Thesis (Ph. D.)--University of Hong Kong, 1989.

Der humanen dermalen Wundheilung liegt ein komplexes Zusammenspiel verschiedener Faktoren zugrunde. Die Bedeutung dieses fein regulierten Gleichgewichts wird deutlich, wenn es durch Fehlregulationen oder Störungen zu chronischen Wundheilungsstörungen oder lokaler Fibrose mit überschießender Narbenbildung kommt. Eine der möglichen Methoden zur Prävention und Behandlung ist die Deckung der Wunde mit einem Hautersatz. Dabei werden zunehmend sogenannte Biomaterialien aus natürlichen Substanzen mit hoher Biokompatibilität und der Möglichkeit zur Interaktion mit dem nativen Gewebe verwendet. In Studien wurde gezeigt, dass vor allem sulfatierte Glykosaminoglykan-Derivate durch die Interaktion ihrer negativ geladenen Sulfatgruppen mit Zytokinen, Wachstumsfaktoren und dermalen Zellen einen positiven Einfluss auf den Wundheilungsprozess haben können. In der vorliegenden Arbeit wurden daher kollagenbasierte artifizielle extrazelluläre Matrizes mit unsulfatierter oder sulfatierter Hyaluronsäure hinsichtlich ihres Einflusses auf humane dermale Fibroblasten als Komponenten der Wundheilung untersucht. Dermale Fibroblasten spielen im Ablauf der Wundheilung eine tragende Rolle und interagieren eng mit der umgebenden Matrix. Anhand ihrer Proteinexpression lassen sich Rückschlüsse auf wichtige Funktionen wie Adhäsion, Proliferation, Differenzierung und Matrixsynthese ziehen. In den durchgeführten Experimenten zeigte sich, dass sulfatierte Matrix in der Kultur mit dermalen Fibroblasten kein entzündliches Milieu förderte. Die Proliferation, Differenzierung und Migration der Fibroblasten schienen gesteigert, während sich die Matrix-Synthese und ihr Remodeling weder pathologisch gehemmt noch überschießend zeigten. Daher wäre die weitere Untersuchung dieses Biomaterials ein vielversprechender Ansatz, um langfristig dem Risiko von Wundheilungsstörungen wie chronischen Wunden oder fibroproliferativen Wundheilungsstörungen effektiv entgegenzuwirken.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type II collagen is the major extracellular matrix (ECM) protein present in cartilage and is detected in non-chondrogenic tissues such as the heart and the neural tube during developmental stages involving rapid tissue morphogenesis indicating an active role played by the collagen in embryogenesis. Type II collagen is synthesized as a procollagen precursor which has amino- and carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central triple helical domain. Two isoforms of type II procollagen are generated by alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous to those present in regulators of the bone morphogenetic protein (BMP) signaling such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv). Previous in vitro binding assays and overexpression studies in frog embryo suggest that the CR domain of IIA antagonized BMP signaling. In order to give a better understanding of the function of IIA in embryonic development and cellular signaling, several approaches including expression pattern analyses, phenotypic analyses of null mutant and gain of function studies are employed in this study. Expression studies of IIA mRNA in early postimplantation mouse embryos find that it is present in the axial mesendoderm (including the anterior definitive endoderm [ADE] and the prechordal plate) which is a critical head organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II collagen (Col2a1) gene by homologous recombination, indeed reveals that the anterior-most neural tissue is deficient at early somitogenesis denoted by reduction/loss of the forebrain/optic cup markers. Marker studies indicate that the ADE may already be affected at the neural plate stage in IIA-/-. The neural phenotype of IIA-/- displays significant similarities with mutants deficient in BMP pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting that IIA plays a role in maintaining the specification and/or regulating the signaling properties of the anterior midline tissue which involves regulation of BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos suggest that IIA regulate BMP and the related Nodal signaling pathways in a context dependent manner which has significant implications in normal anterior neural plate development. Based on the work described in this thesis and the body of existing evidence, a model is presented which suggests that IIA promote/maintain anterior neural plate development by regulating the range and extent of BMP signaling in the anterior neural plate. This study sheds light on the role of an ECM component in regulating tissue patterning and cellular signaling during early mouse development and also provides putative function for the CR domain of other fibrillar procollagens including type I, III and V which is poorly understood currently. This work will provide the framework for the design of subsequent studies in re-examining the role of these fibrillar procollagens in embryogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The extracellular environment of the central nervous system (CNS) through which neuritic processes must traverse during development or after injury is complex, and may vary from stabile conditions to a milieu favorable for neural plasticity and growth. The extracellular space in the CNS accounts for about 20% of brain volume and is composed of aggregating complexes of several different extracellular matrix (ECM) molecules. The ECM supports neural networks and acts as a barrier for neurite extention, depending on the type of molecules involved and the various signals they induce. One mechansim that may produce an environment favoring plasticity is the proteolytic cleavage of ECM. Brevican belongs to the lectican family of aggregating, chondroitin sulfate-containing proteoglycans (CSPGs) and is abundant in brain ECM complexes. It is localized peri-synaptically, inhibits neurite outgrowth, and is thought to stabilize synaptic networks in the adult. Interestingly, a significant proportion of brevican in the CNS is observed as a fragment of the protein core formed by proteolytic cleavage. Endogenous matrix-degrading proteinases, such as the MMPs (matrix metalloproteinases) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), cleave brevican and other lecticans potentially promoting neural plasticity. Cleavage of brevican and similar lectican family members may "loosen" the aggregated complexes and change the extracellular environment to one that is more permissive toward neural plasticity. After injury, during inflammation or with disease, alterations in the ECM may influence development and/or progression of neurological disease. The purpose of these studies was to investigate the catabolism of brevican in the ECM and its potential role in neural plasticity under each of these influences, taking an in depth look at how brevican is processed after (1) undergoing a classical model of neural plasticity, the entorhinal cortex lesion (ECL); (2) a disease state that is thought to have dysregulated neural and synaptic plasticity; and (3) how brevican catabolism and neural plasticity is effected by deleting the protease responsible for the cleavage of lecticans in a mouse model. Overall, these experiments provide evidence that the proteolytic cleavage of brevican, and lecticans in general, may play an important role in the regulation of neural plasticity.

