Wanda F. Reynolds, Ph.D.
Associate Professor, Molecular Biology

Research Interests:
Myeloperoxidase gene regulation by nuclear receptors.
Role of MPO in atherosclerosis and Alzheimer’s Disease.

Wanda Reynolds carried out her Ph.D. studies at the University of California, Davis, and post-doctoral studies at the Research Institute of Scripps Clinic before setting up a laboratory to study the role of myeloperoxidase (MPO) in chronic inflammatory disease at the Sidney Kimmel Cancer Center. The laboratory focuses on aberrant regulation of the human MPO gene in atherosclerosis and Alzheimer’s disease (AD), as well as the protective role of MPO in breast cancer. MPO is an abundant enzyme in leukocytes that reacts with hydrogen peroxide and chloride to generate hypochlorous acid (bleach), a potent microbicidal agent that also oxidizes proteins and lipids, damaging normal tissue at inflammatory sites. The laboratory identified and characterized a promoter polymorphism in an Alu element specific to the human MPO gene which includes binding sites for nuclear receptors such as estrogen receptor, PPARg/a, retinoic acid, and LXR. Epidemiological studies have linked this polymorphism, -463G/A, to risk for a number of inflammatory states such as atherosclerosis and AD, as well as lung, breast, and pancreatic cancers. To enable investigations into the mechanisms by which MPO influences these disease states, transgenic mice were generated carrying the human MPO G and A alleles. The lab is currently investigating the effects of LXR and PPARg ligands on the expression of MPO in mouse disease models. Another project involves the search for small molecule antagonists of MPO that could be therapeutically useful.

Our long term objective is to understand the molecular mechanisms regulating the aberrant expression of the human myeloperoxidase gene (MPO) in chronic inflammatory diseases such as atherosclerosis and Alzheimer’s disease.

MPO is an oxidant generating enzyme that catalyzes a reaction between halide and hydrogen peroxide to produce hypochlorous acid (HOCl, bleach). HOCl is a potent microbicidal agent but can also have deleterious effects, damaging normal cells at inflammatory sites. The MPO-HOCl pathway gives rise to a myriad of reactive intermediates leading to oxidation, chlorination, nitration, carbamylation, lipid peroxides, tyrosyl radicals and dityrosine crosslinks. MPO also consumes nitric oxide, which inhibits the induction of iNOS expression (Kumar, 2005), reducing bioavailable NO and impairing vasodilation.

MPO expression is normally restricted to bone marrow myeloid precursors, but human MPO can also be inappropriately induced in reactive macrophages and other cell types in inflammatory conditions. This aberrant expression is due to the insertion of a primate-specific Alu element in the MPO upstream promoter. This Alu contains a cluster of nuclear receptor binding sites including sites for PPARgamma/alpha, LXR, and estrogen receptor (ER). PPARg ligands can strongly upregulate MPO expression in macrophages, while estrogen blocks this induction, suggesting that ER binding blocks PPARg binding on the adjacent site.

MPO is highly expressed in foam cell macrophages in human atherosclerotic lesions, and MPO-oxidation of LDL and HDL promotes atherosclerosis. Unfortunately, the impact of human MPO expression is not easily examined in existing mouse models of atherosclerosis, such as LDL receptor deficient mice (LDLR-/-), because the mouse MPO gene is not expressed in lesion macrophages, due to the lack of the primate specific Alu with PPARg and other nuclear receptor binding sites. To enable investigation of the role of MPO in atherosclerosis and other chronic inflammatory diseases, we created human MPO transgenic mice in which the entire human MPO gene, including extensive flanking/promoter sequences, was inserted. The huMPO transgene is appropriately expressed at highest levels in bone marrow precursors, yet is also expressed in macrophages in atherosclerotic lesions, leading to increased levels of MPO-oxidized proteins. Similarly, in a mouse model of Alzheimer’s disease, human MPO (but not mouse MPO) is expressed in reactive glia in plaque laden regions of the cerebral cortex.

MPO is a significant contributor to atherosclerosis.

