Tesi sul tema "Extracellular matrix Physiology"

Recently, various proteoglycans (PGs) have been identified in the lung. The first objective of this thesis was to test the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. Lung parenchymal strips were exposed to specific glycosaminoglycans-degradating enzymes to determine whether the mechanical properties of the tissue were affected. The degradation of heparan sulphate and chondroitin/dermatan sulphate glycosaminoglycans caused significant increases in energy dissipation and dynamic resistance relative to control strips. Hyaluronidase treatment did not alter any of the dynamic or static measures. Since PGs were found to be part of the stress bearing structure, the second part of the thesis aimed at examining whether subjecting the lung to excessive mechanical force can cause alteration in PG composition so as to adapt to the altered stress bearing requirement. To address this hypothesis, the effect of different ventilation regimes on lung tissue mechanics and PGs was examined in an in vivo rat model. After 2 h of mechanical ventilation, lung tissue elastance and resistance were significantly increased in rats ventilated with tidal volume of 30 ml/kg at 0 positive end-expiratory pressure (Vt30PEEP0) as compared to controls (Vt8PEEP1.5). Versican, a basement membrane heparan sulphate PG and biglycan, were all increased in rat lungs ventilated with Vt30PEEP0 as compared to control. These data demonstrated that alterations in lung tissue mechanics with excessive mechanical ventilation are accompanied by changes in all classes of ECM PGs. However, whether the alteration seen in PG composition resulted from excessive mechanical ventilation directly was unclear. In addition the cellular source of these PGs was not determined. Therefore, the aim of the third part of the thesis was to investigate and characterize the effect of mechanical strain on lung fibroblast PG production in vitro. We found cell layer associated versican protein in

Airway remodeling in asthma includes alterations in extracellular matrix and airway smooth muscle (ASM) mass. For this study, ASM cells were obtained from rats that were challenged with ovalbumin (OVA) or saline (SAL) as control. OVA and SAL cells were seeded on plastic control (PC) or on plates coated with decorin or biglycan. OVA cell number was significantly increased versus SAL cells, for cells seeded on PC (48 h). A significant decrease in cell number was observed for both OVA and SAL cells seeded on decorin compared to PC cells (48 h). OVA cells, however, showed a more modest reduction in cell number. Furthermore, biglycan decreased SAL cell number only. Compared to no strain (NS), mechanical strain (S) reduced cell number for OVA and SAL cells on all matrices. In addition, S up-regulated expression of beta 1-integrin relative to NS controls. Results suggest an ability of ASM cells to be modulated by matrix and mechanical stimulation.

Synthetic biomedical implants are used to replace diseased tissues and organs. Unfortunately, these implants often fail due to a lack of biocompatibility and poor integration by the recipient. This implant failure is associated with the formation of an avascular fibrous capsule and chronic inflammatory response. Additionally, small diameter vascular grafts have complications associated with surface thrombogenenicity and intimal hyperplasia. Porous polymers are often incorporated in the construction of biomedical devices because they permit tissue integration and improved biocompatibility. While the inclusion of porosity has enhanced device performance, these devices still do not perform optimally. The incorporation of a vascular network in association with and within the pores of these materials is believed to improve tissue integration and long-term device function. Several approaches are actively being studied for their ability to stimulate new vessel growth, angiogenesis, as well as to improve the direct interaction of cells with material surfaces. The process of angiogenesis involves the coordinated involvement of both soluble and insoluble factors such as growth factors and cytokines, and extracellular matrix proteins respectively. Often, growth factors and cytokines are expressed by the inflammatory cells associated with the biomedical implants, but the microenvironment within the polymer remains unstable with respect to the presence of the appropriate extracellular matrix proteins. The overall hypothesis of this dissertation is that the reestablishment of an extracellular microenvironment on and within a porous polymer will provide the appropriate substrates for promoting angiogenesis and neovascularization of porous polymers. The results of the studies within this dissertation demonstrate that extracellular matrix modifications of commercially available expanded polytetrafluoroethylene (ePTFE) successfully promote new vessel growth in the tissue surrounding the implant, termed angiogenesis, and new vessel growth within the pores of the polymer, termed neovascularization. Furthermore, the extracellular matrix protein laminin 5 was determined to promote human microvessel endothelial cell adhesion to ePTFE as well as support angiogenesis and neovascularization when used as a surface modification of ePTFE. Based on these studies, the extracellular matrix protein, laminin 5, could be utilized in the tissue engineering of biomedical implant devices to promote increased new vessel integration and improve the long-term viability of these devices.

All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pisaster ochraceus, consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which fills the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the fluid-filled blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a fluid with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The initial objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pisaster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask filled with liquid nitrogen, a small cup was filled with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fluid with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, justifies its use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies.
Medicine, Faculty of
Graduate

The observation of single biomolecules via optical microscopy eliminates all the implicit averaging of ensemble techniques and thereby provides access to the heterogeneity of molecular systems that will be the key to at least some biological functions. The implementation of photothermal microscopy at the University of Liverpool to achieve the detection of single gold nanoparticles over long times at high signal-to-noise-ratio is presented here, along with the development of Photothermal Raster Image Correlation Spectroscopy, PhRICS. PhRICS was shown to be equally effective as Photothermal Absorption Correlation Spectroscopy, PhACS, in the determination of the hydrodynamic diameter of colloidal gold nanoparticles in solution. The use of gold nanoparticles as labels for biomolecules has been of great interest due to their favorable optical properties and surface chemistry. The development of a new strategy for the covalent biofunctionalisation of gold nanoparticles with a single maleimide group is described. Nanoparticles functionalised this way were used to label FGF-2 protein and heparin-derived oligosaccharides. Both the PhRICS and the new nanoparticles developed in this thesis are combined to investigate the heterogeneity of FGF binding to heparin-derived oligosaccharides and to HS in the pericellular matrix of Rama 27 fibroblasts. The cooperativity of the interaction of FGF-2 with a dodecasaccharide is investigated. Although oligomerization of FGF-2 on the dodecasaccharide is observed, it is not cooperative. The first photothermal imaging of FGF-1 in the pericellular matrix of Rama 27 fibroblasts reveals that its diffusion is quite different from FGF-2. Imaging of FGF-2 on live cells is also revisited and probed with PhRICS. In comparison to photothermal tracking, PhRICS indicates that FGF-2 diffuses faster than first thought, and that the pericellular matrix is remodeling at timescales much shorter than previously observed.

