Leandra N. Ramalho

Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans.

There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild‐type and ghrelin‐deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct–ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin‐deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (−994CT and −604GA) influenced the progression of liver fibrosis. Conclusion: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy. (HEPATOLOGY 2010;51:974–985.)

Cigarette smoking exacerbates nonalcoholic fatty liver disease in obese rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal‐regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase‐1 and procollagen‐alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010.)

Atorvastatin attenuates angiotensin II-induced inflammatory actions in the liver.

Statins exert beneficial effects in chronically damaged tissues. Angiotensin II (ANG II) participates in liver fibrogenesis by inducing oxidative stress, inflammation, and transforming growth factor-beta1 (TGF-beta1) expression. We investigate whether atorvastatin modulates ANG II-induced pathogenic effects in the liver. Male Wistar rats were infused with saline or ANG II (100 ng kg(-1) min(-1)) for 4 wk through a subcutaneous osmotic pump. Rats received either vehicle or atorvastatin (5 mg kg(-1) day(-1)) by gavage. ANG II infusion resulted in infiltration of inflammatory cells (CD43 immunostaining), oxidative stress (4-hydroxynonenal), hepatic stellate cells (HSC) activation (smooth muscle alpha-actin), increased intercellular adhesion molecule (ICAM-1), and interleukin-6 hepatic gene expression (quantitative PCR). These effects were markedly blunted in rats receiving atorvastatin. The beneficial effects of atorvastatin were confirmed in an additional model of acute liver injury (carbon tetrachloride administration). We next explored whether the beneficial effects of atorvastatin on ANG II-induced actions are also reproduced at the cellular level. We studied HSC, a cell type with inflammatory and fibrogenic properties. ANG II (10(-8)M) stimulated cell proliferation, proinflammatory actions (NF-kappaB activation, ICAM-1 expression, interleukin-8 secretion) as well as expression of procollagen-alpha(1(I)) and TGF-beta1. All of these effects were reduced in the presence of atorvastatin (10(-7)M). These results indicate that atorvastatin attenuates the pathogenic events induced by ANG II in the liver both in vivo and in vitro. Therefore, statins could have beneficial effects in conditions characterized by hepatic inflammation.

Sonic Hedgehog Signaling Is Active in Human Adrenal Cortex Development and Deregulated in Adrenocortical Tumors

BACKGROUND The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. OBJECTIVES The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. PATIENTS AND METHODS Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. RESULTS SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability. CONCLUSIONS The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

Resveratrol improves vasoprotective effects of captopril on aortic remodeling and fibrosis triggered by renovascular hypertension

BACKGROUND Renin-angiotensin system triggers vascular remodeling and fibrosis during the renovascular hypertensive two-kidney, one-clip (2K1C) model by oxidative-stress-mediated mechanisms. Thus, we hypothesized that the chronic treatment with the polyphenolic antioxidant resveratrol would improve the vasoprotective effects promoted by the chronic treatment with the angiotensin-converting enzyme inhibitor (ACEi) captopril in 2K1C hypertensive rats. Our main objective was to evaluate the effects of the combined treatment with resveratrol and captopril on vascular remodeling and fibrosis in 2K1C rats. METHODS Male Wistar rats underwent to unilateral renal stenosis by 2K1C Goldblatt model. Six weeks after surgery, rat systolic blood pressure (SBP) was measured by indirect tail-cuff plethysmography. 2K1C rats were considered hypertensive when presenting SBP higher than 160 mmHg and underwent resveratrol (20 mg/kg), captopril (6 or 12 mg/kg), or resveratrol (20 mg/kg) combined with captopril (6 or 12 mg/kg) treatment for 3 weeks. Nine weeks after surgery, rat SBP was measured, and rat thoracic aorta was isolated for histological assays with hematoxylin/eosin or Picrosirius Red to evaluate aortic remodeling and fibrosis, respectively. RESULTS Oral treatment of 2K1C hypertensive rats with resveratrol (20 mg/kg) combined with the dose-dependent ACEi captopril (6 and 12 mg/kg) resulted in lesser aortic thickening and reduced aortic fibrosis. Resveratrol (20 mg/kg) promoted a more expressive hypotensive effect with captopril (12 mg/kg) in 2K1C rats than the treatment with isolated captopril (12 mg/kg). CONCLUSION Resveratrol improves the vasoprotective effects promoted by captopril on aortic remodeling and fibrosis during renovascular hypertension probably by synergic mechanisms involving antioxidant actions and nitric oxide generation.

