Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein-levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1- integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

Eosinophils are end-point effectors of inflammation that contribute to the clinical severity of asthma. Eosinophil homing to the asthmatic lung is primarily guided by eotaxin-1, which is an eosinophil-selective chemokine. The mechanism by which eotaxin-1 augments intracellular calcium during cell migration is incompletely understood but is integral to the extravasation of eosinophils at sites of inflammation. We consequently report here that fluid shear stress, like eotaxin-1, unexpectedly activates human eosinophils in a calcium-dependent manner. We used confocal fluorescence microscopy to study calcium-handling in purified human eosinophils. Application of eotaxin-1 augmented the [Ca2+]i in a concentration-dependent manner. Pre-treatment of cells with ryanodine (10 μM) completely abolished the eotaxin-mediated calcium response, indicating that this phenomenon is dependent on Ca2+-release from the ER. Several SOCC blockers (2-APB, 100 μM; Gd3+, 10 μM; SKF-96365, 100 μM) attenuated SOCE, suggesting that these channels may directly contribute towards the eotaxin-1 calcium response in human eosinophils. In the presence of fluid-perfusion, eosinophils displayed a robust perfusion-induced calcium response (PICR) demonstrating that eosinophils are mechanically sensitive. The PICR rapidly induced adhesion and non-directional migration in eosinophils, suggesting that some hitherto unknown molecular mechanosensor permits these cells to detect and respond to changes in shear-stress. Pre-treatment of eosinophils with the non-selective tripeptide integrin receptor blocker, Arg-Gly-Asp (RGD), abrogated the PICR. The highly selective, dual α4β7/α4β1 integrin receptor blocker, CDP-323, was used to ascertain whether these highly expressed integrin subtypes mediate the PICR in eosinophils. Pre-treatment of cells with CDP-323 completely abolished the PICR, in addition to the eotaxin-mediated calcium response in a shear-dependent manner. Taken together, our results support a novel role for the α4β7/α4β1 integrin receptors as mechanosensors that directly modulate [Ca2+]i, adhesion and migration in human eosinophils. On-going experiments will seek to quantify the shear-response thresholds at which eosinophils activate and the time-course of the associated calcium response. This study suggests that the recruitment and activation of eosinophils are regulated by chemical and mechanical stimuli via overlapping, calcium-dependent signal transduction cascades. Given that the PICR is mediated by the eosinophil-specific α4β7/α4β1 integrin receptors, we conclude that integrin receptors are molecular mechanosensors that may facilitate eosinophil activation, adhesion and non-directional migration independently of, or in conjunction with, chemokine signaling. / Thesis / Master of Science in Medical Sciences (MSMS)

Osseointegration, or the direct integration of an implant into bone tissue, is necessary for implant success. Titanium is commonly used clinically in dental and orthopaedic implants because of its passivating oxide layer, which facilitates osseointegration, and its mechanical properties such as a modulus of elasticity similar to bone. Diverse studies have shown that surface microtopography, chemistry, and surface energy affect osteoblast behavior. The problem with these studies is that they access the average behavior of a culture in response to a substrate and not the behavior of individual cells. The objective of this study was to develop a method for correlating the behavior of individual cells with the characteristics of the surface underneath them. More specifically, this work developed a method to correlate integrin beta-1 (β1) expression with the surface characteristics under individual cells. Integrins are cell surface receptors that bind to specific proteins in the extracellular matrix adsorbed on the implant surface. Previous work has shown that expression of certain integrins is increased when osteoblasts on titanium substrates develop a more differentiated phenotype, and that integrin β1 is necessary for osteoblast response to roughness on titanium substrates. This study used molecular beacons specific to integrin β1 to quantify integrin β1 expression of MG63 cells cultured on titanium disks. A template was designed to coordinate the location of cells using fluorescence microscopy and scanning electron microscopy (SEM) in reference to laser etchings on the disks. After live cell imaging, cells were fixed, dried, and critical point dried for focused ion beam (FIB) milling. Transmission electron microscopy (TEM) sections of cells identified with high and low integrin β1 molecular beacon intensity were milled, and cells with high and low integrin β1 molecular beacon intensity were also serial sectioned. While our TEM results were inconclusive, SEM images from serial sectioning showed contact points between the cell body and the substrate, consistent with previous results. Cells cultured on pretreatment (PT) or sandblasted acid etched (SLA) titanium surfaces were also serial sectioned, showing that cells on SLA surfaces have more regions of contact between the cells and the substrate than cells on PT surfaces. This work is significant as it is the first study to develop a method to correlate individual cell behavior with the substrate surface characteristics under the individual cells. Previous studies have reported the average cell behavior in response to their substrates, while this work allows for the study of substrate surface characteristics that positively affect integrin β1 expression in individual cells. Further optimization of the fluorescence imaging process and FIB milling process could be done, and the method developed in this study could be used in future studies to investigate surface characteristics after using other fluorescent analyses of cell behavior, such as immunocytochemistry.

Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Focal adhesions are important subcellular structures that are composed of many signaling and scaffolding proteins. They serve not only for anchoring the cell to the substratum but they are also important signaling centers that regulate various cellular behavior such as migration, invasiveness, proliferation and survival. Focal adhesion signaling needs to be strictly regulated because alteration in activity or expression of many focal adhesion proteins leads to tumorogenesis and metastasis formation. One of the most important scaffolding protein associated with focal adhesion is p130Cas. The importance of p130Cas in regulation of cell migration and invasiveness has been well established. P130Cas also plays important role in regulation of cell survival and proliferation. Moreover, high protein levels of human ortholog of p130Cas - BCAR1, has been linked to more aggressive breast tumors and poor prognosis. During my doctoral studies, I focused on the role of p130Cas in integrin signaling. At the beginning we characterized the role of tyrosine 12 phosphorylation within its SH3 domain. We confirmed that this phosphorylation is increased in Src527F transformed mouse embryonic fibroblasts compared to non-transformed counterparts and also in some human cancer cell lines. We showed that this phosphorylation...

Key to our understanding of growth regulation in Drosophila would be discovering a ligand that could regulate steroid synthesis. Activins are involved in regulating steroid hormone release in vertebrates. In invertebrates, they most likely function to keep ecdysone levels low to allow the larvae more time to achieve critical weight in order to initiate the metamorphic process. TGF-B(Transforming Growth Factor Beta) is a family of cytokine growth factors. We find that two members of the TGF-B signaling pathway Drosophila Activin (dACT) and Activin-like ligand Dawdle (DAW) signal through the type I receptor Baboon (BABO) and the type II receptor PUNT to primarily activate the transcription factor dSMAD2 and MAD to a lesser extent. One transcription factor brinker (brk) appears to be central to dACT signaling. / In wings dACT signaling is necessary to promote growth however, dACT is not expressed in wings suggesting that dACT is provided through the endocrine system. One possible target tissue of dACT signaling is the ring gland (RG), which synthesizes and secretes the steroid hormone ecdysone (E). Consistent with this idea, using the UAS/GAL-4 system, we find that over-expression of the TGF-B ligand dACT with the neuroendocrine driver 386Y-GAL4 results in an increase in the size of flies. Surprisingly, when we increase the dose with two copies of dACT, it decreases the size of flies also indicating non-autononomous effects. We find that overexpression of the activated form of the dACT type I receptor Baboon (BABO) or brk with the ring gland specific driver phm-GAL4 results in developmental arrest of larvae that stay small and never pupate. The developmental arrest can be overcome by feeding larvae E, suggesting that dACT represses E through brk. These results suggest a model where dACT signaling activates brk which inhibits E. We picked three cytochrome P450 enzymes: phantom (PHM), disembodied (DIS) and spookier (SPKR). / PHM is not regulated by any component in the dACT signaling pathway however, we find DIS and SPKR are down-regulated through brk. MAD and dSmad2 bind to a Smad binding site and MAD out-competes dSMAD2. We find no evidence that Drosophila insulin-like peptides (DILPS)/PI3- Kinase or Ras signal through the dActivin signaling pathway. / by Scott C. Gesualdi. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Objetivo: Medir a expressão da integrina av~3 no endométrio de mulheres usuárias do DIUT200. Desenho: Estudo observacional controlado. Local realizado: Centro de saúde secundário e laboratório universitário. Pacientes: Treze mulheres sadias e férteis (controles) e treze usuárias do DIUT200 (casos). lntervençao: Biópsia endometrial realizada entre o 6°-1 0° dia pós-ovulatório do ciclo menstrual. Principal Desfecho Avaliado: A expressão da integrina av~3 através do HSCORE em amostras endometriais criopreservadas. Resultados: O HSCORE das usuárias do DIUT200 foi 0,9 ± 0,7 (média± DP), enquanto que o dos controles foi 2,13 ± 0,7 (média ± DP) (p = 0.001 Teste-t de Student). Todos os controles foram positivos para a expressão da integrina av~3. mas as usuárias do DIUT200 não apresentou positivdade para a integrina av~3 em 38,5% dos casos (p = 0,03 Teste Exato de Fisher). Conclusao: Os resultados apoiam a teoria que o DIUT200 de cobre também tem um mecanismo de ação que interfere diretamente com a receptividade uterina e a implantação. / Objective: To measure the expression of avf33 integrin in the endometrium of IUDT200 users. Design: Observational controlled study Setting: Secondary health care center and University laboratory Patients: Thirteen healthy fertile women (contrais) and thirteen IUDT200 users (cases). lntervention: Endometrial biopsy on postovulatory day 6-1 O of the menstrual cycle. Main Outcome Measure: The expression of avJ33 by HSCORE on cryopreserved endometrial sections. Results: The HSCORE for IUD users was 0.9 ± 0.7 (mean ± SEM), while for contrais was 2.13 ± 0.7 (mean ±SEM) (p < 0.001 Teste-t de Student). Ali contrais were positiva for avJ33, but women with IUD did not express avl33 integrin in 38.5% of the cases (p < 0.03 Fisher's exact test). Conclusion: These results support the theory that copper IUD T200 also has a mechanism of action that is directed at interference with uterine receptivity and implantation.

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina &alpha;6, &beta;1, &alpha;V e &beta;3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades &alpha;6 do receptor de integrina e &beta;1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades &alpha;V e &beta;3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina &beta;1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting. / Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits &alpha;6, &beta;1, &alpha;V and &beta;3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits &alpha;6 and &beta;1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits &alpha;V and &beta;3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin &beta;1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting.