Wnt signaling in age-related macular degeneration: human macular tissue and mouse model

BACKGROUND: The wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.

METHODS: We examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.

RESULTS: We found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 and Ccl2 (-/-) /Cx3cr1 (gfp/gfp) mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2 (-/-) /Cx3cr1 (-/-) /rd8 retinas.

CONCLUSIONS: Wnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

The host launches an antimicrobial defense program upon infection. A long-held belief is that pathogens prevent host recognition by remodeling their surface in response to different host microenvironments. Yet direct evidence that this happens in vivo is lacking. Here we report that the pathogen Klebsiella pneumoniae modifies one of its surface molecules, the lipopolysaccharide, in the lungs of mice to evade immune surveillance. These in vivo-induced changes are lost in bacteria grown after isolation from the tissues. These lipopolysaccharide modifications contribute to survival in vivo and mediate resistance to colistin, one of the last options to treat multidrug-resistant Klebsiella. This work opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

mRNA Levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue

β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer’s disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbasePLUS, employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

Metabolically-inactive glucagon-like peptide-1(9-36)amide confers selective protective actions against post-myocardial infarction remodelling

BACKGROUND: Glucagon-like peptide-1 (GLP-1) therapies are routinely used for glycaemic control in diabetes and their emerging cardiovascular actions have been a major recent research focus. In addition to GLP-1 receptor activation, the metabolically-inactive breakdown product, GLP-1(9-36)amide, also appears to exert notable cardiovascular effects, including protection against acute cardiac ischaemia. Here, we specifically studied the influence of GLP-1(9-36)amide on chronic post-myocardial infarction (MI) remodelling, which is a major driver of heart failure progression.

METHODS: Adult female C57BL/6 J mice were subjected to permanent coronary artery ligation or sham surgery prior to continuous infusion with GLP-1(9-36)amide or vehicle control for 4 weeks.

RESULTS: Infarct size was similar between groups with no effect of GLP-1(9-36)amide on MI-induced cardiac hypertrophy, although modest reduction of in vitro phenylephrine-induced H9c2 cardiomyoblast hypertrophy was observed. Whilst echocardiographic systolic dysfunction post-MI remained unchanged, diastolic dysfunction (decreased mitral valve E/A ratio, increased E wave deceleration rate) was improved by GLP-1(9-36)amide treatment. This was associated with modulation of genes related to extracellular matrix turnover (MMP-2, MMP-9, TIMP-2), although interstitial fibrosis and pro-fibrotic gene expression were unaltered by GLP-1(9-36)amide. Cardiac macrophage infiltration was also reduced by GLP-1(9-36)amide together with pro-inflammatory cytokine expression (IL-1β, IL-6, MCP-1), whilst in vitro studies using RAW264.7 macrophages revealed global potentiation of basal pro-inflammatory and tissue protective cytokines (e.g. IL-1β, TNF-α, IL-10, Fizz1) in the presence of GLP-1(9-36)amide versus exendin-4.

CONCLUSIONS: These data suggest that GLP-1(9-36)amide confers selective protection against post-MI remodelling via preferential preservation of diastolic function, most likely due to modulation of infiltrating macrophages, indicating that this often overlooked GLP-1 breakdown product may exert significant actions in this setting which should be considered in the context of GLP-1 therapy in patients with cardiovascular disease.

Embracing an integromic approach to tissue biomarker research in cancer: Perspectives and lessons learned

Modern approaches to biomedical research and diagnostics targeted towards precision medicine are generating ‘big data’ across a range of high-throughput experimental and analytical platforms. Integrative analysis of this rich clinical, pathological, molecular and imaging data represents one of the greatest bottlenecks in biomarker discovery research in cancer and other diseases. Following on from the publication of our successful framework for multimodal data amalgamation and integrative analysis, Pathology Integromics in Cancer (PICan), this article will explore the essential elements of assembling an integromics framework from a more detailed perspective. PICan, built around a relational database storing curated multimodal data, is the research tool sitting at the heart of our interdisciplinary efforts to streamline biomarker discovery and validation. While recognizing that every institution has a unique set of priorities and challenges, we will use our experiences with PICan as a case study and starting point, rationalizing the design choices we made within the context of our local infrastructure and specific needs, but also highlighting alternative approaches that may better suit other programmes of research and discovery. Along the way, we stress that integromics is not just a set of tools, but rather a cohesive paradigm for how modern bioinformatics can be enhanced. Successful implementation of an integromics framework is a collaborative team effort that is built with an eye to the future and greatly accelerates the processes of biomarker discovery, validation and translation into clinical practice.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Smart design of scaffolds obtained by biofabrication for tissue engineering applications

