S-adenosyl-L-methionine (SAMe) halts the autoimmune response in patients with primary biliary cholangitis (PBC) via antioxidant and S-glutathionylation processes in cholangiocytes.

S-adenosyl-L-methionine is an endogenous molecule with hepato-protective properties linked to redox regulation and methylation. Here, the potential therapeutic value of SAMe was tested in 17 patients with PBC, a cholestatic disease with autoimmune phenomena targeting small bile ducts. Nine patients responded to SAMe (SAMe responders) with increased serum protein S-glutathionylation. That posttranslational protein modification was associated with reduction of serum anti-mitochondrial autoantibodies (AMA-M2) titers and improvement of liver biochemistry. Clinically, SAMe responders were younger at diagnosis, had longer duration of the disease and lower level of serum S-glutathionylated proteins at entry. SAMe treatment was associated with negative correlation between protein S-glutathionylation and TNFα. Furthermore, AMA-M2 titers correlated positively with INFγ and FGF-19 while negatively with TGFß. Additionally, cirrhotic PBC livers showed reduced levels of glutathionylated proteins, glutaredoxine-1 (Grx-1) and GSH synthase (GS). The effect of SAMe was also analyzed in vitro. In human cholangiocytes overexpressing miR-506, which induces PBC-like features, SAMe increased total protein S-glutathionylation and the level of γ-glutamylcysteine ligase (GCLC), whereas reduced Grx-1 level. Moreover, SAMe protected primary human cholangiocytes against mitochondrial oxidative stress induced by tBHQ (tert-Butylhydroquinone) via raising the level of Nrf2 and HO-1. Finally, SAMe reduced apoptosis (cleaved-caspase3) and PDC-E2 (antigen responsible of the AMA-M2) induced experimentally by glycochenodeoxycholic acid (GCDC). These data suggest that SAMe may inhibit autoimmune events in patients with PBC via its antioxidant and S-glutathionylation properties. These findings provide new insights into the molecular events promoting progression of PBC and suggest potential therapeutic application of SAMe in PBC.

Diverse inhibition of forkhead box O1 activity by linoleic acid isomers - potential role in lipid metabolism in HepG2 cells and livers of C57BL/6J mice.

Conjugated dienes of linoleic acid (CLA) are constitutional and geometric isomers of linoleic acid that are commonly used as dietary supplements during body mass reduction. Their role in the reduction of lipid deposits in liver tissue is not unequivocal. CLA contain an equimolar mixture of two isomers of linoleic acid: trans-10,cis-12 CLA and cis-9,trans-11. Only one isomer - trans-10,cis-12 CLA exhibits fat-reducing properties, cis-9,trans-11 CLA does not. The main goal of this study was to determine if CLA isomers affect the activation of forkhead box O1 (FoxO1) in liver cells and tissue. FoxO1 is a protein that plays a crucial role in regulation of lipid and carbohydrates metabolism. In vitro and in vivo models of our study were HepG2 cells and C57BL/6J mice. Methods used in the study were qPCR - quantification of FoxO1 gene expression, Western blot - posttranslational phosphorylation of FoxO1, Oil Red O (ORO) - lipid staining and ELISA - quantification of apoB100. In both models trans-10,cis-12 CLA diminished FoxO1 gene expression: decrease by 44.1 ± 20.9% SD in the cells and 65.4 ± 29.8% SD in mice. The lowest accumulation of lipids (drop of 37.2 ± 1.7% SD) and the highest increase of apoB100 protein (74 ± 12.8% SD) were detected in the medium of cells cultured with trans-10,cis-12 CLA. Both isomers of linoleic acid have different effects on lipid metabolism. Isomer c9,t11 CLA accelerates lipogenesis, whereas isomer t10,c12 CLA activates secretion of lipids in HepG2 cells. In contrast to the in vitro study, unfortunately this pro-health property of t10,c12 CLA was not confirmed in the in vivo model. This casts a shadow on CLA dietary supplements that are commonly used among people with type 2 diabetes, NAFLD (non-alcoholic liver disease) or a metabolic syndrome in order to lose weight.

The effect of short term treatment with probiotic VSL#3 on various clinical and biochemical parameters in patients with liver cirrhosis.