Several lines of evidence implicate MPO in the initiation and progression of atherosclerosis. MPO oxidizes LDL in the arterial wall, enhancing its uptake by scavenger receptors on macrophages to create foam cells, the major component of atherosclerotic lesions. MPO-oxidation of apoA1 impairs ABCA1-mediated cholesterol efflux, enhancing lesion formation while impairing reverse cholesterol transport to the liver. We are using the huMPO transgenic mice crossed to the LDLR-/- model to investigate the mechanism of MPO expression in lesion macrophages, and the impact of MPO oxidants on lesion complexity as well as serum hyperlipidemia.

We are also examining the effects of PPARgamma and LXR ligands on MPO expression using these mouse models. The figure shows MPO immunostaining in foam cell macrophages in aortic atherosclerotic lesions in the MPO-LDLR-/- mice (Castellani et al, 2006).

	MPO-oxidation products can be detected in human atherosclerosis lesions. Similarly, a recent study detected MPO-dependent oxidation in aortic lesions in the huMPO-LDLR-/- model. We found increased levels of MPO-dependent protein carbamylation in lesions (E), correlating with increased cholesterol deposition in plaques (F) (Wang et al, Nat. Med. 2007)

Alzheimer’s disease and MPO
MPO is also implicated in Alzheimer’s disease, in part through the presence of abundant MPO deposits in amyloid plaques, and by association of a promoter polymorphism, -463G/A, with increased risk. The G/A polymorphism is within the Alu receptor binding sites and influences receptor binding. The -463A mutation enhances binding by estrogen receptor, thereby reducing binding by PPARg, which may explain why the -463G allele is higher expressing. We have created transgenic mice expressing either MPO G or A allele, and crossed these to huAPP overexpressing models of Alzheimer’s disease.

Alzheimer’s disease (AD) is characterized by the presence in brain tissue of extracellular amyloid plaques. These plaques become associated with reactive microglia and astrocytes, which release MPO along with inflammatory cytokines, nitric oxide, and proteases, contributing to neuronal damage and progressive cognitive decline.

In normal aged human brain tissue, MPO is virtually absent (Figure, panel A), yet MPO is abundant in plaques in AD brain (B), indicating the MPO gene is induced by agents present in AD brain. In contrast, in APP-overexpressing models of AD, the mouse MPO gene is not expressed by cells at plaques, apparently due to the lack of the primate specific Alu with PPARg and other receptor binding sites. We crossed the huMPO transgenics to the APP23 model, and detected high levels of huMPO expression in glia in plaque-laden cortex, and high levels of MPO in plaques. As evidence that MPO-oxidants contribute to AD-like pathology, the MPO-APP23 mice exhibit greater memory deficits in water maze studies, as compared to APP23 controls. Also, MPO-APP23 brain tissue has much higher levels of HNE-modified proteins, an indicator of oxidized phospholipids. Mass spectrometry analysis similarly detects increased levels of oxidized phospholipids in MPO-APP brain than control APP brain. We are currently testing the effects of PPARg ligands and estrogen on MPO expression and AD-like pathology in the MPO-APP transgenics.

Oxidation appears to be an important component in the development of both atherosclerosis and Alzheimer’s disease. The humanized MPO mouse model will help identify the role of MPO in these disease processes as well as give us a better understanding of the consequences. It is likely that this model will also be valuable in understanding how MPO contributes to other diseases where inflammation and oxidation are involved.

MPO and cancer
MPO has both protective and deleterious roles in cancer. The -463G/A polymorphism is linked to increased lung cancer risk, due to MPO-conversion of tobacco smoke precarcinogens to carcinogens which form DNA crosslinks. Conversely, the higher expressing MPO -463G/G genotype is linked to reduced recurrence of ovarian and breast cancer in women who have undergone initial treatment. This suggests that MPO-expressing cells, such as macrophages, eliminate resurgent cancer cells. We are interested in investigating the effects of agents that enhance (PPARg) or suppress (statins, ibuprufen) MPO gene expression on metastasis in mouse models of cancer.