Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels.

UNLABELLED: Intestinal bacterial flora may induce splanchnic hemodynamic and histological alterations that are associated with portal hypertension (PH). We hypothesized that experimental PH would be attenuated in the complete absence of intestinal bacteria. We induced prehepatic PH by partial portal vein ligation (PPVL) in germ-free (GF) or mice colonized with altered Schaedler's flora (ASF). After 2 or 7 days, we performed hemodynamic measurements, including portal pressure (PP) and portosystemic shunts (PSS), and collected tissues for histomorphology, microbiology, and gene expression studies. Mice colonized with intestinal microbiota presented significantly higher PP levels after PPVL, compared to GF, mice. Presence of bacterial flora was also associated with significantly increased PSS and spleen weight. However, there were no hemodynamic differences between sham-operated mice in the presence or absence of intestinal flora. Bacterial translocation to the spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and blood vessels were more abundant in colonized and in portal hypertensive mice, as compared to GF and sham-operated mice. Expression of the intestinal antimicrobial peptide, angiogenin-4, was suppressed in GF mice, but increased significantly after PPVL, whereas other angiogenic factors remained unchanged. Moreover, colonization of GF mice with ASF 2 days after PPVL led to a significant increase in intestinal blood vessels, compared to controls. The relative increase in PP after PPVL in ASF and specific pathogen-free mice was not significantly different. CONCLUSION: In the complete absence of gut microbial flora PP is normal, but experimental PH is significantly attenuated. Intestinal mucosal lymphatic and blood vessels induced by bacterial colonization may contribute to development of PH.

Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis.

UNLABELLED: The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation. CONCLUSION: Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

Toxicity of valproic acid in mice with decreased plasma and tissue carnitine stores.

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice.

BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms.

Rapamycin is an immunosuppressant with antiproliferative properties. We investigated whether rapamycin treatment of bile duct-ligated (BDL) rats is capable of inhibiting liver fibrosis and thereby affecting hemodynamics. Following BDL, rats were treated for 28 days with rapamycin (BDL SIR). BDL animals without drug treatment (BDL CTR) and sham-operated animals served as controls. After 28 days, hemodynamics were measured, and livers were harvested for histology/immunohistochemistry. Liver mRNA levels of transforming growth factor (TGF)-beta1, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF)-beta, cyclin-dependent kinase inhibitor p27(kip) (p27), and cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) were quantified by real-time polymerase chain reaction. Liver protein levels of p27, p21, p70 S6 kinase (p70(s6k)), phosphorylated p70(s6k) (p-p70(s6k)), eukaryotic initiation factor 4E-binding protein (4E-BP1), p-4E-BP1 (Thr37/46), and p-4E-BP1 (Ser65/Thr70) were determined by Western blotting. Portal vein pressure was lower in BDL SIR than in BDL CTR animals. Volume fractions of connective tissue, bile duct epithelial, and desmin- and actin-positive cells were lower in BDL SIR than in BDL CTR rats. On the mRNA level, TGF-beta1, CTGF, and PDGF were decreased by rapamycin. p27 and p21 mRNA did not differ. On the protein level, rapamycin increased p27 and decreased p21 levels. Levels of nonphosphorylated p70(s6k) and 4E-BP1 did not vary between groups, but levels of p-p70(s6k) were decreased by rapamycin. Rapamycin had no effect on p-4E-BP1 (Thr37/46) and p-4E-BP1 (Ser65/Thr70) levels. In BDL rats, rapamycin inhibits liver fibrosis and ameliorates portal hypertension. This is paralleled by decreased levels of TGF-beta1, CTGF, and PDGF. Rapamycin influences the cell cycle by up-regulation of p27, down-regulation of p21, and inhibition of p70(s6k) phosphorylation.

Nitric oxide synthase 1 is partly compensating for nitric oxide synthase 3 deficiency in nitric oxide synthase 3 knock-out mice and is elevated in murine and human cirrhosis.

BACKGROUND: The role of endothelial nitric oxide synthase 3 (NOS-3) in the hyperdynamic circulation associated with cirrhosis is established but not that of the neuronal (NOS-1) isoform. We therefore investigated aortic NOS-1 levels in NOS-3 knock-out (KO) and wildtype (WT) mice and in hepatic arteries of patients. METHODS: Mice rendered cirrhotic by bile duct ligation (BDL) were compared with sham-operated controls. Hepatic arteries of cirrhotic patients were collected during liver transplantation; donor vessels served as controls. mRNA levels were quantified by real-time PCR, protein levels by Western blotting and NO production by Nomega-nitro-L-arginine methyl ester inhibitable arginine-citrulline assay. RESULTS: Aortae of NOS-3 KO mice exhibited higher NOS-1mRNA (5.6-fold, P < 0.004) and protein levels (8.8-fold) compared with WT. NO production in aortae of NOS-3 KO mice was 52% compared with WT (P = 0.002). BDL increased NOS-1 mRNA (2.4-fold, P = 0.01) and protein (7.1-fold) levels in aortae of WT, but no further in the NOS-3 KO mice. Hepatic artery NOS-1 mRNA levels in cirrhotic patients were markedly increased compared with controls (24.5-fold, P = 0.0007). CONCLUSIONS: Increased NOS-1 mRNA and protein levels and partially maintained in vitro NO-production in aortae of NOS-3 KO mice suggest that NOS-1 may partially compensate for NOS-3 deficiency. BDL-induced increase in aortic NOS-1 mRNA and protein levels hint that not only NOS-3, but also NOS-1 may be involved in the regulation of systemic hyperdynamic circulation and portal hypertension. Upregulation of NOS-1 mRNA levels in hepatic arteries of portal hypertensive patients suggests possible clinical significance for these experimental findings.