Malgorzata Milkiewicz

Prospective evaluation of ursodeoxycholic acid withdrawal in patients with primary sclerosing cholangitis.

Ursodeoxycholic acid (UDCA) is no longer recommended for management of adult patients with primary sclerosing cholangitis (PSC). We undertook a prospective evaluation of UDCA withdrawal in a group of consecutive patients with PSC. Twenty six patients, all treated with UDCA (dose range: 10‐15 mg/kg/day) were included. Paired blood samples for liver biochemistry, bile acids, and fibroblast growth factor 19 (FGF19) were collected before UDCA withdrawal and 3 months later. Liquid chromatography/tandem mass spectrometry was used for quantification of 29 plasma bile acid metabolites. Pruritus and health‐related quality of life (HRQoL) were assessed with a 10‐point numeric rating scale, the Medical Outcomes Study Short Form‐36 (SF‐36), and PBC‐40 questionnaires. UDCA withdrawal resulted in a significant deterioration in liver biochemistry (increase of alkaline phosphatase of 75.6%; Pu2009<u20090.0001; gamma‐glutamyl transpeptidase of 117.9%, Pu2009<u20090.0001; bilirubin of 50.0%, Pu2009<u20090.001; alanine aminotransferase of 63.9%, Pu2009<u20090.005; and aspartate aminotransferase of 45.0%, Pu2009<u20090.005) and increase of Mayo Risk Score for PSC (change from baseline of +0.5 point; Pu2009<u20090.003). Bile acid analysis revealed a significant decrease in lithocholic acid and its derivatives after UDCA withdrawal, but no effect on concentrations of primary bile acids aside from an increased accumulation of their taurine conjugates. After UDCA removal cholestatic parameters, taurine species of cholic acid and chenodeoxycholic acid correlated with serum FGF19 levels. No significant effect on HRQoL after UDCA withdrawal was observed; however, 42% of patients reported a deterioration in their pruritus. Conclusion: At 3 months, discontinuation of UDCA in patients with PSC causes significant deterioration in liver biochemistry and influences concentrations of bile acid metabolites. A proportion of patients report increased pruritus, but other short‐term markers of quality of life are unaffected. (Hepatology 2014;60:931–940)

Static Strain Stimulates Expression of Matrix Metalloproteinase-2 and VEGF in Microvascular Endothelium Via JNK- and ERK-Dependent Pathways

VEGF and MMP protein production are both required for exercise‐induced capillary growth in skeletal muscle. The underlying process by which muscle activity initiates an angiogenic response is not established, but it is known that mechanical forces such as muscle stretch are involved. We hypothesized that stretch of skeletal muscle microvascular endothelial cells induces production of MMP‐2 and VEGF through a common signal pathway. Endothelial cells were grown on Bioflex plates and exposed to 10% static stretch for up to 24 h. MMP‐2 protein level was measured by gelatin zymography and VEGF, MMP‐2, and MT1‐MMP mRNA levels were quantified by real‐time quantitative PCR. ERK1/2 and JNK phosphorylation and VEGF protein levels were assessed by Western blotting. Effects of mitogen‐activated protein kinases (MAPKs) (ERK1/2, JNK) and reactive oxygen species (ROS) on stretch‐induced expression of MMP‐2 and VEGF were tested using pharmacological inhibitors. Stretching of endothelial cells for 24 h caused significant increases in MMP‐2 protein and mRNA level, but no change in MT1‐MMP mRNA. While MMP‐2 protein production was enhanced by H2O2 in unstretched cells, ROS inhibition during stretch did not diminish MMP‐2 mRNA or protein production. Inhibition of JNK suppressed stretch‐induced MMP‐2 protein and mRNA, but inhibition of ERK had no effect. In contrast, inhibition of ERK but not JNK attenuated the stretch‐induced increase in VEGF mRNA. Our results demonstrate that differential regulation of MMP‐2 and VEGF by MAPK signal pathways contribute to stretch‐induced activation of microvascular endothelial cells. J. Cell. Biochem. 100: 750–761, 2007.

Identification of a Mechanism Underlying Regulation of the Anti-Angiogenic Forkhead Transcription Factor FoxO1 in Cultured Endothelial Cells and Ischemic Muscle

Chronic limb ischemia, a complication commonly observed in conjunction with cardiovascular disease, is characterized by insufficient neovascularization despite the up-regulation of pro-angiogenic mediators. One hypothesis is that ischemia induces inhibitory signals that circumvent the normal capillary growth response. FoxO transcription factors exert anti-proliferative and pro-apoptotic effects on many cell types. We studied the regulation of FoxO1 protein in ischemic rat skeletal muscle following iliac artery ligation and in cultured endothelial cells. We found that FoxO1 expression was increased in capillaries within ischemic muscles compared with those from rats that underwent a sham operation. This finding correlated with increased expression of p27(Kip1) and reduced expression of Cyclin D1. Phosphorylated Akt was reduced concurrently with the increase in FoxO1 protein. In skeletal muscle endothelial cells, nutrient stress as well as lack of shear stress stabilized FoxO1 protein, whereas shear stress induced FoxO1 degradation. Endogenous FoxO1 co-precipitated with the E3 ubiquitin ligase murine double minute-2 (Mdm2) in endothelial cells, and this interaction varied in direct relation to the extent of Akt and Mdm2 phosphorylation. Moreover, ischemic muscles had a decreased level of Mdm2 phosphorylation and a reduced interaction between Mdm2 and FoxO1. Our results provide novel evidence that the Akt-Mdm2 pathway acts to regulate endothelial cell FoxO1 expression and illustrate a potential mechanism underlying the pathophysiological up-regulation of FoxO1 under ischemic conditions.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle.

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre+, FoxO1,3,4L/L, referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

Lipidic last breath of life in patients with alcoholic liver disease

Alcoholic liver disease (ALD) begins with the accumulation of lipid droplets in the liver. Lipids which accumulate in the liver can stimulate inflammation, and the fatty acid derivatives, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs), may play an important role in this process. We evaluated the concentrations of linoleic and arachidonic acid derivatives in the plasma of patients with ALD, non-alcoholic fatty liver disease (NAFLD) and healthy individuals. The groups consisted of 173 subjects: 63 patients with ALD, 90 with NAFLD and 20 healthy volunteers. Plasma 12-, 15-, and 5-HETE as well as 9- and 13-HODE were assessed using HPLC and isoprostane 8-epi-PGF 2α III was evaluated with an ELISA. In addition the mRNA expression of lipoxygenases (5-LOX, 15-LOX-1, 15-LOX-2) in the liver samples of patients with ALD cirrhosis was measured. A significant difference between the plasma concentrations of the analyzed derivatives was found when divided according to gender. The most significant differences were found between healthy individuals and ALD patients, as well as ALD and NAFLD individuals regardless of gender. The increased plasma HODEs and HETEs concentrations were in line with the increase in 5- and 15-LOX-1 and 15-LOX-2 mRNA in liver samples from ALD cirrhosis patients. LOXs expression and peroxidation of polyunsaturated fatty acids by free radical-propagated chemical oxidation may be contributing factors in liver necroinflammatory injury in ALD.

Expression of hepatic Fibroblast Growth Factor 19 is enhanced in Primary Biliary Cirrhosis and correlates with severity of the disease

Cholestasis induces adaptive mechanisms protecting the liver against bile acids (BA) toxicity including modulation of BA synthesis. Whether fibroblast growth factor 19 (FGF19) or farnesoid X receptor (FXR) dependent signaling are involved in the regulation of BA homeostasis in primary biliary cirrhosis (PBC) remains unknown. Here we analyzed hepatic expression of FGF19 and other genes relevant to the adaptive response to cholestasis in tissues from non-cirrhotic (nu2009=u200924) and cirrhotic (nu2009=u200921) patients along with control tissues (nu2009=u200921). Moreover we searched for relationships between serum FGF19 and laboratory/clinical findings in 51 patients. Hepatic FGF19 mRNA expression was increased in non-cirrhotic and cirrhotic tissues (9-fold,pu2009=u20090.01; 69-fold,pu2009<u20090.0001, respectively). Protein levels of FGF19, FGF receptor 4, FXR and short heterodimer partner were increased in cirrhotic livers (9-fold, pu2009<u20090.001; 3.5-fold,pu2009=u20090.007; 2.4-fold,pu2009<u20090.0001; 2.8-fold,pu2009<u20090.0001 vs controls, respectively) which was accompanied by down-regulation of CYP7A1 (50% reduction, pu2009=u20090.006). Serum and liver levels of FGF19 correlated with worse liver biochemistry, BAs, quality of life and Mayo Risk Score. Serum FGF19 was elevated in UDCA non-responders. We conclude that PBC induces characteristic changes in liver expression of BAs synthesis regulatory molecules. FGF19 correlates with severity of liver disease and can potentially serve as an indicator of chronic cholestatic liver injury.

