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Background/Aim: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation., Materials and Methods: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were downregulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule., Conclusion: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues., peer-reviewed

Orientador: João Ernesto de Carvalho Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Astronium fraxinifolium Schott., conhecido popularmente como "Gonçalo Alves", é uma espécie vegetal característica de regiões tropicais e tem sua distribuição no Cerrado Brasileiro, possui o caule e as folhas ricos em substâncias tânicas. Relatados populares indicam a utilização de A. fraxinifolium no tratamento de diarreias, disenterias, anti-séptico, anti-microbiano, antihemorrágico, cicatrizante, anti-inflamatório e antiulcerogênico. Devido o uso popular e a insuficiência de dados na literatura, este trabalho teve por objetivo avaliar as potenciais atividades farmacológicas desta espécie em modelos experimentais in vitro (atividade antiproliferativa em cultura de células) e in vivo (toxicidade aguda, modelos de nocicepção, inflamação e tumor sólido murino) O material vegetal (cascas do caule e folhas) obtido das coletas foram secos e triturados, o pó obtido foi utilizado para obtenção dos extratos por maceração mecânica e sistema Soxhlet. Estes foram avaliados conforme seu rendimento e demonstraram a presença de taninos hidrolisáveis, como compostos majoritários, e flavonoides. No teste realizado em cultura de células tumorais e não tumorais de diferentes origens os extratos apresentaram, em sua maioria, uma ação citostática na maior concentração testada (250µg/mL). Por outro lado, nos testes relacionados com atividade antinociceptiva extrato bruto das folhas demonstrou atividade nas doses de 100 e 200mg/Kg no modelo da formalina em ambas as fases. Nos modelos inflamatórios (úlcera induzida por indometacina, edema de pata induzido por carragenina e edema de orelha induzido por óleo de cróton) as doses de 100 e 200mg/Kg, no modelo de edema de pata induzido por carragenina, e as doses de 150 e 300mg/Kg, no modelo de edema de orelha induzido por óleo de cróton, demonstraram atividade anti-inflamatória. Considerando que aproximadamente 25% dos tumores estão relacionados com inflamações crônicas, o extrato das folhas de A. fraxinifolium foi avaliado no modelo experimental do tumor sólido de Ehrlich no flanco, demonstrando inibição do crescimento tumoral na dose de 100mg/Kg. Sendo assim, os resultados experimentais sugerem que a espécie A. fraxinifolium possui atividade antiinflamatória dos componentes do extrato bruto, corroborando com sua utilização popular. Além de promover inibição do crescimento tumoral indicando que os componentes do extrato podem atuar em outros processos além do inflamatório. Porém, outros estudos necessitam ser realizados para identificar a classe de compostos que promoveram as atividades farmacológicas da espécie Abstract: Astronium fraxinifolium Schott., popularly known as "Gonçalo Alves", is a species characteristic of tropical regions with their distribution in the Brazilian Cerrado, has stems and leaves rich in tannic substances. Reported the use of folk medicinal indicate A. fraxinifolium in the treatment of diarrhea, dysentery, antiseptic, anti-microbial, antihaemorrhagic, wound healing, anti-inflammatory and antiulcerogenic. Due to popular use and insufficient data in the literature, this study aimed to evaluate potential pharmacological activities of this species in vitro experimental models (antiproliferative activity in cell culture) and in vivo (acute toxicity models of nociception, inflammation and tumor solid murine) The plant material (stems and leaves) samples were obtained from dried and crushed, the powder obtained was used to obtain the extracts, mechanical maceration and soxhlet system. These were evaluated according to their yield and showed the presence of hydrolyzable tannins, as main compounds, and flavonoids. Testing conducted on cultured tumor and non-tumor cells of different origins, the extracts showed mostly a cytostatic action at the highest concentration tested (250?g/mL). Moreover, in tests related antinociceptive crude extract of the leaves showed activity at doses of 100 and 200mg/Kg in the formalin model in both phases. In inflammatory models (indomethacin-induced ulcer, paw edema induced by carrageenan and ear edema induced by croton oil) doses of 100 and 200mg/Kg in paw edema induced by carrageenan model, and the doses of 150 and 300mg/kg, in the ear edema induced by croton oil model, demonstrated anti-inflammatory activity. Considering that approximately 25% of tumors are associated with chronic inflammation, the extract of A. fraxinifolium was assessed in the experimental model of Ehrlich solid tumor in the flank, demonstrating inhibition of tumor growth at a dose of 100mg/kg. Thus, experimental results suggest that the species A. fraxinifolium has anti-inflammatory component of the crude extract activity, confirming its popular use. In addition to promoting tumor growth inhibition indicating that extract components can act in addition to other inflammatory processes. However, other studies should be performed to identify the class of compounds that promoted the pharmacological activity of the species Mestrado Fármacos, Medicamentos e Insumos para Saúde Mestre em Biociências e Tecnologia de Produtos Bioativos

[eng] Two decades ago, microfluidic technology begun to make its appearance in the fields of drug delivery and biomedical engineering to irrevocably revolutionize them. It was quickly realized how microchannels can aid formulation of microdroplets, microparticles and nanoparticles (NPs). They offer very small and controlled environment for reaction, that is unreproduced in bulk methods. As a result, the formulation is not limited only to the modification of compounds, but the flowing microvolumes open gates to the unexplored world of controllable mixing time and diffusion region impacting the formation of nanoparticles. Beyond the drug delivery systems formulation, the microfluidic technology is emerging as a gap-bridging element of the in vitro and in vivo tests in preclinical trials. Biocompatible and microscopy- friendly microfluidic chips are used to reconstruct physiological elements of human tissues (organ-on-a- chip). They recapitulate 3D, dynamic in vivo environment, that is lacking in 2D cell culture, revealing their relevance in understanding the development of a disease and screening of drug delivery candidates. This work presents the use of microfluidic technology in the formulation of tunable size amphiphilic block co-polymer nanoparticles for drug delivery. The particle diameter is modified in the response to studied phase flow rates. The impact of fluidic parameters on drug/dyes encapsulation efficiency and NP size are analyzed using traditional bulk methods, as well as techniques with single particle resolution, such as Transmission Electron Microscopy (TEM) and Total Internal Reflection Fluorescence (TIRF). Furthermore, the NPs are bioevaluated with in vitro tests performed on MCF-7 cell line. Following the NPs formulation, a chip for combinatorial mixing of NP precursors is presented. A passive micromixer is designed, prototyped and evaluated with fluorescent dyes, to visualize the mixing efficiency. Finally, the model is microfabricated in glass and re-assessed in terms of mixing and cleaning efficiency, which previously was poor due to the absorption of small molecules by PDMS. The micromixer is built into a platform for NPs formulation and first proof-of-concept experiments are performed, yielding monodisperse nanoparticles with encapsulated fluorescent dyes. The encapsulation of dyes is visualized in single particles with TIRF microscopy. The last part of the thesis takes the microfluidic technology into organ-on-a-chip, where a reconstruction of tumor blood vessel model is presented. It recapitulates elements of tumor 3D microenvironment such as blood vessel, endothelial barrier, extracellular matrix and cancer cell spheroid. Observed in vivo leakiness of endothelial barrier is reproduced here in the presence of cancer cells. In this work the microscopy- friendly chip is used as a platform for time- and space-resolved monitoring of micelles stability followed during their interaction with the reconstructed barriers mentioned above. The special optical properties of perfused micelles allow to distinguish assembled from disassembled form. The results are consulted with previously reported observations in 2D cell culture, revealing significant difference in cellular uptake between the two studies. Overall, this work demonstrates how multidisciplinary approach of incorporation of microfluidic technology into formulation and screening of potential drug nanocarriers can accelerate development of nanomedicine. The proposed solutions deliver tunability of nanoparticle properties, combinatorial formulation to create library of NPs and a complementary method in in vitro screening., [spa] Hace dos décadas, la tecnología microfluídica hizo su aparición en los campos de la industria farmacéutica y la ingeniería biomédica de manera revolucionaria. Rápidamente se descubrió cómo los microcanales pueden ayudar a la formulación de microgotas, micropartículas y nanopartículas. Ofrecen entornos de reacción muy pequeños y controlados comparados con la formulación de los métodos tradicionales. En consecuencia, la formulación no sólo se limita a la modificación de compuestos, sino que los flujos de microvolúmenes posibles con la tecnología abren puertas a un mundo inexplorado para la formulación de nanopartículas a través del control del tiempo de mezcla y el área de difusión. Más allá de la formulación de los sistemas de fármacos, la tecnología microfluídica está emergiendo como un elemento puente de las pruebas in vitro e in vivo en los ensayos preclínicos. Los chips de microfluidica biocompatibles y aptos para microscopía se utilizan para reconstruir elementos fisiológicos de tejidos humanos (órgano en un chip). Recapitulan el entorno dinámico in vivo en 3D, carente en el cultivo celular en 2D, desvelando su relevancia para comprender el desarrollo de una enfermedad y la detección de fármacos candidatos para la administración. Este trabajo presenta el uso de la tecnología microfluídica en la formulación de nanopartículas de copolímeros de bloques anfifílicos de tamaño ajustable en respuesta a los caudales de las fases estudiadas. Se estudia el impacto de los parámetros de flujo sobre la eficiencia de encapsulación de fármacos/colorantes y el tamaño de NP. Además, se presenta un chip para la formulación combinatoria de nanopartículas fluorescentes, con potenciales aplicaciones en medicina personalizada. La última parte de la tesis traslada la tecnología de microfluidos a órgano en un chip, donde se presenta la reconstrucción del modelo de vaso sanguíneo tumoral. Recapitula las fugas observadas in vivo de la barrera endotelial en presencia de células tumorales. En este trabajo, se utiliza como una plataforma para el monitorización en el tiempo y en el espacio de la estabilidad de las micelas, mientras interactúan con las barreras reconstruidas que se encuentran en el cuerpo humano: vasos sanguíneos, barrera endotelial, matriz extracelular y esferoide multicelular de células cancerosas.

