Roberto Roncon-Albuquerque

Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension

We investigated the endogenous production of apelin and the cardiac and pulmonary effects of its chronic administration in monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Wistar rats were injected with MCT (60 mg/kg sc) or vehicle (day 0). One week later, these animals were randomly treated during 17 days with pyroglutamylated apelin-13 (Pyr-AP13; 200 microg*kg(-1)*day(-1) ip) or a similar volume of saline, resulting in four groups: sham (n = 11), sham-AP (n = 11), MCT (n = 16), and MCT-AP (n = 13). On day 25, right ventricular (RV) and left ventricular (LV) hemodynamic and morphometric parameters were assessed. Tissue and plasma samples were collected for histological and molecular analysis. When compared with sham, the MCT group presented a significant increase of RV mass (166 +/- 38%), diameter of cardiomyocyte (40 +/- 10%), myocardial fibrosis (95 +/- 20%), peak systolic pressure (99 +/- 22%), peak rate of ventricular pressure rise (dP/dt(max); 74 +/- 24%), peak rate of ventricular pressure decline (dP/dt(min); 73 +/- 19%), and time constant tau (55 +/- 16%). In these animals, RV expression of apelin (-73 +/- 10%) and its receptor APJ (-61 +/- 20%) was downregulated, whereas mRNA expression of type B natriuretic peptide (9,606 +/- 713%), angiotensinogen (191 +/- 147%), endothelin-1 (RV, 497 +/- 156%; and LV, 799 +/- 309%), plasmatic levels of apelin (104 +/- 48%), and angiotensin 1-7 (161 +/- 151%) were increased. Chronic treatment with Pyr-AP13 significantly attenuated or normalized these changes, preventing apelin-APJ mRNA downregulation and PH-induced neurohumoral activation of several vasoconstrictors, which exacerbates apelin-APJ vasodilator effects. Therefore, apelin delayed the progression of RV hypertrophy and diastolic dysfunction. Together, these observations suggest that the apelin-APJ system may play an important role in the pathophysiology of PH, representing a potential therapeutic target since it significantly attenuates RV overload and PH-induced neurohumoral activation.

The role of lipopolysaccharide/toll-like receptor 4 signaling in chronic liver diseases.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor that functions as lipopolysaccharide (LPS) sensor and whose activation results in the production of several pro-inflammatory, antiviral, and anti-bacterial cytokines. TLR4 is expressed in several cells of healthy liver. Despite the constant confrontation of hepatic TLR4 with gut-derived LPS, the normal liver does not show signs of inflammation due to its low expression of TLR4 and ability to modulate TLR4 signaling. Nevertheless, there is accumulating evidence that altered LPS/TLR4 signaling is a key player in the pathogenesis of many chronic liver diseases (CLD). In this review, we first describe TLR4 structure, ligands, and signaling. Later, we review liver expression of TLR4 and discuss the role of LPS/TLR4 signaling in the pathogenesis of CLD such as alcoholic liver disease, nonalcoholic fatty liver disease, chronic hepatitis C, chronic hepatitis B, primary sclerosing cholangitis, primary biliary cirrhosis, hepatic fibrosis, and hepatocarcinoma.

Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice

AIMS Although toll-like receptors (TLR) are known to mediate the metabolic complications of obesity, the mechanisms underlying its activation remain largely unknown. The present study analyzed a model of diet-induced obesity in mice lacking the TLR4/TLR2 co-receptor CD14. MAIN METHODS Six-week-old male mice lacking CD14 (n = 16) were allocated to either a control diet or a high-fat high-simple carbohydrate diet (5.4 kcal/g; 35% fat; 35% sucrose), and compared with C57BL/6 (WT; n = 15) controls. After 12 weeks, body composition, basal sympathetic activity, non-invasive blood pressure and glucose tolerance were evaluated. Hepatic and adipose tissues were collected for mRNA quantification, histology and LPS incubation. KEY FINDINGS In both WT and CD14 knockout mice, obesity was accompanied by TLR2 and TLR4 upregulation. However, obese mice lacking CD14 presented decreased lipid and macrophage content in hepatic and adipose tissues, lower urinary levels of noradrenaline, decreased systolic blood pressure, reduced fasting plasma glucose and blunted glucose intolerance, compared with obese WT group. In the presence of exogenous sCD14, adipose tissue incubation with LPS-induced TLR2 and TNF-alpha upregulation in both WT and CD14 knockout obese mice. SIGNIFICANCE In our model of diet-induced obesity, mice lacking CD14 showed lower adiposity and hepatic steatosis, improved glucose homeostasis, blunted sympathetic overactivity and reduced blood pressure elevation. This was observed in the presence of preserved TLR4 and TLR2 gene expression, and intact TLR4 signaling pathways. These results suggest that CD14-mediated TLR activation might contribute to the cardiovascular and metabolic complications of obesity.

