Genetic explorers

A link between clogged arteries and uterine fibroids?

Uterine fibroids affect more than one in five American women, and more than 200,000 will have hysterectomies each year because of this painful, chronic condition. Another one million Americans die because of heart and blood vessel diseases, and nearly two million will undergo surgical procedures to widen blocked arteries. Re-clogging of the arteries will occur in several hundred thousand of these patients.

The CCN5 genetic research team, from left, Andrew Lake, John Castellot, professor of anatomy and cellular biology, Jennifer Jones and Holly Mason. © Mark Morelli

But a gene identified in a Tufts laboratory may some day help avoid both these conditions—the growth of fibroids in the uterine lining and the re-clogging of blood vessels (a process called restenosis) after surgery to unblock them.

The gene, CCN5, was discovered in 1997 by Laurie Delmolino when she was a graduate student working in the laboratory of John Castellot, professor of anatomy and cellular biology and of pharmacology and experimental therapeutics.

Delmolino, now at Tufts-New England Medical Center's radiation oncology department, was trying to understand the mechanisms of heparin, a drug widely prescribed to prevent blood clots that also has been shown to reduce the incidence of restenosis in animal models.

She found that heparin activates a particular gene—CCN5—in arterial smooth muscle cells. The protein expressed by this gene stops the proliferation of those cells in laboratory cultures. The protein since has been identified in large quantities in other smooth muscle-rich organs, in particular the uterus.

A family
CCN5 is part of an emerging family of genes involved in the regulation of cell proliferation, motility (movement) and adhesion. So far, six genes in this family have been identified in different labs and given a variety of names. At a conference in France last fall, a group of CCN researchers decided on a collective nomenclature for the family—CCN 1-6—combining previous names (Cystein-rich protein-61, Connective tissue growth factor and Nephroblastoma overexpressed gene).

CCN research, which is under way in about 25 labs around the world, including Castellot's, eventually may yield insight into a variety of pathologies besides restenosis and uterine fibroids. These include persistent pulmonary hypertension of the newborn, thickening of the valve between the stomach and small intestine and thickening of the tube carrying urine from the bladder.

The CCN family also offers potential insights into conditions as diverse as cancer, wound healing and fibrotic diseases such as scleroderma and liver fibrosis. In fact, it may even yield important information about embryonic development. Jennifer Jones, an M.D./Ph.D. student in Castellot's laboratory, is investigating the expression of CCN5 in developing embryos.

A preventive therapy?
The Castellot lab initially focused its attention on whether CCN5 might be used to prevent restenosis, the cause of serious problems in about 20 percent of the two million patients who have vascular surgery annually in the United States.

Whenever the delicate endothelial tissue lining a blood vessel is damaged, as it frequently is in surgery, the smooth muscle cells underneath it begin to proliferate. It can take up to two weeks for the endothelial tissue to heal. During that time, smooth muscle cells often produce a lump of tissue that can lead to a re-clogging of the vessel.

"Many methods—including laser-burning, 'roto-rooter' approaches and angioplasty—have been used to unblock blood vessels," says Castellot. "But all carry the risk of restenosis."

The value of CCN5, he says, is that it appears to be specific to smooth muscle cells. In addition, it could be delivered in a fairly non-invasive manner—during an angioplasty, for example—making it a possible treatment to reduce restenosis. Castellot plans to test this by doing angioplasties in rats and in transgenic mice that either lack the CCN5 gene or make too much of it.

But right now, members of Castellot's laboratory just want to learn as much as they can about CCN5. For instance, Ph.D. candidate Andrew Lake found that CCN5 is secreted by smooth muscle cells and that it adheres tightly to the cells' membranes, probably affecting the matrix, or the material surrounding cells. His work was the cover story of the January 2003 issue of The American Journal of Pathology.

From cardiovascular system to reproductive system
Castellot says he was surprised when Holly Mason, an M.D./Ph.D. student, told him of the extent of uterine fibroids, which are basically benign tumors of smooth muscle cells.

"Between 15 and 20 percent of women have clinically significant fibroids, a figure that increases to 50 to 75 percent among black women," says Castellot. "I had thought about the idea that CCN5 might play a role in uterine fibroids, but I had no idea the problem was as widespread as it is."

Mason decided to focus on uterine fibroids for her doctoral research. Using sets of human tissue samples, she is controlling for individual genetic differences by comparing normal and fibroid tissue samples from the same patient. "So far, it appears that CCN5 inhibits proliferation and motility in both sets of smooth muscle cells—normal and fibroid," says Mason.

Castellot would like to move the research from in vitro to in vivo, possibly using the same sets of human uterine tissue in a special mouse that lacks the ability to reject foreign tissue. Other researchers working in his laboratory include Beatriz Oliveira and Tamar Aprahamian.