Researchers find that unique marks are based on genetics

By Susan Peterson

Gazette Staff

Nathaniel Hawthorne's short story The Birthmark tells of a young doctor's obsession with removing a small red "hand print" mark from his beautiful wife's face. It is a story not so much about appearances, as of pride gone awry -- the woman dies after drinking the potion her husband prescribes to fade the imperfection.

Now we know from recent research at Harvard that beauty -- though still in the eyes of the beholder -- really is more than skin deep.

Locked inside certain genes are the signals that tell cells what to do next. These signals tell a cell where to go when building blood vessels, and to follow certain pathways through muscle, bones, and internal organs.

But sometimes, cells don't get the right message and they go to the wrong place -- or not at all -- and do not develop proper arteries, veins, and capillaries. This is what happens when a vascular birthmark is born. These improperly formed vessels are called vascular malformations and can be as small as a capillary blush on the cheek, to tangles of large vessels that can grow and disfigure, and are sometimes fatal to a child.

A gene has been discovered that is linked to venous malformations, deforming growths that enlarge with age and can invade neighboring tissues in a person's body. This is the most common form of vascular malformation. They can occur sporadically, or be hereditary and appear at birth or later in life.

Bjorn Olsen, Harvard Forsyth Professor of Oral Biology, along with a group of researchers, has identified this gene, which passes on the susceptibility in certain families to develop venous malformations. With this new knowledge, the researchers plan to find out how the cells that make up blood vessels communicate in order to better understand why vascular birthmarks occur and how blood vessels are formed.

"As a surgeon, I started this research with Bjorn because I wanted to know why venous malformations appear, and try to understand the cause of this disorder," said John Mulliken, a plastic surgeon specializing in vascular anomalies at Children's Hospital. "There is a limitation to what we can do for venous malformations right now, and they tend to come back after conventional treatment."

Learning more about birthmarks at a genetic level can enable future treatment to begin at an earlier stage, and could shed light on hyperactive cell growth that can cause cancerous tumors and some diabetics to become blind.

From the cell up

Olsen's laboratory specializes in organogenesis, the processes that genes and molecules go through to form organs. For years, his lab studied bones and the skeletal system. Then, Olsen and some colleagues became interested in how blood vessels form and what determines vascular patterns in a developing organism.

"Together, with a developing skeleton, this determines to a very large extent the final anatomy of animals -- including humans," Olsen said. "The more we learn about blood vessels -- or veins -- and what the important molecules and cell interactions are that make them, the more we'll be able to interfere with their formation or correct blood vessel abnormalities."

Olsen and Mulliken wanted to learn how the different cell types in blood vessels communicate. How do these cells know how to make an artery or a vein? What gives them their direction? What happens in the process? To answer these questions, they started looking at specific genes that express themselves in abnormal vessels.

There are two major cell types in an artery and a vein: the endothelium and smooth muscle cells. The endothelium form a single layer of cells lining a vein, representing a barrier to the blood. Outside of these cells, in the blood vessel wall itself, are smooth muscle cells. Arteries have many layers of smooth muscle cells, and the veins have few layers of muscle cells.

About the time Olsen and Mulliken were beginning to investigate these questions, a surgeon from Belgium, Laurence Boon, came to study plastic surgery under Mulliken. Boon also wanted to do some research, so Mulliken arranged for her to work in Olsen's laboratory on a project related to venous malformations.

Mulliken knew about a family that had an inherited form of venous malformations, and he and Olsen wanted to find the gene that triggers the abnormalities. Current methods of treating the large, jumbled masses of abnormal veins by operations or injections do not always produce lasting results.

"If we have the gene and the mutation, we can engineer a mouse with a venous malformation and study those tissues to see why they come back," explained Mulliken.

At the same time, another postdoctoral researcher who had experience mapping genes, Miikka Vikkula, arrived from Finland to work in Olsen's lab. In only six months, Boon and Vikkula located the defective gene on chromosome nine. But the next step was to find the exact region on the chromosome where the gene sits. To do this, they needed more families who had hereditary venous birthmarks.

Mulliken's clinical experience and knowledge of patients with birthmarks provided access to another family willing to help with the genetic research. By coincidence, this new family and the family already being tested, were related. Armed with this new pool of genetic data, the researchers were able to find the gene, a receptor kinase called TIE-2, which is specific for signaling to endothelial cells.

Olsen and his group sequenced this gene in the unaffected and affected members of the family, and found the affected members had a single, small difference in one part of their DNA on chromosome nine.

"Our question then became whether this change really caused the disease," Olsen explained.

To confirm that this change in DNA really was responsible for an inherited form of vascular birthmarks, Olsen's group collaborated with a postdoctoral researcher, Kermit Carraway, in the laboratory of Lewis Cantley, a professor in the Department of Cell Biology. By inserting the gene into insect cells, they made the protein with and without the specific change. Carraway analyzed the activity of the protein and was able to show that the change in the protein increased its activity compared to the normal protein.

"A signaling increase like this would be very significant," Olsen said. "The increased signaling would alter the characteristics of the endothelial cells."

But the final proof of this genetic link to vascular birthmarks came through another collaboration, with Douglas Marchuk, a researcher in the Department of Genetics in Duke University's Medical Center. Marchuk was working with a family with similar venous malformations, and Boon and Vikkula had helped Marchuk link the defect in the family to chromosome nine. Soon it was shown that Marchuk's family had the same mutation in the same gene.

Now the defect could be explained on the molecular level. But Olsen was still concerned about the dilated, multiple channels of endothelial cells in defective veins, taken from biopsies and seen through a microscope. Somehow the surrounding muscle cells seemed to be having a problem listening to the endothelial cells.

"The defect appeared to be an inability to get smooth muscle cells to come to the vein wall," Olsen said.

"Our prediction is that this mutation, through some feedback loop which is still not really known, prevents the communication between endothelial cells and smooth muscle cells," Olsen said. "We do know that this receptor is hyperactive in these families."

That hyperactivity, he said, causes the mass of improperly developed veins.

Olsen explained that venous malformations in other families might be linked to different genes that have not yet been identified. But the mutation discovered in the first two families, does give a headstart down the road to future treatment beyond current injection therapy and surgical removal.

"Through this type of research, we learn a great deal of fundamental biology, and at the same time, there's hope that the specific genetic disorder these families have might be better treated based on what we learn," Olsen said.

But the story doesn't end here, because there are two happy endings.

Unlike Hawthorne's doctor and his wife, this is more than a story of research and love gone astray. Not only did Vikkula and Boon combine their skills to help locate the gene -- but along the way, they fell in love. They'll be married in Belgium this summer, and Mulliken will be giving the bride away.