Post-translational control of PERIOD stability by Casein Kinase 1δ and ε (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the kinase activation loop and determines which sites on mammalian PER2 are preferentially phosphorylated, thereby directly regulating PER2 stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.

The general goal of this thesis has been to progress oncolytic adenovirus therapy for PDAC, by the incorporation of novel preclinical models to test for patient-specific responses and the generation of oncolytic adenoviruses with enhanced therapeutic index. The two main objectives have been the following: i) Evaluate patients-derived organoids (PDOs) technology as a platform to screen for personalized virotherapy in vitro 1) Establishment of a battery of PDOs from PDAC and normal pancreatic tissues, and evaluation of their applicability in the study of adenoviral infection; 2) Screening of a battery of PDOs to identify individual sensitivities to virotherapies, and the effects derived from the combination with chemotherapy; 3) Study virotherapy-responses in metastasis originated from PDOs xenografted in mice; (ii) Improve oncolytic adenovirus potency by modulation of miRNAs deregulated in PDAC 4) Screening of aberrantly expressed miRNAs sensitizing viral oncolysis in PDAC via CRISPR/Cas9 system; 5) Generation of a miRNA sponge-adenovirus and evaluation of its oncolytic effects in vitro and in vivo; 6) Modulation of miRNA levels with the THZ1 transcriptional inhibitor, and assessment of the effects of its combination with oncolytic adenoviruses.

Background : Many human molecular and genetic studies require the use of a renewable biological material. Although primary fibroblast cell lines can be used for this purpose, there are disadvantages associated with human biopsies including the limited number of cell divisions. Peripheral blood has the advantage of being easier to obtain but also the drawback that blood cells produce only short-term cultures. Epstein-Barr virus is capable of transforming human B lymphocytes into indefinitely proliferating cells that can be maintained in tissue culture. Here, we report a convenient method of B-lymphocyte immortalization using small volumes of freshly collected human blood serum saturated with nucleated blood cells. Aim of the study : The aim of the present study is modification and improvement of the protocol for highly efficient immortalization of human B lymphocytes from small volume of blood samples. Material and methods: Cell line B95-8 was used as Epstein-Barr virus source for viral stock preparation. Immortalizing medium contains RPMI-1640, viral stock and additives. No feeder layer was used. Results: As result we present high efficient method for B lymphocytes immortalization with start blood volume less than 5 ml. Conclusion: The method is applicable for immortalization of B lymphocytes from small blood samples and is essential for studies involving children or patients when large blood volume sampling is impossible. Keywords : Immortalization B lymphocytes Epstein-Barr virus Human cell culture

