Würzburg, Universiẗat, Diss., 2007.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Thrombospondin 1 (TSP1) is a 450 kDa homotrimeric multidomain glycoprotein with fundamental roles in many cell-cell and cell-matrix interactions. These varied, and sometimes conflicting, functions are mediated by specific domains in TSP1. One region with diverse biological roles is the Ca2+ binding loops (or type 3 repeats). The biological activity of this region is determined through a complex assembly of disulfide bonds linking structure and function. Disulfide interchange in a protein is usually very specific and quite slow, unless catalysed. I have found that protein disulfide isomerase (PDI) is expressed on the surface of platelets and endothelial cells in a reduced active conformation. The presence of enzymatically active PDI on the surface of TSP1-secreting cells suggests PDI is well positioned to catalyse disulfide interchange in, and regulate the structure/function relationships of, TSP1. PDI was observed to form disulfide-linked complexes with TSP1. Moreover, incubation of platelet or fibroblast TSP1 with PDI enhanced binding of an isomer-specific anti-TSP1 antibody whose epitope is in the Ca2+ binding loops. These findings suggest that PDI may mediate disulfide bond rearrangement in both the soluble and extracellular matrix-bound forms of TSP1. TSP1 is a tight-binding competitive inhibitor of neutrophil cathepsin G; however, incubation with PDI increased the Ki for the interaction ???10-14-fold. TSP1 bound platelet-derived growth factor (PDGF) tightly in the region of the Ca2+ binding loops and supported binding of PDGF to its receptor. PDI-mediated disulfide interchange in TSP1 ablated PDGF binding, indicating that PDI-catalysed disulfide interchange in TSP1 may modulate PDGF-TSP1 complex formation and the biological activity of PDGF. Finally, PDI-catalysed isomerization of TSP1 potently affected its cell adhesive properties. Treatment of TSP1 with PDI enhanced adhesion and spreading of endothelial cells through the ??v??3 integrin receptor to TSP1, by exposure of a cryptic RGD sequence. Thus, endothelial cell surface PDI may be a physiological regulator of RGD-dependent binding to TSP1. These data suggest that cell-surface PDI may regulate the disulfide-bonded structure and certain biological functions of TSP1. In conclusion, I propose a novel mechanism for the post-translational regulation of TSP1 structure/function, which in turn may regulate certain aspects of TSP1 in vascular biology.

Endometriosis is a gynecological disease defined as the growth of endometrium outside of the uterus. Although linked to 50% of female infertility cases, current medical treatments fail to maintain fecundity. Since the survival of endometriotic lesions is dependent on their early neovascularization, antiangiogenic therapies specifically targeting blood vessel growth could be a promising therapeutic option for the treatment of endometriosis. Angiogenesis, the branching of new blood vessels from existing vasculature, promotes robust vascularization of lesions. ABT-898 (Abbott Laboratories), a thrombospondin-1 analog, induces endothelial cell apoptosis while sequestering pro-angiogenic growth factors. We postulated that ABT-898 would reduce endometriotic lesion vascularization while physiological angiogenesis and pregnancy remained unaffected in a murine model of endometriosis. The antiangiogenic effect of ABT-898 was tested in a human umbilical vein endothelial cell line revealing disruption of endothelial tube branching. Two in vivo experiments were conducted in which endometriosis was induced in female alymphoid BALB/c-Rag2-/-Il2rg-/- mice by adhering sections of human endometrium to the abdominal wall. Lesions from ABT-898 treated mice contained a reduced number of CD31+ endothelial cells and a decrease in blood flow supplying the lesion compared to 5% dextrose controls. Reproductive status was evaluated through maintenance of pregnancies up to gestation day 12 revealing unaffected implantation site structure and physiological angiogenesis. In a trans-generational study, pregnant F0 generation mice received ABT-898 or 5% dextrose injections on gestation days 7, 9, 11, 13, 15, 17, and 19. F1 generation mice were raised to reproductive age and bred resulting in litters (F2 generation) comparable in size to the F0 generation litters. Chronic exposure to ABT-898 did not affect angiogenic plasma cytokine levels in F0 generation mice. In addition, physiological angiogenesis was unaffected within the uteri of ABT-898 treated mice. Histological examination of the kidney, liver, ovary, and uterus revealed no structural abnormalities in F0 and F1 generations exposed to ABT-898. These results suggest that ABT-898 inhibits pathological angiogenesis within endometriotic lesions without affecting physiological angiogenesis involved in pregnancy and organ function across three generations of mice. Further research will establish the effects of ABT-898 on embryonic development, organ toxicity, and physiological angiogenesis in all organs. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2013-05-07 15:19:10.967

Thrombospondin-1 (TSP1) ist ein matrizelluläres, Calcium-bindendes Glykoprotein, das an der Regulation verschiedener zellulärer Prozesse beteiligt ist. TSP1 wird von unterschiedlichen Zelltypen gebildet und ist vor allem in den α-Granula der Thrombozyten zu finden, aus denen es nach deren Aktivierung sekretiert wird. Streptococcus pneumoniae (Pneumokokken) sind Gram-positive humanpathogene Bakterien. Sie besiedeln asymptomatisch den menschlichen Respirationstrakt und können schwerwiegende lokale Infektionen und lebensbedrohliche Erkrankungen, wie z.B. Sepsis, bakterielle Meningitis oder invasive Pneumonien auslösen. Die Anheftung von S. pneumoniae an Wirtsstrukturen ist ein initialer Schritt für die Kolonisierung mukosaler Epitheloberflächen. In dieser Arbeit wird die Bedeutung des humanen TSP1 für die Pathogen-Wirt Interaktion analysiert und der Effekt für die Pathogenese demonstriert. Verschiedene Bindungsstudien und durchflusszytometrische Analysen zeigten eine Assoziation von S. pneumoniae an aktivierte Thrombozyten und an lösliches und immobilisiertes TSP1. In in vitro Infektionsversuchen konnte nachgewiesen werden, dass wirtszellgebundenes TSP1 die Adhärenz an und Invasion in Epithel- bzw. Endothelzellen vermittelt. TSP1 übernimmt die Funktion als Brückenmolekül zwischen S. pneumoniae und eukaryontischen Wirtszellen. Zur Charakterisierung des bakteriellen Adhäsins für TSP1 wurden die Pneumokokken mit dem proteolytischen Enzym Pronase E bzw. mit der Zucker oxidierenden Substanz Natriumperiodat inkubiert. Eine Behandlung mit Natriumperiodat reduzierte die TSP1 vermittelte Adhärenz der Pneumokokken an humane Wirtszellen. Im Gegensatz dazu hatte die Behandlung mit Pronase E keinen Einfluss auf die TSP1 vermittelte Anheftung von S. pneumoniae an eukaryontische Zellen. Diese Ergebnisse deuten an, dass es sich bei dem bakteriellen Adhäsin für TSP1 um eine oberflächenlokalisierte Glykostruktur der Pneumokokken handelt. Die TSP1 vermittelte bakterielle Adhärenz der Pneumokokken an Wirtszellen konnte durch Pneumokokken-spezifisches Phosphorylcholin bzw. durch Lipoteichonsäuren nicht reduziert werden. Im Gegensatz dazu wurde die TSP1 vermittelte Adhärenz von S. pneumoniae an Wirtszellen durch Zugabe von löslichem Peptidoglykan signifikant inhibiert. In verschiedenen Bindungsstudien wurde das Peptidoglykan als Pneumokokken-Adhäsin für TSP1 identifiziert. Weiterhin wurde herausgestellt, dass nicht nur S. pneumoniae, sondern auch andere Gram-positive pathogene Bakterien, wie Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes und verschiedene apathogene Bakterien mit TSP1 interagieren, im Gegensatz zu Gram-negativen Bakterien. Es konnte gezeigt werden, dass TSP1 das Peptidoglykan aller getesteten Gram-positiven Bakterien erkennt. Diese Beobachtung weist auf einen allgemeingültigen Mechanismus der Bakterien-Wirt Interaktion hin, der wahrscheinlich von großer Bedeutung für die Pathogenese Gram-positiver Bakterien ist. Als Rezeptoren für TSP1 auf der Wirtszellseite wurden Proteoglykane auf der Oberfläche von eukaryontischen Zellen identifiziert. Weiterhin konnte herausgestellt werden, dass eine Interaktion der Gram-positiven Bakterien mit TSP1 nicht nur eine Adhärenz an Wirtszellen vermittelt, sondern die Bakterien vor einer Phagozytose durch primäre Granulozyten schützt. Zusammenfassend beweisen diese Ergebnisse eine spezifische Interaktion von Gram-positiven Bakterien mit TSP1, die zur bakteriellen Kolonisierung des Wirtsgewebes beiträgt. Das Peptidoglykan übernimmt die Funktion eines bakteriellen Adhäsins für TSP1, so dass TSP1 als molekulare Brücke die Interaktion von Gram-positiven Bakterien und Wirtszell-Proteoglykanen vermittelt. Diese Untersuchungen tragen in bedeutender Weise zu einem besseren Verständnis der Pathogenese von Infektionen durch S. pneumoniae und anderen Gram-positiven Bakterien bei. / Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. TSP1 is produced by different cell types and is mainly found in the α-granules of human thrombocytes and secreted upon their stimulation. Streptococcus pneumoniae are Gram-positive human bacteria which reside asymptomatically in the human respiratory tract, but can also cause local infections and life-threatening diseases such as sepsis, bacterial meningitis and pneumonia. A prerequisite for pneumococcal colonization of mucosal epithelial cells is the bacterial interaction with host cell structures. This study reports a novel role for human TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that S. pneumoniae specifically interacts with human TSP1. It is shown that S. pneumoniae binds to activated thrombocytes and recruits TSP1 from human plasma. Host-cell bound TSP1 promotes adherence of S. pneumoniae to human epithelial and endothelial cells, thereby acting as a molecular bridge between pneumococci and eukaryotic cells. To identify the bacterial adhesin for TSP1, pneumococci were incubated with the proteolytic enzyme pronase E and with sodium periodate, which oxidizes surface-exposed sugars. Pretreatment of the bacteria with sodium periodate, but not pronase E substantially reduced TSP1 mediated adherence to host cells, suggesting a glycoconjugate as the pneumococcal receptor for TSP1. Pneumococcal phosphorylcholine and lipoteichoic acids did not affect TSP1 mediated adherence of S. pneumoniae to host cells. In contrast, attachment of pneumococci to host cells via TSP1 was blocked by soluble peptidoglycan, indicating the recognition of bacterial peptidoglycan by TSP1. Further studies demonstrate that in addition to S. pneumoniae other Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes and several apathogenic bacteria interact with TSP1. Gram-negative bacteria did not interact with TSP1. Further it is shown that TSP1 recognizes the peptidoglycan of all tested Gram-positive bacteria, suggesting a general mechanism of bacteria-host protein interaction which probably has a significant impact on the pathogenesis of Gram-positive bacteria. Host cell proteoglycans were identified as the cellular receptors for TSP1. It is demonstrated that the bacterial binding to TSP1 does not only support adhesion to host cells, but can also protect the bacteria from phagocytosis by granulocytes. In conclusion, the results demonstrate an exploitation of TSP1 by Gram-positive bacteria which supports bacterial colonization of host tissues. In this scenario peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge, linking Gram-positive bacteria with proteoglycan receptors on the host cells. These studies contribute to a better understanding of infections by S. pneumoniae and other Gram-positive bacteria.

<p>Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an <em>Escherichia coli</em> expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the <em>E.coli</em> by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.</p>

Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an Escherichia coli expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the E.coli by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.

This thesis is an investigation of the use of thrombospondin-1 mimetic peptides for the treatment of epithelial ovarian cancer. The current standard of care for women diagnosed with ovarian cancer is surgical de-bulking followed by chemotherapeutics. Initially, this treatment regimen results in a reduction in the primary tumor, unfortunately chemoresistance and disease recurrence are problematic. Recent data has suggested a potential role for anti-angiogenic therapy for the treatment of various cancers. Therefore, the purpose of this study was to investigate the use of mimetics consisting of the anti-angiogenic domain of thrombospondin-1 (TSP-1) for the treatment of epithelial ovarian cancer (EOC) using a mouse model of the disease. The peptides were applied at various stages of tumor progression and a significant reduction in tumor size following treatment was observed. We found that not only were the peptides capable of slowing down tumor progression but they also played a role in reducing the size of established tumors. Treatment with TSP-1 mimetics also resulted in a significant reduction in secondary lesions and ascites fluid in the peritoneal cavity of animals. A significant increase in disease-free survival was also identified following long-term treatment with the peptide. Various histological studies revealed that the anti-angiogenic peptide was in fact inducing apoptosis of the endothelial cells and also re-organizing the vasculature. To determine whether this resulted in increased blood vessel profusion we applied standard chemotherapeutics in combination with TSP-1 mimetics. Experiments with radiolabelled and fluorescent chemotherapeutics demonstrated that pre-treating with TSP-1 mimetics allowed the vasculature to become normalized and resulted in an increased uptake of chemotherapeutics. Lastly, we investigated the mechanism of action of anti-angiogenic peptides. Most of the anti-tumor effects appeared to be due to the apoptotic effects of TSP-1 mimetics on the vasculature. A direct apoptotic effect on epithelial cells also was observed; however, it is uncertain how much of a role this plays. In conclusion, this study was important for identifying TSP-1 mimetic peptides as a potential therapeutic treatment for women suffering from EOC.

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references (leaves 146-154).

Limb and digit loss poses a significant problem across the animal kingdom. Ambystoma mexicanum, commonly known as the axolotl, however, is one species that has achieved a remarkable ability to bypass the misfortune associated with a lost limb. Viewed as a model organism in regenerative studies, the axolotl retains extraordinary regenerative properties well into adulthood that humans severely lack. While the basics of regeneration have been described, much about the molecular processes of regeneration is still largely unknown. Thrombospondin-1 (TSP-1), an angiogenesis inhibitor, has been identified as a potential factor to play a significant role in the regrowth of limbs. Vascularization of tissues is vital to the survival of biological structures, and TSP-1 has been shown to play a regulatory role in the development and remodeling of tissue vasculature. Here, we study the effect of a loss-of-function mutation in the tsp-1 gene on the process of limb regeneration in the axolotl. Our studies reveal that tsp-1 -/- animals lag in regeneration time, developing smaller blastemas in the first three weeks of regeneration. We show that the loss of TSP-1, however, is not deleterious to the overall process of regeneration as late stage blastemas of the -/- animals catch up in size and development to the wild type animals after three weeks. Our data suggests that while TSP-1 may be important during the initial stages, it may not be required for proper regeneration.