The heat that hurts

Inflammation and the development of heart disease

Editor’s Note:  This story, first published in 2004, has been updated.

Harold Olivey
Published: December, 2004

MacRae Linton, M.D., and Sergio Fazio, M.D., Ph.D., surrounded by images of cholesterol-engorged “foam cells,” have found tantalizing molecular and cellular clues supporting a link between inflammation, heart disease and metabolic syndrome.
Photo illustration by Dean Dixon
When it comes to heart disease, fat in the bloodstream is one of the major culprits. Yet as many as 50 percent of people with atherosclerosis—artery blockage that can lead to a heart attack—do not display traditional risk factors such as high cholesterol.

Thanks to recent technological advances, scientists are now able to take a closer look at what stubbornly remains the nation’s leading disease killer. What they are finding may surprise you.

Inflammation, incited by a plethora of infection-fighting and wound-healing blood cells and molecules, seems to play a major role in atherosclerosis. For example, high levels of C-reactive protein (CRP), a circulating marker of inflammation, are associated with an increased risk for heart attack and stroke.

That doesn’t mean a once-a-day anti-inflammatory pill to prevent heart disease is right around the corner. Researchers are hopeful, however, that their pursuit of inflammation may lead to better ways of treating and preventing not only heart disease and other ailments of the Western lifestyle—including type 2 diabetes.

The Vanderbilt connection

Vanderbilt’s contributions to the field of inflammation and heart disease began more than a decade ago, when, as a resident physician, MacRae Linton, M.D., became interested in atherosclerosis. “I would see all these people having bypass surgery, and nobody was thinking about their risk factors,” recalls Linton, now professor of Medicine and Pharmacology at Vanderbilt.

Linton’s interest led him to pursue an endocrinology fellowship at the renowned Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco. There he met Sergio Fazio, M.D., Ph.D., another research fellow who was studying how the body handles cholesterol.

“The real excitement came from understanding the complexity of lipid metabolism,” recalls Fazio, an Italian native whose doctorate is in Molecular Biology. “But when you look at it from the point of view of clinical relevance, what’s important is the damage that lipid metabolism can do to the vessel wall. It was clear that we needed to become vascular biologists.”

Colorized microscopic image shows monocytes “diving” into an atherosclerotic lesion in the lining of a blood vessel. The monocytes have been “activated” by inflammatory signals on the blood vessel lining. Arm-like pseudopods drag the monocytes into the gaps between endothelial cells and down into the atherosclerotic plaque beneath the endothelium. Here they become macrophages, vacuum up excess lipid and add to the size of the plaque.
Image courtesy of Jay Jerome, Ph.D., director of the Research Electron Microscopy Resource at Vanderbilt; colorization by Deborah Doyle
Linton and Fazio decided early in their careers to take a team approach to their research. Since joining the faculty at Vanderbilt in 1993 (Fazio is a professor of Medicine and Pathology), they have published several seminal papers in the field of atherosclerosis, and they co-direct the medical center’s Atherosclerosis Research Unit.

In one of their highest profile papers, published in the journal Science in 1995, Fazio, Linton and James Atkinson, M.D., Ph.D., professor of Pathology, reported that apolipoprotein E (apoE), a protein important in lipoprotein metabolism, seemed to protect mice from developing atherosclerosis.

The largest supply of apoE comes from the liver, Linton says. But the protein is also made by macrophages, and thus may participate in the inflammatory response.

To determine what, if any, role apoE expressed in macrophages played in the development of atherosclerosis, Linton and Fazio studied a strain of mice that lacked both copies of the apoE gene. These mice develop significant atherosclerosis, unlike their genetically normal—or wild type—counterparts. The researchers irradiated the apoE deficient mice to kill their bone marrow, the source of macrophages, then gave them transplants of bone marrow cells from wild type mice.

Mice deficient in apoE that received the transplants did not develop atherosclerotic plaques. “The small amount of apoE that came from the bone marrow was enough to cure the mice,” says Linton.

After the study published in Science, “we became more interested in genes related to cholesterol homeostasis—enzymes, proteins, receptors,” he continues. “Recently we’ve expanded that into an interest in inflammation and how macrophages and other cells may play a role in the inflammatory process of atherosclerosis.”

