Type IV collagens are components of basement membranes throughout the body and are involved in maintenance of the structural integrity of tissues as well as cellular differentiation, growth, and adhesion. Members of this collagen family are uniquely arranged in pairs in a head-to-head orientation and share a proximal promoter region. The COL4A5-COL4A6 gene pair is involved in numerous human diseases and cancer metastasis. For these reasons, defining the mechanisms that regulate collagen gene expression is of specific interest. To study type IV collagens, an in vitro model system was characterized. Comparative genomics was utilized to identify conserved, non-coding DNA in COL4A5 and COL4A6. These sequences were transfected into cell lines differing in type IV collagen expression and tested for the ability to regulate transcription of a reporter gene. Each cell line was also treated with the epigenetic modifying agents, 5-Aza and TSA. The effects on type IV collagen expression were determined. The COL4A5-COL4A6 promoter region was extensively characterized using ChIP analysis; antibodies against RNAPII, acetylated histone H3, and H3K9me2 were used. Additionally, bisulfite sequencing was carried out on each cell line to determine the methylation status of CpG dinucleotides in the promoter. Cell lines differing in expression of COL4A5 and COL4A6 were identified: 1) SCC-25 keratinocytes and HEK-293 cells transcribed both COL4A5 and COL4A6, 2) HT-1080 cells selectively activated COL4A5, and 3) SK-N-SH neuroblastoma cells did not express either gene. In SK-N-SH cells, histone modifications were shown to facilitate formation of condensed chromatin to prevent transcription initiation; repression was independent of DNA methylation. Activation of COL4A5 and COL4A6 in SCC-25 and HEK-293 cells involved acetylation of histones, although differences between the two cell types were seen. In addition, conserved, non-coding sequences were shown to affect transcription of a reporter gene; these sequences may be interacting with the transcription machinery to modulate collagen expression. Finally, repression of COL4A6 in HT-1080 cells appeared to be mediated through DNA methylation of the promoter; selective activation of COL4A5 may involve conserved, non-coding DNA. In summary, epigenetic modifications as well as conserved sequences are intimately involved in regulation of type IV collagen gene expression.

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.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 99-101).

Osteoarthritis (OA) is one of the most concerned diseases in the field of orthopaedics. During the process of this disease, articular cartilages are degenerated and worn out at the end stage, which create pain and disabilities to the patients. Although multiple mechanical and biochemical factors may initiate and/or enhance the progression of OA, alternation of biomechanical properties of articular cartilage is one of the products. There are several major components in articular cartilage; it is believed that each of their contribution cannot be entirely neglected. Superficial zone is mainly consisted with collagen and it was found that the biomechanical properties of this part of cartilage are also alternated significantly as a result of OA. Hence, degeneration of collagen network also occurs. Alternatively, osteoporosis (OP) is another common disease, which is associated to the decrease of bone mineral density; the effect of OP on articular cartilage is limited. In reverse, increasing bone mineral density in subchondral plate alters the loads distribution on articular cartilage and possibly leads to OA. This current study investigated the morphological and biomechanical properties of individual collagen fibrils extracted from OA, OP and healthy cartilages. A total of ten joint specimens were recruited, 3 OA joints were from 3 OA patients, 3 OP joints were from 3 OP patients, and 4 joints were from 2 healthy individuals. All cartilages were harvested from non weight-bearing zone, and average diameters were calculated from 350 fibrils’ measurements. In addition, 50 fibrils were randomly selected for nano-indentation under ambient condition. However, the representation of biomechanical properties tested at low humidity may be questionable. This current study also investigated the stiffness of hydrated fibrils. The results showed that the collagen fibrils extracted from OA cartilages were thinner than the ones extracted from OP and healthy cartilages. It was believe that the fibrillation and derangement of collagen network spread from superficial zone towards deeper zones. However, the number of thinner collagen fibrils increased in OA specimens could be the reason of the loss of larger fibrils and/or fragmentation occurred in the superficial zone, where contains thinner fibrils. The biomechanical tests showed that fibrils extracted from OA cartilages owned the highest elastic modulus, while the ones from OP had the lowest; significant differences were found between all groups when tested under ambient condition. Alternatively, the same pattern of results could also be found when hydrated fibrils were tested; however, due to the limited amount of samples, only the difference between OA and OP were considered significant. In addition, no individual difference was found; no significant difference between samples within each group could be observed. Since nano-indentations were performed at the center of each fibril, the elastic moduli measured represented the stiffness of the crosslinks and molecules within fibrils. Assuming the triple helix structure of collagen is relatively tough, the decrease of tensile modulus of superficial zone in OA cartilages could due to the changes of the crosslinks between fibrils in collagen network. / published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.

The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.