Ulm, Univ., Diss., 2007.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Mar. 31, 2009). PDF text: x, 186 p. : ill. ; 8 Mb. UMI publication number: AAT 3331460. Includes bibliographical references. Also available in microfilm and microfiche formats.

Yes / Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA‐21 (miR‐21) is a short, non‐coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR‐21 was over‐expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over‐expression of miR‐21 increased the spread cell area and proliferative capacity of SV‐SMC and expression of MMP‐1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet‐derived growth factor BB (PDGF‐BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR‐21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF‐BB induces expression of miR‐21 in SV‐SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. / King Saud University; College of Medicine , Riyadh, Saudi Arabia

Idiopathic Pulmonary Fibrosis (IPF) is a progressive lung disease characterized by altered lung fibroblasts known as myofibroblasts. These myofibroblasts play a crucial role in the development and progression of IPF by exhibiting increased proliferation, migration, and collagen production. One of the fibrogenic factors implicated in these pathological changes is Platelet-derived growth factor-BB (PDGF-BB). However, there is limited knowledge about endogenous factors that counteract these fibrotic processes. Previous research has shown that exogenously administered C-type Natriuretic Peptide (CNP) has a beneficial impact on tissue remodeling, specifically in conditions like heart and liver fibrosis. While CNP and its receptor, guanylyl cyclase B (GC-B), are expressed in the lungs, it remains unknown whether CNP can mitigate lung fibrosis through this pathway. To address this question, we first established murine pulmonary fibroblast isolation and culture and investigated whether PDGF-BB-induces any profibrotic changes in vitro. PDGF-BB stimulation led to an increase in the intracellular levels of the profibrotic proteins Collagen, type I, alpha 1 (Col1a1), Collagen, type III, alpha 1 (Col3a1) and Matrix metalloproteinase 9 (MMP9) indicating transition towards a fibrotic phenotype. Exogenous synthetic CNP at 10 nM and 100 nM could significantly attenuate the PDGF-BB-induced increase in intracellular Col1a1 and Col3a1 levels. CNP increased intracellular cGMP levels in cultured lung fibroblasts, indicating a presence of GC-B-dependent signaling pathway. Even in the presence of pre-existing high PDGF-BB levels, CNP was able to activate GC-B, thus triggering the intracellular signaling cascade. Furthermore, we could confirm, that PDGF-BB acts via activation of the MAPK cascade and PI3K/Akt/mTOR pathway, leading to phosphorylation and nuclear exclusion of forkhead box protein O3 (FoxO3), a transcription factor associated with the progression and severity of multiple diseases, including pulmonary fibrosis. This inactivation of FoxO3 could be attenuated by pretreatment with CNP. Furthermore, stimulation with CNP significantly increased the phosphorylation of VASP at serine 239, unaffected by PDGF-BB, suggesting that CNP mediates its attenuation of PDGF-BB-induced FoxO3 inactivation and decrease of collagen expression via activation of protein kinase G (PKG) I. Thus, in summary, CNP inhibits PDGF-BB-induced Col1a1 and Col3a1 expression which might me mediated by maintenance of FoxO3 activation. / Idiopathische Lungenfibrose (IPF) ist eine fortschreitende Lungenerkrankung, die durch veränderte Lungengewebszellen, sogenannte Myofibroblasten, gekennzeichnet ist. Diese Myofibroblasten spielen eine entscheidende Rolle bei der Entwicklung und dem Fortschreiten der IPF, indem sie eine erhöhte Proliferation, Zellmigration und Kollagenproduktion zeigen. Einer der fibrogenen Faktoren, die an diesen pathologischen Veränderungen beteiligt sind, ist Platelet-derived growth factor-BB (PDGF-BB). Es ist jedoch wenig darüber bekannt, welche endogenen Faktoren diesen fibrotischen Prozessen entgegenwirken. Frühere Forschungsarbeiten haben allerdings gezeigt, dass exogen verabreichtes C-Typ natriuretischem Peptid (CNP) eine schützende Wirkung auf den Gewebeumbau hat, insbesondere bei Erkrankungen wie Herz- und Leberfibrose. Obwohl CNP und sein Rezeptor Guanylylzyklase B (GC-B) in der Lunge exprimiert werden, ist noch unbekannt, ob CNP durch diesen Signalweg die Lungenfibrose mildern kann. Um diese Frage zu beantworten, isolierten und kultivierten wir zunächst murine Lungenfibroblasten und untersuchten, ob PDGF-BB in vitro profibrotische Veränderungen hervorruft. Die Stimulierung mit PDGF-BB führte zu einem Anstieg der intrazellulären Spiegel der profibrotischen Proteine Kollagen Typ I, alpha 1 (Col1a1), Kollagen Typ III, alpha 1 (Col3a1) und Matrix-Metalloproteinase 9 (MMP9), was auf eine Transformation zu einem fibrotischen Phänotyp hin. Exogen zugeführtes synthetisches CNP von 10 nM und 100 nM konnte den durch PDGF-BB verursachten Anstieg der intrazellulären Col1a1- und Col3a1-Spiegel signifikant abschwächen. CNP erhöhte den intrazellulären cGMP-Spiegel in kultivierten Lungenfibroblasten, was auf einen GC-B-abhängigen Signalweg hinweist. Selbst in Gegenwart hoher PDGF-BB-Spiegel konnte CNP seinen Rezeptor GC-B aktivieren und somit die intrazelluläre Signalkaskade auslösen. Darüber hinaus konnten wir bestätigen, dass PDGF-BB über die Aktivierung der MAPK-Kaskade und des PI3K/Akt/mTOR-Signalwegs wirkt, was zur Phosphorylierung und Ausschluss von Forkhead-Box-Protein O3 (FoxO3) aus dem Nukleus führt, eines Transkriptionsfaktors, der mit dem Fortschreiten und der Schwere zahlreicher Krankheiten, einschließlich pulmonaler Fibrose, in Verbindung gebracht wird. Diese Inaktivierung von FoxO3 konnte durch Vorbehandlung mit CNP abgeschwächt werden. Des Weiteren erhöhte die Stimulation mit CNP signifikant die Phosphorylierung von VASP an Serin 239 unbeeinflusst von PDGF-BB. Dies legt nahe, dass CNP seine Abschwächung der PDGF-BB-induzierten Inaktivierung von FoxO3 und Verringerung der Kollagenexpression über die Aktivierung von Proteinkinase G (PKG) I vermittelt. Zusammenfassend hemmt CNP die PDGF-BB-induzierte Expression von Col1a1 und Col3a1, was durch die Aufrechterhaltung der FoxO3-Aktivierung vermittelt werden könnte.

