Mechanism of arsenic-induced vascular disease

 

Project Leader: Aaron Barchowsky Ph.D. (Project Leader), Associate Professor Department of Environmental and Occupational Health Graduate School of Public Health University of Pittsburgh 205 FORBL 3343 Forbes Avenue Pittsburgh, PA 15261 Phone: (412) 383-2085

Decoding the language of cells

Scientists have observed that people living in regions where drinking water contains elevated levels of arsenic have an unusually high likelihood of developing blood vessel diseases. These vascular diseases, which include heart disease and high blood pressure, are caused by a progressive narrowing of blood vessels that diminishes the normal flow of blood. The flow through small vessels in the extremities can be reduced so severely that tissue starves and dies. Untreated, this can lead to gangrene of the feet or blackfoot disease, a condition seen in people from Bangladesh and other regions of the world where arsenic concentrations in drinking water are above 500 parts per billion (500 micrograms per liter). In contrast, people drinking water that contains less than 500 parts per billion may be at a greater risk for hypertension and heart attacks. These forms of environmentally derived vascular diseases may affect millions of individuals through out the world, including those in certain parts of the United States.

Aaron Barchowsky, Ph.D.

Though epidemiological studies point to arsenic as the cause of these diseases, little is known about the mechanisms involved. In a project led by Dartmouth toxicologist Aaron Barchowsky, researchers are examining the way arsenic affects two types of cells: the endothelial cells that line blood vessels and the smooth muscle cells that enable vessels to constrict or to dilate. The group has found that relatively low doses of arsenite — a form of arsenic commonly found in nature — can usurp the chemical messages passed within and among these cells, changing their ability to function. Arsenite does this by tripping a molecular switch on the surface of both types of cells, launching a cascade of chemical signals that alters critical programs within the cells.

Barchowsky and colleagues have found that cell signals activated by environmental levels of arsenite prompt vascular cells to produce growth factors — specifically, proteins that promote angiogenesis, the formation of new microvessels. These tiny vessels supply essential nutrients for cell growth. The researchers believe that the formation of new microvessels may support the growth of successive layers of smooth muscle cells and fibroblasts, enabling vessel walls to thicken and choking off the space within. Narrowing the space inside blood vessels forces the heart to exert more pressure to keep blood flowing. This could lead to chronic hypertension, and as the blood vessels become obstructed, heart attacks.

The researchers are exploring the cell signals involved in this sequence. The goal of Barchowsky’s project is analogous to creating a schematic diagram of a complex electrical system, where one circuit trips another in feedback loops that turn on or off other processes. The group aims first to trace the chemical signals within the cells that are initiated by arsenic at levels found in the environment and then to delineate the downstream effects these signals have on blood vessel cells. They are particularly interested in identifying the chain of chemical events that prompt production of blood vessel growth factors and angiogensis in these cells after arsenic exposure.

This work is helping to shape scientific thinking on the threshold for toxic effects from arsenic. In addition to improving understanding of how drinking-water levels of arsenic may contribute to vascular disease, the research may also provide fundamental insight into mechanisms that underlie other diseases associated with arsenic, such as cancer and diabetes.

A dynamic balance

The cells in our bodies are regulated by countless molecular interactions, each mediated by chemical signals. Some of these signals orchestrate interactions between cells. Others prompt cells to activate genes, to grow or stop growing, to differentiate into particular types, and, when cells become damaged, to commit suicide, a normal cell process called apoptosis. Barchowsky and his colleagues are mapping the different levels of exposure to arsenic that trigger the different molecular signals that account for these wide ranging effects in cells.

Barchowsky with research associate Linda Klei

In laboratory studies of vascular cells the researchers have demonstrated that high doses of arsenic — doses known to be overtly toxic — trigger a different series of cellular signals from those stimulated by arsenic levels found in nature. At high levels, arsenic stimulates signals that prompt vascular cells to destroy themselves. But low levels of arsenic — levels more commonly found in nature — trigger signals that prompt these cells to grow. The investigators found that this growth is linked to increased production of an important class of biological molecules called oxidants.

Oxidants are formed by the simple act of breathing. Aerobic metabolism — the use of oxygen by cells to convert sugar into energy — creates oxidant compounds called “free radicals.” At high levels these compounds can damage proteins and DNA or react chemically with the fats that make up cell membranes to create even more damaging lipid radicals. These reactive compounds destroy the integrity and function of cell membranes and cause other dangerous chemical byproducts to form. But oxidants also have beneficial effects on the body. Oxidants play an important role in the immune system by destroying harmful bacteria or viruses. Oxidants can also prompt cells to produce growth factors — proteins that support cell growth.