AIM: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues. MATERIAL and METHODS: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups' changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays. RESULTS: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrix-like structures. This was statistically significant (p

Cichoric acid (CA) belongs to the group of polyphenols, which occurs in a variety of plant species and it is characterized by anticancer, antibacterial, and antiviral properties. Selected polyphenols have the ability to combine with metal ions to form chelate complexes that reveal greater biological activity than free compounds. In order to study possible antimicrobial and anticancer effect of CA and its complexes with copper(II)/zinc(II)/nickel(II)/cobalt(II) we decided to conduct cytotoxicity tests to estimate the most effective concentrations of tested compounds. The results of the presented study demonstrated, for the first time, that the treatment with newly synthesized CA-metal complexes has anticancer and antimicrobial effects, which were examined in seven different cell lines: MCF-7, MDA-MB-231, and ZR-75-1 breast cancer cell lines, A375 melanoma cell line, DLD-1 cell line, LN-229 cell line, FN cell line, five bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Proteus vulgaris, Lactobacillus rhamnosus, yeast Sacchcaromyces boulardii, and pathogenic yeast-like fungi Candida albicans. The presented study indicates that CA-metal complexes could be considered as a potential supplementary tool in anticancer therapy, however, because of their possible toxic activity on fibroblasts, they should be used with caution. Some of the tested complexes have also preservative properties and positive influence on normal non-pathogenic microorganisms, which was demonstrated in selected microbial strains, therefore they may serve as food preservatives of natural origin with cytoprotective properties.

Plumbagin (1), a naphthoquinone, induces cell death and affects various signaling pathways in cancer cells. Wnt signaling is active constitutively in colorectal cancer and plays an important role in its progression and pathogenesis. It was hypothesized that 1 is likely to modulate Wnt signaling, and this compound was studied for its effect on this pathway in human colorectal cancer cells. Plumbagin (1) was found to downregulate Wnt signaling when assessed by a TOPFlash/FOPFlash reporter activity assay and also decreased the expression of several coactivators and downstream targets of Wnt signaling such as ?-catenin, TCF7L2, p300, Bcl9l, c-Myc, vimentin, and cyclinD1 in SW620 colorectal cancer cells. Using isogenic HCT116p53+/+ and HCT116p53-/- colorectal cancer cells, it was found that compound 1-mediated downregulation of Wnt signaling is p53-independent. Interestingly, treatment with 1 upregulated the expression of HBP1 (a negative regulator of Wnt signaling) in these cells. The results obtained show for the first time that downregulation of Wnt signaling could be one of the molecular mechanisms by which plumbagin exerts its inhibitory effects in human colorectal cancer cells. � 2014 The American Chemical Society and American Society of Pharmacognosy.

Bone tissue regeneration is accomplished by concerted regulation of protein-based extracellular matrix components, glycosaminoglycans (GAGs) and inductive growth factors. GAGs constitute a significant portion of the extracellular matrix and have a significant impact on regulating cellular behavior, either directly or through encapsulation and presentation of growth factors to the cells. In this study we utilized a supramolecular peptide nanofiber system that can emulate both the nanofibrous architecture of collagenous extracellular matrix and the major chemical composition found on GAGs. GAGs and collagen mimetic peptide nanofibers were designed and synthesized with sulfonate and carboxylate groups on the peptide scaffold. The GAG mimetic peptide nanofibers interact with bone morphogenetic protein-2 (BMP-2), which is a critical growth factor for osteogenic activity. The GAG mimicking ability of the peptide nanofibers and their interaction with BMP-2 promoted osteogenic activity and mineralization by osteoblastic cells. Alkaline phosphatase activity, Alizarin red staining and energy dispersive X-ray analysis spectroscopy indicated the efficacy of the peptide nanofibers in inducing mineralization. The multifunctional and bioactive microenvironment presented here provides osteoblastic cells with osteogenic stimuli similar to those observed in native bone tissue. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. © 2011 American Chemical Society.

Valproic acid (VPA) is widely used for the treatment of mood disorders and epilepsy, but its mechanism of action is unclear. In vivo and in vitro studies using rodent models have demonstrated that VPA has both neuroprotective and neurotrophic effects. These beneficial effects are, in part, through modulation of glial cell function. Recently, we and others have shown that VPA selectively induces caspase-3 mediated apoptosis in rodent microglial cells. However, the effect of VPA on human microglia has not been tested. In this study, using microglia derived from adult human brains, we demonstrate that VPA does not induce microglial apoptosis as determined by the absence of caspase-3 cleavage. However, VPA does partially decrease the expression of the microglial markers PU.1 and CD45, as well as dramatically reducing microglial phagocytosis. Due to the many roles of microglia in the brain, these VPA-induced alterations in microglial phenotype could potentially have major effects on physiological and pathological actions of these cells.

Open-source 3D printers mean objects can be quickly and efficiently produced. However, design and fabrication parameters need to be optimized to set up the correct printing procedure, a procedure in which the characteristics of the printing materials selected for use can also influence the process. This work focuses on optimizing the printing process of the open-source 3D extruder machine RepRap, which is used to manufacture poly(&epsilon, caprolactone) (PCL) scaffolds for cell culture applications. PCL is a biocompatible polymer that is free of toxic dye and has been used to fabricate scaffolds, i.e., solid structures suitable for 3D cancer cell cultures. Scaffold cell culture has been described as enhancing cancer stem cell (CSC) populations related to tumor chemoresistance and/or their recurrence after chemotherapy. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. Design and fabrication parameters were first determined with SolidWorks and Slic3r software and subsequently optimized following a novel sequential flowchart. In the flowchart described here, the parameters were gradually optimized step by step, by taking several measurable variables of the resulting scaffolds into consideration to guarantee high-quality printing. Three deposition angles (45&deg, 60&deg, and 90&deg, ) were fabricated and tested. MCF-7 breast carcinoma cells and NIH/3T3 murine fibroblasts were used to assess scaffold adequacy for 3D cell cultures. The 60&deg, scaffolds were found to be suitable for the purpose. Therefore, PCL scaffolds fabricated via the flowchart optimization with a RepRap 3D printer could be used for 3D cell cultures and may boost CSCs to study new therapeutic treatments for this malignant population. Moreover, the flowchart defined here could represent a standard procedure for non-engineers (i.e., mainly physicians) when manufacturing new culture systems is required.

Context: Tetraiodothyroacetic acid (tetrac) blocks angiogenic and tumor cell proliferation actions of thyroid hormone initiated at the cell surface hormone receptor on integrin αvβ3. Tetrac also inhibits angiogenesis initiated by vascular endothelial growth factor and basic fibroblast growth factor. Objective: We tested antiangiogenic and antiproliferative efficacy of tetrac and tetrac nanoparticles (tetrac NP) against human medullary thyroid carcinoma (h-MTC) implants in the chick chorioallantoic membrane (CAM) and h-MTC xenografts in the nude mouse. Design: h-MTC cells were implanted in the CAM model (n = 8 per group); effects of tetrac and tetrac NP at 1 μg/CAM were determined on tumor angiogenesis and tumor growth after 8 d. h-MTC cells were also implanted sc in nude mice (n = 6 animals per group), and actions on established tumor growth of unmodified tetrac and tetrac NP ip were determined. Results: In the CAM, tetrac and tetrac NP inhibited tumor growth and tumor-associated angiogenesis. In the nude mouse xenograft model, established 450–500 mm3 h-MTC tumors were reduced in size over 21 d by both tetrac formulations to less than the initial cell mass (100 mm3). Tumor tissue hemoglobin content of xenografts decreased by 66% over the course of administration of each drug. RNA microarray and quantitative real-time PCR of tumor cell mRNAs revealed that both tetrac formulations significantly induced antiangiogenic thrombospondin 1 and apoptosis activator gene expression. Conclusions: Acting via a cell surface receptor, tetrac and tetrac NP inhibit growth of h-MTC cells and associated angiogenesis in CAM and mouse xenograft models.

Similar to native cardiovascular tissues, the mechanical properties of engineered cardiovascular constructs depend on the composition and quality of the extracellular matrix, which is a net result of matrix remodeling processes within the tissue. To improve tissue remodeling, and hence tissue mechanical properties, various mechanical conditioning protocols, such as strain-based or flow-based conditioning, have been applied to engineered cardiovascular constructs with promising results. We hypothesize that tissue remodeling is dependent on the mode of straining. Therefore, the effects of two modes of straining, being either static or dynamic, were quantified on several indices of tissue remodeling. Differences in matrix composition (collagen and glycosaminoglycans [GAGs]) and maturity (collagen cross-links) were quantified with time on gene expression and protein levels. In addition, the secretion of specific collagen remodeling markers (matrix metalloproteinase-1), collagen synthesis marker (procollagen type I carboxy-terminal propeptide, PIP), and collagen degradation marker (carboxyterminal telopeptide of type I, ICTP) was investigated with time. Static strain stimulated collagen gene expression and production with time. Dynamic straining resulted in (1) lower collagen gene expression and production, but (2) enhanced collagen cross-link expression and density, and GAG production, and (3) stimulated collagen remodeling processes, as expressed by enhanced production of remodeling markers. Thus, despite a lower collagen production, the quality of the neotissue was enhanced by a dynamic straining component. These straining mode-dependent remodeling responses allow us for the first time to balance collagen and cross-link production and, thus, to fine tune tissue mechanical properties via mechanical conditioning protocols. This is of utmost importance for cardiovascular tissue engineering, where insufficient mechanical properties are currently a main limiting factor for present in vivo application. © Copyright 2009, Mary Ann Liebert, Inc. 2009.

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-α, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation. © 2007 Springer Science+Business Media B.V.

The therapeutic management of severe radiation burns remains a challenging issue. Conventional surgical treatment (excision and skin autograft or rotation flap) often fails to prevent unpredictable and uncontrolled extension of the radiation necrotic process. We report here an innovative therapeutic strategy applied to the victim of a radiation accident (December 15, 2005) with an iridium gammagraphy radioactive source (192Ir, 3.3 TBq). The approach combined numerical dosimetry-guided surgery with cellular therapy using mesenchymal stem cells. A very severe buttock radiation burn (2000 Gy at the center of the skin surface lesion) of a 27-year-old Chilean victim was widely excised (10 cm in diameter) using a physical and anatomical dose reconstruction in order to better define the limit of the surgical excision in apparently healthy tissues. A secondary extension of the radiation necrosis led to a new excision of fibronecrotic tissues associated with a local cellular therapy using autologous expanded mesenchymal stem cells as a source of trophic factors to promote tissue regeneration. Bone marrow-derived mesenchymal stem cells were expanded according to a clinical-grade technique using closed culture devices and serum-free medium enriched in human platelet lysate. The clinical evolution (radiation pain and healing progression) was favorable and no recurrence of radiation inflammatory waves was observed during the 11 month patient’s follow-up. This novel multidisciplinary therapeutic approach combining physical techniques, surgical procedures and cellular therapy with adult stem cells may be of clinical relevance for improving the medical management of severe localized irradiations. It may open new prospects in the field of radiotherapy complications.

Background: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. Objective: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. Results: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. Conclusions: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing. © 2006 International Society on Thrombosis and Haemostasis.

Vitamin A, a naturally occuring antioxidant micronutrient, has immunomodulating effect in patients with immunodeficiency, including an influence on cytokine production and lymphocyte growth and functions. Vitamin A deficiency is associated with a shift from type 2 cytokines to predominantly type I cytokines. The aims of this study were to determine Vitamin A status in Common variable immunodeficiency (CVID) patients and the relationship between Vitamin A status and cytokines production. Serum Vitamin A, neopterin, TNF-alpha, IL-2, IL-4, and IL-10 levels were determined in 19 CVID patients and 15 healthy children. Effects of 9-cis retinal, Vitamin A derivative, on cytokines (TNF-alpha, IL-2, IL-4 and IL-10) production in lymphocytes were tested in vitro condition using lymphocyte cultures obtained from CVID patients and healthy children. Serum Vitamin A level in CVDI patients was, 21.1 +/- 1.5 mu g/dL, significantly (p < 0.001) lower than the value, 35.7 +/- 1.8 mu g/dL, observed in healthy children. Serum neopterin level in the patients was, 9.8 +/- 2.9 nmol/L, higher (p < 0.05) than the value, 3.9 +/- 0.7 nmol/L, observed in control group. Common variable immunodeficiency patients, serum IL-4 level was significantly (p < 0.05) lower than the value observed for healthy children. Serum TNF-alpha, IL-2 and IL-10 levels were similar in the patients and healthy children. Vitamin A derivative, 9-cis retinal, increased TNF-alpha and IL-4 production in cultured mononuclear cells obtained from control and CVID patients. Vitamin A derivative, also, increased IL-2 and Il-4 production in cultured mononuclear cells obtained from CVID patients. These results show that CVID patients have low serum Vitamin A levels and high serum neopterin levels. A supplementation with Vitamin A may have role in downregulation of inflammatory responses in CVID patients.