The asthmatic airway wall is characterized by airway remodeling, including changes in the extracellular matrix (ECM) and increased airway smooth muscle (ASM) cell mass. Further, asthmatic ASM has been shown to demonstrate enhanced contractility. Recently, we and others have shown that alterations in the matrix upon which ASM cells are grown in culture, can affect the degree of ASM cell proliferation and apoptosis. Whether changes in matrix can affect ASM cell contractility is less clear. ASM cells were isolated from the trachea of Brown Norway (BN) rats sensitized subcutaneously with ovalbumin (OVA) and challenged with either OVA or saline (SAL) as a control. Cells were grown in culture on plastic as a control, or on plates previously coated with collagen I (col), decorin (dcn) or biglycan (bgn). Contractile protein expression as well as single cell Ca2+ responses to serotonin was measured. Both SAL and OVA ASM cells grown on col had a significant reduction in α-smooth muscle actin (α-SMA) and calponin content. A significant increase in α-SMA and calponin was observed in OVA ASM cells grown on bgn but not in SAL cells. Dcn did not significantly affect α-SMA or calponin in SAL or OVA cells. Ca2+ responses to serotonin were significantly decreased in OVA cells compared to SAL cells grown on plastic, but this was not seen in cells grown on any other matrix. These experiments will help contribute to our understanding of ECM and its potential effects on mechanisms involved in smooth muscle contraction.
Les asthmatiques se caractérisent par un remodelage des voies respiratoires, incluant des changements dans la matrice extracellulaire et une augmentation de muscle lisse des voies respiratoires (MLVR). Aussi, les muscles lisses des asthmatiques ont une contractilité augmentée. Récemment on a montré qu'en changeant la matrice extracellulaire sur laquelle les muscles lisses sont cultivés, on peut affecter leur prolifération et l'apoptose. Mais on ne sait pas encore si la matrice extracellulaire peut affecter la contractilité des muscles lisses. Les muscles lisses ont été isolés des rats « Brown Norway » qui ont été sensibilisés avec l'ovalbumine (OVA) et ont été provoqués avec OVA ou saline (SAL) comme contrôle. Des cellules ont été semées sur un contrôle plastique ou sur des plats enduits de collagène (col), de décorine (dcn), ou de biglycane (bgn). Le niveau des protéines contractiles et la réponse du Ca2+ à l'ajout de serotonine dans une cellule unique a été mesuré. Les cellules OVA et SAL du MLVR qui ont été cultivées sur du col ont montré une réduction substantielle de leur contenu en α-SMA et calponine. Quand cultivées sur du bgn, une augmentation considérable du α-SMA et du calponine a été observée dans les cellules OVA du MLVR mais ceci n'a pas été observé sur des cellules SAL. Cependant quand on a semé les cellules SAL et OVA avec dcn, on n'a pas observé un effet significatif du niveau de calponine et α-SMA. La réaction du Ca2+ à la serotonine a diminué substantiellement dans les cellules OVA en comparaison à l'effet remarqué dans les cellules SAL, quand ces cellules ont été cultivées sur un contrôle plastique. Ces expériences contribuent à notre compréhension de l'importance des matrices extracellulaires dans leur contribution de l'augmentation de la contractilité du MLVR tel que décrit dans l'asthme.

The introduction of necrophagous fly larvae (maggots) into chronic wounds for the purpose of inducing healing is an ancient practice that has recently undergone a renaissance in Western medicine. Through clinical observations, maggots are broadly recognised to debride the wound of necrotic tissue, cleanse the wound of infection and promote granulation tissue formation. Despite such recognition, little research at the biological level has been undertaken to identify the mechanisms by which maggots accomplish such feats. The dermal fibroblast is a major cellular component of granulation tissue and as such, its migration into the wound plays a vital role in new tissue growth. Fibroblast migration is directed by the composition of the extracellular matrix. Maggot secretions contain proteolytic enzymes that are active against a variety of extracellular matrix proteins which are present at the wound site. Hence, this thesis focused upon the effects of maggot secretions on human dermal fibroblast adhesion and migration in the presence of common extracellular matrix proteins. This was with the aim of elucidating the mechanisms by which maggots stimulate tissue formation within the wound and from there, developing new products that may be used to promote wound healing. Experiments showed that maggot secretions modulated fibroblast adhesion to tissue culture plastic surfaces and to surfaces coated with collagen and particularly fibronectin. Modification of the protein-coated surface by enzymes present within the secretion appeared to play a role. Fibroblast migration upon a fibronectin-coated surface was enhanced in the presence of maggot secretions. The same also occurred in the presence of a higher concentration of secretions when the cells were located within a three-dimensional environment comprising collagen gel and fibronectin. Evidence suggested that this may have been associated with enhanced matrix re-modelling.

