Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins αDβ2 and αMβ2 to macrophage migration and development of chronic inflammation. We found that integrin αDβ2 is upregulated on M1-macrophages in vitro and pro-inflammatory macrophages in atherosclerotic lesions. Interestingly, the expression of ligand-sharing integrin αMβ2 remains unaltered. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that robust adhesion of M1-activated macrophages translates to weak 3D migration, which depends on the high expression of αDβ2, since αD-deficiency decreases M1-macrophage adhesion and improves macrophage migration. In contrast, αD- and αM-knockouts decrease M2-macrophages migration, demonstrating that moderate integrin expression supports cell motility. In model of high fat diet-induced diabetes, αD-deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM-deficiency does not affect macrophage accumulation. We detected a new ligand for integrins αMβ2 and αDβ2, 2-(ω-carboxyethyl)pyrrole (CEP). CEP is preferentially generated during inflammation-mediated oxidation and forms adduct with ECM proteins generating novel substrate for αMβ2 and αDβ2. Targeting CEP-dependent macrophage adhesion can be a useful approach to control αDβ2-mediated chronic inflammation. Using specially designed peptide library, protein-protein interaction and adhesion assay, we identified a peptide, called P5, which significantly inhibited αD-CEP binding. P5 peptide regulates macrophage migration in three-dimensional matrix in vitro and reduced macrophage accumulation during thioglycollate-induced peritoneal inflammation. Effect of P5 is completely eliminated in αD-deficient macrophages. Tracking of adoptively-transferred fluorescently-labeled WT and αD-/- monocytes in diabetic mice confirmed that αD-dependent inhibition of macrophage accumulation in adipose tissue is mediated by P5 peptide. Taken together, these results demonstrate the importance of αDβ2 and αDβ2-CEP interaction for the accumulation of infiltrating macrophages during inflammation and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes.

The objectives of this research were to better characterize the protein signaling complexes that form in response to spermatozoa binding to the bovine oocyte vitelline membrane and to elucidate their potential involvement in oocyte activation. Integrins located on the vitelline membrane of bovine oocytes have been implicated in mediating the sperm-oocyte interaction. Anti-integrin function blocking antibodies and immunofluorescence were utilized in order to reveal that the αV and β1 integrin subunits are essential for fertilization in the bovine and could form the integrin heterodimer involved in the sperm-oocyte interaction. Focal adhesion kinase is localized to focal adhesions and is a key component of signal transduction pathways mediated by integrins. The presence of focal adhesion kinase in bovine oocytes was verified by real-time polymerase chain reaction and immunoprecipitation and the localization of focal adhesion kinase at the site of sperm binding to the oocyte plasma membrane was verified using immunohistochemistry. The inhibition of focal adhesion kinase resulted in fewer cleaved embryos in addition to a reduction in the number of oocytes responding with calcium transients. Phospholipase C isoforms regulate the release of calcium from the endoplasmic reticulum and are known to interact with integrins and focal adhesion kinase. The experiments reported in this dissertation explored the involvement of phospholipase C isoforms endogenous to the oocyte in mediating the calcium release associated with fertilization. Reduction in phospholipase C messenger ribonucleic acid levels for the phospholipase C isoforms γ1 and γ2 resulted in significantly lower cleavage rates compared to the controls. Interestingly, the reduction in messenger ribonucleic acid levels for phospholipase ζ failed to impact cleavage. Maximizing protein levels for the phospholipase C isoforms ζ and γ2 resulted in a significantly higher number of oocytes reaching the 2-cell stage compared to all other treatment groups and not significantly different than the activation control. Together these data illustrate the involvement of the αV and β1 integrin subunits, focal adhesion kinase, and the potential involvement of multiple endogenous phospholipase C isoforms (γ1 and γ2) in bovine oocyte activation. A more complete understanding of the molecular players involved in fertilization could have beneficial impacts for human fertility, assisted reproduction, and improved efficiency of animal somatic cell nuclear transfer.

Neutrophils and macrophages accumulate at sites of inflammation and cause chronic inflammation leading to various diseases. Therefore, to better understand chronic disease pathways it is important to investigate the properties of macrophage accumulation in inflamed tissues. The I-domain of the macrophage receptor integrin aDb2 plays a vital role in macrophage retention by binding to CEP (carboxyethyl pyrrole), a ligand available at inflammatory sites. This thesis mainly focuses on evaluating the binding site within integrin aDb2 that binds carboxyethyl pyrrole (CEP)-modified proteins. So, a recombinant plasmid construct containing the integrin I-domain was developed. Seven non-conserved amino acids were mutated by PCR-site-directed mutagenesis to create a mutant construct. After expressing in E. coli, the binding affinities of wild-type and mutant I-domains to CEP were analyzed using biolayer interferometry. It was found that a patch of seven positively charged amino acids contributes to the strong binding of the I domain to CEP.

Bacteria typically adhere to various cell surfaces present in the human body to colonise or invade human tissues. Staphylococcus aureus (S. aureus) can express the fibronectin-binding proteins A and B (FnBP-A, FnBP-B) that can facilitate the binding of multiple copies of fibronectin (Fn). In addition, Fn bound to the bacterium trigger activation of α5β1 integrins found on the cells and facilitate invasion of human cells. Although the invasion mechanisms regarding signalling pathways and overall host cell interactions have been defined, the quantitative relationship between the mediators of invasion and the temporal kinetics has not yet been elucidated. In this thesis, newly developed microscopy-based methods have been used to quantify the interactions between H1299 cells and S. aureus at various Fn concentrations. After an approximate Fn concentration of 15 μg/ml, the S. aureus bacteria strains become saturated both for the wildtype and the negative control strains. Additionally, using the step-by-step protocol developed during this study, adhesion of the wildtype strain of S. aureus with 15 μg/ml Fn is occurring on the H1299 cells. Although adjustments to the protocol are needed, this adhesion mechanism will lead to an internalisation of the S. aureus strains to the H1299 cells.

2-ω-carboxyethylpyrrole (CEP) is a product of Docosahexaenoic acid (DHA) oxidation, which forms covalent adducts with different proteins. CEP-modified proteins can interact with macrophage receptor, integrin αDβ2. This study aims to compare αDβ2 binding to its physiological ligand, fibrinogen, and CEP-modified fibrinogen, which is formed during inflammation. We hypothesize that modification of fibrinogen changes its ligand-binding properties to integrin αDβ2 which can affect macrophage migration and retention. Recombinant αD I-domain and αDβ2-transfected HEK293 cells were used for the experiments. In biolayer interferometry (BLI) assays, fibrinogen was immobilized at pH 5.0 and fibrinogen-CEP at pH 3.5. Our results showed that the affinity of αD I-domain binding to fibrinogen-CEP was higher than fibrinogen, and the binding was inhibited by the anti-CEP antibody. The optimal expression of αD I-domain was found at 25°C. In cell adhesion assays, optimal concentrations were used for the inhibition assay; fibrinogen at 2µg/ml and fibrinogen-CEP at 8µg/ml. αDβ2-transfected cells demonstrated stronger adhesion to fibrinogen-CEP, and this adhesion was significantly inhibited by polyglutamic acid, which mimics CEP-mediated binding. These findings suggest that αDβ2's interaction with DHA-modified extracellular matrix (ECM) proteins significantly increases macrophage adhesion and may serve for macrophage retention during chronic inflammation. Developing small molecules that can inhibit αDβ2-CEP interaction could be a breakthrough in treating inflammatory diseases. The main conclusions drawn from the study are that CEP-modified fibrinogen has a higher affinity for αD I-domain binding than native fibrinogen, and this interaction can be inhibited by targeting CEP. The study provides insights into the potential therapeutic applications of inhibiting αDβ2-CEP interactions in inflammatory diseases.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC50 of < 0.1 µM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation. / Public Authority for Applied Education and Training (PAAET)