The evidence is mounting that alterations of innate immunity and gut microbiota contribute to chronic liver disease and its complications. Modulation of intestinal microbiota is an emerging therapeutic strategy in hepatology. Probiotics through modulation of intestinal milieu have the potential to affect the course of liver disease. The data concerning the influence of probiotics on various plasma molecules and compounds involved in the pathogenesis of hyperdynamic circulatory state in liver cirrhosis is still not confluent and require further evaluation. In our study twenty patients with compensated and decompensated liver cirrhosis and ten healthy controls received probiotic VSL#3 daily for 28 days. Plasma levels of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI), macrophage inflammatory protein 3/α (MIP-3 α/CCL20), monocyte chemotactic protein-1α (MCP-1/CCL2), human myeloperoxidase (MPO), nitric oxide (NO), prostaglandins, thromboxane (TXB2) and big-endothelin were measured at baseline, day 14 and 28 of probiotic administration. The incidence of hepatic encephalopathy was assessed with critical flicker frequency. Changes in clinical, biochemical and microbiological parameters were evaluated. The stage of liver cirrhosis correlated with an increase in plasma levels of pro-inflammatory cytokines (IL-6) and chemotactic chemokines involved in immune cell trafficking (MIP-3α/CCL20). Probiotic administration in patients with liver cirrhosis led to modulation of plasma levels of several molecules and compounds measured (MIP-3α/CCL20, NO, big-endothelin, TXB2 and MPO). The grade of encephalopathy during the course of probiotic supplementation remained unaffected in both groups of patients. VSL#3 treatment was well tolerated and safe in patients with liver disease. In patients with compensated and decompensated liver cirrhosis, VSL#3 manipulates selected plasma molecules and compounds involved in hyperdynamic circulatory dysfunction. Short term VSL#3 administration affects several clinical and biochemical parameters commonly altered in liver cirrhosis.

Apolipoprotein E4 allele is associated with substantial changes in the plasma lipids and hyaluronic acid content in patients with nonalcoholic fatty liver disease.

Fat may affect progression of liver damage in patients with non-alcoholic fatty liver disease (NAFLD). In this study we characterize the state of lipid metabolism in 22 patients with NAFLD and different Apo-E variants. Total concentration of plasma total fatty acids was quantified by gas chromatography, while their derivatives by liquid chromatography/tandem mass spectrometry (LC ESI MS/MS). The ratio of plasma saturated fatty acid to monounsaturated fatty acid increased, whereas the ratio of polyunsaturated fatty acids to saturated fatty acids was reduced in Apo-E4 carriers. Simultaneously, the levels of individual plasma linoleic, arachidonic, and alpha linolenic acids significantly increased in subjects with the Apo-E4 allele. The 15-lipoxygenase metabolite, 13-hydroxyoctadecadienoic acid, was significantly higher in Apo-E3 carriers (p<0.006). 5-oxo-6,8,11,14-eicosatetraenoic acid was significantly elevated in Apo-E4 carriers (p<0.009). A significant difference in hyaluronic acid concentration (p<0.0016) as well as predicted advanced fibrosis (using the BARD scoring system) was found in Apo-E4 carriers (p<0.01). We suggest that a distinct mechanism of fibrosis between Apo E alleles. In Apo-E4 carriers, an elevation in 5-oxo-6,8,11,14-eicosatetraenoic acid synthesis and fatty acid dysfunction may induce fibrosis, while an inflammatory process may be the main cause of fibrosis in Apo-E3 carriers.

Correlation between Endosonographic and Doppler Ultrasound Features of Portal Hypertension in Patients with Cirrhosis.

Purpose. Endoscopic ultrasound (EUS) permits the detailed visualization of clinically significant features of portal hypertension; however, it is an invasive procedure that is not widely available. The aim of this cross-sectional study was to determine whether a correlation exists between the features of portal hypertension detected using both Doppler ultrasound and EUS in subjects with liver cirrhosis. Materials and Methods. Analyzed cohort included 42 patients who underwent a detailed Doppler ultrasound focusing on the parameters of blood flow in the portal/splenic vein as well as an endoscopic/EUS procedure that included the assessment of the size and localization of "deep" varices. Results. The size of "deep" oesophageal varices detected with EUS exhibited no correlation with the parameters assessed by Doppler ultrasound. However, the size of the "deep" gastric varices detected using EUS correlated with the time averaged maximum velocity (T(max) as well as V(min), V(max)) for the portal vein using Doppler ultrasound and exhibited a correlation with the V(max) and T(max) for the splenic vein. No significant correlation was determined between the diameter of the azygous vein and the thickness of the gastric wall when seen on EUS versus the parameters measured with Doppler ultrasound. Conclusion. EUS provides important information regarding the features of portal hypertension, and in the case of "deep" oesophageal varices exhibits a limited correlation with the parameters detected by Doppler ultrasound. Thus, despite its invasiveness, EUS is a method that provides a reliable and unique assessment of the features of portal hypertension in patients with liver cirrhosis.

A new model of peripheral arterial disease: sustained impairment of nutritive microcirculation and its recovery by chronic electrical stimulation.

OBJECTIVES: To develop a model of peripheral arterial disease (PAD) in rat skeletal muscle with sustained impairment of microcirculatory perfusion, and to ascertain whether increased muscle activity can reverse the impairment. METHODS: Three weeks after iliac ligation in rats, the ipsilateral femoral artery was ligated (double ligation, DL), and in some animals, muscle activity was increased by electrical stimulation for 2 weeks (10 Hz, 15 min on, 85 mins off, 7 times per day). Diameter changes of precapillary arterioles to vasoactive agonists and capillary perfusion (flow intermittency, capillary red cell velocity [V(rbc)], and diameters) were measured in extensor digitorum longus muscle and compared with 5 weeks iliac only ligation (single ligation, SL) and controls. Total muscle endothelial nitric oxide synthase (eNOS) was estimated by Western blotting. RESULTS: Whereas single ligation increased intermittency of capillary flow with little effect on V(rbc) and shear stress, DL completely eliminated increases in V(rbc) and shear stress after muscle contractions. Arterial dilation to sodium nitroprusside was attenuated similarly in SL and DL; in SL, acetylcholine induced constriction and bradykinin an attenuated dilation, but in DL vessels were unresponsive to either. Chronic stimulation returned all microcirculatory parameters in DL to normal and increased levels of eNOS protein by 75%. CONCLUSIONS: Femoral artery ligation following iliac ligation impairs arteriolar vasodilator capacity, capillary perfusion, and shear-dependent function of microcirculatory endothelium more than iliac ligation alone and is more representative of long-standing ischemia in PAD. Chronic intermittent electrical stimulation can normalize these derangements.

Differences in local environment determine the site of physiological angiogenesis in rat skeletal muscle.

The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand.

Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia.

OBJECTIVE: To establish the time course and extent of remodeling of terminal microcirculation in ischemic rat skeletal muscle during prolonged low flow that does not lead to inflammation. METHODS: One common iliac artery was ligated via laparotomy in adult Sprague-Dawley rats and extensor digitorum longus (EDL) muscles removed at intervals (1, 2, and 5 weeks) postsurgery. Serial frozen EDL sections were stained to show capillaries (alkaline phosphatase), cell proliferation (antibody to proliferating cell nuclear antigen [PCNA]), terminal microvessels (antibodies to alpha-smooth muscle actin (alpha-SMA) or endothelial nitric oxide synthase [eNOS]), and macrophages (antibodies to infiltrating and resident macrophages). Total muscle eNOS protein was quantified by standard Western blotting techniques. RESULTS: Capillary proliferation was very limited in ischemic EDLs, with a modest 12% increase in the capillary/fiber ratio after 5 weeks, preceded at 2 weeks by increased numbers of PCNA-positive nuclei at capillary sites. There was no muscle necrosis or evidence of inflammation, based on macrophage staining. The number of terminal microvessels that were positive for alpha-SMA and <10 microm in diameter was fewer in ischemic EDLs at all time points, whereas the number of larger positive vessels was unchanged. eNOS-positive vessels <10 microm in diameter were stained similarly throughout ischemic muscles as the controls, and showed a similar increase in vessel/fiber ratio as the capillaries. The total eNOS protein level was similar to that in controls in ischemic EDLs after 1 and 2 weeks, but was 28% lower after 5 weeks. CONCLUSIONS: Prolonged, moderate flow reduction to skeletal muscles does not necessarily lead to inflammation or extensive capillary growth. Based on eNOS staining, the terminal microcirculation remains intact, but the loss of alpha-SMA immunoreactivity may indicate remodeling involving the "deinvestment" of microvessels by smooth muscle.