by Chan Pui, Barbara. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 137-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Objetivos: Comparar dois modelos de hipertensão pulmonar (monocrotalina isoladamente e pneumonectomia com monocrotalina) em relação à gravidade hemodinâmica, estrutura das artérias pulmonares, marcadores inflamatórios - interleucina-1 (IL-1) e fator de crescimento derivado de plaquetas (PDGF) - e sobrevida em 45 dias. Métodos: Total de 80 animais analisados em 2 protocolos de estudo: análise estrutural e análise de sobrevida. Foram divididos em 4 grupos [controle (C), monocrotalina (M), Pneumonectomia com monocrotalina (PM) e pneumonectomia (P)]. Após 28 dias, os animais foram cateterizados, sendo obtidos os valores hemodinâmicos. Após foram sacrificados, sendo obtidos os tecidos cardíaco e pulmonar. O ventrículo direito (VD) foi dissecado do septo interventricular e a relação do seu peso sobre o peso do ventrículo esquerdo (VE+S) com o septo foi obtida como índice de hipertrofia de VD. No tecido pulmonar foram realizadas as análises histológicas (área da camada média das artérias pulmonares) e dosados os peptídeos IL-1 e PDGF através da técnica de ELISA. Para o estudo de sobrevida os animais foram observados por 45 dias. Resultados: Os grupos M e PM apresentaram hipertensão pulmonar em relação aos demais. Houve aumento significativo da relação VD/VE+S no grupo PM em relação aos demais. Não houve diferença significativa entre os grupos M e PM na área da camada média das artérias pulmonares, nas dosagem de IL-1 e PDGF e na sobrevida. Conclusões: Com os resultados obtidos não podemos afirmar que o modelo de pneumonectomia com monocrotalina é superior ao modelo de monocrotalina / Objectives: To compare two models of pulmonary hypertension (monocrotaline and pneumonectomy with monocrotaline alone) in relation to the hemodynamic severity, structure of the pulmonary arteries, inflammatory markers - interleukin-1 (IL-1) factor and platelet-derived growth factor (PDGF) - and survival at 45 days. Methods: Total of 80 animals were analyzed in two study protocols: structural analysis and survival analysis. They were divided into four groups [control (C), monocrotaline (M), Pneumonectomy with monocrotaline (PM) and pneumonectomy (P)]. After 28 days, the animals were catheterized, and the hemodynamic values obtained. Then, they were euthanized and obtained the heart and lung tissues. The right ventricle (RV) was dissected from the interventricular septum and the ratio of its weight on the weight of the left ventricle (LV + S) with the septum was obtained as an index of RV hypertrophy. In lung tissue histological analyzes were performed (area of the middle layer of the pulmonary arteries) and the peptides IL-1 and PDGF measured by ELISA. To the survival study , the animals were observed for 45 days. Results: The groups M and PM show pulmonary hypertension in relation to the others. A significant increase in the RV / LV + S was observed in PM in relation to M, and M and PM in relation to the others. There was no significant difference between groups M and PM in the medial layer of pulmonary arteries, the dose of IL-1 and PDGF, and survival

