Research Suggests New Approaches to Atherosclerosis

By Jason Socrates Bardi

A team of researchers at The Scripps Research Institute has determined that a protein called "TLR2" found on the surface of cells lining the arteries of mammals is involved in the disease atherosclerosis—a significant finding because it supports the concept of a therapeutic approach to heart disease aimed at counteracting the effect of TLR2.

Scripps Research Professor Linda Curtiss, Associate Professor Peter Tobias, and Research Associate Adam Mullick elucidated the effect TLR2 has on atherosclerosis, which is to exacerbate the disease. Their results are described in a recent issue of The Journal of Clinical Investigation.

TLR2 is believed to exacerbate the disease through chronic inflammation, says Curtiss. "We just keep getting new information to suggest that infection and inflammation are related to cardiovascular disease, [which] adds a whole new dimension to how you think about it."

Atherosclerosis and Inflammation

Atherosclerosis is a common vascular disease that increases the risk of heart attacks and strokes, which are leading causes of death in the United States. The name comes from the Greek athero (which means gruel or paste) and sclerosis (which means hardness), and, as the name implies, it is a disease that is characterized by a hardening of the arteries over time due to the buildup of plaques—fibrous tissue, calcium, fat, cholesterol, proteins, cells, and other materials—on the inner "endothelial" walls of an artery.

These plaques feel something like cartilage to the touch, which explains why atherosclerosis is commonly called hardening of the arteries. The open cross-section of the artery shrinks as this buildup occurs, and the reduction in blood flow can become severe. If the surface of the plaque ruptures, clotting can be initiated resulting in complete blockage of the vessel. When this process occurs within the coronary arteries that provide the blood supply to the heart, the result is a myocardial infarction, also known as a heart attack. Heart attacks can also result from rupture of the plaque and obstruction of blood flow. When the carotid arteries are involved, patients may be asymptomatic or they may experience lightheadedness and fainting, transient loss of vision in one eye or the other, weakness of the hands, transient loss of the ability to speak, or even stroke.

High cholesterol is the most obvious risk factor associated with atherosclerosis, but cholesterol is not the complete story. There are plenty of people with high cholesterol who never develop atherosclerosis, and clearly other factors are involved in the development of the disease. But what are these factors?

Over the last several years, evidence has been accumulating that the process of atherosclerosis is linked to infection and inflammation, and a number of infectious agents such as bacteria are known to contribute to high risk for atherosclerosis. In certain rare cases, microbial components have been found in atherosclerotic lesions, and epidemiological evidence suggests that people with chronic infections tend to have more of the disease. The gum disease periodontitis, for instance, is a known risk factor for atherosclerosis.

Wanting to investigate how inflammation is linked to atherosclerosis, Curtiss and Tobias turned their sights on a cellular protein called the Toll-like receptor-2 (TLR2), and after Mullick arrived at Scripps Research to pursue his postdoctoral studies, the three of them designed a project to determine if TLR2 is involved in the disease.

The Toll-like Receptor

TLR2 is named for its similarity to a protein called Toll (thus "toll-like" receptor). The original Toll receptors are a family of proteins that were first discovered in fruit flies and are involved in signaling mechanisms and immune recognition. In the fly, Toll is important for both embryonic development, during which it triggers dorsoventral patterning, and for immune functions of the developed organism—for instance, the protein is a receptor molecule that defends against fungal infections.

Humans and other mammals have these receptors as well. Several mammalian proteins have cytoplasmic domain similarities to Toll and also have leucine-rich ectodomain repeats, displaying gene homology to Drosophila Toll over their entire coding regions. Ten of these have been identified to date, including one essential gene in the innate immune system called Toll-like receptor 2 (Tlr2), which is important in the recognition of yeast and gram positive bacteria—like Staphylococcus aureus, the bacterium that underlies common hospital-acquired infections.

"TLR2 is an innate immune receptor," says Tobias, "and its principal function is to detect [these] microbial pathogens."

How does it work? In the body, TLR2 can be found on macrophages and endothelial cells lining the bloodstream. It is a fairly large protein, about 70,000 Daltons, and it sits on the surface of endothelial cells. There it becomes paired with other TLR molecules, such as TLR6, and when the right ligand molecule comes along and binds to it, a series of events occur involving a cascade of reactions with other proteins. This leads the cell to activate other molecules, called transcription factors, which then turn on the expression of inflammatory genes, attracting immune cells to destroy the pathogens thatrelease inflammatory molecules like tumor necrosis factor and interleukins at the site of infection.

