No description available.

Muscular dystrophy is a disease characterized by skeletal muscle weakness and wasting, but little is known of alterations in the vascular system that occur with this disease. The culprit in many muscular dystrophies is a defective dystrophin-glycoprotein complex (DGC). The DGC is a group of transmembrane proteins that connects the cytoskeleton of muscle cells to the extracellular matrix; it plays a role in mechanotransduction and the maintenance of structural integrity of these cells, and includes the proteins dystrophin and sarcoglycan-delta. The absence of these proteins results in severe muscular dystrophies in humans, and thus knockout mice lacking the genes encoding for dystrophin (mdx mice) and sarcoglycan-delta (sgcd-/- mice) were studied to detect any vascular alterations that occur as a result of a defective DGC. Acute biaxial biomechanical data were obtained through pressure-diameter and axial force-length tests on common carotid arteries of mdx, sgcd-/-, and wild-type mice in the active and passive smooth muscle state. Functional response to the vasoreactive compounds phenylephrine, carbamylcholine chloride, and sodium nitroprusside was also tested. We found significant biomechanical differences between the knockout and wild-type mouse arteries: the mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries also having increased circumferential stresses, and the knockout arteries generated increased axial loads and stresses in the axial force-length tests. The mdx and sgcd-/- arteries also differed from the wild-type in that their ‘homeostatic’ axial stretch, at which the axial force remains constant upon pressurization, was significantly decreased. We conclude that the loss of DGC proteins does trigger changes in vascular smooth muscle cells or their interactions with the extracellular matrix, yet that the altered vascular system was able to adapt and function without the DGC. Knowledge of alterations to the vascular system (and adaptations to these changes) of patients with muscular dystrophy could help physicians customize their treatment to extend and enhance their lives, especially as medical advances extend the lifespan of these patients and they begin to suffer from diseases such as hypertension and atherosclerosis that affect the normal aging population.

Vascular smooth muscle cells (VSMC) in the healthy adult arterial wall are a highlydifferentiated cell type with low levels of proliferation. However, when activated these cells can undergo a phenotypic change to become proliferative, migratory and excrete higher levels of extra-cellular matrix. While this cellular change is an essential element of the adaptable vasculature, excessive proliferation of VSMC underpins the development of a number of disease states, including atherosclerosis and restenosis after balloon angioplasty. The activation of VSMC is dependent on intracellular signalling pathways broadly altering gene expression. A key feature of this process is the initial potent regulation of transcription factors such as Egr-1, c-Jun and Ets-1, which then drive further transcriptional changes resulting in phenotypic change. The aim of this thesis was to discover novel genes, particularly transcription factors, regulated early upon stimulation and to characterise their contribution to the activation of VSMC. A key stimulus for activation of VSMC is the release of fibroblast growth factor 2 (FGF-2). A microarray used to explore the effects of FGF-2 exposure demonstrated the extensive nature of transcriptional modulation. In addition, it highlighted a number of transcription factors that were not previously described in VSMC: p8, ATF-4 and SHARP-2. In particular, SHARP-2 was potently upregulated and was reconfirmed in animal models of vascular injury. The subsequent contribution these factors make to VSMC activation was also demonstrated. p8 strongly induced VSMC proliferation, while ATF-4 contributed to cytokine production and SHARP-2 potently downregulated VSMC differentiation markers. A second area that was explored related to a gene known as YRDC, which was found to be upregulated upon stimulation of VSMC. YRDC is highly conserved across almost all cellular life, however its function remains unknown. A number of novel splice variants of YRDC were discovered and demonstrated to be differentially regulated in VSMC upon stimulation. Further work to commence characterising its function showed that it interacts with key ribosomal proteins and most likely plays a role in regulating translation. The discovery of the relevance of these genes to vascular biology in addition to their transcriptional regulation makes an important contribution to increasing our understanding of the molecular mechanisms behind vascular remodelling.

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 72-82)

Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2006. / Includes vita. Advisory Committee: Lucy Liaw, Scientist, Maine Medical Center Research Institute, Advisor; Carla Mouta-Bellum, Scientist, Maine Medical Center Research Institute; Jeong Yoon, Scientist, Maine Medical Center Research Institute; Dorothy E. Croall, Professor of Biochemistry; Thomas Gridley, Senior Scientist, The Jackson Laboratory. Includes bibliographical references (leaves 97-112 ).

No description available.

Actin dynamics are precisely regulated by a large number of actin binding proteins which collectively alter the rates of actin filament assembly and disassembly. Spectrin, an actin cross-linking protein, forms lateral filamentous networks that are linked to the plasma membrane and are required for membrane stability and resistance to mechanical stress. Adducin binds to spectrin-actin complexes, recruiting additional spectrin molecules, thereby further stabilising the membrane. In addition, adducin can bundle and cap actin filaments, and its actions have been implicated in cytoskeletal rearrangement in a variety of cell types. In vascular smooth muscle there is evidence that rearrangement of the actin cytoskeleton is involved in contraction and transmission of force to the extracellular matrix which leads to tissue remodelling. In addition, cytoskeletal dynamics are involved in vascular smooth muscle cell migration, proliferation and membrane dynamics. Protein kinase C (PKC), Rho-kinase, calmodulin and myosin light chain phosphatase are signalling proteins that are involved in these processes in vascular smooth muscle, and adducin is regulated by these signalling proteins in platelets and epithelial cells. The current study provides evidence for regulation of the actin cytoskeleton by α-adducin in vascular smooth muscle. Both α-adducin and spectrin are associated with the cytoskeleton in vascular smooth muscle cells of rat mesenteric small arteries. In response to activation by noradrenaline (NA), α-adducin becomes rapidly phosphorylated on Ser 724, a site specific for PKC, and dissociates from the actin cytoskeleton and spectrin in a PKC-dependent manner. Longer exposure of vessels to NA results in dephosphorylation of α-adducin on Ser 724 and its Rho-kinase-dependent reassociation with the actin cytoskeleton. Concurrent with this reassociation is enhanced association between the two proteins and an increase in the proportion of spectrin associated with the actin cytoskeleton. In addition, a rise in filamentous actin is observed, which can be blocked by inhibition of PKC or Rho-kinase and also by delivery of the α-adducin antibody into vessels in order to inhibit the function of endogenous a-adducin. These data provide evidence for a model in which α-adducin functions as an actin capping protein in resting vascular smooth muscle cells. Upon vasoconstrictor activation α-adducin becomes phosphorylated by PKC, inducing its dissociation from the actin cytoskeleton allowing elongation of actin filaments and further rearrangement of the actin cytoskeleton. Following this reorganisation, α-adducin re-associates with the actin cytoskeleton, possibly in response to phosphorylation by Rho-kinase, and recruits additional spectrin molecules, thus strengthening the newly formed actin filament network. These data provide further insight into the regulation of the actin cytoskeleton in vascular smooth muscle.

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy