The plasminogen activator (PA) system plays an important role in cell adhesion, migration and invasion, and may require the coordinated expression of various proteins. The human urokinase-type plasminogen activator (uPA) receptor (uPAR) is a central protein component of the PA system. By binding its ligand uPA, uPAR can direct proteolysis of the extracellular matrix. Also, it is now apparent that uPAR can initiate proteolytic independent signal transduction to influence angiogenesis, inflammation, wound repair and tumour progression. To determine whether any novel proteins interacted with uPAR, a yeast two-hybrid screening analysis was undertaken using alternate uPAR domain constructs as baits. These included full-length three domain uPAR (uPAR-DIDIIDIII), two domain uPAR (uPAR-DIIDIII), and each individual uPAR domain (uPAR-DI, uPAR-DII and uPAR-DIII). A number of proteins were identified as putative candidate interactors for the alternate constructs, with two of special interest for uPAR-DIDIIDIII. These were the heat shock protein Mrj, and the extracellular matrix protein fibulin-2. The protein Mrj was shown to bind uPAR both in vitro and in vivo using GST-pull down and co-immunoprecipitation assays respectively. The GST-pull down assay identified the interaction between Mrj and uPAR dependent on the C-terminal domain of Mrj and DI of uPAR. Using in vivo co-immunoprecipitation analysis, Mrj also bound to uPAR. Preliminary data suggest the association between uPAR and Mrj may play a role in the regulation of apoptosis. In regard to the uPAR interactor of fibulin-2, a calcium dependent binding interaction with uPAR was identified using the GST-pull down assay. However due to the large molecular weight and stringent conditions needed to solubilise fibulin-2, it was not possible to co-immunoprecipitate both uPAR and fibulin-2. Together, the identification of both Mrj and fibulin-2 amongst other candidate interactors of uPAR presented here provides further insight into the intricate relationship between uPAR and other proteins which may influence a range of biological functions.

Fibulin-4 is an extracellular matrix protein which plays an essential function in the assembly of elastic fibres, and may be involved in the modulation of TGFβ bioavailability and smooth muscle cell differentiation. Mutations in fibulin-4 can cause autosomal recessive cutis laxa, a frequently lethal connective tissue disorder. Although patient studies have provided some insights into the pathological mechanisms of this disease, a detailed analysis of the consequences of fibulin-4 mutations on a molecular and cellular level was required.The findings presented in this thesis demonstrate that cutis laxa-causing fibulin-4 mutations may lead to reduced extracellular levels of fibulin-4 due to its increased susceptibility to protease degradation and misfolding/aggregation. An accumulation of autophagic vesicles was observed, indicating a blockage of autophagy, possibly due to intracellular accumulation of aggregated/misfolded mutant protein. In the extracellular matrix, mutations affected the ability of fibulin-4 to interact with the major components of the elastic fibre assembly, heparin, LTBP-1 and fibronectin. In addition, fibulin-4 mutations generally reduced expression levels of elastic fibre assembly components. In summary, these findings contribute to the understanding of fibulin-4 associated cutis laxa, and provide a basis upon which future therapeutic interventions may be developed.

Aortic stiffness underlies systolic hypertension, promotes heart failure and is associated with increased cardiovascular morbidity and mortality. It is regarded as a primary driver of left ventricular hypertrophy and aortic aneurysms and is linked to the pathogenesis of cognitive impairment, stroke and renal failure. Like most cardiovascular traits, aortic stiffness is a complex trait and is moderately heritable, yet the precise molecular mechanisms that underpin the stiffening process remain poorly defined. This study aimed to employ multiple approaches to further identify the genetic basis of aortic stiffness in a large repository of human donor aortas that had undergone ex vivo pulse wave velocity (PWV) phenotyping. The first part of this work sought to investigate the molecular basis of Loeys-Dietz type 4 syndrome in a pedigree with multiple cases of aortic aneurysms and dissections. A missense variant p.(Arg320Cys) was identified in a highly evolutionary conserved region of TGFB2. There was striking upregulation of TGFB1, TGFB2 and pSMAD2/3 on imunocytochemical straining and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlighted the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signalling in this emerging familial aortopathy and underscored the significance of TGFβ signalling in aortic extracellular matrix biology. The second part of this work attempted to characterise the biological basis for the susceptibility locus identified in the most recent genome wide analysis of carotid-femoral PWV. While the locus lies within the 14q32.2 gene desert, it contains regulatory elements, with the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) and non-coding RNA DB129663 representing potential targets for these enhancers. The association of five lead SNPs from the genome-wide association studies (GWAS) meta-analysis was examined for ex vivo aortic stiffness and BCL11B and DB129663 aortic mRNA expression. Three of the five SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B suggesting a protective role for BCL11B. Despite the strong association, BCL11B protein was not detected in the human aorta; however, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. In contrast, DB129663 transcripts were detected in 55% of the samples, and of the five SNPs only one showed allele-specific differences in aortic DB129663 transcripts. No significant differences were observed in PWV between samples expressing or lack- ing DB129663, and therefore the implication of this lncRNA in aortic stiffness remains elusive. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association with PWV. For the final part of this work genetic associations with aortic stiffness were explored in a candidate gene-based study utilising tagging SNPs to effectively capture the genetic information from linkage disequilibrium blocks. Association analyses were performed in young, healthy ENIGMA study par- ticipants selected for high and low PWV values then validated in the remaining ENIGMA cohorts. The association of four lead SNPs was then examined for ex vivo aortic stiffness in human donor aortas. The tissue expression of these SNPs and their encoded proteins was also explored. Neither the aggrecan nor the fibulin-1 SNPs showed significant associations with ex vivo PWV in the donor aortas. The exonic aggrecan tagSNP rs2882676 displayed differential transcript abundance between homozygous allele carriers but this did not translate at the protein level. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan, reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.

