Oral Angiotensin-(1-7) Peptide Modulates Intestinal Microbiota Improving Metabolic Profile in Obese Mice.

BACKGROUND: Obesity is a serious health problem that dysregulate Renin-Angiotensin System (RAS) and intestinal microbiota. OBJECTIVE: The present study aimed to evaluate the Angiotensin-(1-7) [ANG-(1-7)] oral formulation effects on obese mice intestinal microbiota. METHODS: Mice were divided into four groups: obese and non-obese treated with ANG-(1-7) and obese and non-obese without ANG-(1-7) during four weeks. RESULTS: We observed a significant decrease in the fasting plasma glucose, total cholesterol, triglycerides, and Low-density lipoprotein levels and increased High-density lipoprotein in animals treated with ANG-(1-7). The histological analysis showed intestinal villi height reduction in mice treated with ANG-(1-7). Additionally, increased Bacteroidetes and decreased Firmicutes (increased Bacteroidetes/ Firmicutes ratio) and Enterobacter cloacae populations were observed in the High-Fat Diet + ANG-(1-7) group. Receptor toll-like 4 (TLR4) intestinal mRNA expression was reduced in the HFD+ANG-(1-7) group. Finally, the intestinal expression of the neutral amino acid transporter (B0AT1) was increased in animals treated with ANG-(1-7), indicating a possible mechanism associated with tryptophan uptake. CONCLUSION: The results of the present study suggest for the first time an interaction between oral ANG-(1-7) and intestinal microbiota modulation.

Oral Probiotic Bifidobacterium Longum Supplementation Improves Metabolic Parameters and Alters the Expression of the Renin-Angiotensin System in Obese Mice Liver.

BACKGROUND: Obesity and non-alcoholic fatty liver disease (NAFLD) have been increasing at an alarming rate worldwide. Bifidobacterium longum (BL), a common member of the human gut microbiota, has important health benefits through several mechanisms. OBJECTIVES: We evaluated the BL supplementation effects on body metabolism and renin-angiotensin components hepatic expression in mice fed a high-fat diet. METHODS: Thirty-two male mice were divided into four groups: standard diet + placebo (ST), standard diet + Bifidobacterium longum (ST + BL), high-fat diet + placebo (HFD) and high-fat diet + Bifidobacterium longum (HFD + BL). Following the obesity induction period, the ST + BL and HFD + BL groups were supplemented with Bifidobacterium longum for 4 weeks. Then, body, biochemical, histological and molecular parameters were evaluated. RESULTS: HFD + BL mice had a significant decrease in adipose tissue mass and blood glucose levels, as well as a significant reduction in blood glucose during an intraperitoneal glucose tolerance test. The treatment also resulted in reduced levels of total cholesterol and hepatic fat accumulation. Moreover, we observed an increase in angiotensin converting enzyme 2 (ACE2) and Mas receptor (MASR) expression levels in BL-treated obese mice. CONCLUSIONS: These data demonstrate that BL may have the potential to prevent obesity and NAFLD by modulating the mRNA expression of renin-angiotensin system components.

Arterial stiffness in black adults from Angola and Brazil.

Ethnicity is an important determinant of blood pressure levels, being black individuals affected more than any other ethnic group. Arterial stiffening, an independent risk factor for hypertension, is also influenced by ethnicity. However, whether black individuals from different continents would have different patterns of arterial stiffening is still unknown. Thus, the authors aimed to compare pulse wave velocity (PWV) in black subjects living in Angola and Brazil. A total of 677 black individuals from two independent cross-sectional studies conducted in Brazil and Angola were included in this analysis. Carotid-to-femoral PWV was measured following the same protocols for both studies, as well as clinical and anthropometric variables. Adjusted PWV was higher in Brazilian blacks than in Angolans, regardless of sex (men from Brazil: 10.7 ± 1.8 vs men from Angola: 9.9 ± 1.8 m/s, P < .001; women from Brazil: 10.3 ± 1.5 vs women from Angola: 9.2 ± 1.3 m/s, P < .001). Although the cf-PWV was higher in Brazilian blacks, the age-related increase in cf-PWV was higher in Angolan men compared to Brazilians, but not in women. SBP showed the strongest association with cf-PWV, regardless of sex and country. However, age was associated with cf-PWV in all groups, except in Brazilian men. Our results clearly show a difference in PWV between two black populations, and highlight for sex differences in the hemodynamic parameters that might affect blood pressure levels in these populations.

Retraction notice to Plexin-B1 and Semaphorin 4D Cooperate to Promote Perineural Invasion in a RhoA/ROK-Dependent Manner Am J Pathol 180 (2012) 1232-1242.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article is being retracted following correspondence from the Office of Accountability and Compliance at the University of Maryland, Baltimore. An internal investigation into this manuscript by the University of Maryland, Baltimore, found evidence that there are errors with the presentation of the standard deviations and statistical significance shown in Figure 6 which are not supported by the original data, and that these inaccuracies warrant retraction to correct the scientific record. Despite extensive efforts, the journal was unable to contact Dr. Ying-hua Yang and Dr. Hua Zhou with regard to this retraction.

Regulation of Amyloid ß Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex.

The prion protein (PrPC) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrPC, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrPC and mGluR5 are co-receptors also for ß-amyloid oligomers (AßOs) and have been shown to modulate toxicity and neuronal death in Alzheimer's disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrPC-mGluR5 or AßO-PrPC-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrPC-mGluR5 has an important role in AßO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrPC (Ln-γ1) binds to PrPC and induces intracellular Ca2+ increase in neurons via the complex PrPC-mGluR5. Ln-γ1 promotes internalization of PrPC and mGluR5 and transiently decreases AßO biding to neurons; however, the peptide does not impact AßO toxicity. Given that mGluR5 is critical for toxic signaling by AßOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrPC-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrPC-mGluR5 may modulate signaling intensity by different PrPC ligands.

Evaluation of the antineoplastic activity of gallic acid in oral squamous cell carcinoma under hypoxic conditions.

The purpose of the current study was to develop and test a theoretical model that could explain the mechanism of action of gallic acid (GA) in the oral squamous cell carcinoma context for the first time. The theoretical model was developed using bioinformatics and interaction network analysis to evaluate the effect of GA on oral squamous cell carcinoma. In a second step to confirm theoretical results, migration, invasion, proliferation, and gene expression (Col1A1, E-cadherin, HIF-1α, and caspase-3) were performed under normoxic and hypoxic conditions. Our study indicated that treatment with GA resulted in the inhibition of cell proliferation, migration, and invasion in neoplastic cells. Observation of the molecular mechanism showed that GA upregulates E-cadherin expression and downregulates Col1A1 and HIF-1α expression, suggesting that GA might be a potential anticancer compound. In conclusion, the present study demonstrated that GA significantly reduces cell proliferation, invasion, and migration by increasing E-cadherin and repressing Col1A1.

Leptin Receptor Gene Gln223Arg Polymorphism Is Not Associated with Hypertension: A Preliminary Population-Based Cross-Sectional Study.

Hypertension is responsible for high morbidity and mortality as one of the most important cardiometabolic risk factors. The aim of the study was to investigate whether the Gln223Arg in the leptin receptor (LEPR) influences the prevalence of hypertension. A cross-sectional study was carried out in individuals aged ≥ 18 years. Polymorphism identification was performed using PCR-RFLP analysis. Participants with blood pressure ≥ 140/90 mmHg or medication use were considered hypertensive. Frequencies, means, cross-tabulations, and multivariate models were produced to study differences in hypertension prevalence by genotypes. The study includes 470 participants. The frequency of GG polymorphism variant was 10.43%, 46.81% AG, and 42.77% AA. The distribution of hypertension frequency by LEPR genotypes was the following: AA 43.8%, AG 40.4%, and GG 40.8%; there were no significant differences between groups. Comparative analysis which used multivariate Poisson regression adjusted by many potential confounders (age, sex, schooling, smoking, alcohol intake, obesity, and family history of parental obesity) did not modify this result. In this large sample of population-based study, the association of the LEPR Gln223Arg gene polymorphism with hypertension was not observed.

Relationship between microRNA expression levels and histopathological features of dysplasia in oral leukoplakia.

BACKGROUND: Increased expression of microRNAs (miRNAs), miR-21, miR-345, and miR-181b has been demonstrated in oral leukoplakia (OL) that progresses to oral squamous cell carcinoma (OSCC), suggesting a miRNA signature with potential prognostic value. On the basis of these findings, this pilot study aimed to investigate the cytological and histopathological features that are used to grade oral dysplasia and determine associations with the expression of these 3 potentially cancer-related miRNAs. We also compared the expression levels of these miRNAs in OL with normal oral mucosa and OSCC. METHODS: We evaluated miRNA expression by qPCR in 22 samples of OL demonstrating different grades of dysplasia, as well as 17 cases of OSCC, and 6 samples of normal oral mucosa. We associated the miRNAs expression profiles with cytological and histopathological features of OL. RESULTS: OSCC cases showed increased expression of all 3 miRNAs when compared with OL and normal oral mucosa. Increased expression of miR-21 was also observed in OL when compared with normal oral mucosa. We found a higher expression of miR-21 and miR-181b in OL that presented with an increased number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromasia. Increased expression of miR-21 was also detected in OL with abnormally superficial mitosis. Higher expression of miR-345 was observed in OL with an increased number and size of nucleoli or increased nuclear/cytoplasmic ratio. CONCLUSIONS: In conclusion, the present study shows that some cytological and histopathological parameters used to grade dysplasia are associated with altered miRNA expression.

Immunohistochemical analysis of TIMP-3 and MMP-9 in actinic keratosis, squamous cell carcinoma of the skin, and basal cell carcinoma.

The expression of metalloproteinases and their inhibitors has been related to different invasive and metastatic potentials in cancer. This study aims to investigate the immunohistochemical expression of TIMP-3 and MMP-9 in samples of basal cell carcinoma (BCC), squamous cell carcinoma of the skin (SCC), and actinic keratosis (AK). Immunohistochemistry was performed to evaluate the expression of TIMP-3 and MMP-9 in samples of BCC (n=22), SCC (n=10), and AK (n=15). Ten fields of both tumor parenchyma and tumor stroma were photographed and counted in image software. The ratio of positive cells to total cells was used to quantify the staining. A higher expression of MMP-9 was found in tumor stroma of SCC compared to BCC and AK. No significant differences in TIMP-3 expression were observed among the groups. Considering the well-described differences between these neoplasms, these results provide additional evidence of the role of MMP-9 in tumor invasiveness of keratinocyte-derived tumors.

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein.

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild-type astrocytes by 3-fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia-mediated neuronal death in a prion protein-dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.

Increased prion protein processing and expression of metabotropic glutamate receptor 1 in a mouse model of Alzheimer's disease.

Prion protein (PrP(C) ), a glycosylphosphatidylinositol-anchored protein corrupted in prion diseases, has been shown recently to interact with group I metabotropic glutamate receptors (mGluRs). Moreover, both PrP(C) and mGluRs were proposed to function as putative receptors for ß-amyloid in Alzheimer's disease. PrP(C) can be processed in neurons via α or ß-cleavage to produce PrP(C) fragments that are neuroprotective or toxic, respectively. We found PrP(C) α-cleavage to be 2-3 times higher in the cortex of APPswe/PS1dE9 mice, a mouse model of Alzheimer's disease. A similar age-dependent increase was observed for PrP(C) ß-cleavage. Moreover, we observed considerable age-dependent increase in cortical expression of mGluR1, but not mGluR5. Exposure of cortical neuronal cultures to ß-amyloid oligomers upregulated mGluR1 and PrP(C) α-cleavage, while activation of group I mGluRs increased PrP(C) shedding from the membrane, likely due to increased levels of a disintegrin and metalloprotease10, a key disintegrin for PrP(C) shedding. Interestingly, a similar increase in a disintegrin and metalloprotease10 was detected in the cortex of 9-month-old APPswe/PS1dE9 animals. Our experiments reveal novel and complex processing of PrP(C) in connection with mGluR overexpression that seems to be triggered by ß-amyloid peptides. Prion protein (PrP(C) ) and metabotropic glutamate receptors (mGluR) are implicated in Alzheimer's disease (AD). We found age-dependent increase in PrP(C) processing, ADAM10 and mGluR1 levels in AD mouse model. These changes could be reproduced in cultured cortical neurons treated with Aß peptide. Our findings suggest that increased levels of Aß can trigger compensatory responses that may affect neuronal toxicity.

