstriction in human placental vasculature. A) In placental perfusions, infusion of a 1 μM TC bolus into the fetal circulation (P < 0.01, n = 4 perfusions) and subsequently 20 nM angiotensin II (P < 0.011, n = 4 perfusions) significantly increased fetoplacental pressure from baseline. Data are presented as means and sem and were analyzed using an unpaired Student’s t test in which data were compared with a hypothetical value of 0. B) In chorionic plate arteries from human term placenta, TC (n = 5) produced a modest constriction that was significantly less potent than the thromboxane mimetic U46619 (1 μM) (n = 3, P < 0.01). Although this was a modest response, it was abolished by both nifedipine (1 μM, = 2) and ES (100 μM, n = 3, P < 0.01).
	Figure 5. TC-induced concentration-dependent vasoconstriction in rat aortas is inhibited by OATP substrates, UDCA, and the calcium channel blocker nifedipine. A) Vasoconstriction is induced by phenylephrine and TC (n = 13). B) TC responses were abolished in the presence of 1 μM nifedipine, a calcium channel blocker (n = 3, *P < 0.005). C) TC constriction is abolished in the presence of UDCA (n = 5, P < 0.01). D) TC constriction is abolished in the presence of OAT inhibitor BSP (n = 7, *P < 0.005) and ES (n = 4, *P < 0.005).
	Figure 6. A) GO analysis of the differentially expressed proteins following 8 h exposure of placental villous fragments to TC vs. controls showed significant enrichment for GO terms related to metabolism, cell death, oxidative stress, and vesicle-mediated transport. The dotted line indicates a value of P = 0.05. B) Differentially expressed proteins that map to significant pathways (cellular response to oxidative stress, cell death, and vesicle-mediated transport) presented in heatmap format representing the ratio of expression in treated vs. control samples for each placenta.
	Figure 7. A) In cholestasis, high maternal bile acid concentrations lead to reversal of the normal direction of bile acid transfer being reversed. Placental OATP4A1 activity inhibition via UDCA prevents toxic bile acid uptake across the microvillous membrane of the placental syncytiotrophoblast. B) We propose that vasoconstriction of placental smooth muscle results from altered calcium signaling induced by a rise in intracellular calcium from M2 receptors and l-type calcium channels.