Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains are well-known inhibitors of neural plasticity and associate with hyaluronan and tenascin-R to form a complex of extracellular matrix (ECM) in the central nervous system (CNS). Little is known about whether proteolytic cleavage of the core protein affects neural plasticity. Several members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of metalloproteinases are glutamyl-endopeptidases that cleave aggregating PGs. Our initial studies determined that neural cultures secrete a brevican-containing matrix, and that these neural cultures also produced ADAMTS4, a protease that cleaves brevican. Furthermore, this brevican-containing matrix in astrocytes could be modulated by treatment with transforming growth factor beta (TGFbeta) through the inhibition of the activity of the ADAMTSs.Once it was established that neural cultures produce a brevican-rich matrix, we s ought to utilize this matrix to determine whether cleavage of aggregating PGs, especially brevican, by the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of rat neurons with ADAMTS4 cDNA induced longer neurites, and interestingly, this effect proved to be independent of the proteolytic action of the ADAMTSs. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures similarly enhanced neurite extension, an action dependent on the activation of extracellular signal-related kinase (ERK)1/2 (MAP kinase 42/44), resulting in the first evidence that ADAMTSs may induce intracellular signaling events. Studies of dendritic spine morphology and levels of synaptic proteins in response to ADAMTS4 treatment were also undertaken. Neuronal cultures treated with ADAMTS4 showed increased length of dendritic spines and increased percent of immature spines detected. A concurrent decrease in post-synaptic protein staining was detected on the neurites of yo ung neurons overexpressing ADAMTS4 or expressing proteolytically-inactive mutant ADAMTS4 protein. Thus, ADAMTS4 may promote plasticity in neurons in vitro by preventing the formation, maturation, and/or stabilization of synapses. Overall, these experiments provide evidence that implicate the ADAMTSs as mediators of neural plasticity, and while primarily known only as proteases, these studies demonstrate that the ADAMTSs exert actions distinct from these proteolytic properties that require the induction of intracellular signaling events.

<p>Basement membranes are a dense, sheet-like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the BM that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the BM component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this aims to understand how SPARC overexpression promote invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies.</p> / Dissertation

Some epithelial cancers can be induced to revert to quiescent differentiated tissues when combined with embryonic mesenchyme; however, the mechanism of this induction is unknown. This dissertation is based on the hypothesis that because extracellular matrix (ECM) plays a critical role during organ development in the embryo, it also may mediate the differentiation-inducing effects of embryonic mesenchyme on cancer cells. To test this hypothesis, I first optimized methods to isolate ECMs from whole tissues or cultured cells, and to repopulate them with cultured cells, using embryonic tooth as a model system. In Chapter 2, I describe these studies and use them to demonstrate that embryonic ECM is sufficient to regulate odontogenic signaling, cell fate decisions and histodifferentiation during normal tooth development. In Chapter 3, I adapt these methods to show that culture of breast cancer cells with ECM derived from embryonic mammary mesenchyme decreases tumor cell proliferation, and stimulates differentiation, including formation of hollow acini and ducts as well as enhanced expression of estrogen receptor-alpha and decreased migration. Further, when the inductive ECMs were injected into fast-growing breast tumors in mice, they significantly inhibited cancer expansion. Critically, the differentiation observed with ECM was the same as that observed in co-culture with mammary mesenchyme cells, showing that ECM is playing a dominant role in tumor cell normalization. In Chapter 4, I then set out to determine the mechanism by which embryonic ECM normalizes tumor cells, I analyzed the contributions of bound cytokines, ECM composition and mechanics. Western blot analysis revealed several bound growth factors, which remained following decellularization; however, removal of these growth factors using high salt washes had no effect on ECM-mediated normalization of tumors. Further, using proteomics analysis I identified eleven ECM proteins present only within inductive ECMs and by testing these proteins in 3D culture, I found three proteins -- collagen III, biglycan and SPARC -- that increased lumen formation to a similar extent as embryonic ECM. These data confirm that mesenchyme-induced tumor cell normalization is mediated by the insoluble ECM, and reveal the identity of some of the inductive molecules responsible for these effects.

The total annual incidence of arthritis in this prospective cross-sectional study on adults was 115/100 000. The annual incidence of rheumatoid arthritis (RA) was 24/100 000, 29/100 000 for women, and 18/100 000 for men. For reactive arthritis (ReA) the annual incidence was slightly higher, 28/100 000, and for undifferentiated arthritis 41/100 000. The annual incidence of Lyme disease and sarcoid arthritis was low. The annual incidence of arthritis in this study compares well with findings in earlier reports from both registers and case review studies. Almost 50% of the patients in the series of 71 patients with arthritis of less than 3 months’ duration had a preceding infection. Campylobacter jejuni ReA dominated the enteric ReA group. We found only a few patients with preceding Chl. trachomatis, Chl. pneumoniae, Borrelia burgdorferi or parvovirus B19 infections. The arthritis patients with a preceding infection went into remission more often than the patients without a preceding infection. The disease specificity of anti-CCP antibodies for RA was high, 96%, confirming earlier results. Anti-CCP antibodies differentiated RA from other arthritides. Several patients in the different diagnosis groups had raised serum COMP levels, indicating cartilage involvement very early in the disease, even in mild and self-limiting disease with good prognosis. The economic burden of early joint inflammation was found to be considerable already during the first few months of the arthritis irrespective of diagnosis. Surprisingly, patients with ReA generated almost as high costs as patients with RA during thefirst few months of the disease, even though most of the ReA patients had a relatively mild disease. Sick leave accounted for about 50% of the costs. The distribution of costs in the different patient groups was skewed. The median cost per patient for the group of patients with RA was US$4385, for ReA US$4085, for other types of specified arthritis US$3361, and for undifferentiated arthritis US$1482. This underlines the necessity of quick referral and therapy, not only to decrease the inflammation and prevent functional impairment, but also to decrease the costs of early arthritis.

Heart failure, predominately caused by myocardial infarction (MI), is the leading cause of death in the United States. Currently the only treatment for heart failure is cardiac transplantation, but studies show that progenitor cell, biomaterial, or combined therapies have improved cardiac function post-MI. The endogenous environment of CPCs is drastically different from commonly used culture conditions. Further the endogenous environment changes with age and disease state. We evaluated the behavior of CPCs cultured on a naturally-derived, cardiac extracellular matrix (cECM) as compared to the standard culture coating collagen I, that also mimics fibrotic tissue. In this study, CPCs cultured on cECM had improved cell numbers and cardiomyogenic maturation. However, the microenvironmental cues responsible for stimulating CPC activation are largely unknown. During development, aging and disease the myocardium changes in matrix composition and stiffness exposing endogenous cells to a wide variety of stimuli. In a combinatorial study, we evaluated the effect of cyclic strain and extracellular matrix composition on CPC behavior. The response of CPCs to signals from the microenvironment is complex, with more matrix-dependency observed at lower strains. Alignment, cell division and paracrine signaling are extracellular matrix and strain dependent. Extracellular matrix conditions affect CPC maturation and calcium signaling. Mechanotransduction pathways, including focal adhesion kinase and extracellular signal-regulated kinase, are activated through adhesion and maintained under cyclic strain. Insights from this work will advance pragmatic cell therapy attempts to regenerate healthy myocardium post-MI.

A better understanding of these structural changes that occur before Hypertension (HTN) could ultimately result in a treatment that can prevent or reverse this disease state before its onset. T cells have been shown essential for the development of HTN. The aim of these murine studies was to investigate the role of the T-helper CD4⁺ lymphocytes in initiating vascular remodeling and HTN in the absence of an increased mechanical load and to investigate the role of T-helper 17 (Th17) CD4⁺ lymphocyte initiating vascular remodeling and HTN by stimulating Lysyl Oxidase (LOX). LOX is known to cross-link collagen and elastin and. Excess synthesis of collagen and elastin results in a stiffer artery and hypertension. We established L-NAME-induce HTN in wild type (WT) mice. CD4⁺ splenic lymphocytes were isolated from these mice and adoptively transferred into naïve syngeneic severe combined immunodeficient (SCID) mice. The SCID mice receiving these cells became hypertensive. Cytokine analysis demonstrated an increase in both Th1 and Th17 cytokine in HTN donor mice and of lymphocytes in the aortic infiltrates of the HTN recipient mice. The increased collagen and LOX expression in recipient mice suggest that the adoptively transfer CD4⁺ lymphocytes are associated with vascular extracellular matrix remodeling. Furthermore we examined the role of Th17 lymphocyte in aortic LOX regulation in Angiotensin II-induced hypertension. The Increase in blood pressure and Velocity Time Integral (VTI) was measured in WT Angiotensin II treated mice whereas no change was detected in the Th17 deficient (RORT KO) Angiotensin II treated group. When compared to the control group the WT group infused with Angiotensin II had higher LOX protein expression, LOX fluorescent Immunohistochemical stain and LOX activity. This group also had increased hydroxyproline levels, collagen stain, hyperplasia and aortic thickening. In contrast, the Th17 deficient mice Angiotensin II treated group had no changes in these parameters. The results provide evidence that IL-17 mediates Angiotensin II-induced hypertension and vascular dysfunction by the overstimulation of LOX. Potentially targeting T17 cells will allow for a drug-specific therapeutic approach and delay the progression of hypertension pathology.