In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis. In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK). During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ. In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay.  Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.

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published_or_final_version / abstract / toc / Anatomy / Doctoral / Doctor of Philosophy

Acute injury of major epithelial organ systems (kidney, liver, lung, etc.) is collectively a principal cause of death worldwide. Regenerative medicine promises to meet these human health challenges by harnessing intrinsic cellular processes to repair or replace damaged tissues. Epithelial morphogenesis is a hard-wired, multicellular differentiation program that dynamically integrates microenvironmental cues to coordinate cell fate processes including adhesion, migration, proliferation, and polarization. Thus, epithelial morphogenesis is an instructive mode of tissue assembly, maintenance, and repair. Three-dimensional epithelial cell cultures in natural basement membrane (BM) extracts produce hollow, spherical cyst structures and have indicated that the BM provides the critical cell adhesion ligands to facilitate cell survival, stimulate proliferation, and promote polarization and lumen formation. However, the utility of natural BMs for detailed studies is generally limited by lot-to-lot variations, uncontrolled cell adhesive interactions, or growth factor contamination. The goal of this thesis was to engineer bioartificial extracellular matrices (ECM) that would support and modulate epithelial cyst morphogenesis. We have engineered hydrogels, based on a multi-arm maleimide-terminated poly (ethylene glycol) (PEG-4MAL), that present cell adhesive molecules and enzymatic degradation substrates and promote polarized epithelial cyst differentiation in vitro. To investigate the influence of matrix physical and biochemical signals on cyst morphogenesis, we independently varied the polymer weight percentage (wt%), the density of a cell adhesion ligand (RGD), and crosslink degradation rates of the hydrogels. Then, we evaluated functional outcomes including Madin-Darby canine kidney (MDCK II) epithelial cell survival, proliferation, cyst polarization, and lumen formation. We found that cell proliferation, but not cell survival, was sensitive to the polymer wt%, which is related to elastic modulus and crosslink density. This result defined a working range of PEG-4MAL concentration (3.5% - 4.5%) that promotes robust proliferation. Analysis of mature cysts indicated that 4.0% and 4.5% gels produced cysts resembling those typically grown in type I collagen gels while 3.5% gels produced cysts with higher incidence of inverted polarity and multiple lumens. Perturbation of matrix degradability using a slow-degrading crosslink peptide or matrix metalloproteinase inhibitors showed that the rate of matrix degradation exerts major influence on cyst growth in PEG-4MAL gels. We employed 4.0% PEG-4MAL hydrogels with RGD ligand density ranging over 0 – 2000 uM to discover that (1) lumen formation was eliminated in the absence of RGD, (2) extent of lumen formation increased with increasing RGD concentration, and (3) cyst polarity was inverted below a threshold of integrin binding to RGD. Together, these results show that the biochemical and physical properties of the matrix, particularly integrin binding and matrix degradability, effectively modulate establishment of apico-basal polarity and lumen phenotypes in MDCK II epithelial cyst structures. Furthermore, these studies validate PEG-4MAL hydrogels as a powerful culture platform to enable detailed investigation of matrix-directed modulation of epithelial morphogenesis.

Ο ρόλος της εξωκυττάριας ουσίας στην καρδιακή ανεπάρκεια. / The functional role of extracellular matrix in heart failure.