Angiogenesis is a highly regulated process orchestrated by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Heparin/heparan sulfate (HS) proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors for VEGF-A, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors and the two major VEGF-A isoforms, using surface plasmon resonance (SPR) and in vitro binding assays. We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its “decoy” receptor, VEGF receptor-1 (VEGFR-1). We identified a potential mechanism for co-receptors to decrease the dissociation rate and prolong the signaling complex lifetime. Using a systematic approach, we obtained kinetic parameters for each individual interaction in an intercomparable way to measure the effect NRP-1 and HS have on complex stability. Additionally, we demonstrated that these binding events influence VEGF activity within endothelial cells. These parameters can be used in mathematical models to predict therapy outcomes in defined cellular contexts. Furthermore, we optimized a competition-based technique using SPR and structurally defined HS oligosaccharides and demonstrated that it can be used to rapidly measure affinities to HS-binding proteins. We used this method to define interactions and structural and length requirements for heparin/HS interactions with VEGFR-1, NRP-1, and VEGF165, the most relevant VEGF-A isoform, in complex with VEGFR-2 and NRP-1. We show that the structural requirements were distinct for each interaction. We further found that VEGF165, VEGFR-2 and monomeric NRP-1 bound weakly to heparin alone, yet binding to heparin increased synergistically when presented together. This enhanced binding correlated with alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increased VEGF binding and activated phosphorylation of VEGFR-2 and Erk1/2 in endothelial cells, and that these effects required sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.

This study quantified mechanical and structural responses to loading conditions at subtendon hierarchical levels. Tensile tests were performed at three strain rates on three groups of rabbit patellar tendon specimens. For each rate, tangent modulus (E) was computed from the stress-strain curves and the following structural responses were evaluated: (i) Area percent of collagen fibrils (FAR) and (ii) Skewness angle formed between proteoglycans and collagen fibrils. For 0.1%/s, 10%/s, and 70%/s, E was 48.8±20.3MPa, 64.7±29.3MPa, and 78.6±31.7MPa, respectively. For control, 0.1%/s, 10%/s, and 70%/s, the mean FAR was 0.7552±0.1476, 0.6628±0.1190, 0.6335±0.1013, and 0.6047±0.0384, respectively; and proteoglycan skewness angles were 14.70º±11.01º, 12.76º±10.13º, 15.08.0º±11.66º, and 16.68º±12.07º, respectively. For increased E, interfibrillar components had less time for effective fluid flow, energy dissipation, and structural rearrangement. The inverse relationship of FAR to strain rate may be due to broken fibrils and the Poisson effect. Proteoglycan skewness angle increase is likely due to stretched fibrils.

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published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

Development of the visual system involves the precise orchestration of neural connections between the retina of the eye, the thalamus (dorsal lateral geniculate nucleus; dLGN) and the superior colliculus (SC). During early development, receptor molecules on the growth cones of retinal ganglion cell (RGC) axons sense molecular guidance cues in the extra cellular matrix (ECM) that define their route and branching behaviour within the visual system. Heparan sulphate proteoglycans (HSPGs) are ECM molecules composed of a core protein and a variable number of disaccharide residues that have been implicated in mediating axon guidance. HSPGs are modified by a number of enzymes that contribute to their structural diversity. Based on this structural diversity; the “heparan sulphate code” hypothesis of Bulow and Hobert (2004) postulated that different HSPG modifications confer different axon navigation responses as the growth cones traverse the local environment. To investigate the roles played by specific modifications of HSPG molecules in the guidance of axons, we examined two lines of mutant mice harbouring mutations in the genes encoding HSPG modifying enzymes, Heparan sulphate-6-O-sulphotransferase-1 (Hs6st1) and Heparan sulphate-2-O-sulphotransferase (Hs2st). These two mutant lines were generated through the use of gene trapping. Previous observations of RGC axon development in the two mutant lines revealed distinct axon guidance errors at the optic chiasm. Loss of Hs6st1 sulphation resulted in RGC axons navigating ectopically into the contralateral eye. Loss of Hs2st sulphation resulted in RGC axons navigating outside the normal boundary of the optic chiasm. Early observations suggested that both Hs2st sulphation and Hs6st1 sulphation have distinct, non-overlapping actions and thus, influence different axon guidance signalling pathways at the optic chiasm. Based on our findings and previous work describing the expression patterns and functions of the chemo-repellent axon guidance molecules, Slit1 and Slit2 at the optic chiasm and their Robo2 in the retina, we formulated the hypothesis of an HSPG sulphation code where Hs2st sulphation is specifically required for Slit1-Robo2 signalling and Hs6st1 sulphation is specifically required for Slit2-Robo2 signalling at the optic chiasm. To further our understanding of the roles Hs2st sulphation and Hs6st1 sulphation have on axon guidance, we looked at a number of key choice points that navigating axons encounter and are known to be influenced by Slit signalling. Further observations of RGC axons at the optic chiasm of Hs2st-/- mutants and Hs6st1-/- mutants showed distinct axon guidance phenotypes, both resulting in statistically significant increases in the width of the optic chiasm at the midline. While Hs6st1 sulphation had no effect on RGC axon navigation within the eye (possibly due to 6-O-sulphation compensation by Hs6st3); the loss of Hs6st1 sulphation at the dLGN resulted in a significant increase in the defasciculation of the optic tract. Observations of other axonal tracts influenced by Slit signalling revealed the importance of Hs2st and Hs6st1 sulphation in aiding callosal axons to successfully traverse the midline in corpus callosum development. Observations of the thalamocortical (TCA)/corticothalamic (CTA) tracts revealed that neither Hs2st sulphation nor Hs6st1 sulphation was required for the development of the mouse TCA tract (the latter may be explained by 6-O-sulphation compensation by Hs6st2). To test whether Hs2st and Hs6st1 enzymes have redundant functions in optic chiasm development, we attempted to create Hs2st-/-/Hs6st1-/- double mutants. A PCR genotyping strategy was developed for the identification of Hs6st1 animals and showed that Hs6st1-/- mutants had high postnatal lethality with only 3% of the offspring surviving to weaning while Hs2st-/-/Hs6st1-/- double mutants all died very early during embryonic development. Observations of Hs2st-/-/Hs6st1+/- mutants and Hs2st+/-/Hs6st1-/- mutants that lacked three of the four Hst alleles showed no differences when compared to single Hst knockouts. Finally, we showed that altered Slit expression at the optic chiasm and Robo expression in the retina could not explain the mutant phenotypes observed in Hs2st-/- mutants and Hs6st1-/- mutants, and therefore we hypothesized that Hs2st sulphation and Hs6st1 sulphation regulate distinct aspects of Slit-Robo signalling at the surface of the navigating axon growth cone.

Includes bibliographies. / 1 v. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A collection of 27 published journal articles representing work carried out between 1983 and 1995, the major thrust being an extension of the author's PhD studies. Centres on detailed investigations into the nature of proteoglycans in various periodontal compartments and what factors might influence their structure and synthesis. / Thesis (D.D.Sc.)--University of Adelaide, Dept. of Dentistry, 1996

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy