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Research Newsletter-Fall/Winter 2008

COPPER CHELATION AND VASCULAR INFLAMMATION



Hao Wei
LPI Graduate Research Assistant

Copper is a double-edged sword: small amounts are required for health, but excess copper can generate free radicals and cause inflammation or toxicity. Tetrathiomolybdate (TTM) chelates copper, helping to remove excess amounts from the body. Copper is required for angiogenesis, or blood vessel formation. Tumors depend on angiogenesis for growth, and copper chelation may be an effective treatment strategy. Treating mice with TTM reduced copper levels by over 50% and inhibited inflammatory molecules that contribute to the development of atherosclerosis.

Copper, an essential mineral, can be toxic


Copper is one of the most abundant trace elements found in the human body. It is an essential nutrient and intricately involved in catalyzing biochemical reactions and stabilizing proteins. Copper serves as a cofactor in enzymes involved in cellular energy generation (cytochrome C oxidase), free radical detoxification (copper-zinc superoxide dismutase, or SOD), connective tissue production (lysyl oxidase), iron mobilization (ceruloplasmin), and neurotransmission (dopamine β-hydroxylase). However, excessive copper can be toxic, primarily by causing oxidative damage to the body. Copper is involved in both generation of and defense against reactive oxygen species (ROS). As a transition metal, copper can change its redox status by accepting and donating electrons, shifting between the cuprous (Cu+) and cupric (Cu2+) forms. Therefore, copper can participate in reactions that generate superoxide radicals and hydrogen peroxide, which are major ROS in the body. If the level and distribution of copper are not carefully regulated, its uncontrolled redox activity can lead to the harmful generation of these ROS, compromising cellular functions. Precise regulatory mechanisms must be in place to prevent the accumulation of copper to toxic levels. Free copper, unbound to proteins, rarely exists in cells, and there are multiple proteins that serve as copper chaperones by binding to copper and facilitating its distribution. Copper can also exert toxic effects by displacing other essential metal ions from their natural ligands (molecules bound to the metal). The replacement of zinc by copper in the human estrogen receptor renders the protein defective, altering its role in hormone-dependent cell signaling. One proposed mechanism for amyotrophic lateral sclerosis (ALS) is that the loss of zinc from Cu,Zn SOD in motor neurons generates defective SOD, which then produces superoxide radicals instead of scavenging them as a functional antioxidant.

The human body contains 50 to 120 mg of copper, most of which is stored in the liver. Copper is readily absorbed from the diet through the small intestine and is usually excreted via bile through the gastrointestinal tract. The average dietary intake of copper by human adults in the U.S. varies from 1.0 to 1.6 mg/day. The main sources are seeds, grains, nuts, and beans (concentrated in the germ and bran), shellfish, and liver. The recommended copper intake for adults is 0.9 mg/day, and an intake of copper exceeding 10 mg/day may lead to symptoms of toxicity, such as weakness and nausea.

Copper is also required for normal embryonic development and the growth of new blood vessels (angiogenesis). Interest in copper's role in angiogenesis dates back several decades. Researchers found that copper promoted angiogenesis in the rabbit cornea and that copper deficiency reduced angiogenesis produced by a known angiogenic stimulant. Because tumors require new blood vessel growth to obtain nutrients and remove waste, anti-angiogenic strategies are important in cancer therapy. Clinical studies have demonstrated the potential of copper chelators for the treatment of cancer in humans.

Recent studies show that copper is also implicated in inflammatory conditions, such as pulmonary fibrosis and arthritis. Using copper chelators, researchers demonstrated that production of many cellular growth factors, interleukins, and pro-inflammatory cytokines, such as tumor necrosis factor-alpha, are affected by copper. Reduction of intracellular copper leads to a decrease of these growth factors, interleukins, and cytokines. These copper-regulated molecules are involved in multiple physiological functions, such as tissue growth, angiogenesis, and inflammation. Angiogenic and inflammatory signaling pathways are related, and the critical regulator orchestrating both pathways is the transcription factor NF-κB, a protein that can regulate gene expression by binding to a specific DNA sequence. Although the mechanism of copper's role in promoting angiogenesis and mediating inflammatory events is not totally understood, it has been suggested that NF-κB may be the critical factor affected by copper.