Objetivos: Comparar dois modelos de hipertensão pulmonar (monocrotalina isoladamente e pneumonectomia com monocrotalina) em relação à gravidade hemodinâmica, estrutura das artérias pulmonares, marcadores inflamatórios - interleucina-1 (IL-1) e fator de crescimento derivado de plaquetas (PDGF) - e sobrevida em 45 dias. Métodos: Total de 80 animais analisados em 2 protocolos de estudo: análise estrutural e análise de sobrevida. Foram divididos em 4 grupos [controle (C), monocrotalina (M), Pneumonectomia com monocrotalina (PM) e pneumonectomia (P)]. Após 28 dias, os animais foram cateterizados, sendo obtidos os valores hemodinâmicos. Após foram sacrificados, sendo obtidos os tecidos cardíaco e pulmonar. O ventrículo direito (VD) foi dissecado do septo interventricular e a relação do seu peso sobre o peso do ventrículo esquerdo (VE+S) com o septo foi obtida como índice de hipertrofia de VD. No tecido pulmonar foram realizadas as análises histológicas (área da camada média das artérias pulmonares) e dosados os peptídeos IL-1 e PDGF através da técnica de ELISA. Para o estudo de sobrevida os animais foram observados por 45 dias. Resultados: Os grupos M e PM apresentaram hipertensão pulmonar em relação aos demais. Houve aumento significativo da relação VD/VE+S no grupo PM em relação aos demais. Não houve diferença significativa entre os grupos M e PM na área da camada média das artérias pulmonares, nas dosagem de IL-1 e PDGF e na sobrevida. Conclusões: Com os resultados obtidos não podemos afirmar que o modelo de pneumonectomia com monocrotalina é superior ao modelo de monocrotalina / Objectives: To compare two models of pulmonary hypertension (monocrotaline and pneumonectomy with monocrotaline alone) in relation to the hemodynamic severity, structure of the pulmonary arteries, inflammatory markers - interleukin-1 (IL-1) factor and platelet-derived growth factor (PDGF) - and survival at 45 days. Methods: Total of 80 animals were analyzed in two study protocols: structural analysis and survival analysis. They were divided into four groups [control (C), monocrotaline (M), Pneumonectomy with monocrotaline (PM) and pneumonectomy (P)]. After 28 days, the animals were catheterized, and the hemodynamic values obtained. Then, they were euthanized and obtained the heart and lung tissues. The right ventricle (RV) was dissected from the interventricular septum and the ratio of its weight on the weight of the left ventricle (LV + S) with the septum was obtained as an index of RV hypertrophy. In lung tissue histological analyzes were performed (area of the middle layer of the pulmonary arteries) and the peptides IL-1 and PDGF measured by ELISA. To the survival study , the animals were observed for 45 days. Results: The groups M and PM show pulmonary hypertension in relation to the others. A significant increase in the RV / LV + S was observed in PM in relation to M, and M and PM in relation to the others. There was no significant difference between groups M and PM in the medial layer of pulmonary arteries, the dose of IL-1 and PDGF, and survival

In this study the aim was to optimize the production of the Affibody fusion-protein Z03358- ABD094-(S4G)3-IL2 with regard to the amount of soluble protein produced. However, problems with reproducibility with this protein and the chosen expression system were encountered. Therefore, expression of the His-tagged Affibody His6-(Z05477)2 was evaluated using the same expression system as well as expression in another well characterized expression system. Both target proteins are of therapeutic interest. One of the proteins is an IL2 fusion protein (Z03358-ABD094-(S4G)3-IL2) that bind the platelet-derived growth factor receptor β (PDGFR-β). PDGF signaling is of interest in cancer treatment where, among other things, the effects of PDGF on tumor angiogenesis is researched. The His6-(Z05477)2 protein has a classified target but is developed as a therapeutic in the area of inflammation and autoimmune disease. Both model proteins are known to be difficult to purify due to low solubility. The two E. coli expression systems investigated and compared were BL21(DE3) and Lemo21(DE3). The fusion protein Z03358-ABD094-(S4G)3-IL2 was produced in BL21(DE3) in inclusion bodies with a yield of 4.95 g/l. An optimized process for the expression of His6-(Z05477)2 using BL21(DE3) was developed with a yield of 6.6 g/l soluble protein after expression at 30°C for 6 h.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 147-164.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.