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[EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engineered to display the III 7-10 fragment of the fibronectin fused to GFP as reporter protein. Fibronectin is a ubiquitous protein present in the extracellular matrix, a complex mesh of structural and adhesive proteins which serve as mechanical support and development niche for cells of a wide variety of tissues. This fragment contains two important sequences, RGD and PHSRN. RGD is an adhesive sequence that interacts with a wide range of integrins, membrane-bound receptors that play a role in cellular processes such as adhesion, migration, proliferation and differentiation. On the other hand, PHSRN binds synergistically with RGD to some integrins such as alpha-5-beta-1 and others, increasing the specificity of this interaction. Genetically engineered L. lactis has been thoroughly characterized to test its capabilities as a living interface. This strain was found to express the FNIII 7-10-GFP fragment covalently linked to the cell wall and biological activity and expression levels of this fragment was assessed with techniques such as Western blot, ELISA and immunofluorescence. Moreover, this strain still holds the ability to develop biofilms, communities of sessile, attached bacteria to abiotic surfaces which helps greatly in the generation of a stable monolayer of bacteria between synthetic substrates and mammalian cells. Mammalian cell behaviour in response to the expressed fibronectin fragment on L. lactis membrane was also assessed. Several cell lines were tested, such as Fn-/Fn- and NIH3T3 fibroblasts, C2C12 myoblasts and human bone-marrow derived mesenchymal cells. This living biointerface was found to trigger cell adhesion and FAK phosphorylation, a marker for intracellular integrin-mediated signalling in all of the tested cell lines. It also triggered myoblast-to-myotube differentiation on C2C12 cells. In hMSCs, the cell-wall exposed fibronectin fragment was found to enhance the phosphorylation of ERK1/2, a kinase involved in the MAPK pathway, which is deeply involved in a multitude of cellular processes related to differentiation, proliferation and migration. Nevertheless, this thesis is a proof of concept that this novel system can be further exploited to express almost any desired protein or small molecule to help in the development of new tissues from progenitor cells. These molecules can be either secreted in the medium or displayed in the membrane, and can also be constitutively expressed or in-demand, due to the great flexibility of L. lactis and the wide variety of expression systems available. This interface based on living bacteria establishes a new paradigm in surface functionalization for biomedical engineering applications. / [ES] Esta tesis aborda el desarrollo de una biointerfase viviente entre materiales sintéticos y células vivas con el objetivo de dirigir la interacción célula-material en aplicaciones de ingeniería tisular. Esta biointerfase está compuesta de Lactococcus lactis, una bacteria láctica no patógena, ampliamente usada en la industria láctea como inóculo, y, recientemente, en la expresión heteróloga de proteínas para su uso como vacunas de administración oral o su expresión en membrana. L. lactis ha sido genéticamente modificado para expresar el fragmento III 7-10 de la fibronectina, unida a GFP como reporter. La fibronectina es una proteína presente de forma ubicua en la matriz extracelular, una compleja red de proteínas adhesivas y estructurales cuyo propósito es servir como soporte estructural y como nicho de desarrollo para diversos tejidos. Este fragmento contiene dos secuencias importantes, RGD y PHSRN. RGD es una secuencia adhesiva de unión que interacciona con una amplia variedad de integrinas, receptores de membrana que juegan muchos e importantes papeles en diferentes procesos celulares, como adhesión, proliferación, migración o diferenciación. Por otra parte, PHSRN se une a las integrinas de forma sinérgica con RGD facilitando aún más estos procesos y aumentando la especificidad de esta interacción. Esta cepa de L. lactis modificada ha sido ampliamente caracterizada para estudiar su idoneidad como interfaz funcional viviente. Se ha demostrado que L. lactis es capaz de expresar el fragmento FNIII7-10-GFP covalentemente anclado a la pared celular bacteriana, habiéndose caracterizado también su actividad biológica con técnicas como Western blot, ELISA e inmunofluorescencia. Esta cepa mantiene la capacidad de desarrollo de biofilms presente en la gran mayoría de microorganismos. Los biofilms son comunidades de bacterias sésiles adheridas a un sustrato que pueden ser usadas como interfase física entre células de mamífero y sustratos abióticos. También se ha estudiado la respuesta celular a la fibronectina expuesta en la membrana de L. lactis. Se estudiaron varias líneas celulares, como fibroblastos Fn-/Fn- y NIH3T3, mioblastos C2C12 y células mesenquimales humanas derivadas de médula ósea. Esta interfase viviente fue capaz de provocar respuesta