Orientador: Edson Rosa Pimentel
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-06T17:20:03Z (GMT). No. of bitstreams: 1 Almeida_FernandaMartinsde_M.pdf: 1290451 bytes, checksum: c386eaff7996cd60bd0cc59338f8718f (MD5) Previous issue date: 2006
Resumo: Os tendões servem para realizar a transferência de força dos músculos para os ossos, sendo capazes de suportar altas forças de tensão. Muitos trabalhos descreveram alterações nas propriedades estruturais, bioquímicas e biomecânicas dos tendões de animais que foram submetidos a exercícios prolongados, porém pouco se sabe sobre o que ocorre no tendão que passa por um processo de alongamento, um procedimento bastante comum em academias e clínicas de Fisioterapia. Assim, nosso trabalho teve como objetivo avaliar os aspectos morfológicos e bioquímicos dos tendões de ratos submetidos ao alongamento três e cinco vezes por semana. Os ratos tiveram seus músculos alongados por um período de 30 segundos intercalados com 30 segundos de relaxamento, com 10 repetições, 3 e 5 vezes por semana durante 21 dias. Os tendões foram removidos e utilizados para os procedimentos de morfologia e de bioquímica. Os tendões também foram submetidos ao ensaio mecânico sob tração a fim de avaliar suas propriedades biomecânicas. Nos cortes corados com AT, na entese dos tendões dos grupos alongados, observou-se um aumento na quantidade de células com morfologia arredondada. Já na região próxima à entese pode ser observado metacromasia mais intensa nos grupos alongados. Observações feitas nos cortes corados com HE, nas regiões de tensão, mostraram que as células apresentaram-se mais alinhadas. Nos grupos alongados, em ambas regiões ocorreu aumento na quantidade de células. Nos cortes que foram submetidos à Reação de Von Kossa, observou-se uma região calcificada em todos os grupos, porém esta apresentou uma MEC mais densa nos tendões dos grupos alongados três e cinco vezes. Análise do gel de SDS-PAGE revelou uma maior quantidade de colágeno nos grupos alongados e a presença do componente de 65 kDa nas regiões de tensão e compressão em todos os grupos. A quantidade de proteínas, de glicosaminoglicanos e de hidroxiprolina também foi superior nos animais alongados. O gel de agarose revelou a presença de dermatam sulfato nas regiões de tensão e de compressão e de condroitim sulfato somente nesta última. Durante o ensaio mecânico, os tendões dos grupos alongados suportaram valores de tensão máxima superiores, com deslocamentos semelhantes, sugerindo que estes tendões são mais resistentes à ruptura. Esses resultados mostraram que o estímulo do alongamento acarretou modificações nas características estruturais, bioquímicas e biomecânicas, confirmando o caráter adaptativo do tendão em resposta à aplicação dos procedimentos de alongamento
Abstract: The tendons are structures that transmit forces from the muscles to the bone, and are capable of supporting high tensile strenghts. Many studies have shown alterations in tendons of animals submitted to strenuous exercises, however, just a little is known about what happens in tendons when they are under a stretching program, a common procedure in academies and phisiotherapy institutes. So our objective was to evaluate the morphological, biochemical and biomechanical aspects of tendons submitted to stretching exercises. Rats had their muscles stretched for a period of 30 seconds with 30 seconds of resting, with 10 repetitions, three and five times a week during 21 days. The tendons were used for morphological and biochemical procedures. They were also submitted to mechanical tensile strain test and their mechanical properties were evaluated. Analysis of AT stained sections of enthesis from stretched tendons, showed a high amount of rounded cells. In the region next to enthesis, which passes close to the calcaneous, the metachromasy was more intense in stretched groups. In the tension region, in the HE stained sections, it was found a larger alignment of the cells in the stretched group. In both regions occurred an increase on the amount of cells. In the sections submitted to Von Kossa reaction, was observed, a calcified region in all groups, but the extracellular matrix were denser in stretched ones. Analysis of SDS-Page showed a high amount of collagen and the presence of a polidisperse component of 65 kDa in the tension and compression regions in all groups. The amount of proteins, glycosaminoglycans and hydroxiproline was higher in the tendons of three and five times stretched groups. The agarose gel revealed the presence of dermatan sulfate in the tension and compression regions and chondroitin sulfate only in this last one. During the mechanical tensile strain test, the stretched tendon supported high values of maximum stress with the same strain, when compared to the control group, suggesting that the stretched tendons were more resistant to the failure. These results show that the stimulus of stretching leads to alterations in the structural, biochemical and biomechanical characteristics, confirming the adaptative character of tendons, in response to the application of stretching exercises
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

Cardiovascular disease (CVD) is the leading cause of death in the United States. A common feature in most cardiac pathologies is the dysregulation of beta-adrenergic receptors (β-AR) and changes in the extracellular matrix (ECM). The ECM maintains strength and normal organization of cardiac tissue, while fibrosis (connective tissue scarring) is necessary for repair of damaged cardiac tissue. However, the dysregulation of the ECM leads to a number of cardiac disease pathologies. Osteopontin (OPN) is a protein with diverse biological functions in regulating the ECM such as bone resorption and calcification, wound healing, cell adhesion, cell survival, and apoptosis. OPN is expressed at low levels in the heart but increases with injury by promoting collagen synthesis, cardiac fibroblast growth, and adhesions to ECM proteins. Furthermore, as the heart ages, increases in ECM reorganization leads to cardiac damage and failure. Several studies have examined the role of OPN in the heart, but to date, no studies exist on the role of OPN in response to β-AR signaling and cardiac remodeling or the role that aging plays in this response. The goal of this study was to examine the effects of OPN on cardiac ECM remodeling following chronic beta-adrenergic stimulation. We proposed that OPN expression increases cardiac remodeling and dysfunction following ISO treatment in the aging heart evidenced by increased fibrosis. For this study, young (4 months) and middle age (14 months) mice with (WT) and without (KO) the OPN gene were treated with isoproterenol (ISO) for 28 days. Echocardiography was used to assess cardiac function. Mice were euthanized, and the hearts were analyzed for fibrosis using Masson’s Trichrome Staining. Results showed ISO increased fibrosis in the WT-ISO, but not KO-ISO compared to the respective controls (SHAM, no ISO treatment) for the middle age mice (p≤0.05). Furthermore, the aged WT-ISO group exhibited a 3-fold increase in fibrosis compared to the young WT-ISO group. Results from echocardiography will be analyzed and we expect to see compromised cardiac function in the WT-ISO groups compared to KO-ISO groups. OPN is currently being examined as a potential biomarker in heart failure. The results from this study will provide new insight on changes in the cardiac vasculature in the aging heart following injury and the role OPN plays in this process.

Includes bibliographical references (leaves 68-76) Aims to observe changes in the expression of syndecan-1 in both the developing epithelium of the rat oral mucosa, and in epithelial cell rests of Malassez in the developing periodontium of normal rat molars, from late crown development through to early eruption.

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.

In cattle, the oviduct plays an important role in the reproductive process. Oviductal secretions characterize the environment where storage and sperm capacitation, fertilization, and early embryo development take place. Because molecular control of bovine oviduct receptivity is poorly understood, this Thesis proposed a model of receptivity based on the manipulation of pre-ovulatory follicle growth (POF) used to study the effects of periovulatory endocrine profile on oviductal physiology. Growth of POF in Nelore cows (Bos indicus) was manipulated to produce two groups: cows with large POF and large corpus luteum (LF-LCL; higher fertility) and cows with small POF and small CL (SF-SCL; Lower fertility). Ampulla and isthmus samples were collected on day 4 after induction of ovulation with GnRH. In the first study, the transcriptome of the ipsilateral to CL ampulla and isthmus was determined by RNAseq, the regional expression of genes was studied by qPCR, and the distribution of the PGR and ER proteins was assessed by immunohistochemistry. Greater abundance of PGR and ER was found in the oviduct of the LF-LCL animals indicating that there is a greater availability of receptors and, possibly, of signaling-mechanisms stimulated by steroids in both oviductal regions. The transcripts profile showed enriched oviductal functional characteristics that could affect its embryo receptivity. These characteristics include changes in morphology i.e. branching morphogenesis, and changes in cell functioning i.e. cell secretion, that were enriched in the LF-LCL group. In the second study, after morphological analyses, it was concluded that the ampulla of the LF-LCL animals presented more primary folds, a larger perimeter of the luminal epithelium, and a higher proportion of secretory and proliferating cells, when compared to SF-SCL group. There was no difference in isthmus morphology between groups. In the third study, the extracellular matrix remodeling was reserched. It was concluded that in the isthmus region of the LF-LCL animals, there is less type 1 collagen fibers and greater abundance of proteins involved in extracellular matrix remodeling. In the fourth study, it was determined that the periovulatory endocrine milieu affects the expression of components of the microRNAs biosynthesis pathway and the microRNAs profile, both different between groups. Finally, in the fifth study, 205 metabolites were quantified in the oviductal fluid and 37 were found to be in different concentrations when both groups were compared. It was concluded that oviduct of cows of higher fertility presents a profile of transcripts, proteins, and metabolites that is associated with morphological and functional characteristics favorable to the survival and development of the embryo.
Em fêmeas bovinas, o oviduto apresenta um importante papel no processo reprodutivo. As secreções ovidutais representam o ambiente onde ocorrem o armazenamento e a capacitação espermática, a fecundação e o desenvolvimento embrionário inicial. O controle molecular da receptividade do oviduto em bovinos é pouco conhecido. Na presente tese, empregou-se um modelo de receptividade baseado na manipulação do crescimento do folículo pré-ovulatório (FPO) para o estudo dos efeitos do perfil endócrino periovulatório na fisiologia do oviduto. O crescimento do FPO de vacas Nelore (Bos indicus) foi manipulado com o objetivo de produzir dois grupos: vacas com FPO e corpo lúteo (CL) grandes (FG-CLG; maior fertilidade) e vacas com FPO e CL pequenos (FP-CLP; menor fertilidade). Amostras da ampola e istmo foram coletadas no dia 4 após da indução da ovulação com GnRH. No primeiro estudo, o transcriptoma da ampola e istmo do lado ipsolateral ao CL foi determinado por RNAseq, à expressão gênica regional e a distribuição das proteínas PGR e ER foram analisadas por qPCR e imunohistoquímica, respectivamente. Houve maior abundância de PGR e ER no oviduto dos animais do grupo FG-CLG, o que indica uma maior disponibilidade de receptores e possivelmente, de mecanismos intracelulares de sinalização estimulados pelos esteroides em ambas as regiões. O perfil global de transcritos mostrou enriquecimento de características funcionais do oviduto que poderiam afetar sua receptividade ao embrião. Tais características incluem mudanças morfológicas, como a ramificação morfogênica, e celulares, como a secreção, que foram aumentadas no grupo FG-CLG. No segundo estudo, após analisarem-se características morfológicas dos tecidos, concluiu-se que a ampola dos animais FG-CLG apresentou maior número de pregas primárias, maior perímetro do epitélio luminal, e maior proporção de células secretoras e de células em proliferação quando comparado aos animais do grupo FP-CLP. Não houve diferença na morfologia do istmo entre os grupos. No terceiro estudo, foi analisado o processo de remodelamento de matriz extracelular. Concluiu-se que no istmo dos animais do grupo FG-CLG existe menor quantidade de fibras de colágeno tipo 1 e maior abundância de proteínas envolvidas no remodelamento de matriz. No quarto estudo, determinou-se que o perfil endócrino periovulatório afeta a expressão de componentes da via de biossíntese e o perfil de microRNAs, que são diferentes entre os grupos. Finalmente, no quinto estudo, foram quantificados 205 metabólitos no fluido ovidutal dos animais. Destes, 37 encontram-se em concentrações diferentes entre os grupos. Concluiu-se que o oviduto de vacas de maior fertilidade apresenta um perfil de transcritos, proteínas e metabólitos que está associado a características morfológicas e funcionais favoráveis à sobrevivência e desenvolvimento do embrião.

Decorin, is an extracellular matrix proteoglycan with important biological functions. Decorin deficiency affects collagen fibrillogenesis, airway mechanics, airway-parenchymal interdependence, and airway smooth muscle proliferation and apoptosis. We questioned whether decorin deficiency would alter allergen-induced asthma in a mouse model. Decorin-/- and decorin+/+ mice (C57Bl/6) were sensitized and challenged with ovalbumin. Control animals received saline. Responsiveness was assessed at baseline and after delivery of increasing concentrations of methacholine. Histological analyses were also performed. Decorin deficiency resulted in more modest hyperresponsiveness. Respiratory resistance and elastance along with tissue damping and tissue elastance, were increased in ovalbumin decorin +/+ and decorin-/-, but more so in decorin+/+ . Airway resistance was increased in ovalbumin decorin+/+ only. Inflammation and collagen staining within the airway wall, were increased in ovalbumin decorin+/+ mice only; whereas biglycan was significantly increased in ovalbumin decorin-/- mice only. These results reflect the role of decorin in the development of allergen-induced asthma.

