Functional shift with maintained regenerative potential following portal vein ligation

Selective portal vein ligation (PVL) allows the two-stage surgical resection of primarily unresectable liver tumours by generating the atrophy and hypertrophy of portally ligated (LL) and non-ligated lobes (NLL), respectively. To evaluate critically important underlying functional alterations, present study characterised in vitro and vivo liver function in male Wistar rats (n = 106; 210-250 g) before, and 24/48/72/168/336 h after PVL. Lobe weights and volumes by magnetic resonance imaging confirmed the atrophy-hypertrophy complex. Proper expression and localization of key liver transporters (Ntcp, Bsep) and tight junction protein ZO-1 in isolated hepatocytes demonstrated constantly present viable and well-polarised cells in both lobes. In vitro taurocholate and bilirubin transport, as well as in vivo immunohistochemical Ntcp and Mrp2 expressions were bilaterally temporarily diminished, whereas LL and NLL structural acinar changes were divergent. In vivo bile and bilirubin-glucuronide excretion mirrored macroscopic changes, whereas serum bilirubin levels remained unaffected. In vivo functional imaging (indocyanine-green clearance test; (99mTc)-mebrofenin hepatobiliary scintigraphy; confocal laser endomicroscopy) indicated transitionally reduced global liver uptake and -excretion. While LL functional involution was permanent, NLL uptake and excretory functions recovered excessively. Following PVL, functioning cells remain even in LL. Despite extensive bilateral morpho-functional changes, NLL functional increment restores temporary declined transport functions, emphasising liver functional assessment

Assessing the effects of Ang-(1-7) therapy following transient middle cerebral artery occlusion

The counter-regulatory axis, Angiotensin Converting Enzyme 2, Angiotensin-(1-7), Mas receptor (ACE2/Ang-1-7/MasR), of the renin angiotensin system (RAS) is a potential therapeutic target in stroke, with Ang-(1-7) reported to have neuroprotective effects in pre-clinical stroke models. Here, an extensive investigation of the functional and mechanistic effects of Ang-(1-7) was performed in a rodent model of stroke. Using longitudinal magnetic resonance imaging (MRI) it was observed that central administration of Ang-(1-7) following transient middle cerebral artery occlusion (MCAO) increased the amount of tissue salvage compared to reperfusion alone. This protective effect was not due to early changes in blood brain barrier (BBB) permeability, microglia activation or inflammatory gene expression. However, increases in NADPH oxidase 1 (Nox1) mRNA expression were observed in the treatment group compared to control. In order to determine whether Ang-(1-7) has direct cerebrovascular effects, laser speckle contrast imaging (LSCI) was performed to measure dynamic changes in cortical perfusion following reperfusion. Delivery of Ang-(1-7) did not have any effect on cortical perfusion following reperfusion however; it showed an indication to prevent the ‘steal phenomenon’ within the contralateral hemisphere. The comprehensive series of studies have demonstrated a moderate protective effect of Ang-(1-7) when given alongside reperfusion to increase tissue salvage

Drug trafficking in mice: In vivo functions of OATP uptake and ABC efflux transporters

