Chapter 1: Periodontitis is a chronic inflammatory disease wherein microbial factors induce complex inflammatory and immune responses in a susceptible host. In periodontitis host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in the destruction of tooth supporting structures and alveolar bone. TWEAK (TNF-like weak inducer of apoptosis), one of the members of the TNF superfamily, has recently been identified as an important inflammatory mediator. Fn14 (fibroblast growth factor-inducible 14) protein/TWEAKR has been identified as the cell surface receptor for TWEAK. TWEAK/Fn14 signaling results in multiple biologic effects including induction of inflammatory cytokines, modulating immune response angiogenesis and stimulation of apoptosis. TWEAK has also been shown to promote osteoclastic differentiation of cells from the monocyte/macrophage lineage. Expression of TWEAK and its receptor Fn14 is elevated in tissues and cells cultured from a number of chronic inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, inflammatory skin, kidney and airway diseases. This review considers the biology of TWEAK and its receptor Fn14 in periodontitis. Chapter 2: Periodontitis is a chronic inflammatory disease wherein microbial factors induce complex inflammatory and immune responses in a susceptible host. In periodontitis host derived enzymes, cytokines and other proinflammatory mediators play an integral role in the destruction of tooth supporting structures and alveolar bone. TWEAK (TNF-like weak inducer of apoptosis) is one of the newest members of the TNF superfamily to be identified. Fibroblast growth factor-inducible 14 (Fn14) protein/TWEAKR has been identified as the cell surface receptor for TWEAK. TWEAK/Fn14 signaling results in multiple biologic effects including induction of inflammatory cytokines, modulating immune response angiogenesis and stimulation of apoptosis. Recently, TWEAK has also been shown to promote osteoclastic differentiation of cells from the monocyte/macrophage lineage. Expression of TWEAK and its receptor Fn14, is elevated in tissues and cells cultured from a number of chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, inflammatory skin, kidney and airway diseases. Accordingly, we hypothesised that the expression of TWEAK and Fn14/TWEAKR will be increased in tissue samples from periodontitis patients. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367201 / Thesis (D.Clin.Dent.) - University of Adelaide, School of Dentistry, 2009

The liver is the largest solid organ in the body and is frequently the site of injury. During disease, liver injury is usually compensated for by exceptionally efficient regeneration which occurs both from differentiated epithelia and also from an undifferentiated cell population with stem cell like qualities known as hepatic progenitor cells (HPCs). HPCs are particularly active during massive or chronic liver injury and therefore are an attractive target for much needed novel therapies to enhance regeneration in patients for whom the only current effective therapy is liver transplantation. Stem cells in other organs systems are believed to reside in a specialised microenvironment or niche which supports their maintenance and function. To investigate the hypothesis that HPCs are supported by a functional niche and are capable of regenerating hepatocytes, we commenced by establishing a number of murine in vivo models. Having shown a stereotypical niche, consisting of macrophages, myofibroblasts and laminin exists in both animal models and human disease, we investigated the active recruitment of extrahepatic cells into this niche and showed that macrophages are actively recruited from the bone marrow during liver injury. Macrophages were shown to influence HPC behaviour during injury. Furthermore using macrophages as a cellular therapy, induced HPC activation with corresponding changes to liver structure and function. Investigation of signalling pathways revealed and confirmed a TWEAK dependent activation of HPCs following macrophage transfer. Having demonstrated the potential for macrophage therapy via HPC activation, we aimed to study the ability of HPCs to regenerate the hepatic parenchyma. To do so we developed and characterised a novel model of hepatocellular injury and HPC activation. Using the genetic labeling of hepatocytes in this model we were able to show rapid and large scale repopulation of hepatocytes from a precursor source with HPCs being the critical precursor source of hepatocellular regeneration. In addition this process is again dependent on TWEAK signalling, without which HPC mediated regeneration fails resulting in mortality. Therefore HPCs are an attractive biological target for regenerative medicine, and both TWEAK signalling and autologous macrophage infusion offer genuine potential to manipulate these cells as future therapies.