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.

[spa] La obesidad se asocia con un estado de inflamación crónica de bajo grado y es considerada uno de los mayores factores de riesgo cardiovascular y de otro tipo de enfermedades como son la diabetes, hipertensión, arteriosclerosis y trombosis. Se han descrito diversos procesos que tienen lugar en el tejido adiposo y que determinan el desarrollo de la obesidad. Sin embargo, el tejido adiposo no solo se considera un órgano de almacenamiento energético o endocrino, sino que hoy en día también es considerado como una fuente de células madre mesenquimales (ASC). Recientemente las ASC se han convertido en una alternativa de gran interés en terapia celular para regenerar el corazón dañado. Sin embargo se ha sugerido que una alteración en la homeostasis de las células madre desencadenada por un estímulo nocivo, constante y repetitivo, como podría ser el caso de una enfermedad crónica, podría provocar un desequilibrio persistente en el reservorio de éstas células produciendo un cambio prematuro e irreversible del potencial regenerador de las células madre. De hecho, estudios previos han demostrado que la presencia de factores de riesgo cardiovascular, como la hipertensión, la enfermedad coronaria o la diabetes, alteran y merman las propiedades de las células progenitoras circulantes. Del mismo modo, se ha visto que la edad y el género modifican la funcionalidad de las ASC. El objetivo central de esta tesis ha sido el estudiar y caracterizar las ASC derivadas de pacientes obesos. En esta tesis doctoral se ha observado que la presencia de desórdenes metabólicos disminuye el porcentaje de células positivas para ciertos marcadores característicos de ASC, como es el caso de CD90, en el tejido adiposo. Sin embargo, una vez aisladas las ASC, independientemente del índice de masa corporal del donante de las ASC, éstas presentan el 100% de los marcadores característicos confirmándonos la obtención de una población homogénea. No obstante, hemos observado que algunas características importantes y necesarias para el uso de las ASC en la terapia celular se ven afectadas cuando las células se aíslan de pacientes que presentan obesidad mórbida y factores de riesgo cardiovascular. A nivel clínico y en la terapia celular es beneficioso poder obtener el máximo número de células en el menor tiempo posible. El hecho de que observemos una mejora en la velocidad de crecimiento celular al cultivar las ASC en condiciones de hipoxia refuerza el uso de bajas concentraciones de oxígeno a la hora de su expansión para fines clínicos. Sin embargo, el uso de las ASC de pacientes obesos no parece ser tan beneficioso. Estas células presentan un crecimiento celular más lento, una menor capacidad proangiogénica al liberar el factor antiangiogénico trombospondina-1, así como una capacidad de diferenciación disminuida respecto a las ASC de pacientes no-obesos. De hecho, parece ser que las ASC de pacientes obesos se encuentran en un estado de desarrollo menos multipotencial que las de individuos no-obesos, más dirigido hacia la adipogénesis, y a un estado más proinflamatorio. Una de las ventajas del uso de las ASC en la terapia celular es su uso autólogo, sin embargo esta ventaja desaparece en el caso de los pacientes obesos. Aunque se conocen bastante bien los cambios que se producen en el tejido adiposo de los obesos, no está muy estudiado como afectan estos cambios a la población endógena de células madre mesenquimales de éste tejido. Los resultados obtenidos en este trabajo nos ayudan a comprender mejor como la obesidad, y los desórdenes metabólicos que derivan de ella, afectan y modifican a la población de ASC., [eng] The adipose tissue is an endocrine regulator and a risk factor for atherosclerosis and cardiovascular disease when by excessive accumulation induces obesity. Moreover, it has been demonstrated that the adipose tissue is a reservoir of mesenchymal stem cells. The potential use of adipose-derived stem cells (ASCs) from white adipose tissue for organ repair and regeneration has been considered because of their obvious benefits in terms of accessibility and quantity of available sample. However, age, adipose tissue depot site, and gender have been shown to modify the number and the proliferation, differentiation, and angiogenic capacity of ASCs. Our aim was to investigate the mechanisms by which obesity affects subcutaneous white adipose tissue stem cells. Here we report that ASCs from white adipose tissue of patients with obesity have lower proliferative and angiogenic potential than ASCs of nonobese metabolically normal individuals. Moreover, the transcriptomic profile of the stem cells reservoir in obese subcutaneous adipose tissue is highly modified with significant changes in genes regulating stemcellness, lineage commitment and inflammation. In addition to body mass index, cardiovascular risk factor clustering further affect the ASC transcriptomic profile inducing loss of multipotency and, hence, capacity for tissue repair. In summary, obesity produces a detrimental effect on its resident stem cells. In fact, the ASC undifferentiated multipotent state is impaired in obese patients with respect to non-obese individuals. These effects may negatively influence their regenerative potential when used in cell therapy and also in spontaneous repair of minor organ damage. Indeed, ASCs have already been tested in several clinical trials, from repair of heart ischemic injury to Crohn’s disease and multiple sclerosis. However, our observations indicate that the therapeutic strategies based on autologous ASC implantation would be impaired in patients with obesity and metabolic syndrome.