Vitamin D Receptor Polymorphisms Predispose to Primary Biliary Cirrhosis and Severity of the Disease in Polish Population

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver condition characterized by the immune-mediated damage of the intrahepatic bile ducts. Polymorphisms of vitamin D receptor (VDR) are considered to contribute to its pathogenesis however their incidence varies in different populations and their potential association with the course of the disease has not been studied. In this paper we investigated the incidence and correlation of three VDR polymorphisms (BsmI, ApaI or TaqI) with various clinical, biochemical, and serological factors in a homogenous group of 143 Caucasian patients with PBC. Control group comprises 306 DNA samples from umbilical cord blood of healthy newborn children. When compared to controls, we observed a significant dominance of the b allele in the BsmI (ORu2009=u20091.69 [1.27–2.24]; P = 0.0003) and t allele in the TaqI (ORu2009=u20090.62 [0.47–0.82], P = 0.0001) in patients with PBC. Moreover the BsmI and TaqI polymorphisms were associated with the presence of advanced fibrosis/liver cirrhosis at the diagnosis of PBC. Pairwise linkage disequilibrium (LD) calculations proved that the analyzed SNPs are within an LD block (100% of LDs were D>0.9). Our study showed, for the first time, that the analyzed polymorphisms of VRD may exert an effect on a natural history of PBC.

Activation of FoxO3a/Bim axis in patients with Primary Biliary Cirrhosis.

Impaired regulation of apoptosis has been suggested to play a role in the pathogenesis of Primary Biliary Cirrhosis (PBC). In this study, we analysed a signalling pathway that comprises the transcription factor FoxO3a and its downstream target Bim, a Bcl‐2 interacting mediator of apoptosis.

Angiogenesis within the duodenum of patients with cirrhosis is modulated by mechanosensitive Kruppel-like factor 2 and microRNA-126

The mechanism involved in neovascularization in splanchnic circulation and the main trigger that induces angiogenesis in patients with cirrhosis are not fully recognized.

Serum Autotaxin is a Marker of the Severity of Liver Injury and Overall Survival in Patients with Cholestatic Liver Diseases

Autotaxin (ATX) is involved in the synthesis of lysophosphatidic acid. Both have recently been linked to cholestatic pruritus and liver injury. We aimed to investigate whether ATX is an indicator of cholestatic liver injury, health-related quality of life (HRQoL) and prognosis based on a group of 233 patients, 118 with primary biliary cholangitis (PBC) and 115 with primary sclerosing cholangitis (PSC). Patients were followed for 1–60 months, cumulative survival rates were calculated. ATX activity was significantly higher in both groups than in the 103 controls, particularly in patients with cirrhosis and in patients with longer disease duration. Ursodeoxycholic acid (UDCA) non-responders with PBC exhibited increased ATX activity. ATX activity was correlated with liver biochemistry, MELD, Mayo Risk scores and was associated with worse disease-specific HRQoL aspects. In both groups, Cox model analysis indicated that ATX was a negative predictor of survival. Increased ATX levels were associated with a 4-fold higher risk of death/liver transplantation in patients with PBC and a 2.6-fold higher risk in patients with PSC. We conclude that in patients with cholestatic conditions, ATX is not only associated with pruritus but also indicates impairment of other HRQoL aspects, liver dysfunction, and can serve as a predictor of survival.