Abstract Cardiac remodeling is defined as changes in the size, shape and function of the heart, caused most commonly by hypertension-induced left ventricular (LV) hypertrophy and myocardial infarction (MI). It is characterized by changes in cellular and extracellular compartments regulated by e.g. neurohumoral and inflammatory factors. In the present study the expression of novel, load induced factors, thrombospondin (TSP)-1 and -4, matrix Gla protein (MGP), tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14, was investigated during cardiac remodeling. Their expression in the heart was characterized using experimental models of pressure overload, hypertensive hypertrophy and MI, and the effect of hypertrophic agonists and cellular stretch was studied in vitro. The effect of beta-blocker treatment on TSP expression was also examined. TSP-1 and -4 were rapidly upregulated in response to pressure overload, and the induction of TSP-4 gene expression was attenuated in hypertrophied heart. After MI, TSP-1 and -4 mRNA and TSP-1 protein levels were increased, and the induction was attenuated by metoprolol. TSP-1 and -4 expression correlated with natriuretic peptide expression and LV remodeling after MI. In hypertensive hypertrophy, only TSP-4 expression decreased after metoprolol treatment and was correlated with LV remodeling. MGP gene expression was increased in response to pressure overload and MI both in the early and late phase of cardiac remodeling. MGP protein levels were increased in the acute phase of post-MI remodeling and in hypertensive hypertrophy. In vitro, angiotensin II increased MGP gene expression in myocytes and fibroblasts, whereas expression decreased in response to mechanical stretch. In response to increased cardiac load Fn14 expression was upregulated both acutely and chronically while TWEAK expression remained relatively constant. Fn14 localized mainly to fibroblasts in the inflammatory area while TWEAK localized to myocytes and endothelial cells. In myocytes, Fn14 expression was induced by hypertrophic agonists and mechanical stretch in contrast to stabile or decreased TWEAK expression. This study provides new insights into the expression of the studied novel factors in cardiac remodeling. The distinct expression of TSPs in pressure overload and post-MI suggests that TSP-1 and -4 may have unique roles in the remodeling process. The results also imply that MGP is part of the common gene program of hypertrophic remodeling in vivo and contributes to the molecular basis of cardiac hypertrophy. Finally, the study demonstrates differential regulation of TWEAK and Fn14 expression in the heart and emphasizes the importance of Fn14 as a mediator of TWEAK/Fn14 signaling and as a potential target of therapeutic interventions.

Skeletal muscle atrophy is a debilitating condition caused by pathological conditions including cancer cachexia, disuse and denervation. Disuse atrophy is characterized by reduction in fiber size, fiber-type change and induction of markers of atrophy such as MuRF1 and Fn14. Recent studies have focused on understanding the fundamental role of signalling pathways and the proteolytic system in response to muscle atrophy. Unfortunately the exact mechanisms behind atrophy remain poorly understood. I recently demonstrated that cIAP1 and/or cIAP2 proteins are critical regulators of NF-kB activation, which has been shown to be involved in skeletal muscle atrophy. Here, I used genetic and pharmacological means to investigate the role of cIAP1 in a denervation-induced skeletal muscle atrophy model. Interestingly, I found that upon denervation loss of cIAP1 rescues muscle fiber size, prevents fiber-type changing and inhibits the expression of MuRF1 and Fn14. Moreover, treatment of mice with Smac mimetic compounds (SMC), a novel class of small molecule IAP antagonists, showed successful knockdown of cIAP1 in muscle and protects against denervation-induced muscle atrophy. Taken together, these data reveal that cIAP1 is both a novel mediator of skeletal muscle atrophy and an important therapeutic target.

The NF-κB family of transcription factors can be activated through canonical (classical) or non-canonical (alternative) signalling pathways, which are regulated by the redundant ubiquitin ligases, cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2). While the canonical NF-κB pathway is needed for myoblast proliferation, it is inactivated during myoblast differentiation. However, the non-canonical NF-κB pathway is a major factor in promoting myoblast fusion, which is crucial to the processes of myogenesis and muscle repair. Ablation of cIAP1 levels through a chemical antagonist such as a SMAC- mimetic compound (SMC) activates non-canonical signalling to enhance myogenesis. The cytokine TNF-like weak inducer of apoptosis (TWEAK) has also been shown to activate primarily the alternative NF-κB pathway when signalling through its receptor Fn14. Here I show that alternative NF-κB signalling activity, stimulated by the addition of TWEAK or loss of cIAP1, can promote myogenesis. I also demonstrate that TWEAK is an endogenous myokine produced by myoblasts to promote their own differentiation, and suggest that targeting the alternative NF-κB pathway, with SMAC-mimetics or recombinant TWEAK for example, would be of therapeutic value in the repair and regeneration of muscle for various myopathies.