Investigations into the effects of chlamydia pneumoniae and helicobacter pylori on atherogenesis in mice

Abstract

Chronic bacterial infections such as the respiratory pathogen, Chlamydia pneumoniae (C. pneumoniae), and the gastric pathogen, Helicobacter pylori (H. pylori), have been implicated as contributors to the development of atherosclerosis, on the basis of sero-epidemiological studies and the detection of the bacteria in atherosclerotic plaque samples. The purpose of this study was to investigate potential mechanisms by which chronic pathogens promote atherosclerotic changes that can ultimately contribute to disease progression. In particular we investigated whether NAPDH oxidase and oxidative stress is an important link between bacterial infection and atherosclerosis. In Chapter 3 of this thesis, we investigated whether H. pylori infection triggers early systemic inflammation and exacerbates disease parameters in atherosclerosis-prone Apolipoprotein E-null (ApoE-/-) mice. H. pylori had no effect on serum levels of pro-inflammatory markers, vascular oxidative stress, NADPH oxidase expression or on atherosclerotic lesion burden in the aorta. From these findings, we concluded that H. pylori infection is unlikely to play a significant role in the early or intermediate stages of atherosclerosis, at least in this disease model. Due to the lack of effect of H. pylori on atherosclerotic lesion burden, the remainder of this thesis focused on C. pneumoniae. In Chapter 4 of this thesis, we tested the hypothesis that C. pneumoniae infection of cultured vascular smooth muscle cells (VSMCs) is associated with an increase in NADPH oxidase activity and elevated reactive oxygen species (ROS) production, leading to activation of redox-sensitive pro-inflammatory signalling pathways. C. pneumoniae infection of VSMCs led to an increase in expression of the Nox2 isoform of NADPH oxidase, as well as an increase in superoxide (O2•-) production. This latter effect was inhibited by the reputed NADPH oxidase inhibitor apocynin. C. pneumoniae infection of VSMCs caused an increase in the expression of pro-inflammatory markers including MCP-1, TNF-α and ICAM-1, however these effects were not prevented with apocynin treatment, suggesting that they occurred in parallel with (as opposed to downstream of) NADPH oxidase activation. In Chapter 5 we investigated another potential pro-atherogenic role for C. pneumoniae in VSMCs, by examining whether the bacterium enhanced VSMC-derived foam cell formation. We established a novel in vitro model of lipid accumulation in VSMCs using serum from normocholesterolemic wild-type (C57Bl6/J) and hypercholesterolemic ApoE-/- mice. The accumulation of serum lipids by VSMCs was insensitive to pharmacological inhibitors of traditional scavenger receptor-mediated lipid-uptake pathways, but was blocked by antagonists of macropinocytosis-dependent endocytosis including LY294002 and cytochalasin D. In addition, VSMCs treated with mouse sera did not appear to adopt a pro-inflammatory phenotype and C. pneumoniae infection did not appear to enhance lipid accumulation by VSMCs. Hence, while we found no pro-atherogenic role for C. pneumoniae in our model of lipid accumulation by VSMCs, these studies are nonetheless important as they highlight a hitherto unrecognized atheroprotective function of VSMCs, namely as an inert cellular ‘sink’ for unmodified lipids. Finally in Chapter 6, we looked into the potential mechanisms by which C. pneumoniae disseminates from the lung and into the vascular wall in vivo. Following respiratory infection in ApoE-/- mice, C. pneumoniae was detected by flow cytometry in circulating monocytes, neutrophils and T lymphocytes. C. pneumoniae led to sustained activation of these leukocyte subsets, as indicated by upregulation of the adhesion molecule L-selectin. We speculated that this may enhance the attachment of leukocytes to activated endothelium. However, at 4 weeks post-infection, we found no evidence of an increase in leukocyte infiltration into the vascular wall, nor did we observe an increase in atherosclerotic lesion size. In summary, this thesis has contributed to the understanding of how C. pneumoniae may promote atherosclerosis. We have demonstrated that C. pneumoniae induces pro-atherogenic events such as increasing oxidative stress and inflammation in VSMCs and that after establishment of a respiratory infection, C. pneumoniae can be detected in circulating leukocytes and promote their activation. Hence, as discussed in Chapter 7, our study may help to inform on novel future therapeutic strategies that aim to reduce the burden of cardiovascular disease in society by removing the link between bacterial infections and atherosclerosis.