How the Heart Hardens

In patients with advanced atherosclerosis, heart valves and arteries often become calcified. As soft tissue literally turns into bone, the risk of heart attack soars. For many patients, heart surgery is the only available treatment.

Now, new work from HMS has identified the very earliest signs that calcification is imminent. The evidence becomes apparent at the molecular level long before the patient begins to show symptoms. This research, described in the April 7 Circulation, may lead to novel treatments that prevent heart damage and eliminate the need for valve replacement surgery.

Acute blockage of the coronary artery is one of the most common causes of death in the Western world. In addition, each year in the United States nearly 20,000 patients with aortic valve calcification—typically people over 60 years old—undergo life-extending valve replacement. As the population ages, and as more and more younger people have risk factors such as high cholesterol, renal failure and diabetes, this number is sure to rise.

To better understand the disease and its progression, first author Elena Aikawa, HMS assistant professor of radiology at Massachusetts General Hospital, put modern molecular imaging tools to work. She combined optical imaging with near-infrared fluorescent molecular probes. Developed at the Center for Molecular Imaging at MGH, the probes bind to various molecules or cells in the diseased tissue and light up when exposed to different wavelengths of laser light.

Because it is possible to apply more than one probe at a time, this multimodal imaging reveals potential molecular interactions. Aikawa showed in 2007, for example, that inflammation precedes and advances along with calcification.

“Aikawa has been able to use multimodal imaging to look at the very earliest stages of aortic valve calcification,” said Dwight Towler, the Ira M. Lang professor of medicine at the Washington University School of Medicine. Not an author on the paper, he is a self-proclaimed “bonehead” who studies bone and mineral diseases.

That 2007 work added support for a paradigm shift in thinking about calcification. “I often hear practitioners refer to ‘degenerative’ valve disease, as if the epidemic of [valve calcification] were an inevitable consequence of aging,” wrote coauthor Peter Libby, the Mallinckrodt professor of medicine at Brigham and Women’s Hospital and HMS, in an e-mail. “We counter this fatalistic notion.” The new concept put forth by Aikawa and her colleagues suggests that cardiovascular calcification is regulated at the molecular level and may be controlled by targeted therapeutics.

Aikawa’s current paper illuminates the path by which inflammation leads to calcification. To explore these molecular steps, she applied her imaging tools to a mouse model of chronic renal disease. The progress of calcification in mice prone to atherosclerosis—the model in her 2007 work—takes a year to build up. In mice with both atherosclerosis and end-stage renal failure, it takes just 30 weeks.

According to clinical studies, 50 percent of patients with chronic renal disease will die of heart disease. “Patients with end-stage renal disease are a sort of perfect storm of vascular calcification,” said Towler.

Though Aikawa had found this clinically relevant model to study, she did not immediately know what to look for; a molecular probe can only be effective if the researcher knows which molecules to target. She got her answer while carrying out a routine validation of a new probe for an enzyme called cathepsin S (CatS), which breaks down an elastic protein in connective tissue called elastin. When Aikawa recognized that CatS knockout mice never develop calcification, suggesting that the enzyme might promote it, she thought, “Oh my God, this is exactly what I need.”

When Aikawa used the CatS probe to look for the enzyme in her renal-disease mice, she found that a complex regulatory cycle leads to calcification and that CatS plays a crucial role in driving it.

This vicious cycle begins with inflammation, typically associated with atherosclerosis. Aikawa probed for inflammation by targeting macrophages. As these pro-inflammatory immune cells ingest cholesterol, bloat like balloons and form plaques, they also take up the fluorescent probes, illuminating the inflamed tissues.

Using the new probe, Aikawa demonstrated that the macrophages also produce CatS activity, which degrades elastin in the artery and valve. The elastin fragments are bioactive molecules that attract more macrophages, and these macrophages produce more CatS. “It’s a positive feedback loop,” said Towler.

When the cycle reaches a tipping point, it begins to affect the smooth muscle cells in the artery or the myofibroblasts in the valves. According to a third probe that targets calcification, these heart tissue cells start to behave like bone cells. They accumulate calcium and crystallize it into bony masses.

In mice with chronic renal disease, the process accelerates. Excess phosphate in the blood combines with calcium to form bone. In Aikawa’s study, mice with chronic renal disease that lack CatS developed less calcification, suggesting that a CatS inhibitor might prevent calcification.

By watching this cycle progress in stages, Aikawa made a crucial observation. Calcification is preceded by an early, presymptomatic stage. During this stage, when cells in the arteries and valves begin to act like bone cells, they are able to deposit calcium crystals, but they do not. “This is where we can introduce therapy that reprograms the cells back,” said Aikawa. Later, after the cells start to deposit crystals, “calcification is difficult to reverse.”

Though no such therapy has been identified yet, statins seemed a promising option for nipping calcification in the bud. But in a 2008 clinical trial published in The New England Journal of Medicine, statins did not significantly slow calcification. Aikawa hypothesizes that patients selected for this trial may have already progressed beyond the optimal time for intervention.

At present, there is no way to test this hypothesis, however. Imaging tools in use today in the clinic cannot detect these earliest stages of calcification. Moreover, the imaging tools Aikawa uses are not yet approved for use in humans. “It is hoped that some of these tools will be tested clinically in the next year,” wrote senior author Ralph Weissleder, director of the Molecular Imaging Center at MGH, in an e-mail.

Meanwhile, Aikawa is applying the tools to another disease that increases the risk of calcification: diabetes.

Students may contact Elena Aikawa at eaikawa@mgh.harvard.edu for more information.

Conflict Disclosure: Senior author Ralph Weissleder and co-author Farouc A. Jaffer hold shares of VisEn Medical, Woburn, Mass., maker of the OsteoSense 680 probe, which detects calcification.

Funding Sources: The Donald W. Reynolds Foundation, the Translational Program of Excellence in Nanotechnology, the Leducq Foundation, and the American Heart Association; the content of the work is the responsibility solely of the authors.