At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of this form remains unknown as does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

A variety of proteases have been shown to promote the progression of cancer by virtue of their ability to degrade extracellular proteinaceous barriers, such as basement membrane and interstitial stroma. At the outset of this study available evidence strongly implicated cathepsin D in breast cancer metastasis. It was envisaged that an antibody inhibitory to the activity of this enzyme might retard invasion, and restrain a tumour from spreading. To this end anti-peptide antibodies were generated against a peptide sequence derived from the substrate capturing "flap" of the enzyme. Inhibition of enzyme activity by these antibodies could not be demonstrated, probably due to the lack of a suitably sensitive enzyme assay. However, the rationale of this study and the expertise gained from it could be applied, in the future, to enzymes that have since been found to be more relevant to tumour invasion. A feature of many transformed cells is an anomalous lysosomal enzyme trafficking system, and concomitant hyper-secretion of some enzymes. The distribution of low pH compartments and lysosomal enzyme-containing compartments was investigated in human breast epithelial cells, and their c-Ha-ras- transformed counterparts. Immunofluorescence and immunoelectron microscopy showed that these compartments have a more peripheral cellular distribution with respect to normal cells, and cathepsins B and D were cell surface-associated. Studies were undertaken to reveal the extracellular matrix degrading ability of c-Ha- ras-transformed cells. Transformed cells exhibited increased degradation of fluorescein-labelled extracellular matrix in serum free medium, and increased motility, and degradation and disruption of extracellular matrix in serum-containing medium. In vitro invasion through artificial basement membrane by transformed cells was investigated using scanning electron microscopy, and was further used to preliminarily identify the proteases involved in invasion by specific inhibition. By this means, greatest inhibition of in vitro invasion was obtained using a specific metalloproteinase inhibitor. Overexpression by transformed cells of a metalloproteinase was detected by gelatin zymography. Together these results suggest that the increased invasive capacity of ras-transformed breast epithelial cells may be largely due to increased metalloproteinase activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg , 1996.

Aggrecan is one of the most important structural components of the extracellular matrix (ECM) of articular cartilage, where it contributes to the hydration of the tissue and its ability to resist compressive loads during joint movement. Increased aggrecan degradation and loss occurs in joint diseases and is thought to be mediated by enzymes such as the matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). It has also been proposed that aggrecan release from the cartilage can be mediated by a non-proteolytic mechanism which involves the degradation of hyaluronan (RA) to which the aggrecan is bound. As aggrecan degradation and loss is known to be induced by pro-inflammatory cytokines, IL-1, TNFalpha, IL-6, IL-17 and OSM were used to investigate the mechanisms involved in proteoglycan catabolism in organ cultures of bovine articular cartilage. Irrespective of the cytokine, all aggrecan fragments generated were characteristic of aggrecanase action, and no additional aggrecan-degrading enzymatic activity was detected. In the presence of OSM, more rapid aggrecan release was observed, due to both proteolysis and fragmentation of HA by hyaluronidase activity. Moreover, addition of OSM resulted in the cleavage of aggrecan at a non-canonical aggrecanase site near its carboxy-terminal globular domain. Such cleavage could be reproduced in vitro by the action of either ADAMTS-4 or ADAMTS-5. Gene expression analysis revealed that both aggrecanases were highly induced by the cytokines, and while ADAMTS-4 was the major aggrecanase to be stimulated in all conditions, ADAMTS-5 remains the predominant aggrecanase to be expressed in cartilage. Thus, the present study shows that aggrecanase activity is primarily responsible for aggrecan degradation in the early stages of cytokine stimulation, and that in the presence of OSM, aggrecanase substrate specificity can be differentially modulated and hyaluronidase-mediated RA degradation can be induced.

Smooth muscle is a primary determinant of physiology as its ability to contract affords dynamic control of diameter of the hollow organs it encircles including the airways. Mature airway smooth muscle (ASM) cells are phenotypically plastic, enabling them to subserve contractile, proliferative, migratory and secretory roles that relates to its function in health and disease. ASM cells can control airway diameter both acutely, via reversible contraction, and chronically, by driving fixed changes in structure and function properties of the airway wall. However, the scope of research on ASM biology and function has broadened greatly in the past two decades, embracing the now recognized dynamic and multifunctional behavior, but there is always a need to investigate the role of new proteins regulating ASM phenotype in vitro and lung function in vivo. The multimeric dystrophin-glycoprotein complex (DGC) links the extracellular matrix (ECM) and actin cytoskeleton while caveolae form membrane arrays on ASM cells. Using ASM cells and tissues from human and canine and intact mouse for lung physiology, we investigated the role of DGC in phenotype maturation. We also investigated the mechanism for the organization of DGC with caveolae and further tested whether this is functionally important in mobilizing intracellular calcium in ASM cells, contraction of ASM tissue and finally its role in airway physiology. Our data demonstrate that the expression of DGC is an integral feature and a key determinant for phenotype maturation of human ASM cells. Our new data reveals an interaction between caveolin-1 and DGC and indicate that this association, in concert with anchoring to the actin cytoskeleton, underpins the spatial organization of caveolae on the membrane and has a functional role in receptor-mediated calcium release in ASM in vitro, ASM contraction ex vivo and lung function in vivo. Collectively our study indicates that the organization of caveolae and DGC, and its link from ECM to the actin cytoskeleton with in caveolae are a determinant of phenotype and functional properties of ASM, which underpins its role in physiology and pathophysiology of chronic airway diseases such as asthma.