Faculty Bibliography

RATIONALE: Diabetes mellitus increases cardiovascular disease risk in humans and remains elevated despite cholesterol-lowering therapy with statins. Consistent with this, in mouse models, diabetes mellitus impairs atherosclerosis plaque regression after aggressive cholesterol lowering. MicroRNA 33 (miR33) is a key negative regulator of the reverse cholesterol transport factors, ATP-binding cassette transporter A1 and high-density lipoprotein, which suggested that its inhibition may overcome this impairment. OBJECTIVE: To assess the effects of miR33 inhibition on atherosclerosis regression in diabetic mice. METHODS AND RESULTS: Reversa mice, which are deficient in the low-density lipoprotein receptor and in which hypercholesterolemia is reversed by conditional inactivation of the microsomal triglyceride transfer protein gene, were placed on an atherogenic diet for 16 weeks, then either made diabetic by streptozotocin injection or kept normoglycemic. Lipid-lowering was induced by microsomal triglyceride transfer protein gene inactivation, and mice were treated with anti-miR33 or control oligonucleotides. Although regression was impaired in diabetic mice treated with control oligonucleotides, anti-miR33 treatment decreased plaque macrophage content and inflammatory gene expression in these mice. The decreased macrophage content in anti-miR33 treated diabetic mice was associated with a blunting of hyperglycemia-induced monocytosis and reduced monocyte recruitment to the plaque, which was traced to an inhibition of the proliferation of bone marrow monocyte precursors associated with the upregulation of their Abca1. CONCLUSIONS: miR33 inhibition overcomes deleterious effects of diabetes mellitus in atherosclerosis regression in mice, which suggests a therapeutic strategy in diabetic patients, who remain at elevated cardiovascular disease risk, despite plasma cholesterol lowering.

OBJECTIVE: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. APPROACH AND RESULTS: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. CONCLUSIONS: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.

Hypothiocyanous acid (HOSCN) is produced in biological systems by the peroxidase-catalyzed reaction of thiocyanate (SCN(-)) with H(2)O(2). This oxidant plays an important role in the human immune system, owing to its potent bacteriostatic properties. Significant amounts of HOSCN are also formed by immune cells under inflammatory conditions, yet the reactivity of this oxidant with host tissue is poorly characterized. Traditionally, HOSCN has been viewed as a mild oxidant, which is innocuous to mammalian cells. Indeed, recent studies show that the presence of SCN(-) in airways has a protective function, by preventing the formation of other, more damaging, inflammatory oxidants. However, there is an increasing body of evidence that challenges this dogma, showing that the selectivity of HOSCN for specific thiol-containing cellular targets results in the initiation of significant cellular damage. This propensity to induce cellular dysfunction is gaining considerable interest, particularly in the cardiovascular field, as smokers have elevated plasma SCN(-), the precursor for HOSCN. This review will outline the beneficial and detrimental aspects of HOSCN formation in biological systems.