A engenharia de tecidos é um domínio tecnológico emergente em rápido desenvolvimento que se destina a produzir substitutos viáveis para a restauração, manutenção ou melhoria da função dos tecidos ou órgãos humanos. Uma das estratégias mais predominantes em engenharia de tecidos envolve crescimento celular sobre matrizes de suporte (scaffolds), biocompatíveis e biodegradáveis. Estas matrizes devem possuir não só elevadas propriedades mecânicas e vasculares, mas também uma elevada porosidade. Devido à incompatibilidade destes dois parâmetros, é necessário desenvolver estratégias de simulação de forma a obter estruturas optimizadas. A previsão real das propriedades mecânicas, vasculares e topológicas das matrizes de suporte, produzidas por técnicas de biofabricação, é muito importante para as diversas aplicações em engenharia de tecidos. A presente dissertação apresenta o estado da arte da engenharia de tecidos, bem como as técnicas de biofabricação envolvidas na produção de matrizes de suporte. Para o design optimizado de matrizes de suporte foi adoptada uma metodologia de design baseada tanto em métodos de elementos finitos para o cálculo do comportamento mecânico, vascular e as optimizações topológicas, como em métodos analíticos para a validação das simulações estruturais utilizando dados experimentais. Considerando que as matrizes de suporte são estruturas elementares do tipo LEGO, dois tipos de famílias foram consideradas, superfícies não periódicas e as superfícies triplas periódicas que descrevem superfícies naturais. Os objectivos principais desta dissertação são: i) avaliar as técnicas existentes de engenharia de tecidos; ii) avaliar as técnicas existentes de biofabricação para a produção de matrizes de suporte; iii) avaliar o desempenho e comportamento das matrizes de suporte; iv) implementar uma metodologia de design de matrizes de suporte em variáveis tais como a porosidade, geometria e comportamento mecânico e vascular por forma a auxiliar o processo de design; e por fim, v) validar experimentalmente a metodologia adoptada.

Soft-computing techniques applied to artificial tissue temperature estimation

Tese dout., Engenharia electrónica e computação - Processamento de sinal, Universidade do Algarve, 2008

Endocrine regulation of extracellular matrix proteins in calcified tissue in teleost fish

Tese dout., Biologia, Universidade do Algarve, 2005

Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor alpha gene

Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants.

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

Tissue temperature estimation with pulse-echo in blood flow presence

Aiming at time-spatial characterization of tissue temperature when ultrasound is applied for thermal therapeutic proposes two experiments were developed considering gel-based phantoms, one of them including an artificial blood vessel. The blood vessel was mimicking blood flow in a common carotid artery. For each experiment phantoms were heated by a therapeutic ultrasound (TU) device emitting different intensities (0.5, 1, 1.5, 1.8 W/cm2). Temperature was monitored by thermocouples and estimated through imaging ultrasound transducer's signals within specific special points inside the phantom. The temperature estimation procedure was based on temporal echo-shifts (TES), computed based on echo-shifts collected through image ultrasound (IU) transducer. Results show that TES is a reliable non-invasive method of temperature estimation, regardless the TU intensities applied. Presence of a pulsatile blood flow vessel in the focal point of TU transducer reduces thermal variation in more than 50%, also affecting the temperature variation in the surrounding area. In other words, vascularized tissues require longer ultrasound thermal therapeutic sessions or higher TU intensities and inclusion of IU in the therapeutic procedure enables non-invasive monitoring of temperature. © 2013 IEEE.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.