The overall aim of the present thesis is to clarify the control of intracellular chloride homeostasis in central neurons, because of the critical role of chloride ions (Cl–) for neuronal function. Normal function of the central nervous system (CNS) depends on a delicate balance between neuronal excitation and inhibition. Inhibition is, in the adult brain, most often mediated by the neurotransmitter γ-aminobutyric acid (GABA). GABA may, however, in some cases cause excitation. GABA acts by activating GABA type A receptors (GABAARs), which are ion channels largely permeable to Cl–. The effect of GABAAR-mediated neuronal signaling - inhibitory or excitatory - is therefore mainly determined by the Cl– gradient across the membrane. This gradient varies with neuronal activity and may be altered in pathological conditions. Thus, understanding Cl– regulation is important to comprehend neuronal function. This thesis is an attempt to clarify several unknown aspects of neuronal Cl– regulation. For such clarification, a sufficiently sensitive method for measuring the intracellular Cl– concentration, [Cl–]i, is necessary. In the first study of this thesis, we examined two electrophysiological methods commonly used to estimate [Cl–]i. Both methods, here called the interpolation and the voltage-ramp method, depend on an estimate of the Cl– equilibrium potential from the current-voltage relation of GABA- or glycine-evoked Cl– currents. Both methods also provide an estimate of the membrane Cl– conductance, gCl. With a combination of computational and electrophysiological techniques, we showed that the most common (interpolation) method failed to detect changes in [Cl–]i and gCl during prolonged GABA application, whereas the voltage-ramp method accurately detected such changes. Our analysis also provided an explanation as to why the two methods differ. In a second study, we clarified the role of the extracellular matrix (ECM) for the distribution of Cl– across the cell membrane of neurons from rat brain. It was recently proposed that immobile charges located within the ECM, rather than as previously thought cation-chloride transporter proteins, determine the low [Cl–]i which is critical to GABAAR-mediated inhibition. By using electrophysiological techniques to measure [Cl–]i, we showed that digestion of the ECM decreases the expression and function of the neuron-specific K+ Cl– cotransporter 2 (KCC2), which normally extrudes Cl- from the neuron, thus causing an increase in resting [Cl–]i. As a result of ECM degradation, the action of GABA may be transformed from inhibitory to excitatory. In a third study, we developed a method for quantifying the largely unknown resting Cl– (leak) conductance, gCl, and examined the role of gCl for the neuronal Cl– homeostasis. In isolated preoptic neurons from rat, resting gCl was about 6 % of total resting conductance, to a major part due to spontaneously open GABAARs and played an important role for recovery after a high Cl– load. We also showed that spontaneous, impulse-independent GABA release can significantly enhance recovery when the GABA responses are potentiated by the neurosteroid allopregnanolone. In a final commentary, we formulated the mathematical relation between Cl– conductance, KCC2-mediated Cl– extrusion capacity and steady-state [Cl–]i. In summary, the present thesis (i) clarifies how well common electrophysiological methods describe [Cl–]i and gCl, (ii) provides a novel method for quantifying gCl in cell membranes and (iii) clarifies the roles of the ECM, ion channels and ion transporters in the control of [Cl–]i homeostasis and GABAAR-mediated signaling in central neurons.

Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.

Les protéoglycanes (PGs) sont des protéines présentes au niveau de la matrice extracellulaire et à la surface des cellules. Ils sont constitués d’une protéine sur laquelle sont attachées des chaînes de glycosaminoglycanes. Ils jouent un rôle essentiel dans plusieurs processus biologiques. Des mutations au niveau des gènes codant pour la protéine porteuse ou les enzymes impliquées dans la biosynthèse des GAGs sont associées à plusieurs syndromes et pathologies chez l’homme. L’initiation de la synthèse des GAGs est catalysée par la xylosyltransférase I (XT-I). La XT-I joue un rôle clé dans la régulation de la synthèse des PGs au niveau du cartilage et il a été montré récemment que les mutations hypomorphiques de la XT-I sont associées au syndrome du Desbuquois de type II (DBQD2) caractérisé par des anomalies squelettiques (ostéochondrodysplasie). Afin d’élucider le rôle de la XT-I dans le développement ostéoarticulaire, nous avons généré une souris transgénique conditionnelle Col2α1-CreERTM ;XylT1flox/flox permettant l’invalidation de la XT-I au niveau du cartilage. De façon intéressante, l’invalidation de la XT-I induit des anomalies du développement ostéoarticulaire caractérisées par un nanisme important et des défauts des éléments squelettiques. Des études histologiques et la microscopie SHG (génération de seconde harmonique) de la plaque de croissance ont permis de montrer l’importante de la XT-I dans la formation de la matrice extracellulaire (MEC), la fibrillation du collagène II, la maturation des chondrocytes et leur organisation en colonne dans la plaque de croissance. L’analyse des mécanismes moléculaires impliqués indique la perturbation de la voie de signalisation du TGF-β dans la plaque de croissance. D’autre part, des études histomorphométriques et histologiques des os ont révélé que la déficience en XT-I entraîne une accélération du processus d’ossification avec une stimulation de l’activité des ostéoclastes au niveau de l’os spongieux conduisant à une résorption osseuse importante et à une ossification accrue de l’os cortical. Ces travaux ont permis de révéler le rôle de la XT-I dans le développement ostéoarticulaire et dans le maintien de l’homéostasie du cartilage et du tissu osseux et ont mis en évidence le rôle de la voie du TGF-β dans les anomalies du développement. Ces travaux ouvrent également la voie pour le développement de thérapeutiques potentielles pour le traitement des patients atteints du syndrome de Desbuquois type II
Proteoglycans (PGs) are proteins present in the extracellular matrix and on the surface of cells. They consist of a protein to which chains of glycosaminoglycans (GAGs) are attached. PGs play an essential role in many biological processes and in the homeostasis of different tissues including cartilage, bone and skin. Mutations in the genes encoding PG core proteins or the enzymes involved in GAG biosynthesis are associated with several syndromes and pathologies in human. Initiation of GAG synthesis is catalyzed by xylosyltransferase I (XT-I). XT-I plays a key role in the regulation of the synthesis of PGs in cartilage and it has been shown recently that hypomorphic mutations of XT-I are associated with the Desbuquois syndrome type II (DBQD2), characterized by skeletal abnormalities (osteochondrodysplasia). To elucidate the role of XT-I in skeletal development, we generated a conditional transgenic mouse, Col2α1-CreERTM; XylT1flox/flox allowing the invalidation of XT-I gene in the cartilage. Interestingly, the invalidation of XT-I induces skeletal developmental abnormalities characterized by significant dwarfism, and defects in many skeletal elements. Histological studies and SHG microscopy (second harmonic generation) of the growth plate showed the importance of XT-I in extracellular matrix formation, fibrillation of collagen type II, maturation of chondrocytes and their organization in column in the growth plate. The analysis of the molecular mechanisms involved indicates the disruption of the TGF-β signaling pathway in the growth plate. On the other hand, histomorphometric and histological studies of the bones revealed that the XT-I deficiency causes an acceleration of the ossification process with a stimulation of the osteoclasts activity in spongy bone leading to bone resorption, and increased ossification of the cortical bone. This work revealed the role of XT-I in skeletal development and in the maintenance of cartilage and bone homeostasis and highlighted the role of the TGF-β pathway in developmental abnormalities. This work also paves the way for the development of potential therapeutics for the treatment of patients with Desbuquois syndrome type II

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem.
In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix (ECM), located on the blastocoelic side of the trophectoderm, to form a continuous layer of extraembryonic endoderm. Cell migration events depend on a family of cell surface proteins known as integrins that bind specific ECM proteins. In an effort to understand the mechanisms involved in bovine endodermal cell migration, two experiments were conducted. In the first experiment, expression of the ECM proteins fibronectin, laminin and vitronectin was evaluated by immunofluorescent staining in in vivo and in vitro developing embryos during Day 6-10 and Day 7-10, respectively (Day 0=onset of estrus). Fibronectin was detected in all stages of in vivo and in vitro embryos, however no difference (P>0.10) was observed due to day or developmental stage. Laminin staining was moderately expressed in all stages of in vivo embryos, with an increase (P<0.05) in Day 10 in vivo embryos. Laminin staining in Day 9 in vitro embryos was less intense (P<0.05) than Day 7 and 8 in vitro embryos. Higher (P<0.05) expression of laminin was observed in Day l0 in vivo embryos as compared to Day 10 in vitro. Vitronectin staining was expressed throughout all stages of development. Day 6 in vivo embryos exhibited more intense (P<0.05) staining compared to Day 8 in vivo embryos. Day 10 in vivo embryos expressed more (P<0.05) vitronectin than Day 10 in vitro embryos. In the second experiment, the effects of ECM-type and inhibitors of integrin binding on bovine endodermal cell outgrowth from the ICM were evaluated. Day 7 embryos were nonsurgically collected and cultured for 96 h on either fibronectin-layered microdrops containing 0 (control), 0.5 or 1.0 mg/ml RGD and/or EILDV peptides or vitronectin-layered microdrops containing 0, 0.5 or 1.0 mg/ml RGD peptides. At 24-h intervals, ICM were photographed and the numbers of cells leaving the ICM were counted. Areas of cellular outgrowth were calculated from the photomicrographs. Compared to the control, addition of 0.5 or 1.0 mg/ml RGD, EILDV or RGD and EILDV did not (P>0.10) reduce the areas of cellular outgrowth from the ICM on matrices of fibronectin. Numbers of cells in outgrowths were greater (P<0.05) in control ICM compared to 0.5 mg/ml RGD, but this effect was eliminated (P>0.10) when the inhibitor concentration was increased to 1.0 mg/ml. Addition of 0.5 or 1.0 mg/ml RGD did not reduce (P>0.10) the area of cellular outgrowth from the ICM on vitronectin and had no effect (P>0.10) on numbers of cells in the outgrowths. Detection of fibronectin, laminin and vitronectin by immunofluorescence suggests these proteins are present in the developing bovine embryo to support endodermal cell migration and stabilization in extraembryonic endoderm formation. Because cell migration over fibronectin and vitronectin was not inhibited by the RGD and EILDV peptides, endodermal cells must use either an integrin that recognizes alternative binding sites in fibronectin and vitronectin or an alternative cell adhesion system.
Graduation date: 2003

Basic fibroblast growth factor (bFGF) plays an instrumental role in the cascade of events leading to restenosis, vascular re-occlusion due to excessive smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) deposition following arterial intervention procedures such as balloon angioplasty. The mechanism of bFGF activation following vascular injury has remained elusive. bFGF is stored bound to heparan sulfate proteoglycans in the ECM of the arterial media; release from extracellular sequestration may activate bFGF and initiate SMC proliferation and migration. bFGF mobilization at injured sites may be induced by platelet degranulation products. We have carried out in vitro studies demonstrating that platelet-derived heparanase and platelet factor-4 (PF4) liberate bFGF from the ECM of vascular SMCs, resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. These increases in SMC proliferation and migration were completely inhibited by the addition of a bFGF-neutralizing antibody. In order to investigate the role of heparanase and PF4 in vivo, each was delivered to the uninjured rat carotid artery. Heparanase and PF4 were both found to release bFGF, induce substantial SMC proliferation and increase the expression of several growth factor receptors thought to promote restenosis. An antibody that neutralizes platelet-derived heparanase was developed and evaluated in a rat carotid balloon injury model. Perivascular delivery of anti-heparanase IgG was found to inhibit bFGF depletion from the arterial wall by approximately 60% (p < 0.001) at 4 days. This correlated with the reduction in intimal thickening observed at 14 days. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to luminal narrowing following vascular injury. In addition, platelet-derived heparanase is likely to play a key role in initiating events leading to restenosis via bFGF mobilization.

Aims to observe changes in the expression of syndecan-1 in both the developing epithelium of the rat oral mucosa, and in epithelial cell rests of Malassez in the developing periodontium of normal rat molars, from late crown development through to early eruption.
Thesis (M.D.S.) -- University of Adelaide, School of Dentistry, 2001

Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1beta. Since these gene products are regulated by the transcription factor NF-kappaB, we investigated the effects of resveratrol on IL-1beta-induced NF-kappaB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1beta-induced proteasome function and the degradation of IkappaBalpha (an inhibitor of NF-kappaB) without affecting IkappaBalpha kinase activation, IkappaBalpha-phosphorylation or IkappaBalpha-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-kappaB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1beta-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-kappaB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

Tese de doutoramento em Ciências da Saúde, no ramo de Medicina, na especialidade de Cirurgia, apresentada à Faculdade de Medicina da Universidade de Coimbra
Despite recent progresses in surgical technique and perioperative care, failure of intestinal anastomotic healing remains one of the most feared complications in digestive surgery, exerting a profound adverse impact on the operative morbidity and mortality rates, oncologic, and functional outcomes and socioeconomic costs. Teduglutide is an enterotrophic analogue of glucagon-like peptide 2 (GLP-2) approved for the pharmacological rehabilitation of short-bowel syndrome. Present study aims to clarify the potential of teduglutide as a promoting strategy for the improvement of intestinal anastomotic healing, on an animal model, through the influence on the cellular, humoral and molecular mediators of repair. An experimental rat model of standard small-bowel anastomosis was used with evaluation at the third and seventh postoperative days. Structural assessment of the anastomosis included the macroscopic integrity and the histological and immunohistochemical examination of healing parameters, comprising reepithelialization, neoangiogenesis and fibroplasia. Cellular and molecular mediators of anastomotic healing were analyzed, including: putative epithelial stem cells response (using Lgr5, Bmi1 and the panel CD24/CD44/CD166/Grp78 surface markers by flow cytometry); cellular viability and death (with double staining with annexin-V/propidium iodide by flow cytometry); oxidative stress [quantification of cytosolic peroxides with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH2) probe, mitochondrial reactive species with dihydrorhodamine 123 (DHR 123) probe, total intracellular reduced glutathione with mercury orange staining and mitochondrial membrane potential with 5,5',6,6'-tethrachloro-1,1',3,3'-tethraethylbenzimidazolcarbocyanine iodide (JC-1) probe, by flow cytometry]; local and systemic inflammatory response (tissue and plasma concentrations of interleukine-1α, macrophage chemo-attractant protein-1, tumor necrosis factor-α, interferon-γ and interleukine-4 by flow cytometric multiplexed bead assay); gene expression of main extracellular matrix components (Collagen, type I, alpha 1: Col1a1; Collagen, type III, alpha 1: Col3a1; Collagen, type IV, alpha 1: Col4a1; Collagen, type V, alpha 1: Col5a1) and remodeling factors, matrix metalloproteinases (Mmp; Mmp1 and Mmp13, Mmp2 and Mmp9, Mmp3, Mmp12 and Mmp14) and tissue inhibitors of metalloproteinases 1 and 2 (Timp; Timp1 and Timp2); gene expression of growth factors and receptor potentially implicated on anastomotic repair (Insulin-like growth factor 1, transcript variant: Igf1; Vascular endothelial growth factor A, transcript variant 2: Vegfa; Transforming growth factor, beta 1: Tgfb1; Connective tissue growth factor: Ctgf; Fibroblast growth factor 2: Fgf2; Fibroblast growth factor 7: Fgf7; Epidermal growth factor: Egf; Heparin-binding epidermal-like growth factor: Hbegf; Platelet-derived growth factor beta polypeptide: Pdgfb; Glucagon-like peptide 2 receptor: Glp2r) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR); and Glp-2 plasma levels (by competitive enzyme immunoassay). Teduglutide had no apparent relevant impact on the rate or severity of intestinal anastomotic leakage. A favorable influence of teduglutide on the reepithelialization and neoangiogenesis events of the proliferative phase of anastomotic repair was documented. Teduglutide was associated with an increase of subepithelial myofibroblasts density score, but no significant effect on the goblet, Paneth and glial cellular indexes was observed. This growth factor was associated with an enhancement of type III collagen deposition on the submucosa at the seventh postoperative day, although with simultaneous reduction of type I collagen level in that layer, and a non-significant reduction of global anastomotic collagen content. Teduglutide inhibited the gene modulation of fibrolysis in the predominantly inflammatory phase of anastomotic repair, while stimulated the fibrolysis in the proliferative stage. Teduglutide induced the upregulation of gene expression of Timp1, Timp2 and Col4a1, and the downregulation of Mmp3 and Mmp12, at the third postoperative day; and the repression of gene expression of Timp1, Col3a1, Col4a1 and Col5a1, at the seventh day. Teduglutide contributed to the expansion of the putative crypt base columnar stem cells pool at the seventh day and to the concomitant depletion of the putative “position +4” stem cells fraction. An increase (non-significant) of the overall putative intestinal epithelial stem cells was also observed in teduglutide-treated animals. Teduglutide was associated with a non-significant prooxidative effect, with an increase of the cytosolic peroxides level and mitochondrial reactive species levels and a reduction of the cellular reduced glutathione content. Those effects were coincident with an increase of cellular viability indexes and a non-significant decrease of early apoptotic events. No relevant influence on mitochondrial membrane potential was verified. A non-significant increase of tissue levels of the anti-inflammatory interleukin-4 at the seventh day, and a significant reduction of plasma levels of interferon-γ at the third day were observed in teduglutide-treated animals. Teduglutide induced the upregulation of the gene expression of Igf1, Vegfa and Ctgf and the downmodulation of Fgf2, Fgf7, Tgfb1 and Glp2r. To conclude, the present study reflects the complexity of the intestinal anastomotic repair and points to a favorable influence of teduglutide on this process that deserves additional investigation.