celular en forma de adhesión en todas las líneas estudiadas, además de inducir diferenciación de mioblastos a miotubos en C2C12 y de provocar la fosforilación de FAK, un marcador de señalización celular mediada por integrinas. En células mesenquimales humanas se demostró la capacidad del fragmento de fibronectina expuesto para fosforilar ERK1/2, una kinasa perteneciente a la ruta de señalización MAPK, ruta que forma parte de muchos procesos celulares importantes como diferenciación, proliferación y migración. Pese a todo, esta tesis es sólo una prueba de concepto de un sistema que puede ser utilizado para expresar casi cualquier proteína o molécula pequeña deseada, que puede ser muy útil en el desarrollo de nuevos tejidos a partir de sus células progenitoras. Estas moléculas pueden ser secretadas en el medio o ancladas en la pared celular, de forma constitutiva o bajo demanda, debido a la flexibilidad y amplia variedad de sistemas de expresión disponibles para L. lactis. Esta biointerfase basada en bacterias vivas establece un nuevo paradigma en el campo de la funcionalización de superficies para aplicaciones de ingeniería biomédica. / [CA] Aquesta tesi aborda el desenvolupament d'una interfase viva entre materials sintètics i cèl·lules vives amb l'objectiu de dirigir la interacció cèl·lula-material, per al seu ús en aplicacions d'enginyeria tissular. Aquesta interfase està composta de Lactococcus lactis, un bacteri làctic, no patogènic i àmpliament utilitzat en l'industria làctica com a inòcul, i, recentment, en l'expressió heteròloga de proteïnes per al seu ús com vacunes d'administració oral o per a la seva expressió en membrana. L. lactis ha sigut genèticament modificada per a expressar el fragment III7-10 de la fibronectina, unida a GFP com a reporter. La fibronectina és una proteïna present de forma ubiqua en la matriu extracel·lular, una complexa xarxa de proteïnes adhesives i estructurals que s'utilitzen com a suport estructural i com a nínxol de desenvolupament per a diversos teixits. Aquest fragment conté dos seqüències importants, RGD i PHSRN. RGD és una seqüència adhesiva d'unió a integrines, receptors de membrana que juguen molts i molt importants papers en diferents processos cel·lulars, com poden ser adhesió, proliferació, migració o diferenciació. Per altra banda, PHSRN s'uneix a les integrines de forma sinèrgica amb RGD facilitant encara més aquests processos i augmentant l'especificitat d'aquesta interacció. Aquesta modificació genètica de L. lactis ha estat àmpliament caracteritzada per provar les seves característiques com a interfase funcional vivent. S'ha demostrat que L. lactis és capaç d'expressar el fragment FNIII 7-10-GFP covalentment ancorat a la paret cel·lular bacteriana, havent-se caracteritzat també la seva activitat biològica amb tècniques com Western blot, ELISA i immunofluorescència. A més, aquest cep manté la capacitat de desenvolupament de biofilms, comunitats de bacteris sèssils adherits a un substrat que poden ser utilitzades com a interfase física entre cèl·lules de mamífer i substrats abiòtics. També s'ha estudiat la resposta cel·lular a la fibronectina expressada en la paret cel·lular de L. lactis. El estudi es va fer utilitzant diverses línies cel·lulars, com fibroblasts Fn-/Fn- i NIH3T3, mioblasts C2C12 i cèl·lules mesenquimals humanes derivades de medul·la òssia. Aquesta interfase vivent va ser capaç de provocar resposta cel·lular en forma d'adhesió a totes les línies estudiades, a més d'induir diferenciació de mioblasts a miotubs en C2C12 i de provocar la fosforilació de FAK, un marcador de senyalització cel·lular mediat per integrines, en les línies assajades. En cèl·lules mesenquimals humanes es va demostrar la capacitat del fragment de fibronectina exposat per fosforilar ERK1/2, una kinasa pertanyent a la ruta de senyalització MAPK, ruta que forma part de molts processos cel·lulars importants com diferenciació, proliferació i migració. Malgrat tot, aquesta tesi mostra només una prova de concepte d'un sistema que pot ser utilitzat per expressar gairebé qualsevol proteïna o molècula petita desitjada, que pot ser molt útil en el desenvolupament de nous teixits a partir de les seves cèl·lules progenitores. Aquestes molècules poden ser secretades en el medi o ancorades a la paret cel·lular, de manera constitutiva o sota demanda, a causa de la flexibilitat i àmplia varietat de sistemes d'expressió disponibles per L. lactis Aquesta biointerfase basada en bacteris vius estableix un nou paradigma en el camp de la funcionalització de superfícies per a aplicacions d'enginyería biomèdica. / Rodrigo Navarro, A. (2015). Functional living biointerfaces to direct cell-material interaction [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51461