Atherosclerosis is a slow-developing disease of large and medium sized arteries, and is the premier cardiovascular disease that underlies myocardial and cerebral infarction, aneurysm, stroke and gangrene of the extremities. At least 17 million people die of atherosclerotic complications each year worldwide, with another 15 million surviving unstable events. Despite therapeutic advances such as drug-eluting stents and statins, which reduce cardiovascular events by around 25%, there is an urgent need for additional strategies to complement these treatments and further reduce morbidity and mortality. Inflammation plays a fundamental role in mediating all stages of atherogenesis. The innate immune response has long been implicated in atherogenesis, and activation of the complement system has been associated with all stages of disease. In particular, C5b-9 (membrane attack complex) has been detected in human plaques and may be pathogenic. Since C5b-9 is produced in plaques then the complement activation product 5a (C5a) must also be generated. However, very little is known about the role of C5a in atherogenesis. Indeed, elevated levels of serum C5a have been detected in patients with advanced atherosclerosis and recently the classical C5a receptor, CD88, has been detected on most of the cells found in human atherosclerotic plaques. To date, no studies examining specific C5a receptor antagonism in an animal model of atherosclerosis have been performed. This thesis explored the potential therapeutic benefits of inhibiting C5a, using the C5a receptor antagonist, PMX53, in the ApoE knockout (ApoE-/-) mouse model of atherosclerosis. In Chapter 2, expression of both receptors to C5a, CD88 and C5L2, in aortae of ApoE -/- mice was explored. CD88 and C5L2 mRNA expression was detected in the aorta of ApoE -/- mice at 3, 5, 9,12, 17 and 25 weeks of age. CD88 expression in ApoE -/- mice increased with time, and with macrophage accumulation within the plaque, as indicated by an increase in expression of the macrophage marker, F4/80. Expresssion of CD88 was significantly increased at 17 and 26 weeks of age, compared with age-matched wild-type controls. C5L2 was also expressed albeit at much lower levels compared with wild-type controls. Having established the presence of C5a receptors in ApoE -/- mice, in Chapter 3, the effects PMX53-treatment on ApoE -/- mice on a normal chow diet was examined. PMX53 treatment (3 mg/kg; tri-weekly s.c., plus ~1mg/kg/day p.o. for 20 weeks) resulted in a significant reduction in neointimal area and therefore the intima:media ratio in the brachiocephalic artery compared to untreated controls (P < 0.05; n = 6-8). PMX53 treatment also reduced collagen content and outward remodelling of the brachiocephalic artery. In Chapter 4, studies exploring the effects of PMX53-treatment in the more inflammatory environment created by a high fat (or Western-type diet) were explored. Male ApoE -/- mice were treated with PMX53 from 5 – 25 weeks of age (3 mg/kg; tri-weekly s.c., plus v ~1mg/kg/day p.o.). Mice were placed on a high fat diet from 10 weeks of age. While PMX53- treated did not affect neo-intimal area, it did result in a significant increase in cell density (P<0.01; n=12) and a significant reduction in buried caps (P < 0.05; n = 12) in the brachiocephalic artery compared with untreated animals. Interestingly, PMX53-treated mice also had significantly reduced total cholesterol compared with untreated controls (P < 0.05; n = 12). These results provide the first evidence for a role for C5a in plaque destabilisation and cholesterol metabolism. Finally, Chapter 5 described the expression of CD88 and C5L2 in cultured primary rat vascular SMC was explored. Expression of CD88 and C5L2 was detected by Western blot; immunocytochemical analysis demonstrated intracellular expression of both C5L2 and CD88. Conversely, radioligand binding experiments suggested the presence of ~25000 cell surface receptors with a high affinity to C5a (KD = 0.3 nM). After establishing the presence of receptors to C5a, experiments were conducted to determine whether C5a has any functional effects on these cells. C5a induced a moderate increase in TNF-α release after 4 hours of treatment (P < 0.05, n = 3), but did not affect SMC proliferation (n = 3). In summary, this study is the first to demonstrate the benefits of specifically inhibiting C5a in a mouse model of atherosclerosis. These findings suggest that C5a plays a role in atherogenesis in ApoE -/- mice and that the C5a receptor antagonist PMX53 may have therapeutic potential in human atherosclerotic disease.
| Identifer | oai:union.ndltd.org:ADTP/291169 |
| Creators | Helga Manthey |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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