Doctor of Philosophy (PhD) / It is well recognised that both genetic and environmental factors determine an individual’s predisposition to asthma. In recent years, the airway smooth muscle (ASM) cell has come to the attention of researchers to, not merely be a contractile cell of the airway, but one that orchestrates events affecting airway remodelling and proliferation. Experiments described in this thesis have, for the first time, examined genes that are associated with various aspects of the pathogenesis of asthma by using the candidate gene approach and a genome wide search. Genes have not only been identified to be differentially expressed in ASM cells derived from asthmatic and non-asthmatic participants, but have also been linked with a functional consequence of asthma. The three genes found to be differentially regulated between ASM cells derived from asthmatic and non-asthmatic participants were Peroxisome Proliferator-Activated Receptor- gamma (PPARγ), mimecan and fibulin-1. Expression of the anti-proliferative transcriptional factor PPARγ, found by the candidate gene approach, was elevated in ASM cells derived from asthmatic participants. Whilst elevated, the anti-proliferative effect of PPARγ was absent in ASM cells derived from asthmatic participants. By microarray analysis, mimecan, an anti-proliferative agent was identified. Mimecan levels, although not different basally in ASM cells, were upregulated by transforming growth factor β (TGFβ) only in asthmatic derived ASM cells. Silencing mimecan, by the use of specific oligonucleotides, increased proliferation of ASM cells. This suggested that by increasing mimecan expression, the proliferation of ASM cells may be halted. Fibulin-1, also found by microarray analysis and the final gene examined in this thesis, was found in elevated levels in BAL fluid, serum and ASM cells obtained from asthmatic participants. In addition, ASM cells derived from asthmatic participants, for the first time were shown to have faster wound healing rates compared with nonasthmatics. The elevated fibulin-1 levels in ASM cells derived from asthmatic participants, in the presence of TGFβ, were demonstrated to contribute to this increased wound healing. Specifically, fibulin-1 was found to affect wound healing by increasing proliferation rather than migration. The current available treatments for asthma, target the contractility and inflammatory conditions in the airway. Through this thesis, novel genes discovered to be associated with proliferation may be potential therapeutic targets to treat asthma. In particular, the fibulin-1 gene is outstandingly promising, as it was shown that silencing fibulin-1 resulted in slower wound healing rates through decreased cell proliferation, to possibly inhibit the airway remodelling observed in asthma, and furthermore, corticosteroid therapy was demonstrated not to affect to this gene.

GABAB receptors are G-protein coupled receptors for the major inhibitory neurotransmitter in the mammalian central nervous system, γ-aminobutyric acid (GABA). The receptor is linked to a variety of disorders including epilepsy, pain, spasticity, drug addiction and cognitive impairment and is, therefore of major importance for drug discovery. The most abundant receptor isoforms GABABR1a and R1b differ by the presence in R1a of a pair of Nterminal extracellular complement control protein modules (CCP1 and CCP2) which - in other proteins - are generally involved in mediating specific protein-protein recognition. The CCP1 module contains disulphides but is natively disordered. In the current work, the yeast two-hybrid system was used to confirm an interaction of CCP1 of GABABR1a with the extracellular protein fibulin-2. Further work with the yeast twohybrid system extablished the novel interaction of the abundant extracellular matrix protein laminin, with GABABR1a CCP1, via its laminin globular (LG) domains. The laminin interaction was further characterised by surface plasmon resonance, demonstrating that several different domains are involved in the binding to the GABAB receptor CCPs. The primary binding site is located on laminin α5 LG4-5, but the E10 domains of the β1 chain and LG1-3 on α1 may also be involved. The pharmacological properties of the GABABR1a and R1b isoforms were studied by transient expression in Xenopus laevis oocytes. It was demonstrated that the agonist baclofen, as well as the antagonist CGP55845, appear to be more potent at GABABR1b compared to GABABR1a. Intriguingly, when recorded in the precence of laminin, GABABR1b/R2 expressing oocytes exhibited an increased baclofen-evoked response while the response in GABABR1a/R2 was completely abolished. In conclusion, the work demonstrates that laminin is a binding partner for GABABR1a CCPs. Such an interaction between the metabotropic GABA receptor and the extracellular matrix may lie behind the recently reported roles of GABA in neuronal migration and the laying down of neuronal circuitry during the development of parts of the central nervous system.