In recent years, there has been increasing attention for drug uptake transporters of the Organic Anion-Transporting Polypeptide (human OATP, mouse Oatp, gene names SLCO, Slco) superfamily. Especially the OATP1A and OATP1B subfamilies turn out to have important physiological and pharmacological functions. Members of the OATP1A/1B subfamilies have received most interest because of their localization in tissues important for detoxification and pharmacokinetics (i.e. liver, small intestine and kidney) and their ability to mediate the cellular uptake of a wide variety of endogenous compounds but also many xenobiotics. OATP1A/1B transporters play important roles in the physiological detoxification by the liver of endogenous compounds, but also in determining tissue distribution, the rate and route of elimination, systemic exposure and oral bioavailability of drugs. In this thesis we studied OATP transporters in vivo using knockout mouse strains lacking all Oatp1a/1b transporters, and humanized transgenic mouse strains with liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3. The concomitant use of these different mouse models is required because mouse and human OATP1A/1B proteins are not straightforward orthologues and their tissue localization and substrate specificity can differ substantially. Using these mice, we gained more insight into the physiological functions of OATP1A/1B, for instance the plasma clearance of unconjugated bilirubin and bile acids by facilitating their liver uptake. Further, we focussed on the pharmacological functions of these transporters in the plasma clearance (by efficient hepatic uptake) of statin drugs (pravastatin, rosuvastatin) and anticancer drugs (irinotecan/SN-38 and docetaxel). These findings have pharmacogenetic implications because there are several low-activity polymorphisms of OATP1A/1B transporters known. Also, complete deficiencies of either OATP1B1 or OATP1B3 transporters have been found in humans in addition to the Rotor syndrome patients, which completely lack both OATP1B1 and OATP1B3. All these individuals might be at risk of developing toxicities when treated with OATP1B substrates. Also co-administration of these drugs with OATP1A/1B inhibiting drugs might lead to clinically relevant drug-drug interactions. Human OATP1A/1B transporters are also expressed in several types of tumors and can thus confer sensitivity to anticancer drugs in cell lines overexpressing these transporters. Based on our findings that OATP1A/1B transporters transport several anticancer drugs in vivo we can speculate that they might mediate the tumor uptake of these anticancer drugs, and therefore modulate response to chemotherapy. We also studied the main ATP binding cassette (ABC) efflux transporters, ABCB1 (P-gp) and/or ABCG2 and their effect on the brain accumulation and oral bioavailability of tamoxifen and axitinib. We have found that endoxifen, a 100-fold more active metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo. High expression of P-gp in breast tumors treated with tamoxifen might thus lead to insufficient intratumoral endoxifen concentrations and therefore insufficient therapeutic response. In the case of the tyrosine kinase inhibitor axitinib, we observed that Abcg2 has a role in limiting its bioavailability after oral administration, while at the blood-brain barrier, P-gp is the main determinant of axitinib brain penetration. These results might have clinical relevance for brain tumors positioned behind an intact and functional blood-brain barrier

Drug trafficking in mice: In vivo functions of OATP uptake and ABC efflux transporters

In recent years, there has been increasing attention for drug uptake transporters of the Organic Anion-Transporting Polypeptide (human OATP, mouse Oatp, gene names SLCO, Slco) superfamily. Especially the OATP1A and OATP1B subfamilies turn out to have important physiological and pharmacological functions. Members of the OATP1A/1B subfamilies have received most interest because of their localization in tissues important for detoxification and pharmacokinetics (i.e. liver, small intestine and kidney) and their ability to mediate the cellular uptake of a wide variety of endogenous compounds but also many xenobiotics. OATP1A/1B transporters play important roles in the physiological detoxification by the liver of endogenous compounds, but also in determining tissue distribution, the rate and route of elimination, systemic exposure and oral bioavailability of drugs. In this thesis we studied OATP transporters in vivo using knockout mouse strains lacking all Oatp1a/1b transporters, and humanized transgenic mouse strains with liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3. The concomitant use of these different mouse models is required because mouse and human OATP1A/1B proteins are not straightforward orthologues and their tissue localization and substrate specificity can differ substantially. Using these mice, we gained more insight into the physiological functions of OATP1A/1B, for instance the plasma clearance of unconjugated bilirubin and bile acids by facilitating their liver uptake. Further, we focussed on the pharmacological functions of these transporters in the plasma clearance (by efficient hepatic uptake) of statin drugs (pravastatin, rosuvastatin) and anticancer drugs (irinotecan/SN-38 and docetaxel). These findings have pharmacogenetic implications because there are several low-activity polymorphisms of OATP1A/1B transporters known. Also, complete deficiencies of either OATP1B1 or OATP1B3 transporters have been found in humans in addition to the Rotor syndrome patients, which completely lack both OATP1B1 and OATP1B3. All these individuals might be at risk of developing toxicities when treated with OATP1B substrates. Also co-administration of these drugs with OATP1A/1B inhibiting drugs might lead to clinically relevant drug-drug interactions. Human OATP1A/1B transporters are also expressed in several types of tumors and can thus confer sensitivity to anticancer drugs in cell lines overexpressing these transporters. Based on our findings that OATP1A/1B transporters transport several anticancer drugs in vivo we can speculate that they might mediate the tumor uptake of these anticancer drugs, and therefore modulate response to chemotherapy. We also studied the main ATP binding cassette (ABC) efflux transporters, ABCB1 (P-gp) and/or ABCG2 and their effect on the brain accumulation and oral bioavailability of tamoxifen and axitinib. We have found that endoxifen, a 100-fold more active metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo. High expression of P-gp in breast tumors treated with tamoxifen might thus lead to insufficient intratumoral endoxifen concentrations and therefore insufficient therapeutic response. In the case of the tyrosine kinase inhibitor axitinib, we observed that Abcg2 has a role in limiting its bioavailability after oral administration, while at the blood-brain barrier, P-gp is the main determinant of axitinib brain penetration. These results might have clinical relevance for brain tumors positioned behind an intact and functional blood-brain barrier

Bradykinin Protects Against Oxidative Stress-Induced Endothelial Cell Senescence

Premature aging (senescence) of endothelial cells might play an important role in the development and progression of hypertension and atherosclerosis. We hypothesized that bradykinin, a hormone that mediates vasoprotective effects of angiotensin-converting enzyme inhibitors, protects endothelial cells from oxidative stress-induced senescence. Bradykinin treatment (0.001 to 1 nmol/L) dose-dependently decreased senescence induced by 25 mu mol/L of H2O2 in cultured bovine aortic endothelial cells, as witnessed by a complete inhibition of increased senescent cell numbers and a 34% reduction of the levels of the senescence-associated cell cycle protein p21. Because H2O2 induces senescence through superoxide-induced DNA damage, single-cell DNA damage was measured by comet assay. Bradykinin reduced DNA damage to control levels. The protective effect of bradykinin also resulted in a significant increase in the migration of H2O2-treated bovine aorta endothelial cells in an in vitro endothelial injury model, or "scratch" assay. The protective effect of bradykinin was abolished by the bradykinin B2 receptor antagonist HOE-140 and the NO production inhibitor N-omega-methyl-L-arginine acetate salt. Therefore, we conclude that bradykinin protects endothelial cells from superoxide-induced senescence through bradykinin B2 receptor-and NO-mediated inhibition of DNA damage. (Hypertension. 2009; 53[part 2]: 417-422.

Synergistic interaction between genetics and disease on pravastatin disposition

BACKGROUND & AIMS: A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Nonalcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters. METHODS: To determine how genetic alteration in one Oatp transporter can interact with NASH-associated changes in Oatp expression we measured the disposition of intravenously administered pravastatin in Slco1b2 knockout (Slco1b2−/−) and wild-type (WT) mice fed either a control or a methionine and choline deficient (MCD) diet to induce NASH. RESULTS: Genetic loss of Oatp1b2, the rodent ortholog of human OATP1B transporters, caused a modest increase in pravastatin plasma concentrations in mice with healthy livers. Although a diet-induced model of NASH decreased the expression of multiple hepatic Oatp transporters, it did not alter the disposition of pravastatin compared to WT control mice. In contrast, the combination of NASH-associated decrease in compensatory Oatp transporters and Oatp1b2 genetic loss caused a synergistic increase in plasma area under the curve (AUC) and tissue concentrations in kidney and muscle. CONCLUSIONS: Our data show that NASH alters the expression of multiple hepatic uptake transporters which, due to overlapping substrate specificity among the OATP transporters, may combine with the pharmacogenetic loss of OATP1B1 to increase the risk of statin-induced adverse drug reactions

Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro–in vivo correlations

BACKGROUND: Several tyrosine kinase inhibitors (TKIs) can decrease docetaxel clearance in patients by an unknown mechanism. We hypothesised that these interactions are mediated by the hepatic uptake transporter OATP1B1. METHODS: The influence of 16 approved TKIs on transport was studied in vitro using HEK293 cells expressing OATP1B1 or its mouse equivalent Oatp1b2. Pharmacokinetic studies were performed with Oatp1b2-knockout and OATP1B1-transgenic mice. RESULTS: All docetaxel-interacting TKIs, including sorafenib, were identified as potent inhibitors of OATP1B1 in vitro. Although Oatp1b2 deficiency in vivo was associated with increased docetaxel exposure, single- or multiple-dose sorafenib did not influence docetaxel pharmacokinetics. CONCLUSION: These findings highlight the importance of identifying proper preclinical models for verifying and predicting TKI–chemotherapy interactions involving transporters