Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.

In the absence of ligand, certain growth factor receptors can be activated via G protein-coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate the receptor tyrosine kinase (RTK) platelet-derived growth factor (PDGF) ?? receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here, it is shown that 5-HT can transiently increase the phosphorylation of PDGF?? receptors in a time- and concentration-dependent manner in SH-SY5Y neuroblastoma cells. This transactivation pathway was pertussis-toxin sensitive, and was dependent on phospholipase C activity, intracellular calcium signaling and subsequent protein kinase C activation. Exogenous application of non-lethal concentrations of H2O2 induced the phosphorylation of PDGF?? receptors in a concentration-dependent fashion, similar to that observed with 5-HT. Further investigation revealed reactive oxygen species (ROS) production as a necessary component in the transactivation pathway, as scavenging ROS eliminated PDGF?? receptor phosphorylation. NADPH oxidase was determined to be the likely source of ROS given that the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin abrogated PDGF?? receptor transactivation. The role of Src tyrosine kinase was also investigated, and its location in this signaling cascade was determined to be downstream of calcium signaling, but upstream of NADPH oxidase activity. In addition, the activation of ERK1/2 in this system was elucidated to be independent of PDGF?? receptor transactivation. Interestingly, 5-HT also transactivated TrkB receptors, another RTK whose function is implicated in clinical depression. Expectedly, the enzymes in this mechanism were consistent with those revealed in 5-HT-to-PDGF?? receptor signaling. This cross-talk between 5-HT and RTKs such as TrkB and PDGF?? receptors identifies a potentially important signaling link between the serotonergic system and neurotrophic factor signaling in neurons that could have implications in mental health disorders including depression. Furthermore, although transactivation pathways are commonly initiated by a GPCR, recent reports have demonstrated that selective serotonin reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of PDGF?? receptors, suggesting that in addition to GPCRs, monoamine transporters may also be involved in RTK transactivation. SH-SY5Y cells pretreated with the SSRI fluoxetine blocked 5-HT-induced transactivation of the PDGF?? receptors, but not PDGF-induced PDGF?? receptor activation. Upon further examination, it was discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGF?? receptor via 5-HT2 receptors. By the end of the pretreatment period, the effects of fluoxetine on PDGF?? receptor phosphorylation had returned to baseline, and a subsequent transactivating stimulus (5-HT) failed to ???re-transactivate??? the PDGF?? receptor. Additional investigations demonstrated that 5-HT pretreatment can block dopamine-induced PDGF?? receptor transactivation, but not PDGF-induced PDGF?? receptor activation. This is the first demonstration of the heterologous desensitization of an RTK via a transactivation pathway, and this phenomenon is specific for transactivation pathways because in all cases the PDGF?? receptor ligand PDGF-BB was able to directly stimulate receptor activity in spite of GPCR agonist pretreatment. Heterologous desensitization in transactivation signaling reveals a previously unknown short-term ???blackout??? period wherein no further transactivation signaling can occur to potentially exploit the mitogenic effects of RTK activation.

Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

The replacement of missing teeth by osseointegrated dental implants is a commonly utilised treatment option in dentistry. However, successful treatment outcomes are dependent on sufficient bone quantity in the proposed surgical site for implant placement (Buser et al., 2004). Surgical augmentation of bone defects is commonly performed prior to or during implant placement. Bone augmentation procedures of the maxillary sinus or guided bone regeneration (GBR) procedures of alveolar ridge defects have utilised a variety of bone graft materials in block or particulate form, either alone or in combination with resorbable or non-resorbable barrier membranes. Objective: The aim of this study was to determine whether Hydroxyapatite β-Tricalcium Phosphate (HA-TCP) either alone or combined with Enamel Matrix Derivative (EMD) or recombinant human Platelet Derived Growth Factor-BB (rhPDGF-BB) is osteoinductive when implanted into a nonosseous site. Methods: Twenty CD-1 adult male mice underwent intramuscular implantation into both hindlimbs of an empty gelatine capsule or a gelatine capsule containing one of the following: 10 mg of uncoated particulate HA-TCP, (Straumann Bone Ceramic®, HA-TCP), EMD coated HA-TCP, (Emdogain®, HATCP + EMD) or rhPDGF-BB coated HA-TCP (HA-TCP + PDGF). Ten animals were sacrificed at four and eight weeks with five specimens from each group retrieved at each time point. The area of graft placement was radiographed and after graft retrieval, a semi-quantitative histological examination was performed with the aim of assessing the inflammatory changes, reparative processes and osteoinduction within the graft site. Results: At both 4 and 8 weeks, histological analysis failed to demonstrate any osteoinductive activity in any of the specimens from the three experimental groups. A minimal chronic inflammatory response and foreign body reaction was seen in the experimental groups which reduced over time. The particles were embedded within fibrous connective tissue and were encapsulated by a dense cellular layer consisting of active fibroblasts and occasional macrophages with the thickness of this layer decreasing over time. At 4 weeks, a greater density of the fibrous connective tissue was demonstrated in the HA-TCP + EMD group (P<0.001) while a greater thickness in the capsule thickness was seen in the HA-TCP group (P=0.022) although no differences were seen after 8 weeks. Greater neovascularisation was seen in the HA-TCP + PDGF group after 8 weeks (P=0.043) while greater amounts of adipose tissue surrounding the particles were detected in the HA-TCP + PDGF group at 4 weeks (P=0.002) and in the HA-TCP + EMD group at eight weeks (P=0.002). Conclusions: The results of this study suggest that the use of commercially available HA-TCP alone or in combination with EMD or rhPDGF-BB is biocompatible but not osteoinductive in the murine model. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522641 / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2010

The process of osseointegration described by Brånemark (Brånemark et al 1969, 1977) and Schroeder (Schroeder et al 1981) plays an integral role in dental rehabilitation. Since the first observation 40 years ago, osseointegrated titanium implants have been used predictably in the dental rehabilitation of fully edentulous patients. The application of dental implants has evolved, and from the 1980s dental implants have been used increasingly in the treatment of partially edentulous patients, with equal or better long-term success (Buser et al 1990, 1997, Lekholm et al 1994, Behneke et al 2000, Bornstein et al 2005). The surgical procedures for the placement of endosseous dental implants are based on the original work by Brånemark and colleagues approximately 40 years ago. The two-stage surgical procedure was originally advocated to obtain an optimal process of new bone formation and remodelling after implant placement (Brånemark et al 1977). Osseointegration and good long-term success was also found to be achievable with non-submerged implants (Buser et al 1990, 1992, 1997, Ericsson et al 1997) with the added advantage of avoiding a second surgical procedure (Buser et al 1999). Implant dentistry has evolved over the last 15 years and has benefited from significant progress made in associated treatment protocols and the development of bone augmentation procedures (guided bone regeneration (GBR) and sinus floor elevation) allowing for correction of alveolar bone deficiencies. Additionally, improved osteophilic microtextured implant surfaces have been developed to accelerate healing, significantly reducing treatment time. Research and clinical focus in dental implantology in the last two decades has primarily concentrated on the bone-to-implant interface of osseointegrated implants. The soft tissue profile and seal around implants have been investigated to a much lesser degree. This interest has been largely due to the fact that a successfully osseointegrated implant depends on anchorage in bone and requires a direct bone-to-implant interface to provide long-term support for a prosthesis. Both bone and soft tissue integration onto dental implants are wound healing processes whereby several stages of tissue formation and degradation are involved (Berglundh et al 2003, Abrahamsson et al 2004). Osseointegration is the result of the modelling and remodelling of bone tissue that occurs after implant placement whilst the wound healing that occurs following the closure of mucoperiosteal flaps during implant surgery results in the establishment of a mucosal attachment (transmucosal attachment) to the implant. The establishment of the mucosal barrier around the implant is characterised by the gradual shift from a coagulum to granulation tissue followed by the formation of a barrier epithelium and the maturation of the connective tissue (Berglundh et al 2007). Like natural teeth, osseointegrated implants are transmucosal “masticatory devices” that penetrate the oral mucosa with the periodontal and peri-implant tissues expected to perform a protective function (Weber & Cochran 1998). Background: Peri-implant tissues form a crucial but fragile seal between the oral environment, the bone and the implant surface. Enhancing the seal formed by the peri-implant soft tissues at the titanium/connective tissue interface may be an important factor in implant survival. Additionally, enhancing soft tissue adherence to the implant surface when implants are placed in dehiscence type defects may mean that simultaneous osseous grafting procedures will not always be required. Objective: The aim of this study was to investigate the effect of implant surface modification with either platelet-derived growth factor (PDGF) or enamel matrix derivative (EMD) on the connective tissue attachment to moderately roughened titanium implants. Material and Methods: 18 moderately roughened titanium implants were subcutaneously implanted into 14 rats. 6 implants each were coated with PDGF and EMD immediately prior to implantation and 6 implants were left uncoated. The implants were retrieved with a sample of surrounding tissue at 4 and 8 weeks. The specimens were resin-embedded and sections viewed under confocal microscopy for collagen autofluorescence and prepared for qualitative and histomorphometric analysis under light microscopy. ANOVA and t-tests were used to compare the thickness of fibroblast encapsulation on the implant surface and the depth of connective tissue penetration onto the implant grooves. Results: Qualitative analysis under confocal and light microscopy showed encapsulation of all implants by fibroblasts and good soft tissue integration at the end of 4 and 8 weeks. Coating of the implants with growth factors did not alter the orientation of fibroblasts and collagen fibres. Histomorphometric analysis demonstrated that the depth of connective tissue penetration into the implant grooves was significantly greater for the implants coated with PDGF at 4 weeks (ANOVA, P value 0.0014). The thickness of the fibroblast encapsulation on the implant surface was significantly less for the implants coated with PDGF at 8 weeks (ANOVA, P value 0.0012). Conclusion: Good soft tissue integration can be achieved on a moderately roughened titanium implant surface. Coating the implant surface with rhPDGF-BB could increase the speed of soft tissue healing around an implant surface but this increased rate of healing with rhPDGF-BB coating could also result in a less robust titanium/connective tissue interface. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2010

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.