The human body has evolved checks that keep these reactive compounds in balance, and to eliminate damage they produce. Healthy tissue accumulates antioxidants, such as vitamins E and C, and produces enzymes that scavenge and break down excess radicals. When these defenses are overwhelmed, threatening the integrity or function of cells, chemical stress signals trigger damaged cells to die.

Research by Barchowsky’s group suggests that environmental levels of arsenic may do their harm by usurping normal cell signals, tipping the dynamic balance of oxidants. The investigators have found that the master key to the actions of low-level exposure to arsenite is an enzyme that sits on the membranes of vascular cells called NADPH oxidase. In the presence of low levels of arsenic this enzyme produces oxidants that promote the growth of smooth muscle cells and fibroblasts in blood vessels. This growth is a major contributor to cardiovascular disease. Scientists have found that people exposed to arsenic through drinking water are known to have elevated oxidant levels in their blood. This enzyme-mediated process may explain higher rates of cardiovascular disease in regions where drinking water arsenic levels are elevated.

A chain of molecular events

In cell studies, Barchowsky and colleagues found that levels of arsenite above 100 parts per billion stimulate the NADPH oxidase to enhance production of two oxidants — superoxide and hydrogen peroxide. These oxidants initiate cell signals to activate important transcription factors, such as HIF-1alpha, AP-1 and NF-kappa B. These transcription factors bind to and turn on genes that produce inflammatory proteins and vascular cell growth factors — proteins that support the proliferation of cells that line arteries. The investigators believe that a continued stream of inflammatory and growth signals caused by these proteins overwhelms natural checks that curb excessive cell growth such as contact inhibition — signals that stop cells from growing when they are surrounded by similar cells. In addition, the superoxide produced by NADPH oxidase combines with the nitric oxide in vessel walls, inhibiting its actions. Normally, nitric oxide is required to dilate or keep blood vessels open and blood flowing.

Barchowsky explains his work to some middle school science teachers

Barchowsky’s group has demonstrated that when cells are exposed to levels of arsenite that exceed 600 parts per billion, both the enzyme and the growth promoting signals are turned off. The excessive oxidants produced in response to these high levels come from damaged mitochondria, the cell’s energy producing bodies. This activates genes that induce the cells to commit suicide. The combination of damage to the mitochondria and production of death genes leads to damaged tissue. Fortunately, the investigators and others have found that many of these damaging effects can be prevented by increasing the antioxidant capacity of the cell by vitamin E or lipoic acid.

Arsenic and angiogenesis

New work by Barchowsky and colleagues is examining arsenic's effects in living systems. The group has found that the growth factors stimulated by low levels of arsenic promote the formation of new microvessels, a process called angiogenesis. New blood vessels are essential in providing nutrients for rebuilding or repairing tissue, a process called tissue remodeling. Angiogenesis also plays a role in proliferative diseases such as cancer. Enhanced angiogenesis has been found to fuel the expansion of tumors by increasing their access to the nutrients in blood.

Graduate student Nicole Soucy

When the researchers exposed chicken embryos to low doses of arsenite the density of new blood vessels in the chorioallantoic membranes, the outer protective layer of the egg, increased. Higher doses triggered the opposite responses — preventing vessel growth or destroying blood vessels. Researchers found the loss of blood vessels could be prevented by adding the antioxidant vitamin E to the cultures.

To see if arsenic would have this same effect on tumors, the researchers treated mice with arsenite and then placed a plug of tissue containing a growth factor — a model that mimics a tumor — under their skin. Arsenite was found to accelerate the growth of new microvessels in the tissue plug. In mice treated with antioxidants this effect was inhibited. The blood vessels that grew into this tissue were leaky and abnormal. This type of vessel supports the growth of tumors, but will not support proper function in an organ, such as the heart.

Looking ahead

Barchowsky and colleagues have begun a new study investigating whether the blood vessels of mice exposed to arsenic in their drinking water are affected by the same changes in oxidant formation, nitric oxide availability and cell signaling observed in cell studies. An important focus of this research is to demonstrate whether growth factors and proteins identified in these studies thicken the vessel walls in these animals. The key question is whether arsenite promotes the growth of poorly functioning or abnormal blood vessels, which would contribute to hypertension and heart attacks.

The researchers will also follow changes in the animals’ blood pressure and the ability of their blood vessels to dilate. The tissues of these animals will be also be examined to determine whether arsenic-laden drinking water leads to angiogenesis, and whether this angiogenesis contributes to the thickening of blood vessel walls. The researchers will also investigate whether feeding the animals antioxidants improves their ability to defend against the effects of oxidants stimulated by exposure to arsenic.