Cataloged from PDF version of article. New blood vessel formation (angiogenesis) is one of the most important processes required for functional tissue formation. Induction of angiogenesis is usually triggered by growth factors released by cells. Glycosaminoglycans (e.g., heparan sulphates) in the extracellular matrix aid in proper functioning of these growth factors. Therefore, exogeneous heparin or growth factors were required for promoting angiogenesis in previous regenerative medicine studies. Here we report for the first time induction of angiogenesis by a synthetic nanofibrous peptide scaffold without the addition of any exogenous growth factors or heparin. We designed and synthesized a self-assembling peptide amphiphile molecule that is functionalized with biologically active groups to mimic heparin. Like heparin, this molecule has the ability to interact with growth factors and effectively enhance their bioactivity. The nanofibers formed by these molecules were shown to form a 3D network mimicking the structural proteins in the extracellular matrix. Because of heparin mimicking capabilities of the peptide nanofibers, angiogenesis was induced without the addition of exogenous growth factors in vitro. Bioactive interactions between the nanofibers and the growth factors enabled robust vascularization in vivo as well. Heparin mimetic peptide nanofibers presented here provide new opportunities for angiogenesis and tissue regeneration by avoiding the use of heparin and exogenous growth factors. The synthetic peptide nanofiber scaffolds enriched with proper chemical functional groups shown in this study can be used to induce various desired physiological responses for tissue regeneration. © 2011 American Chemical Society.

Objective-: Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). Methods and Results-: In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)(-/-) mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS(-/-) mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage.

Mesenchymal stem cells (MSCs) have gained much interest to be used as targeting vehicle in cancer therapy due to the intrinsic tumor-homing behavior associated with them. In this scenario, superparamagnetic nanoparticles are emerging as an ideal probe for noninvasive cell tracking for different stem cell applications. In the study, it is demonstrated that the formulated aqueous dispersible glyceryl monooleate coated magnetic nanoparticles (MNPs) can act as a better labeling and efficient tracking agent without affecting the inherent properties of MSCs. The MNPs-MSCs facilitate the stem cell tracking by magnetic resonance imaging at a very low cell number having high T2 relaxivity and potentiates the use of MNPs-MSCs as a prospective diagnostic tool. Most importantly, the homing of MNPs-MSCs toward inflammation site, subcutaneous prostate tumor (small as well as large tumor), and in orthotopic prostate tumor suggests the clinical relevance of the system. In addition, intraperitoneal delivery of MNPs-MSCs shows enhanced tumor accumulation and less sequestration in liver as revealed by in vivo imaging and histological studies. The results here demonstrate that MNPs-MSCs may prove as a better targeted delivery agent for early diagnosis of tumors even of smaller size. � 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.