A model of complex plaque formation: 7,8-Dihydroneopterin protects human monocyte-derived macrophages from oxidised low density lipoprotein-induced death

Abstract

Plasma neopterin is an excellent marker of inflammation and is found in elevated levels in plasma of patients with cardiovascular disease. Neopterin originates as the oxidation product of 7,8-dihydroneopterin (7,8-NP), which is secreted by human macrophages when stimulated with interferon-y during inflammation. 7,8-NP has been shown to be a very efficient free radical scavenger and a potent antioxidant which can protect macrophages from a range of oxidative stresses. The uptake of oxidised low density lipoprotein (oxLDL) by macrophages which lead to the formation of foam cells is a hallmark of early atherosclerotic lesions. OxLDL-induced cell death is also considered to be an important process in the formation of necrotic lipid rich plaques and in atherosclerotic plaque destabilisation. This thesis examined the extent of oxLDL-induced damaged to HMDMs and whether 7,8-NP can inhibit oxLDL-mediated cell death in HMDMs. Foam cells had previously been defined as cholesteryl ester (CE) macrophages that stained positive with oil red-O. This thesis shows that the foamy appearance and presence of lipid droplets stained with oil red-O was not dependent on accumulation of CE which raises the suitability of using oil-red-O staining to identify the foam cells. In addition, HPLC but not GC analysis showed an increased in CE levels of the macrophages when the macrophages were incubated with oxLDL. The HPLC approach spared the samples of lengthy manipulations that might cause ex vivo oxidation. It also avoided subjecting the samples to high temperature treatment that could alter the lipid composition and therefore quantification of the lipid contents. Previous studies showed that 7,8-NP is a potent antioxidant and cytoprotective agent. Exposure of HMDMs to 1 mg/ml oxLDL caused 50% loss of cell viability as measured by the MTT reduction and trypan blue exclusion assays. The development of apoptotic features including caspase-3 activity, cytochrome c release from mitochondria and phophatidyserine (PS) exposure was examined. OxLDL did not cause caspase-3 activation as shown by Western Blot analysis and did not cause DEVD-AMC cleavage in HMDMs. However, cytochrome c release and phosphatidylserine exposure were observed when HMDMs were incubated with oxLDL as shown by Western Blot analysis and Annexin V-FITC staining respectively. Dihydroethidium (DHE) staining showed that oxLDL treatment caused mitochondrial superoxide generation in HMDMs. OxLDL-induced oxidative stress appeared to cause a rapid loss of HMDMs' intracellular glutathione (GSH) as analysed by HPLC technique. Incubation of HMDMs' with buthionine sulfoximine (BSO) and diethyl maleate (DEM) caused similar loss in GSH as incubation with oxLDL but did not result in HMDMs' death. This showed that oxLDL-induced decrease in GSH alone was not sufficient to cause cell death. The loss of cell viability by oxLDL was inhibited by 7,8-NP in the concentration range of 50 to 200 lM. HMDMs' GSH loss caused by oxLDL was similarly inhibited by 7,8-NP supporting the idea that preventing the cellular GSH loss will protect the HMDMs from death. Incubation of HMDMs with 7,8-NP showed reduction in DHE fluorescence intensity staining suggesting that 7,8-NP inhibited or scavenged oxLDL-dependent generation of superoxide. 7,8-NP also effectively inhibited oxLDL-induced PS externalisation to the outer membrane but failed to inhibit the oxLDL-induced release of cytochrome c from mitochondria to the cytosol. The labelling of oxLDL with DiI showed that 7,8-NP significantly inhibited the uptake of oxLDL. However, the inhibitory effect was only measured at non-toxic concentration of oxLDL. The ability of 7,8-NP to inhibit oxLDL uptake raised the possibility that 7,8-NP protective effect against oxLDL involved modulation of the scavenger receptors'expression in particular SRA and CD36. The Western Blot analysis showed that incubation of HMDMs with 7,8-NP did not affect HMDMs' SRA protein expression. In 50% of the experiments, it was demonstrated that certain isoforms of CD36 protein were significantly down regulated by 7,8-NP suggesting that various factors might interact with 7,8-NP or CD36. The ability of 7,8-NP to protect HMDMs from oxLDL-induced death provides further evidence that this antioxidant is secreted by HMDMs to protect them against the oxidative damage in the highly oxidative environment of atherosclerotic plaque.