Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 137-153). Also available online through Digital Dissertations.

Sprouting angiogenesis is a multi-step process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and stabilization. Such complexity reveals that the orchestration of individual genes and multiple signaling pathways are required. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to multicellular invading sprouts, we analyzed differential gene expression with time using an in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate (S1P) and angiogenic growth factors. Gene expression changes were confirmed by real-time PCR and Western blot analyses. In addition, we have undertaken a proteomic screen to dissect downstream targets of the S1P receptors that possibly regulate EC invasion. Gene silencing or overexpression were used to examine the involvement and role of downstream targets of S1P in EC invasion into three-dimensional collagen matrices. We demonstrated that various cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated; whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, A Disintegrin and Metalloproteinase with Thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt (wingless) and Notch pathways were observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. In addition, we identified annexin 2 as a regulator of endothelial morphogenesis. We observed that S1P triggered annexin 2 translocation from cytosol to plasma membrane and its association with vascular endothelial (VE)-cadherin. Moreover, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced EC invasion observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.

Etwa 15% aller Paare bleiben ungewollt kinderlos. Männliche Faktoren sind in circa einem Drittel der Fälle als ursächlich anzusehen. Jedoch sind die Erfolgsraten der Therapie männlicher Infertilität durch assistierte Reproduktion auch nach über 30 Jahren seit deren Einführung unbefriedigend. Bestehende Spermienaufbereitungsmethoden wie einfaches Waschen, swim up oder die Dichtegradientenzentrifugation basieren auf makroskopisch-funktionellen Parametern wie Motilität und Morphologie. Spezifische Eigenschaften wie etwa eine aktivierte Apoptosesignalkaskade der Spermien werden dabei nicht berücksichtigt. Die wesentlichen Elemente verschiedener Signalwege der aus somatischen Zellen bekannten Apoptose konnten auch am humanen ejakulierten Spermatozoon nachgewiesen werden. Über die (negativen) Auswirkungen der Apoptose auf die männliche Fruchtbarkeit gibt es einen Konsens. Ziel der vorliegenden Arbeit war es, Selektionsmethoden zu entwickeln, welche auf subzellulärer Ebene intakte Spermien mit dem größtmöglichen Fertilisationspotential aus dem Ejakulat extrahieren. In einem ersten Versuchskomplex konnte gezeigt werden, dass durch die Kombination von Dichtegradientenzentrifugation und swim-up (Standardmethoden in Reproduktionskliniken und andrologischen Laboren) zur Aufbereitung der Spermien von subfertilen Patienten eine akzeptable Reduktion der Spermien mit aktivierter Apoptosesignalkaskade erreicht werden kann. Jedoch gaben die großen interindividuellen Unterschiede im Separationseffekt Anlass zur Entwicklung innovativer Untersuchungs- und Separationsmethoden. So wurden unter anderem fluoreszenzbasierte Tests zur Evaluation von Spermiendefekten, wie beispielsweise einer gestörten Integrität des mitochondrialen Membranpotentials, eingeführt. In den Untersuchungen wurde die Praktikabilität dieser neuen Analyseverfahren im Routineeinsatz unter Standardbedingungen getestet und bestätigt. Die innovative Selektionsmethode der Annexin V-MACS Separation basiert auf der Bindung von Annexin V-MicroBeads an apoptotische Spermien, womit eine Subpopulation reifer, motiler und vitaler Spermien mit inaktivierter Apoptosesignalkaskade gezielt angereichert wird. Das Konzept wurde zudem erfolgreich auf ein (Glaswoll-) Festphasen-Filtersystem ohne frei schwimmende Microbeads übertragen. Dadurch gelang die Minimierung eines potentiellen Transmissionsrisikos der Microbeads bei der Anwendung im Rahmen der künstlichen Befruchtung. Den hohen Stellenwert dieser Verfahren belegen die Ergebnisse zweier in-vitro Modelle, an denen erstmalig gezeigt werden konnte, dass durch die Selektion von Spermien mit inaktivierter Apoptose-Signalkaskade höhere Fertilisationsraten erreichbar sind.

Diabetes is a complex disease characterised by hyperglycaemia, which often leads to microvascular complications including diabetic nephropathy and cardiomyopathy. In this thesis, I have investigated the role of Annexin-A1 (ANXA1), an endogenous anti-inflammatory peptide, in two experimental murine models of diabetes caused by streptozotocin (STZ) or high-fat high-sugar diet (HFD), which mimic type-1 (T1DM) and type-2 diabetes (T2DM) respectively. I have also investigated the levels of ANXA1 in patients with either T1DM or T2DM. Patients with T1DM have increased plasma ANXA1 levels. In a murine models of type 1 diabetes loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice. Specifically, I have shown that key mediators of the MAPK pathway (p38, JNK and ERK1/2) are constitutively activated in ANXA1-/- mice, and activation of these pathways is exacerbated in diabetic ANXA1-/- mice. Administration of human recombinant (hr) ANXA1 did not alter the diabetic phenotype in diabetic WT mice, but attenuated the cardiac and renal dysfunction caused by STZ. Interestingly, late administration of ANXA1 (after significant cardiac and renal dysfunction had already developed) halted the progression of both cardiac and renal dysfunction. Patients with T2DM have increased plasma ANXA1 levels. HFD-fed ANXA1-/- mice have a more severe diabetic phenotype compared to HFD-fed WT mice. Therapeutic administration of hrANXA1 prevented the development of a diabetic phenotype. Specifically, I have shown that the insulin signalling pathway is further perturbed in diabetic mice resulting in severe insulin resistance, and that these signalling abnormalities were prevented by therapeutic administration of hrANXA1. In addition, loss of endogenous ANXA1 aggravates both cardiac and renal dysfunction in mice with experimental T2DM. The GTPase RhoA is constituently activated in ANXA1-/- mice leading downstream activation of MYPT1. Feeding a HFD also activated the small GTPase RhoA, leading to increased MYPT1 activity, which could be attenuated with treatment with hrANXA1. Mice subjected to HFD for 12 weeks had a more 'leaky' blood brain barrier (BBB), which is further exacerbated in ANXA1-/- mice fed a HFD. Compared to mice fed a chow diet, mice fed a HFD had an augmented CD4+ T-cell profile; with a clear decline in CD4+FoxP3+ (anti-inflammatory) and increase in CD4+RORgt+ (pro-inflammatory) cells. Administration of hrANXA1 to mice fed on HFD restored BBB integrity and CD4+ T-cells profile similar to mice fed on normal chow diet. Mice fed a HFD also had more activated CD4+ T-cells, which adhered more readily and transmigrated through a brain endothelial mono-layer ex vivo. In contrast, administration of hrANXA1 to mice fed on HFD reduced re-activity of CD4+ T-cells, reducing the number of adherent CD4+ T-cells to the brain endothelial mono-layer.

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.

Les annexines sont définies comme étant des protéines qui se lient de manière calcium-dépendante aux phospholipides membranaires chargés négativement. Elles ont été associées à différents processus biologiques tels les réponses des plantes aux stress biotiques et abiotiques. Nous avons identifié une annexine végétale, appelée Ntann12, dont l’expression est induite après infection des plantes par la bactérie Rhodococcus fascians. Ntann12 possède les domaines caractéristiques des annexines et se lie aux phospholipides chargés négativement, de manière calcium-dépendante. L’expression de Ntann12 est très abondante dans les cellules différentiées des racines, où la protéine a été détectée par immunolocalisation dans le cytosol et dans le noyau. Des analyses par western blot ont montré que l’accroissement relatif de la quantité de protéines liées aux membranes est positivement corrélé à l’augmentation de la concentration en Ca2+. Au niveau physiologique, l'expression de Ntann12 est induite par l’apport exogène d’auxine. Elle est contrôlée dans les racines par un signal induit par la lumière, et provenant des parties aériennes. Le transport polaire de l'auxine a été identifié comme étant le processus cellulaires nécessaires à l'expression de Ntann12 dans les racines. En outre, cette expression est réprimée par les stress salin, osmotique et hydrique. Ces résultats suggèrent que l’annexine Ntann12 est impliquée dans le métabolisme de l’auxine. / Annexins are defined as calcium-binding proteins, and they have been associated in plants with different biological processes such as responses to biotic and abiotic stress. Ntann12 expression is induced upon infection of tobacco plant by R. fascians. Ntann12 possesses the conserved annexin repeat with the sequence for type II Ca2+-binding site and recombinant as well as native Ntann12 binds to negatively charged phospholipids in a Ca2+-dependent manner. It is mainly expressed in root differentiated cells where the protein was immunolocalized in the cytosol and in the nucleus. Ntann12 was examined by western blot in both microsomal and cytosolic fractions from tobacco roots cells, and was detected in both the cytosol and microsome. The relative increase of Ntann12 proteins associated with the microsome is coupled with an increase in Ca2+ concentration. At the physiological level, Ntann12 expression is induced by exogenous application of auxin, and was found to be regulated in the root system by a light-induced signal coming from plant aerial part and polar auxin transport was identified to be the cellular process required for Ntann12 expression in root cells. Furthermore, Ntann12 expression is down-regulated by salt, osmotic and water stress. These results collectively suggest that the annexin Ntann12 is implicated in auxin metabolism.

