Carvedilol impairs bile acid homeostasis in mice: implication for nonalcoholic steatohepatitis.

Carvedilol is a widely used beta-adrenoreceptor antagonist for multiple cardiovascular indications; however, it may induce cholestasis in patients, but the mechanism for this effect is unclear. Carvedilol also prevents the development of various forms of experimental liver injury, but its effect on nonalcoholic steatohepatitis (NASH) is largely unknown. In this study, we determined the effect of carvedilol (10 mg/kg/day p.o.) on bile formation and bile acid (BA) turnover in male C57BL/6 mice consuming either a chow diet or a western-type NASH-inducing diet. BAs were profiled by liquid chromatography-mass spectrometry and BA-related enzymes, transporters, and regulators were evaluated by western blot analysis and qRT-PCR. In chow diet-fed mice, carvedilol increased plasma concentrations of BAs resulting from reduced BA uptake to hepatocytes via Ntcp transporter downregulation. Inhibition of the ß-adrenoreceptor-cAMP-Epac1-Ntcp pathway by carvedilol may be the post-transcriptional mechanism underlying this effect. In contrast, carvedilol did not worsen the deterioration of BA homeostasis accompanying NASH; however, it shifted the spectra of BAs toward more hydrophilic and less toxic α-muricholic and hyocholic acids. This positive effect of carvedilol was associated with a significant attenuation of liver steatosis, inflammation, and fibrosis in NASH mice. In conclusion, our results indicate that carvedilol may increase BAs in plasma by modifying their liver transport. In addition, carvedilol provided significant hepatoprotection in a NASH murine model without worsening BA accumulation. These data suggest beneficial effects of carvedilol in patients at high risk for developing NASH.

Phox2B is a sensitive and reliable marker of paraganglioma-Phox2B immunohistochemistry in diagnosis of neuroendocrine neoplasms.

Phox2B is a transcription factor responsible for chromaffin cell phenotype. Although it is used routinely for diagnosis of neuroblastoma, previous reports concerning its utility in the diagnosis of neuroendocrine neoplasms have been conflicting. We assessed Phox2b immunoreactivity in different neuroendocrine neoplasms. Tissue microarrays or whole sections of 36 paragangliomas (PGs), 91 well-differentiated neuroendocrine tumours of different organs (WDNETs), 31 neuroendocrine carcinomas (NECs), and 6 olfactory neuroblastomas (ONBs) were stained with Phox2B antibody (EP312) and GATA3. The percentage of positive cells and intensity was analysed using H-score. Phox2B immunoreactivity was seen in 97.2% (35/36) PGs, 11% (10/91) WDNETs, 9.7% (3/31) NECs, and 16.7% (1/6) ONBs. PGs were significantly more often positive (p < 0.001, χ2) than other neuroendocrine tumours, showing highest H-score (mean 144.9, SD ± 75.1) and percentage of positive cells (median 81.3%, IQR 62.5-92.5%). Compared to Phox2B-positive WDNETs, PGs showed significantly higher H-score (median 145 vs 7.5, p < 0.001) and percentage of positive cells (median 82.5% vs 4.5%, p < 0.001). Phox2B positivity was 97.2% sensitive and 89% specific for the diagnosis of PG. GATA3 was 100% sensitive and 88% specific for the diagnosis of PG. When combined, any Phox2B/GATA3 coexpression was 97.1% sensitive and 99.1% specific for the diagnosis of paraganglioma. Widespread Phox2B immunoreactivity is a highly characteristic feature of PGs and it can be used as an additional marker in differential diagnosis of neuroendocrine tumours.

Pitx2 is a useful marker of midgut-derived neuroendocrine tumours - an immunohistochemical study of 224 cases.

Pitx2 is a transcription factor responsible for establishment of the right-left axis and development of the gut and pituitary. In mouse embryos, Pitx2 is expressed in the greater curvature of the stomach and midgut. Previously, Pitx2 was studied in pituitary neuroendocrine tumours but not in other NETs. Pitx2 expression was immunohistochemically assessed in whole sections and tissue microarrays in a cohort of 224 neuroendocrine neoplasms, and was analysed in 29 cases. The cohort included 18 cauda equina NETs, 38 paragangliomas, 98 cases of primary visceral NETs from different organs, 23 metastases of visceral NETs and 47 neuroendocrine carcinomas (NECs). Pitx2 expression was observed in 29.5% (29 of 98) NETs and 14.9% (7 of 47) NECs, but was not observed in any paraganglioma or cauda equina NET. Pitx2 was observed only in tumours of midgut-derived organs, including the small intestine (100%, 20 of 20), appendix (88.9%, eight of nine) and large intestine (9.1%, one of 11 - only caecal NET). The NETs of remaining locations were negative. Pitx2 was 96.7% sensitive and 100% specific for NETs of midgut origin. In NECs, Pitx2 positivity was observed in goblet cell adenocarcinoma (75%, three of four), medullary thyroid carcinoma (42.9, three of seven) and one Merkel cell carcinoma (25%, one of four). In metastatic NETs, Pitx2 was observed in all the tumours originating in the small intestine (n = 17) or caecum (n = 1). No positivity was observed in tumours from other locations (four pancreas, one lung). We observed no correlation between immunoreactivity and mRNA expression. Thus, Pitx2 immunohistochemistry can be helpful in assessing the midgut origin of NETs.

Atorvastatin Modulates Bile Acid Homeostasis in Mice with Diet-Induced Nonalcoholic Steatohepatitis.

Bile acids (BA) play a significant role in the pathophysiology of nonalcoholic steatohepatitis (NASH). The present study evaluates the modulation of bile acid metabolomics by atorvastatin, a cholesterol-lowering agent commonly used to treat cardiovascular complications accompanying NASH. NASH was induced in mice by 24 weeks of consuming a high-saturated fat, high-fructose, and high-cholesterol diet (F), with atorvastatin administered orally (20 mg/kg/day) during the last three weeks. Biochemical and histological analyses confirmed the effectiveness of the F diet in inducing NASH. Untreated NASH animals had significantly reduced biliary secretion of BA and increased fecal excretion of BA via decreased apical sodium-dependent bile salt transporter (Asbt)-mediated reabsorption. Atorvastatin decreased liver steatosis and inflammation in NASH animals consistently with a reduction in crucial lipogenic enzyme stearoyl-coenzyme A (CoA) desaturase-1 and nuclear factor kappa light chain enhancer of activated B-cell pro-inflammatory signaling, respectively. In this group, atorvastatin also uniformly enhanced plasma concentration, biliary secretion and fecal excretion of the secondary BA, deoxycholic acid (DCA). However, in the chow diet-fed animals, atorvastatin decreased plasma concentrations of BA, and reduced BA biliary secretions. These changes stemmed primarily from the increased fecal excretion of BA resulting from the reduced Asbt-mediated BA reabsorption in the ileum and suppression of synthesis in the liver. In conclusion, our results reveal that atorvastatin significantly modulates BA metabolomics by altering their intestinal processing and liver synthesis in control and NASH mice.

Metformin impairs bile acid homeostasis in ethinylestradiol-induced cholestasis in mice.

Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

Pre-operative management of Pleomorphic and florid lobular carcinoma in situ of the breast: Report of a large multi-institutional series and review of the literature.

BACKGROUND: Pleomorphic and Florid Lobular carcinoma in situ (P/F LCIS) are rare variants of LCIS, the exact nature of which is still debated. AIM: To collect a large series of P/F LCIS diagnosed on preoperative biopsies and evaluate their association with invasive carcinoma and high grade duct carcinoma in situ (DCIS). Data obtained were compared with those reported in the literature. METHODS: A multi-institutional series of P/F LCIS was retrieved. All cases were diagnosed on pre-operative biopsies, which was followed by an open surgical excision. Data on post-operative histopathology were available. A literature review was performed. RESULTS: A total of 117 cases were collected; invasive carcinoma and/or DCIS was present in 78/117 cases (66.7%). Seventy cases of P/F LCIS were pure on biopsy and 31 of these showed pathological upgrade in post-surgical specimens. Pre-operative biopsy accuracy was 47/78 (60.3%); pre-operative biopsy underestimation of cancer was 31/78 (39,7.%). In the literature review papers, invasive carcinoma or DCIS was associated with 274 of 418 (65.5%) cases of P/F LCIS. Pre-operative biopsy accuracy was 66% (181/274) whereas pre-operative biopsy underestimation of cancer was 33.9% (93/274). CONCLUSIONS: The data presented here indicate that P/F LCIS is frequently associated with invasive carcinoma or high grade DCIS and that pre-operative biopsy is associated with an underestimation of malignancy. Open surgery is indicated when P/F LCIS is diagnosed pre-operatively.