“Living wounds” that will not heal

Atherosclerotic plaques form when blood vessels are injured by chemicals (such as those found in cigarette smoke), high blood pressure or high levels of plasma lipids (fats, like cholesterol).

These plaques are living wounds that can trigger clot formation inside the blood vessel. When a clot forms in an artery that supplies the heart with blood, a heart attack ensues, leading to death of heart muscle. Understanding the cellular and molecular events that lead to atherosclerosis will be critical to making progress against the disease.

Atherosclerotic plaques contain a variety of cell types (see illustration). These include endothelial cells that line the blood vessels and make up the endothelium, and vascular smooth muscle cells that give form and resilience to the blood vessels. Other cells found in plaques, such as pro-inflammatory macrophages and lymphocytes, do not normally reside in the vessel wall. Instead, they remain in the bloodstream and stand ready to mediate inflammatory responses at sites of injury and infection.

As part of the innate immune response system, macrophages are among the first line of defense at sites of injury. Derived from circulating monocytes, these specialized cells engulf and destroy pathogenic organisms and damaged cells. When circulating monocytes encounter injured endothelium, they migrate underneath the endothelium. This invasion of monocytes starts the formation of the atherosclerotic plaque.

Once inside the vessel wall, monocytes differentiate into macrophages that become “activated” and recruit other monocytes and T helper lymphocytes to enter the plaque. They also ingest cholesterol. As macrophages become engorged with cholesterol, they take on a characteristically foamy appearance, and thus are referred to as “foam cells.”

As an atherosclerotic lesion becomes more advanced, an increasing number of foam cells are found in the plaque due to the continual recruitment of macrophages into the lesion. A thin fibrous cap of smooth muscle cells and collagen forms over the plaque, and smooth muscle cells underlying the damaged endothelium begin to proliferate, expanding the volume of the plaque.

Stable atherosclerotic plaques are less likely to cause an acute cardiovascular event such as a heart attack or stroke. Although they restrict blood flow through the lumen of the blood vessel, they rarely cause total occlusion. Instead, plaques provide a site within the vessels where clots can form. Platelets, blood cells involved in clotting, do not attach to the wall of healthy blood vessels. However, they will attach to atherosclerotic lesions.

As foam cells within a lesion die, the center of the plaque becomes necrotic, weakening the overlying fibrous cap and increasing the risk of rupture. The interior of an atherosclerotic plaque contains molecules that attract platelets and provide ample sites for attachment.

Thus, when an atherosclerotic plaque ruptures, a clot can quickly form and completely occlude the blood vessel. Acute cardiovascular events are most often precipitated by the rupture of the thin fibrous cap that covers the atherosclerotic plaque.

Clot blocker

Aspirin, the classic anti-inflammatory drug, can prevent clot formation over atherosclerotic plaques by inhibiting the enzyme cycoloxygenase-1 (COX-1) in platelets. That, in turn, reduces formation of the prostaglandin Thromboxane A2 (TxA2), a powerful pro-coagulant molecule.

Aspirin also inhibits the related COX-2 enzyme, which produces other pro-inflammatory prostaglandins at sites of inflammation. Long recognized for its role in chronic inflammatory processes like arthritis, COX-2 is also expressed by cells within atherosclerotic plaques, but not elsewhere in the circulatory system.

Linton and Fazio have reported that COX-2 contributes to the pathology of atherosclerosis in mouse models of the disease. Inhibiting the enzyme in mice with high cholesterol levels, either pharmacologically or genetically, retards early atherosclerotic plaque formation. These data suggest that blocking inflammation could suppress the progression of atherosclerosis.

But Linton cautions that the tale is not so cut-and-dry. “It’s tough to say (whether COX-2) is just good or bad. It probably depends on which cell is expressing it and at what time.” For example, “COX-2 is expressed by basically all the players in the artery wall—smooth muscle cells, endothelial cells, macrophages,” he says.

In addition, macrophages down-regulate their pro-inflammatory activities and lose COX-2 expression when they become foam cells. Other studies have suggested that blocking COX-2 activity does little to ameliorate the symptoms of more advanced atherosclerotic lesions.

This change in macrophage gene expression may come out of necessity: “When it’s overloaded with cholesterol, the macrophage has to focus on getting rid of cholesterol,” Linton explains. “Before that, it may be more important to be an inflammatory cell involved in the recruitment of other cells and propagation of the inflammatory pathway.”

Experiments on atherosclerotic mice have provided significant insight into the mechanisms behind cardiovascular disease, including the recent findings on the role of inflammation. Even so, Fazio is quick to point out that the mouse models of atherosclerosis offer only a pale reflection of the disease state in human beings.

“There is an issue in quality and in the extent and topography (of lesions in mice),” Fazio cautions. The majority of human cases of atherosclerosis, according to Fazio, are due to a combination of risk factors. This is in sharp contrast to atherosclerosis in mice induced experimentally by the targeted disruption of one or two genes.

Talking back to fat

During the past decade, two new classes of drugs were developed to relieve pain and inflammation in patients with rheumatoid arthritis—specific inhibitors of the COX-2 enzyme, and blockers of the pro-inflammatory tumor necrosis factor (TNF). It remains to be seen, however, whether they also will be useful in preventing heart disease.

Although COX-2 is expressed in atherosclerotic lesions, chronic use of high doses of one of the COX-2 inhibitors has been linked to an increase in blood pressure, edema and serious heart problems in some patients. As for TNF-inhibitors, animal studies and at least one report in a patient suggest that TNF blockade may actually destabilize atherosclerotic plaques and precipitate heart attacks.

Statins, the blockbuster cholesterol-lowering drugs, also have anti-oxidant and anti-inflammatory properties that may protect against cardiovascular disease. To test this hypothesis, investigators in the multi-center JUPITER study are administering the statin drug Crestor to participants who have elevated circulating inflammatory markers including CRP, but normal LDL and triglyceride levels.

The newest targets for pharmacological treatment of atherosclerosis may come from studies of how adipose tissue (fat) regulates cholesterol metabolism and inflammation. Fazio and Linton point to recent studies that suggest adipose tissue, composed of fat cells, and macrophages in atherosclerotic plaques lead the inflammatory response in atherosclerosis and cardiovascular disease.

Some genes originally reported to be expressed primarily or exclusively in adipose tissue are also expressed in activated macrophages. Some of these genes, including the fatty acid binding protein aP2, are believed to be involved in insulin resistance, an early hallmark of type II (non-insulin dependent) diabetes. These recent reports suggest inflammation as a critical link between diabetes and atherosclerosis.

Individuals showing symptoms of insulin resistance are more likely to develop cardiovascular disease as well as diabetes. Patients with diagnosed diabetes are at elevated risk for adverse cardiovascular events such as heart attack and stroke. By targeting proteins expressed in fat cells and macrophages that seem to play a dual role in reducing insulin sensitivity and increasing inflammation, new therapies may reduce the incidence of both atherosclerosis and diabetes in at-risk populations.

“I wouldn’t be surprised if we develop a panel of 20-30 genes that we routinely look at in individuals to define and predict risk,” predicts Douglas Vaughan, M.D., chair of Medicine at Northwestern University Feinberg School of Medicine and former chief of Cardiovascular Medicine at Vanderbilt. “It’s going to be a multi-factorial approach that includes … biochemical, physiological and genetic parameters.”

Tale of a thrombus
Endothelium that is injured, in this case by high levels of lowdensity lipoprotein (LDL) cholesterol, releases factors that attract blood cells called monocytes. Once inside the tissue, the monocytes differentiate into macrophages. Part of their job is to sweep up excess cholesterol. As they become engorged with cholesterol, the macrophages take on a foamy appearance, and now are called foam cells.
Interactions between foam cells and white blood cells trigger a chronic inflammatory process. Smooth muscle cells migrate to the arterial wall, proliferate and secrete proteins that form a fibrous plaque. As the foam cells die, the plaque weakens and can rupture. Platelets are attracted to the site of the rupture, and can quickly form a clot that blocks the vessel completely.
Illustrations by Dominic Doyle

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