Apesar dos recentes progressos da técnica cirúrgica e suporte peri-operatório, a falência da cicatrização anastomótica intestinal constitui, ainda, uma das mais temíveis complicações da cirurgia digestiva, com um importante impacto adverso na mortalidade e morbilidade operatórias, resultados oncológicos e funcionais e custos económico-sociais. O teduglutide é um análogo enterotrófico do glucagon-like peptide 2 (GLP-2) aprovado para a reabilitação farmacológica da síndroma do intestino curto. Este estudo procurou analisar as potencialidades do teduglutide como estratégia adjuvante da cicatrização anastomótica intestinal, num modelo animal, através da sua influência nos mediadores celulares, humorais e moleculares do processo reparativo. Foi utilizado um modelo experimental de anastomose intestinal estandardizada, em rato, com avaliação ao terceiro e ao sétimo dias pós-operatórios. A avaliação estrutural da anastomose incluiu a integridade macroscópica e os exames histológico e imunohistoquímico dos parâmetros de cicatrização, tais como reepitelização, neoangiogénese e fibroplasia. Foram analisados os seguintes mediadores celulares e moleculares da cicatrização anastomótica: resposta das putativas células estaminais epiteliais (usando os marcadores de superfície Lgr5, Bmi1 e o painel CD24/CD44/CD166/GrpP78 por citometria de fluxo); viabilidade e morte celular (com marcação dupla com anexina V/iodeto de propídeo, por citometria de fluxo); stresse oxidativo [quantificação de peróxidos citoplasmáticos com sonda de diacetato de 2’,7’-diclorodihidrofluoresceína (DCFH2), espécies reactivas mitocondriais com sonda de dihidrorodamina 123 (DHR 123), glutatião reduzido intracelular com marcação com alaranjado de mercúrio e potencial de membrana mitocondrial com sonda de iodeto de 5,5',6,6'-tetracloro-1,1',3,3'-tetraetilbenzimidazolcarbocianina (JC-1), por citometria de fluxo]; resposta inflamatória local e sistémica (concentrações tecidulares e plasmáticas de interleucina-1α, macrophage chemo-attractant protein-1, factor de necrose tumoral-α, interferon-γ e interleucina-4 por citometria de fluxo; expressão génica de componentes da matriz extracelular (Collagen, type I, alpha 1: Col1a1; Collagen, type III, alpha 1: Col3a1; Collagen, type IV, alpha 1: Col4a1; Collagen, type V, alpha 1: Col5a1) e respectivos factores de remodelação, metaloproteinases (Mmp) da matriz 1, 13, 2, 9, 3, 12 e 14 (Mmp1 e Mmp13, Mmp2 e Mmp9, Mmp3, Mmp12 e Mmp14) e inibidores tecidulares das Mmp 1 e 2 (Timp1 and Timp2); assim como dos factores de crescimento potencialmente implicados na reparação anastomótica (Insulin-like growth factor 1, transcript variant: Igf1; Vascular endothelial growth factor A, transcript variant 2: Vegfa; Transforming growth factor, beta 1: Tgfb1; Connective tissue growth factor: Ctgf; Fibroblast growth factor 2: Fgf2; Fibroblast growth factor 7: Fgf7; Epidermal growth factor: Egf; Heparin-binding EGF-like growth factor: Hbegf; Platelet-derived growth factor beta polypeptide: Pdgfb; Glucagon-like peptide 2 receptor: Glp2r) por transcrição reversa quantitativa da reacção em cadeia da polimerase em tempo real; e da concentração plasmática do Glp-2 (por imunoensaio enzimático competitivo). O teduglutide não teve impacto relevante aparente na incidência e gravidade da deiscência anastomótica mas exerceu uma influência favorável nos processos de reepitelização e de neoangiogénese da fase proliferativa da cicatrização. Associou-se, ainda, a um aumento da densidade de miofibroblastos subepiteliais, sem modificação significativa dos índices de células caliciformes, de Paneth e gliais. Este factor de crescimento associou-se ao aumento da deposição de colagéneo III na submucosa ao sétimo dia pós-operatório, embora com redução concomitante do colagéneo I na mesma camada, e à redução não significativa do teor global de colagéneo na anastomose. O teduglutide inibiu a modulação génica da fibrólise na fase predominantemente inflamatória da reparação enquanto, pelo contrário, reprimiu a fibrogénese na fase proliferativa. O teduglutide aumentou a expressão génica de Timp1, Timp2 e Col4a1 e, reduziu a de Mmp3 e Mmp12, ao terceiro dia pós-operatório; e diminuiu a expressão génica do Timp1, Col3a1, Col4a1 e Col5a1, ao sétimo dia. O teduglutide induziu a expansão das putativas células estaminais colunares basais das criptas e, concomitantemente, a depleção das células da “posição +4”. Nos animais tratados com teduglutide, observou-se, ainda, um aumento global não significativo das putativas células epiteliais intestinais. O teduglutide associou-se a um efeito pro-oxidativo não significativo, com aumento dos níveis de peróxidos citoplasmáticos e das espécies reactivas mitocondriais e redução do glutatião reduzido celular. Estes efeitos foram acompanhados por um aumento do índice de viabilidade celular e uma redução não significativa dos eventos apoptóticos precoces. Não se verificou influência significativa no potencial de membrana mitocondrial. Nos animais tratados com teduglutide, observou-se um aumento não significativo dos níveis tecidulares da citocina anti-inflamatória interleucina-4 ao sétimo dia, assim como uma redução significativa da concentração plasmática de interferon-γ ao terceiro dia. O teduglutide promoveu o aumento da expressão génica do Igf1, Vegfa e Ctgf e a repressão do Fgf2, Fgf7, Tgfb1 e Glp2r. Em conclusão, o presente estudo reflecte a complexidade da cicatrização anastomótica intestinal e sugere uma influência favorável do teduglutide neste processo que justifica uma investigação adicional.