Functional polymorphisms of Toll-like receptors 2 and 4 alter the risk for colorectal carcinoma in Europeans

BACKGROUND Colon carcinogenesis is associated with increased expression levels of Toll-like receptor 2 and Toll-like receptor 4. AIM To determine in a Caucasian population the role of Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in colorectal cancer development. METHODS Hospital based multicentre case control study involving 193 colorectal cancer patients and 278 healthy individuals. DNA samples were extracted from blood cells and genotyping of TLR2+597T>C, TLR2-4760T>C, TLR4-3745A>G, TLR2Arg753Gln, TLR4Asp299Gly was performed. Functionality of risk polymorphisms was evaluated through production of TNF-α in cell culture and Toll-like receptors levels quantified by real-time RT-PCR. RESULTS TLR2+597CC homozygous had 5-fold decreased risk (odds ratio (OR)=0.21, 95% CI: 0.09-0.50, p<0.001) and TLR4 299Gly homozygous 3-fold increased risk of colorectal cancer (OR=3.30, 95% CI: 1.18-9.28, p=0.015). In stratified analysis, TLR2+597CC genotype protective effect was even higher in overweight individuals (OR=0.17, 95% CI: 0.06-0.53, p<0.001) and in never smokers (OR=0.11, 95% CI: 0.02-0.51, p=0.001). Also, the increased risk effect for TLR4 299Gly homozygous genotype was higher in overweight individuals (OR=8.67, 95% CI: 1.11-87.85, p=0.011). TLR2+597T>C polymorphism conferred 41% less (p=0.03) and TLR4Asp299Gly 65% more TNF-α production (p=0.02) with no differences in Toll-like receptors levels. CONCLUSION Functional Toll-like receptor 2 and Toll-like receptor 4 polymorphisms significantly alter the risk to have colorectal cancer. Obesity and smoking may influence the risk for colorectal cancer in individuals presenting these genetic profiles.

Increased Expression of Toll-like Receptors (TLR) 2, 4 and 5 in Gastric Dysplasia

TLRs are important innate immunity receptors. Even though TLR2, 4 and 5 appear to be important for Helicobacter pylori (HP) recognition, their role in the evolution of gastritis to more advanced lesions is still unknown. To compare the expression of TLR2, 4 and 5 in normal gastric mucosa, HP+ gastritis, intestinal metaplasia, dysplasia and adenocarcinoma. Immunohistochemistry for TLR2, 4 and 5 was performed with anti-TLR2-TLR4-TLR5 antibodies in 117 histological samples of normal gastric mucosa (n = 22), HP+ gastritis (n = 20), intestinal metaplasia (n = 33), dysplasia (mucosectomy specimens, n = 20) and intestinal type adenocarcinoma (surgery specimens,n = 22); quantification of expression was performed independently by two pathologists taking into account the percentage of positive epithelial cells and the degree of expression (zero to three score). A statistically significant trend for progressive increase of TLRs expression from normal mucosa to gastric dysplasia was found (mean expression: normal mucosa 0.1; gastritis 1.0; metaplasia 2.2; dysplasia 2.8, p < 0.01). All dysplasia samples presented more than 90% positive epithelial cells with strong expression (2.8;95%CI2.7–3). There was less TLRs expression in carcinomas (TLR2:1.0; TLR4:2.0 and TLR5:1.2, p < 0.05) when compared with dysplasia, with TLR4 being more expressed than TLR2 and 5 in these lesions (p = 0.03). A score of all markers’ expression of eight leads to a low (4%) false positive rate in patients with precancerous conditions. Progression of gastric lesions associated with gastric carcinogenesis is associated with increased TLRs expression. Gastric dysplasia presents a high level of TLRs expression, suggesting that these receptors may play a role in adenocarcinoma development.

Helicobacter pylori Induces Increased Expression of Toll‐Like Receptors and Decreased Toll‐Interacting Protein in Gastric Mucosa that Persists Throughout Gastric Carcinogenesis

Toll‐like receptors (TLR) are essential for Helicobacter pylori (HP) recognition. Their role in the progression of gastric lesions leading to cancer is not established.

Ghrelin expression in human and rat fetal lungs and the effect of ghrelin administration in nitrofen-induced congenital diaphragmatic hernia

Ghrelin is a strong physiologic growth hormone secretagogue that exhibits endocrine and non-endocrine actions. In this study, ghrelin expression in humans and rats was evaluated throughout development of normal and hypoplastic lungs associated with congenital diaphragmatic hernia (CDH). Additionally, the effect of antenatal treatment with ghrelin in the nitrofen-induced CDH rat model was tested. In normal lungs, ghrelin was expressed in the primitive epithelium at early stages of development and decreased in levels of expression with gestational age. In hypoplastic lungs ghrelin was overexpressed in both human and rat CDH fetuses when compared with controls. Exogenous administration of ghrelin to nitrofen-treated dams led to an attenuation of pulmonary hypoplasia of CDH pups. Furthermore, the growth hormone, secretagogue receptor (GHSR1a), could not be amplified from human or rat fetal lungs by RT-PCR. In conclusion, of all the lungs studied so far, the fetal lung is one of the first to express ghrelin during development and might be considered a new source of circulating fetal ghrelin. Overexpression of ghrelin in hypoplastic lungs and the effect of exogenous administration of ghrelin to nitrofen-treated dams strongly suggest a role for ghrelin in mechanisms involved in attenuation of fetal lung hypoplasia, most likely through a GHSR1a-independent pathway.

Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence:

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66 ± 0.69; TLR4: 3.11 ± 0.79; P < 0.05) and cirrhosis (TLR2: 2.14 ± 0.5; TLR4: 1.74 ± 0.27; P < 0.05) and decreased in hepatocarcinoma (TLR2: 0.48 ± 0.15; TLR4: 0.54 ± 0.10; P < 0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24 ± 0.79; COX-2: 2.47 ± 0.36; P < 0.05) and cirrhosis (TNF-α: 1.73 ± 0.28; COX-2: 1.8 ± 0.35, P < 0.05), whereas NF-κB mRNA was increased in hepatitis (2.42 ± 0.31; P < 0.05) and unchanged in cirrhosis (1.34 ± 0.17; P = 0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63 ± 0.15; P < 0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52 ± 0.12) and TNF-α (0.52 ± 0.12; P < 0.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence.

Thymulin inhibits monocrotaline-induced pulmonary hypertension modulating interleukin-6 expression and suppressing p38 pathway

The pathogenesis of pulmonary hypertension (PH) includes an inflammatory response. Thymulin, a zinc-dependent thymic hormone, has important immunobiological effects by inhibiting various proinflammatory cytokines and chemokines. We investigated morphological and hemodynamic effects of thymulin administration in a rat model of monocrotaline (MCT)-induced PH, as well as the pattern of proinflammatory cytokine gene expression and the intracellular pathways involved. Adult Wistar rats received an injection of MCT (60 mg/kg, sc) or an equal volume of saline. One day after, the animals randomly received during 3 wk an injection of saline, vehicle (zinc plus carboxymethyl cellulose), or thymulin (100 ng/kg, sc, daily). At d 23-25, the animals were anesthetized for hemodynamic recordings, whereas heart and lungs were collected for morphometric and molecular analysis. Thymulin prevented morphological, hemodynamic, and inflammatory cardiopulmonary profile characteristic of MCT-induced PH, whereas part of these effects were also observed in MCT-treated animals injected with the thymulins vehicle containing zinc. The pulmonary thymulin effect was likely mediated through suppression of p38 pathway.

Ghrelin and ghrelin receptor inhibitors: agents in the treatment of obesity.

Background: Current medical treatment of obesity is highly ineffective. Soon after its discovery as the endogenous ligand for the growth hormone secretagogue-receptor (GHS-R), ghrelin was shown to stimulate food intake (including in humans) and promote body weight gain and adipogenesis. Objectives: This review discusses the role of the ghrelin/GHS-R pathway in energy homeostasis regulation and its role as a novel molecular target for the treatment of obesity. Methods: Medline was searched for relevant articles published in English. Results/conclusion: A large series of animal studies shows that inhibition of the ghrelin/GHS-R pathway reduces food intake, body weight and adiposity, through reduction of appetite and augmentation of energy expenditure and fat catabolism. This suggests that inhibition of this novel pathway may be used to treat/prevent obesity and its complications.