Nádia Gonçalves

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.

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.

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.

Attenuation of toll-like receptor 2-mediated innate immune response in patients with alcoholic chronic liver disease.

Background: Alcoholic chronic liver disease (ACLD) is a common form of acquired immunodeficiency.

Interleukin-1 signaling pathway as a therapeutic target in transthyretin amyloidosis

Abstract Introduction: Inflammation is a key pathological hallmark of several neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and familial amyloidotic polyneuropathy (FAP). Among all inflammatory cytokines associated with FAP, IL-1β, in particular, has been implicated in playing a key pathogenic role. In the present study, we sought to investigate whether blocking IL-1β signaling provides disease-modifying benefits in an FAP mouse model. Methods: We assessed the effect of chronic administration of Anakinra, an IL-1 antagonist, on FAP pathogenesis in vivo, using real-time polymerase chain reaction (qPCR), semi-quantitative immunohistochemistry (SQ-IHC), western blot and nerve morphometric analyses. Results: We found that treatment with Anakinra prevents transthyretin (TTR) extracellular deposition in sciatic nerve, protecting unmyelinated nerve fibers from aggregate-induced degeneration. Moreover, Anakinra administration significantly suppressed IL-1 signaling pathway and inhibited apoptosis and nitrative stress. Conclusions: The present work highlights the relevance of the IL-1 signaling pathway in the pathophysiology of FAP. Our results bring to light the importance of non-amyloid targets in the therapeutic strategies for this disorder. Thus, we propose the use of Anakinra as a potential therapeutic agent for TTR-related amyloidosis.

Correlation between plasma levels of apelin and myocardial hypertrophy in rats and humans: possible target for treatment?

Objective: To investigate the effects of left ventricular (LV) pressure overload and diabetes on the apelinergic system. Research design/methods: Pressure overload was established in rats by supra-renal aortic-banding. Six weeks later, diabetes was induced by streptozotocin (65 mg/kg, intraperitoneal), resulting in four groups: sham, banded (BA), diabetic (DM) and diabetic-banded (DM-BA). Twelve weeks later, LV function and structure were evaluated by echocardiography and biopsies and plasma samples collected. Furthermore, plasma samples and LV-endomyocardial biopsies were procured from aortic stenosis and mitral stenosis patients during surgery to evaluate myocardial expression of apelin and APJ-receptor and plasma levels of apelin. Results: Direct correlations between apelin plasma levels and LV-mass index and between apelin and APJ myocardial expression were observed both in humans and rats. Expression of apelin and APJ was not significantly altered by pressure-overload in humans, being downregulated by pressure overload and even more by diabetes in rats. Finally, an inverse correlation between apelin rat plasma levels and its myocardial expression was observed. Conclusions: While apelin/APJ myocardial expression decreases, apelin plasma levels increase in LV hypertrophy. Considering apelins positive inotropic and vasodilator properties, this elevation in apelin plasma levels may represent a compensatory mechanism to maintain inotropism and cardiac output during pressure-overload or diabetic cardiomyopathy.

Fibroblasts endocytose and degrade transthyretin aggregates in transthyretin-related amyloidosis

Transthyretin (TTR)-related amyloidosis is a fatal disorder characterized by systemic extracellular deposition of TTR amyloid fibrils. Mutations in the TTR gene cause an autosomal dominant form of the disease—familial amyloidotic polyneuropathy (FAP). Wild-type (WT) TTR can also form amyloid fibrils in elderly patients with senile systemic amyloidosis. Regression of amyloid deposits in FAP patients who undergo liver transplantation to remove the main source of mutant TTR suggests the existence of mechanisms for the clearance of TTR deposits from the extracellular matrix (ECM), but the precise mechanisms are largely unknown. Because fibroblasts are abundant, playing a central role in the maintenance of the ECM and because the skin is one of the major sites of soluble TTR catabolism, in the present study, we analyzed their role in clearance of TTR aggregates. In vitro studies with a fibroblast cell line revealed that fibroblasts endocytosed and degraded aggregated TTR. Subcutaneous injection of soluble and aggregated TTR into WT mice showed internalization and clearance over time by both fibroblasts and macrophages. Immunohistochemical studies of skin biopsies from V30M patients, asymptomatic carriers, recipients of domino FAP livers as well as transgenic mice for human V30M showed intracellular TTR immunoreactivity in fibroblasts and macrophages that increased with clinical status and with age in transgenic mice. Overall, the present in vitro and in vivo data show that fibroblasts endocytose and degrade TTR aggregates. The function or dysfunction of TTR clearance by fibroblasts may have important implications for the development, progression, and regression of TTR deposition in the ECM.

Curcumin: A multi-target disease-modifying agent for late-stage transthyretin amyloidosis.

Transthyretin amyloidoses encompass a variety of acquired and hereditary diseases triggered by systemic extracellular accumulation of toxic transthyretin aggregates and fibrils, particularly in the peripheral nervous system. Since transthyretin amyloidoses are typically complex progressive disorders, therapeutic approaches aiming multiple molecular targets simultaneously, might improve therapy efficacy and treatment outcome. In this study, we evaluate the protective effect of physiologically achievable doses of curcumin on the cytotoxicity induced by transthyretin oligomers in vitro by showing reduction of caspase-3 activity and the levels of endoplasmic reticulum-resident chaperone binding immunoglobulin protein. When given to an aged Familial Amyloidotic Polyneuropathy mouse model, curcumin not only reduced transthyretin aggregates deposition and toxicity in both gastrointestinal tract and dorsal root ganglia but also remodeled congophilic amyloid material in tissues. In addition, curcumin enhanced internalization, intracellular transport and degradation of transthyretin oligomers by primary macrophages from aged Familial Amyloidotic Polyneuropathy transgenic mice, suggesting an impaired activation of naïve phagocytic cells exposed to transthyretin toxic intermediate species. Overall, our results clearly support curcumin or optimized derivatives as promising multi-target disease-modifying agent for late-stage transthyretin amyloidosis.