Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

No description available.

In pigs and other mammals, embryonic losses often occur during implantation when the conceptus (embryo plus its extra-embryonic membranes) attaches to the maternal uterine epithelium. Mechanisms controlling this process are not completely understood. Integrins and growth factors are among many molecules likely involved in controlling implantation. Numerous integrins (ITG), including subunits ITGAV (alpha v), ITGB1 (beta 1), ITGB3 (beta 3), and ITGB5 (beta 5), and transforming growth factor betas (TGFBs), in both latent and active forms, are present at the porcine conceptus-maternal interface. TGFBs are released as latent precursors which cannot interact with TGFBRs prior to their activation. Latency associated peptide (LAP), part of the TGFB latent complex, contains an amino acid sequence Arg-Gly-Asp (RGD) that is found in other extracellular matrix molecules and may interact with and signal through integrins. We hypothesize that LAP will bind to and activate ITGAV-containing heterodimers at the conceptus-maternal interface and that these interactions are a functional component of implantation. We also hypothesize that TGFB acting via TGFBRs has critical roles during peri-implantation, and such roles may include promoting conceptus development, survival, and adhesion. Immunofluorescence was used to colocalize TGFB, LAP, and integrins in porcine peri-implantation uterus and conceptus; immunohistochemistry of phosphorylated SMAD2/3 provided evidence of TGFB activity. Affinity chromatography identified cell surface integrins on porcine trophectoderm that are capable of binding LAP. In vivo, intrauterine infusions of LAP with its native RGD site (LAP-RGD) resulted in inhibition of conceptus elongation; LAP-RGE infusions yielded normal-appearing filamentous conceptuses at d13 of pregnancy. At d24, allantois length and fetal weights were greater in gilts which received LAP-RGE infusions compared to controls which received vehicle only. Results provide evidence for 1) active and latent TGFB in porcine conceptus and uterus; 2) receptor-ligand interactions of integrins and LAP; 3) integrin aggregation and potential focal adhesion formation at the conceptus-maternal interface; and 4) TGFB- and/or integrin-associated mechanisms which regulate conceptus elongation and placental and fetal size. Collectively, results suggest that TGFB and integrins are extensively involved in communication at the porcine conceptus-maternal interface, particularly regulating conceptus development, adhesion, and placental and fetal development.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-103).

Thesis (M.S. in Bio. E.)--School of Biomedical Engineering, Georgia Institute of Technology, 2004. Directed by Cheng Zhu. / Includes bibliographical references (leaves 79-84).

Τα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, FAK, Src και MAP κινάσες. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Τα σφαιρίδια λάτεξ -αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες- παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Τα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and positive bacteria E. coli and S. αureus respectively, are recognized and binded on C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways involving the kinases Ras, FAK, Src and MAP. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin, leads to a regulated secretion that is a prerecuisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes being a new product of human knowledge, are recognized and binded on hemocyte surface by so far unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and promote -with the participation of actin and tuboulin cytoskeleton- their phagocytosis. LPS is recognized and binded on hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕΡΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, does not get involved in the process of its internalization. All the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.

Angiogenese, die Bildung neuer Blutgefäße aus bereits existierenden Blutgefäßen, ist ein essentieller Prozess bei der Embryonalentwicklung, Wundheilung und Reproduktion. Dieser Prozess wird von Wachstumsfaktoren wie z. B. dem vascular endothelial growth factor (VEGF) und Zelladhäsionsmolekülen wie den Integrinen beeinflusst. Die Integrine avb3, avb5 und a5b1, sind in der Tumorangiogenese involviert, wobei die während des Tumorwachstums neu gebildeten Blutgefäße den Zugang einzelner Tumorzellen zum Blutgefäßsystem erleichtern. Zwar liegen Integrine auch auf Zellen in gesundem Gewebe vor, allerdings sind die Integrine avb3 und a5b1 besonders auf der Oberfläche aktivierter Endothelzellen und auf Tumorzellen verstärkt präsentiert. Dennoch gibt es noch immer kontroverse Ergebnisse bezüglich ihrer Rolle als pro- oder antiangiogene Moleküle. Zur Untersuchung der Funktionsweise der Integrine avb3, avb5 und a5b1 in der Tumorangiogenese wurden in dieser Arbeit intrazelluläre Antikörper, sogenannte Intra(dia)bodies, verwendet, um die Integrine innerhalb der Zelle zurückzuhalten und damit einen phänotypischen Knockout zu erzielen. Damit die Intra(dia)bodies in die Zellen gelangen konnten, wurden adenovirale Vektoren als Transfervehikel eingesetzt. In der vorliegenden Arbeit konnte mit Hilfe von Intra(dia)bodies, sowohl bei humanen Nabelschnurendothelzellen (HUVEC), als auch bei humanen Melanomzellen (M21), ein phänotypischer Knockout von avb3 erzielt werden, sowie ein gleichzeitiger Knockout/Knockdown von avb3/a5b1. Es konnte bei beiden Zelllinien gezeigt werden, dass dies eine verminderte Oberflächenpräsentation von avb5 und der b1-Untereinheit zur Folge hat. Die Endothelzellen waren durch den Verlust der genannten Integrine weder in der Lage, die Matrixmoleküle Fibronektin und Vitronektin zu binden, noch war es ihnen möglich, weiter zu proliferieren. Zudem konnten sich diese Endothelzellen in vitro nicht mehr dreidimensional anordnen und kapillarähnliche Strukturen bilden. Die Proliferation der Melanomzellen in vitro hingegen war trotz verminderter Adhäsion an die Matrixmoleküle Fibronektin und Vitronektin nicht beeinträchtigt. Zusätzlich zeigten die Melanomzellen in dem durchgeführten CAM-Assay, dass sie den Verlust der Integrine weitestgehend tolerieren können. Das Tumorwachstum war durch die fehlenden Integrine unbeeinflusst, wohingegen der phänotypische Knockout von avb3 zu einer beeinträchtigten Angiogenese führte. Um das Wirkungsspektrum zur Untersuchung der Integrine in der Angiogenese zu erweitern, konnten in dieser Arbeit erstmals bindende Fab-Fragmente gegen das Integrin avb5 selektiert werden. Diese können, nach Umwandlung in scFvs und genauerer Charakterisierung, als Intrabodies oder Intradiabodies verwendet werden.