The development of animal infection models and antifungal efficacy assays against clinical isolates of Trichosporon asahii, T. asteroides and T. inkin

The present study developed Galleria mellonella and murine infection models for the study of Trichosporon infections. The utility of the developed animal models was demonstrated through the assessment of virulence and antifungal efficacy for 7 clinical isolates of Trichosporon asahii, T. asteroides and T. inkin. The susceptibility of the Trichosporon isolates to several common antifungal drugs was tested in vitro using the broth microdilution and the E-test methods. The E-test method depicted a lower minimal inhibitory concentration (MIC) for amphotericin and a slightly higher MIC for caspofungin, while MICs observed for the azoles were different but comparable between both methods. All three Trichosporon species established infection in both the G. mellonella and immunosuppressed murine models. Species and strain dependent differences were observed in both the G. mellonella and murine models. T. asahii was demonstrated to be more virulent than the other 2 species in both animal hosts. Significant differences in virulence were observed between strains for T. asteroides in the murine model. In both animal models, fluconazole and voriconazole were able to improve the survival of the animals compared to the untreated control groups infected with any of the 3 Trichosporon species. In G. mellonella, amphotericin was not able to reduce mortality in any of the 3 species. In contrast, amphotericin was able to reduce murine mortality in the T. asahii or T. inkin models, respectively. Hence, the developed animal infection models can be directly applicable to the future deeper investigation of the molecular determinants of Trichosporon virulence and antifungal resistance.

Altered expression of noncanonical Wnt pathway genes in paediatric and adult adrenocortical tumours

The role of planar cell polarity (Wnt/PCP) and calcium‐dependent (Wnt/Ca) noncanonical Wnt pathways in adrenocortical tumours (ACTs) is unknown.

Rosmarinic Acid Suppresses Subconjunctival Neovascularization in Experimental Glaucoma Surgery

Abstract Purpose: Excessive subconjunctival scarring is associated with increased angiogenesis and leads to filtration failure in glaucoma surgery. In this study, we describe that rosmarinic acid (RA) has anti-angiogenic activity during wound healing in a rabbit model of glaucoma surgery. Methods: Forty New Zealand rabbits underwent an experimental trabeculectomy and were randomly allocated into two treatment groups: RA group – treated with subconjunctival injections of 0.1 ml RA (15 mg/ml; n = 20) – and control group – treated with subconjunctival injections of 0.1 ml balanced salt solution (n = 20). The in vivo effect of RA was investigated after 5 and 15 d by measuring the intraocular pressure (IOP; with Tonopen) and bleb area and vascularity (using the Moorfields Bleb Grading System). Vascularization was also studied by counting histological blood vessels and by immunohistochemistry of vascular endothelial growth factor (VEGF) at the surgical site and by quantification of vessels in chicken’s chorioallantoic membrane (CAM), treated with AR 500 μg/ml for 48 h. Results: On the fifth day, eyes of RA group displayed higher bleb area (3.6 ± 0.2 versus 1.8 ± 0.2; p = 0.004) and lower vascularity (3.0 ± 0.5 versus 4.0 ± 0.4; p = 0.009) than controls; however, difference in IOP reduction was not significant (−1.4 ± 0.3 versus −0.8 ± 0.3 mmHg; p = 0.226). Proportion of vessels/field (4.6 ± 0.5 versus 10.4 ± 0.9; p = 0.008) and VEGF immunostaining (15,347 ± 3788 versus 31,043 ± 3230; p = 0.019) also declined with RA treatment. However, at the 15th day, none of the parameters were different between the groups, except for vessels/field proportion (5.4 ± 1.0 versus 10.6 ± 1.6; p = 0.035). CAM exposed to AR inhibited vascularization (−45.67 ± 4.74%; p < 0.001). Conclusion: These data indicate RA has a short-term anti-angiogenic effect and could be a potential modulator of neovascularization during subconjunctival healing at glaucoma filtration surgical sites.