Prognostic value of microvessel density and p53 expression on the locoregional metastasis and survival of the patients with head and neck squamous cell carcinoma.

Cancer cells need to develop microvessels in order to grow and to establish metastatic foci. A role for the p53 protein in the regulation of the angiogenic process is suggested. This study aimed to investigate the relationship between immunohistochemical expression of microvessel density (MVD), measured by CD31 staining, and p53 protein with clinicopathologic factors, and survival in head and neck squamous cell carcinoma (n=70). Tumor angiogenesis was estimated by determining MVD in areas with the highest number of stained microvessels (hot spots). Clinicopathologic factors and immunohistochemical data were evaluated by χ statistical test and were submitted to binary logistic regression to analyze the risk of presence of lymph node metastasis. Factors that might predict survival were investigated using Cox proportional hazards tests. Differences were considered statistically significant when P<0.05. The percentage of p53-positive cells showed no association with clinicopathologic parameters and MVD. Patients with locoregional metastasis presented statistically significant higher MVD (P=0.043). Individuals presenting head and neck squamous cell carcinoma in posterior sites (P=0.022; OR=3.644) and higher MVD (P=0.039; OR=3.247) had a significant increase in risk of metastasis occurrence. Multivariate analysis showed that presence of lymph node metastasis was statistically significant for overall survival of head and neck carcinoma patients (P=0.006; OR =2.917). The present data suggest that MVD represents a promising diagnostic tool to identify individuals with increased risk for the development of metastatic disease, which is very indicative of poor prognosis.

Increased circulating angiotensin-(1-7) protects white adipose tissue against development of a proinflammatory state stimulated by a high-fat diet.

INTRODUCTION: The aim of the present study was to evaluate the effect of a transgenic-induced chronic increase of Ang-(1-7) on the expression of inflammatory markers in adipose tissue and the metabolic profile in rats treated with high-fat diet. RESEARCH DESIGN AND METHODS: Transgenic rats expressing an Ang-(1-7)-producing fusion protein (TGR L-3292) and Sprague Dawley (SD) control rats 4 weeks old were treated for 8 weeks with a high-fat diet. Food intake and body weight were measured once a week. Glucose-tolerance and insulin sensitivity tests were performed one week before the sacrifice. At the end of the experiment plasma lipid concentrations were measured in TGR and SD rats. Adipose tissue were weighted and corrected by the body weight. Proinflammatory markers in adipose tissue were analyzed using Western-blotting, real time-PCR and immunohistochemistry. RESULTS: High-fat diet TGR rats presented increased HDL cholesterol levels and decreased abdominal fat mass, without changes in food intake. In addition, rats with increased Ang-(1-7) levels had lower body weight. Molecular analysis revealed decreased IL-1ß and COX-2 in adipose tissue. CONCLUSIONS: Taken together, these results show that chronic high circulating angiotensin-(1-7) levels protect against metabolic stress induced by a high-fat diet decreasing the proinflammatory profile of adipose tissue.

Plexin-B1 and semaphorin 4D cooperate to promote perineural invasion in a RhoA/ROK-dependent manner.