This thesis is focused on neural stem cell (NSC) and glioma biology. I discuss how NSCs interact with extracellular matrix (ECM) proteins in the stem cell niche, and investigate the consequences of deregulated Platelet-derived growth factor (PDGF) signaling for embryonic NSCs in transgenic mice. Furthermore I present cell cultures of human glioblastoma multiforme (GBM) that models human disease, taking into account the heterogeneity of GBM. Finally, interactions between brain tumors and mast cells are studied using the glioma cultures. In paper I, the importance of NSC interactions with the ECM in the stem cell niche during development is discussed. Contacts between NSCs and the ECM in the subventricular zone (SVZ) are emerging as important regulatory mechanisms. We show that early postnatal neural stem and progenitor cells (NSPC) attach to collagen I, and that the adhesion is explained by higher expression of collagen receptor integrins compared to adult NSPC. Further, blood vessels in the SVZ express collagen I, indicating a possible functional relationship. Growth factors, e.g. PDGF, regulate NSC proliferation and differentiation. Aberrant activation of growth factor signaling pathways also plays a role in brain tumor formation. Paper II demonstrates that transgenic mice expressing PDGF-B at high levels in embryonic NSCs displayed mild neurological defects but no hyperplasia or brain tumors. This suggests that a high level of PDGF is not sufficient to induce brain tumors from NSCs without further mutations. Paper III presents a novel panel of human glioma stem cell (GSC) lines from GBM that display NSC markers in vitro and form secondary orthotopic tumors in vivo. GBM has recently been categorized in molecular subclasses and we demonstrate, for the first time, that these subclasses can be retained in vitro by stem cell culture conditions. We have thus generated models for research and drug development aiming at a focused treatment depending on GBM subtype. Interactions with the immune system are integral parts of tumorigenesis. Mast cells are found in glioma and in paper IV we demonstrate that the grade-dependent infiltration of mast cells is in part mediated by macrophage migration inhibitory factor and phosphorylation of STAT5.

Neural crest cells go through an epithelial-mesenchymal transition (EMT) before they migrate. B1 integrins are necessary during these phases of neural crest development, but it is unclear if integrins are required for both EMT and neural crest migration. Chimeric integrin B1 subunit and mutant Rho GTPases are used in this study to assess function during neural crest emigration. Cultures of chick embryonic cells, transfected with these constructs, are used to confirm the effects and expression in conjunction with a green fluorescent protein (GFP) reporter. In control experiments targeting the neural ectoderm of the hindbrain by localized electroporation. GFP-expressing cells release from the neural tube, migrate along neural crest pathways and express the HNK-1 neural crest marker. Immunolabeling of Sox9 and Slug neural crest markers shortly after electroporation confirms transfection of prospective neural crest cells. Electroporation with a chimeric hemagglutinin-B1 integrin subunit inhibits release of transfected cells from the neural tube. Embryos electroporated with constitutively active RhoA have a few transfected cells outside the neural tube that express N-cadherin. but they fail to migrate to the branchial arch. Electroporation with constitutively active Rac1 results in numerous cells near the neural tube, none of which express N-cadherin. Embryos electroporated with Cdc42 mutants are not distinguishable from control embryos expressing GFP alone. In embryos co-electroporated with chimeric integrin and dominant negative RhoA together, co-transfected cells migrate along neural crest pathways. The conclusion is that integrin signaling, transduced through RhoA, is necessary for the EMT of cranial neural crest. Key to this investigation of neural crest emigration is the methodology of localized electroporation. This technique introduces transgenes to targeted patches of cells in the embryo. Localized electroporation employs a double-barreled suction electrode to deliver plasmid and produce an electric field. Parameters for localized electroporation are optimized for transfecting a range of cells in the chick embryo, and expansion of the technique to mammals is demonstrated. Localized electroporation has improved reliability and higher efficiency than existing in vivo transfection techniques.

Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC. EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber. Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC. After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells. Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.