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. Olfactory ensheathing cells (OECs), specialized glial cells within the olfactory nerve, are thought to play an important role in the ongoing growth and replenishment of sensory connections in this system. To gain insight into novel molecules that could mediate OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments revealed very high expression of the fibulin-3 glycoprotein in OECs. To date, research on fibulin-3 has been limited and mainly focused on its involvement in Doyne honeycomb retinal dystrophy, vasculogenesis and tumor formation. As the extracellular matrix associated with OECs is thought to be an important contributor to a growth-permissive environment, the main aim of this thesis was to define a putative role for fibulin-3 during olfactory receptor neuron replacement and regeneration. This hypothesis was investigated in a series of in vitro and in vivo experiments that involved lentiviral vectors to manipulate fibulin-3 gene expression in OECs as well as the use of knock-out mice. Using genetically-modified OECs, experimental data showed that increased levels of fibulin-3 induced morphological changes in OECs and also impeded their migration. Lentiviral vector-mediated expression of fibulin-3 in OECs also had an inhibitory effect on neurite outgrowth from dorsal root ganglion explants. On the other hand, knock-down of fibulin-3 levels via siRNA technology resulted in reduced proliferation. Comparative lesioning experiments in fibulin-3 knock-out and wild-type mice allowed for further assessment of a role for fibulin-3 in olfactory nerve repair in vivo. Two experimental injury models, i.e. epithelial (Triton-X) lesioning and olfactory bulbectomy, were employed. The results obtained were in line with in vitro observations. A lack of fibulin-3 in knock-out mice resulted in a seemingly augmented regeneration of the olfactory epithelium at 10 days post-injury. However, at the latest recovery time point of 42 days post-injury, an impaired recovery of the olfactory epithelium from the experimental insults was observed. Although the precise mechanism for the latter phenomenon is not yet fully understood, our data point towards several factors which include vascular abnormalities and altered cell proliferation within the olfactory epithelium. Additionally, the precise protein distribution of another wide-spread family of extracellular matrix molecules, the laminins, was investigated in this thesis. It was of interest to investigate the spatiotemporal expression of laminin isoforms during iii olfactory nerve development and regeneration as these molecules may have distinct roles in promoting olfactory sensory neuron growth and patterning. In situ hybridization and immunohistochemical studies concluded that laminin-211 and laminin-411 were the most likely candidates to play such a role. In summary, this thesis provides new insights into the role of the extracellular matrix, fibulin-3 in particular, in regulating cell migration, division and axonal growth in the primary olfactory pathway. Such knowledge also gives a greater understanding of the molecular mechanisms by which OEC transplants may enhance axonal regeneration elsewhere in the CNS.

The evolution of biomechanical behavior of arteries plays a key role in the onset and progression of cardiovascular disease. Biomechanical behavior is governed by the content and organization of the key structural constituents (e.g., collagen, elastin, and smooth muscle) and vessel geometry. The evolution of biomechanical behavior of arteries is governed by biologically-mediated synthesis, degradation, and reorganization of these key structural constituents. A hallmark goal in biomechanics is quantifying the relationship between the microstructure of tissues and their mechanical response throughout tissue growth and remodeling; this will provide a crucial link in understanding the tissue level effects of biological processes involved in disease and normal growth Fibulin-5 (fbln5) is an ECM protein that binds tropoelastin and interacts with integrins. Arteries from fbln5 knockout mice lack functional elastic fibers and provide a system for investigating the link between an artery's microstructure and its mechanical response. The overall goal of this project was to develop multi-scaled theoretical and experimental frameworks to quantify the relationship between microstructural content and organization and tissue level material properties of arteries from fbln5 null mice and littermate controls and to quantify the effects of fbln5 on the in vivo maturation of mouse carotid arteries. We found significant differences in the mechanical properties of carotid arteries of fbln5 null mice, and these differences were correlated with altered extracellular matrix organization. We also developed a microstructurally-motivated 3-dimensional constrained mixture model for vascular growth and remodeling. Using physiological rates of constituent growth and turnover, the model captured the salient findings found in the literature. Incorporating experimentally measured fiber angle data into constitutive relations yielded greater predictive accuracy. This dissertation incorporates experimental data quantified at the micro (microstructural-level fiber distributions) and macro (tissue-level mechanical response) scale and incorporates these data into microstructurally motivated constitutive relations. The use of structurally motivated constitutive relations and experimentally measured microstructural data provides a foundation for future work in further understanding the relationship between processes governing microstructure and the tissue level effects of disease and normal growth.

PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1(ki/ki) mice carrying this mutation or in Efemp1(-/-) mice. METHODS. Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (R-s) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1(ki/ki) Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1(ki/ki) Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1(-/-) Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1(+/+) mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1(-/-) Bruch's membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

No description available.

Background: Fibulin-1, an extracellular matrix component and mediator in cardiac fibrosis, is expressed in cardiac valves, heart muscles and blood vessels and may contribute to different cardiovascular pathological conditions such as hypertension, aortic valve stenosis, atrial fibrillation and coronary artery disease. The most conspicuous functions of fibulin-1 include cell adhesion and cell migration within the extracellular matrix (ECM). This was found to reflect vascular dysfunction contributing to the development of fibrosis in the myocardium by means of changes in the ECM, possibly as a result of inflammation. Inflammatory mediators such as C-reactive protein (CRP) and albumin have been investigated over the years for the role they play in the inflammatory processes. However, one inflammatory mediator, soluble urokinase-type plasminogen activator receptor (suPAR), only emerged as a potential biomarker in the development of sclerotic disease. SuPAR is a soluble bioactive form of the urokinase-type plasminogen activator receptor (uPAR) secreted by inflammatory cells such as macrophages, endothelial cells and monocytes. The most profound functions of suPAR such as cell migration and cell adhesion contribute to the development of diseases such as infection, autoimmune diseases, cancer and atherosclerosis. Motivation and aim: This study was motivated by an awareness of the limited data on the potential link between fibulin-1 and suPAR, along with other markers of inflammation (CRP and albumin). We aimed to compare the levels of a marker of cardiac fibrosis (fibulin-1) and inflammatory mediators (suPAR, CRP and albumin) in African and Caucasian men and women. A second aim was to explore fibulin-1 and its potential association with these inflammatory markers independent of haemodynamic and metabolic risk factors in a bi-ethnic cohort from South Africa. Methodology: Data from the cross-sectional SAfrEIC study (South African study regarding the role of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function) were used, which initially included 756 participants. Our study population comprised 290 Africans (men: n=130; women: n=160) and 343 Caucasians (men: n=160; women: n=183). We excluded HIV-infected participants (n=115) as well as those with missing data (n=8). Traditional cardiovascular measurements together with the relevant biochemical analyses were done. T-tests and Chi-square tests were used to compare means and proportions between groups, respectively. Single and partial correlations were performed to determine the relationship of fibulin-1 with suPAR, CRP and albumin, with adjustments for age. SuPAR, CRP and albumin were divided into tertiles to explore the association with fibulin-1 levels, while adjusting for age, body mass index (BMI) and diastolic blood pressure (DBP) by using analysis of covariance (ANCOVA). Multiple regression analysis was performed to explore independent associations. Results: Participants were divided into African and Caucasian men and women due to significant interactions of the main effects of ethnicity and gender on the association of fibulin-1 with suPAR (ethnicity: F(633)=7.29; p<0.001 and gender: F(633)=7.99; p<0.001). Fibulin-1 levels were higher in African men (p=0.010), whereas CRP was higher in African women (p<0.001) compared to their Caucasian counterparts. In both gender groups suPAR levels were higher and albumin lower in Africans compared to Caucasians (p<0.006). In single regression analyses, a positive correlation existed between fibulin-1 and suPAR in African (r=0.19; p=0.028) and Caucasian men (r=0.37; p<0.001), also in African (r=0.193; p=0.028) and Caucasian women (r=0.14; p=0.036). After adjustments were applied for age, this correlation remained in African (r=0.23; p=0.010) and Caucasian men (r=0.22; p=0.005) only. An inverse correlation was found between fibulin-1 and albumin in African men (r=-0.28; p=0.002), but not in Caucasian men (r=-0.09; p=0.245). No significant correlation was found between fibulin-1 and CRP in any group. Forward stepwise regression analysis was performed in men and the previous associations between fibulin-1 and suPAR were confirmed in African and Caucasian men; along with the inverse relationship of fibulin-1 with albumin (Adj. R2=0.217; β=–0.210; p=0.013) in African men only. Conclusion: Fibulin-1 was positively associated with suPAR in African and Caucasian men, but not in women. We also found fibulin-1 to be negatively associated with albumin in African men only. These results are indicative of the presence of potential subclinical low-grade inflammation as depicted by suPAR within the extracellular matrix. This low-grade inflammation may contribute to the potential onset of cardiac fibrosis or vascular sclerosis among these South African men with lower albumin levels. / MSc (Physiology), North-West University, Potchefstroom Campus, 2014