This mechanism is essential for eliminating pathogens that TLR2 is able to detect. However, says Tobias, "we knew that it had a role in responding to infectious agents. Now, we have found another role in which it is [involved]."

That role is also to induce inflammation, but of a different type. In some cases when the infection is chronic, normal process go awry and inflammation and injury are followed by repair followed by further inflammation and injury and more repair, and on and on. When this happens, atherosclerotic plaques can form.

Before the recent study by Curtiss, Mullick, and Tobias, some evidence suggested that TLR2 might be linked to the formation of atherosclerotic plaques—such as the fact that the protein can be found at the sites where the lesions occur.

More compelling is the fact that most endothelial cells do not express TLR2 at all. It tends to be expressed at sites of disturbed blood flow, such as the inner radii of the aortic arch where the shape of the blood vessel creates uneven fluid dynamics. Basic research has backed up this physiological observation. In Tobias' lab, Stefan Dunzendorfer looked at the expression of TLR2 on endothelial cells in flow chambers and found that the nature of the physical flow of blood to which the endothelial cells were subjected determined whether there was any TLR2 on their surface. Normal flow tends to inhibit the expression of the protein whereas disturbed flow initiates its expression.

Atherosclerosis is also highly localized and atherosclerotic plaques tend to appear at places where there is disturbed flow, such as the inner radii of the aortic arch.

Tracking Down Clues

But Curtiss, Mullick, and Tobias wanted to look at the effect of TLR2 on atherosclerosis directly, and this meant looking at conditions where the protein was activated, deactivated, or gone altogether. And they wanted to look in vivo, in rodents that may develop atherosclerosis. This was not straightforward. Mice are usually very resistant to atherosclerosis and can eat the equivalent of several fast food hamburgers a day their entire lives and never get the disease.

However, there is a special mouse that has a condition known as diet-induced hyperlipidemia, a condition that makes them highly susceptible to atherosclerosis. By looking at this model in situations where TLR2 was absent or activated, Curtiss, Mullick, and Tobias were able to observethe effect of the protein on the disease.

When they examined the role of TLR2 activation and deletion in mice, they found that TLR2 exacerbates the disease in a distinct way: when you knock out the TLR2 receptor, you have a lot less atherosclerosis. Without TLR2, the mice got less disease; this suggests that normally, when the receptor is there, it is proatherogenic, says Mullick.

Moreover, the scientists wanted to differentiate between the TLR2 that is expressed on endothelial cells (which line the blood vessels) and the TLR2 that is expressed on the surface of macrophages, since the receptor is expressed in both cell types and since both are involved in lesion development. They expected the latter to be significant, since macrophages are the cells that release the inflammatory chemicals during an immune reaction and therefore play a major role in atherosclerosis. Surprisingly, the expression of TLR2 on these cells seemed not to play a role.

The researchers were able to distinguish between the two types by performing a bone marrow transplant with TLR2 knockout mice as either the bone marrow donors or recipients. This procedure created chimeric mice with tissue-selective deletion of TLR2, such as TLR2 deficient macrophages. They found that the loss of TLR2 in bone marrow cells did not have an effect on the disease, which suggests that it is only the TLR2 receptor in endothelial cells that matters for atherosclerosis. This work was done in the absence of any administered microbial agonist for TLR2.

The work is interesting for several reasons, say the researchers, because it suggests that activation of atherosclerosis may be linked to a natural compound in the body that activates the TLR2 receptor on endothelial cells. If the TLR2 is detecting this natural compound, then identifying these "endogenous ligands" could help in the design of a drug that would block the action of TLR2 and prevent the inflammation that contributes to atherosclerosis. Additionally, the progression of the disease may be linked to activation of macrophages via molecules generated by pathogenic organisms, which may be a common scenario in individuals with chronic inflammatory disorders.

Moreover, the research suggests a starting point for further studies into the genetic background of families that are more or less susceptible to atherosclerosis than the general population.

The article, "Modulation of Atherosclerosis in Mice by Toll-Like Receptor 2" by Adam E. Mullick, Peter S. Tobias, and Linda K. Curtiss appears in the November 1 2005 issue of The Journal of Clinical Investigation115 (3149-3156). See: http://dx.doi.org/10.1172/JCI25482.

This work was supported by the National Institutes of Health and the Tobacco-Related Disease Research Program.

Send comments to: mikaono[at]scripps.edu

(Left to right) Associate Professor Peter Tobias, Professor Linda Curtiss, and Research Associate Adam Mullick. Photo by Kevin Fung.