Hypoxia and expression of VEGF-A protein in relation to capillary growth in electrically stimulated rat and rabbit skeletal muscles.

To investigate the role of hypoxia as a stimulus to the early upregulation of vascular endothelial growth factor (VEGF) in fast skeletal muscles during chronic low frequency stimulation, blood flow, oxygen consumption, VEGF expression and capillary : fibre ratio were measured in chronically stimulated tibialis anterior and extensor digitorum longus (EDL) muscles in rabbits and rats. No differences were found in blood flow, oxygen consumption and extraction between rabbit muscles stimulated for 2 or 4 days (8 h on-16 h off) and controls. Muscle P(O(2)) polarographically measured immediately at the end of stimulation on day 2 was also no different from control under resting conditions (10.7 +/- 1.6 vs. 9.5 +/- 1.2 Torr, n.s.). Unlike control muscles, however, P(O(2)) in 2 day stimulated muscles did not increase immediately after a further acute bout of contractions. This difference was not apparent after similar acute contractions in 4 day stimulated muscles. The involvement of VEGF in early angiogenesis in stimulated muscles was studied in serial cryosections of rat EDL. The proportion of capillaries positively immunostained for VEGF increased from 25 +/- 1 % to 40 +/- 1 % (P < 0.05) in muscles removed on day 2 immediately at the end of chronic stimulation; it decreased slightly after 16 h rest, and increased again after 4 days of stimulation. Capillary : fibre ratio was unchanged throughout the experiment. Capillary cell proliferation increased only after the rest period on day 2 (20-fold increase) and day 4 (12-fold increase), indicating angiogenesis in progress. Thus the timing of transient hypoxia and increase in capillary-linked VEGF in stimulated muscles, albeit in different species, was similar, and increased VEGF staining and capillary cell proliferation occurred even after the hypoxia had resolved. This suggests (1) a connection between hypoxia and VEGF during the early stages of stimulation, although ensuing capillary proliferation may thereafter rapidly correct for local hypoxia, and (2) that the subsequent angiogenesis and VEGF expression are dependent on factors other than hypoxia.

Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo.

OBJECTIVE: To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long-term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF). METHODS: Adult rats received prazosin in drinking water ( approximately 2 mg per day) or had extensor digitorum longus (EDL) muscles stimulated by implanted electrodes for up to 14 days. At intervals, serial frozen sections of EDL were stained for alkaline phosphatase to identify capillaries, proliferating cell nuclear antigen (PCNA), and VEGF-A protein. Shear stress was estimated from capillary red blood cell velocities and diameters, measured by direct observation of epi-illuminated EDL. RESULTS: Chronic stimulation and prazosin treatment both increased capillary: fiber ratio by approximately 40% after 14 days. In stimulated muscles, the percentage of capillaries positively stained for VEGF increased within 3 to 4 days, while the density of PCNA-positive capillaries had increased 20-fold after 2 days. With prazosin, VEGF-positive capillaries increased after 2 and 4 days, accompanied by a threefold increase in PCNA. By 14 days, PCNA labeling and VEGF were still high in stimulated muscles, but no longer different from controls with prazosin. After 3 to 4 days of treatment, capillary shear stress in resting muscle was 57% higher than in controls as a result of stimulation, but 4 times higher with prazosin. CONCLUSIONS: Higher capillary shear stress with prazosin than with stimulation may upregulate VEGF expression in the early stages of treatment. Greater proliferation of capillaries preceding a higher proportion of VEGF-positive capillaries in stimulated muscles, in the presence of a modest increase in shear stress, suggests that angiogenesis was initiated by other factors in addition to shear stress.

Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle.

Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 +/- 0.7- and 1.5 +/- 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 +/- 0.08 in GM-6001-treated muscles compared with 1.69 +/- 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 +/- 0.27% in GM-6001-treated muscles compared with 3.72 +/- 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 +/- 9.3 nuclei in control and 63.5 +/- 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.