Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Platelets are small discoid blood cells that regulate hemostasis and wound healing. Normal platelet function is largely contingent on the shape change reaction that occurs following the cells’ exposure to external stimuli and/or soluble agonists. Agonist-driven platelet shape change is mediated by reorganization of the actin cytoskeleton (the cell’s structural framework) although the underlying molecular mechanisms are not precisely defined. Moreover, platelet lifespan is partly determined by apoptosis (programmed cell death); how the actin cytoskeleton regulates platelet apoptosis is also undefined. In response to ABT-737 (an apoptosis-inducing drug that targets Bcl-xL pro-survival protein), the 3 apoptotic markers of mitochondrial depolarization, surface phosphatidylserine (PS) exposure and caspase-3 activation were all unaffected by the actin-depolymerizing drug cytochalasin D or the actin polymerizing drug jasplakinolide. Similar results were observed in platelets isolated from mice deficient in gelsolin, an actin-binding protein that is essential for normal cytoskeletal remodeling, suggesting that ABT-737 induces established markers of platelet apoptosis in an actin-independent manner. I then studied another actin-binding protein, filamin A (FLNA), which crosslinks actin filaments and also serves as an intracellular signalling scaffold. ABT-737 induced caspase-3 cleavage and PS exposure in control mouse platelets but these effects were essentially abrogated in platelets obtained from FLNA-null mice. These data point to a novel role for FLNA in the regulation of platelet apoptosis that is independent of the actin cytoskeleton. Furthermore, I studied the role of FLNA in regulating thrombin-driven platelet shape change. Preliminary data suggest that FLNA-null platelets show impaired early phosphorylation of myosin light chain (MLC), which is a key determinant of the shape change reaction. Collectively, my findings serve to delineate the respective roles for the actin cytoskeleton, and of FLNA, with respect to platelet shape change and apoptosis.
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Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronicinflammation of the joint tissues caused by activation of locally recruited immune cells. Withinthe joints, the resident fibroblast-like synoviocytes (FLS) play an important role in RAassociatedtissue degradation in part due to their secretion of pro-inflammatory cytokines andtissue-degrading matrix metalloproteinases (MMPs) into the synovium, as well as the cells’ability to invade nearby structures. Platelets also function as immune cells that contain andsecrete pro-inflammatory molecules but their role in RA is not understood. Of particular interestis platelet factor 4 (PF4), a major constituent of platelet alpha-granules.Methods: Cultured SW982 cells were used as a model for FLS. Cells were cultured in thepresence or absence of recombinant PF4. The secretion of MMP-1 (interstitial collagenase) wasmeasured by enzyme-linked immunosorbent assay (ELISA). Cell adhesion was determined bywash-off assays.Results: Cells cultured with recombinant PF4 secreted more MMP-1 relative to controls. PF4treatment also increased the FLS production of fibronectin, a matrix adhesion molecule. PF4-treated cells also exhibited greater adhesion and spreading.Conclusion: I conclude that PF4 contributes to an invasive/destructive phenotype in FLS, bypromoting MMP-1 release and increased cell adhesion.
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Objective: Periodontitis (gum disease) is characterized by the degradation of tooth- supporting connective tissues, largely due to matrix metalloproteinase (MMP) release by fibroblasts residing in the gingival (gum) tissue. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. Here we studied the role of GzmB in the context of periodontitis.Methods: Gingival fibroblasts were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to quantify GzmB-induced release of collagenase (MMP-1). MMP-1 expression was quantified by qPCR. In some experiments, cells were pre-treated with PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Human samples of gingival crevicular fluid (GCF), an inflammatory transudate released at the dento-gingival (tooth- gum) interface, were obtained from teeth with periodontal disease and healthy control teeth. GzmB was quantified in the GCF ([GzmB]GCF) by ELISA.Results: Recombinant GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2 which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 gene expression/secretion were also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the GzmB- induced increase in MMP-1 secretion by GF. The [GzmB]GCF was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and matrix degradation.Conclusions: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 expression and secretion are upregulated in a PAR1- and Erk1/2- dependent manner.
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Background and Objective: Periodontitis is a highly prevalent chronic inflammatory disease that causes tooth loss, morbidity and confers an increased risk for systemic disease. Tissue destruction during periodontitis is due in large part to collagen-degrading matrix metalloproteinases (MMPs) released by resident cells of the periodontium in response to pro-inflammatory cytokines. Platelets are immune-competent blood cells with a newly recognized role in chronic inflammation, however their role in the pathogenesis of periodontitis is undefined. Consequently, the objective of this study was to assess the effect of platelet factor 4 (PF4), a major platelet-derived cytokine, on MMP-1 (collagenase) expression in human gingival fibroblasts (HGFs).Methods: HGFs were cultured in the presence or absence of recombinant PF4. Pro-MMP-1 secretion was quantified by enzyme-linked immunosorbent assay (ELISA) analysis of the cell culture supernatants. MMP-1 transcription was quantified by real-time polymerase chain reaction (qPCR). Regulation of MMP-1 production by the p44/42 MAP kinase (MAPK) signaling pathway was examined in the presence or absence of PF4.Results: Exposure to PF4 caused a ~2-3-fold increase in MMP-1 transcription and secretion from cultured human gingival fibroblasts (HGFs). PF4 treatment also enhanced phosphorylation of p44/42 MAP kinase (MAPK), which has been previously shown to induce MMP-1 expression in fibroblasts. Blockade of p44/42 MAPK signaling with the cell-permeant inhibitors PD98059 and PD184352 abrogated PF4-induced pro-MMP-1 transcription upregulation and release from cultured HGFs. Conclusion: We conclude that platelet factor 4 upregulates MMP-1 expression in human gingival fibroblasts in a p44/42 MAPK-dependent manner. These findings point to a previously unidentified role for platelets in the pathogenesis of periodontal diseases.
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Aim: Platelets contribute to chronic inflammation but their role in periodontitis is not well understood. The aim of this study was to compare platelet recruitment and activation in healthy and inflamed periodontium. Materials and Methods: Gingival crevicular fluid (GCF) samples were obtained from sites of healthy periodontium, gingivitis and periodontitis. Platelets were quantified in the GCF by staining and microscopy. GCF concentrations of platelet factor 4 (PF4) [PF4]GCF and glycoprotein IIbIIIa ([GPIIbIIIa]GCF) were determined by ELISA. Blood samples were obtained from the 3 patient groups. Platelets were isolated from whole blood and stimulated with lipopolysaccharide (LPS) from P. gingivalis to evaluate and compare the LPS-induced PF4 release. Results: Compared to controls, platelet recruitment was increased at gingivitis and periodontitis sites, based on platelet counts and [GPIIbIIIa]GCF. [PF4]GCF was elevated in periodontal pockets but not at gingivitis or healthy sites. Circulating plasma levels of PF4 were higher in patients with generalized severe periodontitis (SP), compared to patients with gingivitis or healthy periodontium. Platelets isolated from SP patients contained and released more PF4 in response to P. gingivalis LPS than platelets from gingivitis or periodontally healthy patients. Conclusions: Periodontitis is associated with increased platelet activation and PF4 release, both locally and systemically.
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