Neutrophil extravasation into tissues is an essential process required for the inflammatory response. Upon receiving an inflammatory cue, neutrophils begin accumulating on the luminal surface of the endothelium. Neutrophil recruitment is initiated by selectin-mediated tethering and rolling of neutrophils along the endothelial monolayer, followed by integrin-mediated firm adhesion. Adherent neutrophils then traverse the endothelium in a process known as transendothelial migration. The events mediating the rolling and adhesion steps are well characterised, but research into the molecular mechanisms regulating transendothelial migration is an area of intense focus. A previous study conducted in our laboratory found that the activation of endothelial extracellular signal-regulated kinase (ERK) 1/2 was required for neutrophil transmigration. Furthermore, it was found that endothelial ERK was activated in response to a soluble protein produced by fMLP- or IL-8-stimulated neutrophils. In the present study, the soluble ERK-activating neutrophil protein was identified as annexin A1, which was selected as a possible candidate following mass spectrometry analysis of proteins secreted from activated neutrophils. Annexin A1 antibodies (Abs) were found to block endothelial ERK activation induced by conditioned medium harvested from stimulated neutrophils. Annexin A1 Abs were additionally able to inhibit neutrophil transmigration across human umbilical vein endothelial cell (HUVEC) monolayers in an in vitro transmigration assay. Following the purification of recombinant annexin A1, it was demonstrated that it could activate endothelial ERK in a similar manner to neutrophil conditioned medium. Upon further investigation, ERK activation was found to be induced by a truncated form of annexin A1 present in the protein preparation rather than the full length protein. Calpain I, a calcium dependent protease that is activated upon neutrophil stimulation and is known to cleave annexin A1 within the N-terminal domain, was shown to process full length inactive recombinant annexin A1 into an unidentified product that could activate endothelial ERK. A calpain I inhibitor was also found to prevent stimulated neutrophils from secreting an ERK-activating protein, thus further suggesting a role for calpain I in this process. As full length annexin A1 has been reported to signal through the formyl peptide receptor (FPR) family, a pan-FPR antagonist was incubated with endothelial cells and was found to inhibit ERK activation induced by neutrophil conditioned medium, indicating that pro-inflammatory annexin A1 is also a FPR ligand. Endothelial projections termed “transmigratory cups” form around neutrophils during extravasation, of which ICAM-1 is a major component. Using an assay that examined transmigratory cups during neutrophil transmigration, it was found that annexin A1 Abs could inhibit neutrophil adhesion and transmigration through HUVEC monolayers by interfering with transmigratory cup formation around neutrophils, as shown by monitoring ICAM-1 during the process. Quantification of transmigrating neutrophils highlighted that the majority of neutrophils were emigrating via a transcellular pathway, which is in opposition to many in vitro studies where paracellular transmigration predominates. The results generated from this study identified a novel pro-inflammatory role for annexin A1 in neutrophil transendothelial migration. Preliminary experiments suggested that the pro-inflammatory annexin A1 responsible for endothelial ERK activation was a truncated form. Calpain I appears to be a likely candidate responsible for the generation of this uncharacterised, truncated annexin A1 product, however further experiments are required to confirm this hypothesis. Pro-inflammatory annexin A1 represents a new target for the treatment of inflammatory disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374554 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.

Proefschrift Universiteit Maastricht. / Auteursnaam op omslag: Sylvia Jans. Met bibligr., lit. opg. - Met een samenvatting in het Nederlands.

Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI . No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses. / Master of Science