Perineural invasion (PNI) is a tropism of tumor cells for nerve bundles located in the surrounding stroma. It is a pathological feature observed in certain tumors, referred to as neurotropic malignancies, that severely limits the ability to establish local control of disease and results in pain, recurrent growth, and distant metastases. Despite the importance of PNI as a prognostic indicator, its biological mechanisms are poorly understood. The semaphorins and their receptors, the plexins, compose a family of proteins originally shown to be important in nerve cell adhesion, axon migration, and proper central nervous system development. Emerging evidence has demonstrated that these factors are expressed in tissues outside of the nervous system and represent a widespread signal transduction system that is involved in the regulation of motility and adhesion in different cell types. We believe that the plexins and semaphorins, which are strongly expressed in both axons and many carcinomas, play a role in PNI. In this study, we show that plexin-B1 is overexpressed in tissues and cell lines from neurotropic malignancies and is attracted to nerves that express its ligand, semaphorin 4D, in a Rho/Rho kinase-dependent manner. We also demonstrate that nerves are attracted to tumors through this same system of proteins, suggesting that both plexin-B1 and semaphorin 4D are important in the promotion of PNI.

Amyloid-beta oligomers increase the localization of prion protein at the cell surface.

In Alzheimer's disease, the amyloid-ß peptide (Aß) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aß. We show here that Aß oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aß oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aß oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aß oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aß oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aß oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aß oligomers. Our experiments show for the first time that Aß oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma.

AIMS: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. METHODS AND RESULTS: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n=15), actinic cheilitis (AC) (n=30), and lip squamous cell carcinoma (LSCC) (n=27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. CONCLUSIONS: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

Association of mast cell, eosinophil leucocyte and microvessel densities in actinic cheilitis and lip squamous cell carcinoma.

AIMS: To determine the contributions of mast cells (MC), eosinophil leucocytes (EL) and microvessel density (MVD) in lip carcinogenesis, and to establish the relationships between these biomarkers and their possible prognostic value in lip squamous cell carcinoma (LSCC). METHODS AND RESULTS: Archived specimens of lip mucosa (n=13), actinic cheilitis (n=29) and LSCC (n=29) were formalin-fixed, paraffin-embedded, sectioned and stained with toluidine blue and haematoxylin and eosin (H&E) in order to identify MC and EL and to measure their densities. Tumour angiogenesis was estimated by determining, with the use of CD31 antibody MVD in areas with the highest number of stained microvessels ('hot spots'). Progressive increases of MC, EL and MVD were observed during lip tumour development. Correlation analysis revealed positive associations between the biomarkers during tumour progression. In LSCC samples, significant associations were found between MVD values and metastatic disease. On multivariate analysis, MVD was a predictor of risk of cervical metastasis. CONCLUSIONS: The densities of MC, EL and microvessels increase during lip carcinogenesis, and for MC and EL this may be related to the stimulation of tumour angiogenesis. MVD could be a useful predictor of cervical metastasis in LSCC.

P21/ WAF1 and cyclin D1 variants and oral squamous cell carcinoma.

BACKGROUND: Genetic factors are known to be involved in oral squamous cell carcinoma (OSCC) development. METHOD: We evaluated a possible association between CCND1 A870G and P21/WAF1 C98A polymorphisms and OSCC, as well as the impact of the genotypes on protein immunoexpression. The study group consisted of 80 individuals with histopathological diagnosis of OSCC and the control group consisted of 80 healthy individuals without oral lesions and matched by age, sex and tobacco usage. The genotypes were studied by the polymerase chain reaction and restriction fragment length polymorphic analysis. Paraffin-embedded sections were used for immunohistochemical analysis. RESULTS: No statistical association between CCND1 and/or P21/WAF1 genotypes and OSCC was demonstrated, although we found that people harbouring the combined presence of at least one variant allele of both genes showed a 1.8 times more chance of developing OSCC compared to the referent genotype. OSCC tumours from individuals with P21 heterozygous genotype showed a significantly higher immunopositivity than tumours from wild-type individuals. CONCLUSION: The present study did not demonstrate a significant association between CCND1 and / or P21 / WAF1 genotypes and OSCC. However, P21 protein expression in OSCC tumours is affected by P21 / WAF1 genotype.