Atherosclerosis is an inflammatory disease


Atherosclerosis is the leading cause of mortality in the Western world and is increasing in developing countries. Atherosclerotic lesions begin to form early in life, develop progressively, and most often remain clinically insignificant until the acute rupture of a plaque and the ensuing thrombosis, leading to myocardial infarction or stroke. Although atherosclerosis was formerly considered as simply a lipid storage disease, we now appreciate the role of inflammation in atherosclerosis. Compelling evidence indicates that inflammation plays a major role in all stages of atherosclerosis—from initiation to thrombotic complications—that are characterized by intense immunological activity. Levels of inflammatory markers also predict the outcome of patients with acute coronary syndrome.

These inflammatory markers include adhesion molecules secreted by cells in the vascular wall, chemokines, and pro-inflammatory cytokines. The earliest development of atherosclerosis is characterized by inflammatory events and the recruitment of blood cells, such as monocytes, into the vascular wall of the major arteries. Adhesion molecules and chemokines secreted by vascular cells cause monocytes to roll along the vascular wall and then migrate across the endothelial layer into the intima, the innermost layer of the vessel wall. There, the monocytes differentiate into macrophages and absorb oxidized lipids. These lipid-laden macrophages, also known as foam cells, are the major component of the fatty streak, which is the hallmark of early atherosclerosis. During these processes, NF-κB is significantly activated and, in turn, increases the levels of other inflammatory molecules to mediate further monocyte recruitment to the vessel wall. The progressive development of lesions is characterized by inflammation, lipid accumulation, cell death, and fibrosis. Over time, these lesions develop into mature atherosclerotic plaque that can cause a heart attack or stroke.

Copper chelation with tetrathiomolybdate


The term chelate, from the Greek "chele" for "claw", refers to the ability of a substance to combine with metals in the body so that they can be excreted. Metal chelation therapy is traditionally used in occupational medicine, as it effectively removes toxic heavy metals from the body. Chelation therapy is also used as a complementary or alternative treatment in an attempt to inhibit oxidative stress, atherosclerosis, or tumor growth. Metal chelation is also used to study the biological function of metal ions in living systems.

Tetrathiomolybdate (TTM) is an anti-copper drug initially developed to treat Wilson's disease, a genetic disease characterized by excessive accumulation of copper in the liver and consequent liver and brain damage. The copper-chelating property of TTM was discovered decades ago in Australia and New Zealand, where ruminants developed copper deficiency due to naturally formed TTM in the sulfur-rich rumen. TTM is a small compound with high affinity and specificity to bind copper. It forms a stable complex with copper and protein, and one TTM molecule can chelate up to three copper ions (see figure below). TTM is fast acting and has relatively low toxicity among copper chelators. Its toxicity is mainly due to copper deficiency, which can be easily reversed by short-term copper supplementation. Administered orally, TTM can bind copper in food and prevent it from being absorbed. Recently, TTM has been shown to have anticancer activity in rodents through the inhibition of angiogenesis. As indicated above, tumor angiogenesis requires copper, so the strategy is to lower copper to levels that impair angiogenesis in tumors but still meet copper requirements in normal cells. During TTM treatment, the level of ceruloplasmin, a copper-containing protein in blood, is commonly used as a surrogate marker to monitor body copper status.

Tetrathiomolybdate (TTM)

Our hypothesis is that copper promotes NF-κB activation, which in turn causes the production of adhesion molecules, chemokines, and pro-inflammatory cytokines during vascular inflammation, and, therefore, copper chelation by TTM ameliorates inflammatory conditions by inhibiting NF-κB activation. We first studied TTM's anti-inflammatory effects on human aortic endothelial cells (HAEC) in culture. By treating HAEC with TTM, we successfully suppressed the inflammatory response induced by exposure to the endotoxin lipopolysaccharide (LPS). We observed inhibition of the production of adhesion molecules, chemokines, and proinflammatory cytokines, as well as inhibition of NF-κB activation. We then fed TTM to mice every day for three weeks. Following an acute inflammatory stimulation with LPS, we checked toxicity, inflammatory response, copper, and the activity of inflammation-related transcription factors. TTM-treated mice showed no side effects of any kind, and no liver toxicity was observed. Following TTM treatment, serum copper was reduced by 57%. We found that TTM inhibited the expression of adhesion molecules, chemokines, and pro-inflammatory cytokines, and that NF-κB activity was also significantly decreased by TTM. Our studies support the concepts that copper plays a role in mediating vascular inflammation by promoting NF-κB activation and that TTM can inhibit acute inflammation by decreasing NF-κB activity. TTM chelation of excessive copper associated with pathological development has the potential to treat atherosclerosis and ameliorate other inflammatory conditions in the vascular system. We are currently investigating the anti-atherosclerotic potential of TTM in a mouse model of human atherosclerosis.

Last updated January 2009