Blood clots are the bane of human existence. They cause heart attacks and strokes, and they cut off blood supply to the legs. Physicians refer to the process of their formation as thrombosis. Scientists have long studied vascular biology in an attempt to prevent thrombosis and treat its consequences, yet it remains the culprit for the leading cause of death in the Western world. Mayo cardiovascular researchers broadened their studies of vascular biology about five years ago after discovering that thrombosis is influenced by the interaction of cells present in the vessel wall and those circulating in the bloodstream. "The major focus of our lab is to understand the biological process that narrows blood vessels, both in artherosclerosis and in re-stenosis," says Dr. Simari. "Our aim is to take the biological clues that we learn from our research and use them to generate biological markers and treatments that can predict, prevent and treat thrombosis at the site of vascular injury."

Which Blood Cells Will Become Vascular Cells?

Dr. Simari's lab is on a quest to isolate true endothelial vascular progenitor cells from the blood stream. Why? Because these blood cells have the ability to become cells that make up vessel walls and would be ideal candidates for applying to an injured vessel to promote its healing. Dr. Simari has published studies disputing the claims of other groups who say they have isolated such cells.

"We have shown that what is referred to as endothelial progenitor cells in vitro is neither endothelial or progenitor," explains Dr. Simari." They are not proliferative and cannot, therefore, be progenitor. And they are not fully endothelial because they are primarily of myelomonocytic lineage."

Unsatisfied with these results, Dr. Simari's lab continues searching cell surface markers on circulating cells in the quest for providing true progenitor cells for a treatment they call a vascular bandage

Direct Cell Delivery: The Vascular Bandage

Every time there's injury to a blood vessel, whether the result of artherosclerosis, surgical grafting or from a balloon injury during angioplasty, endothelial cells are injured. Since endothelial cells work to heal the injured vessel, Dr. Simari speculates that a good supply of them would heal the vessel faster. But where to get them?

"There is a large population of cells that can become endothelial in culture that's different than becoming endothelial in a living organism," says Dr. Simari. "In animal studies, we removed cells from animal models of vascular injury, turned them into endothelial cells in culture then redelivered each animal's own cells directly to the site of vessel injury."

Such studies require close collaboration with Mayo's translational research and graft engineering team; stem cell biologists, Stanimir Vuk-Pavlovic, Ph.D., and Allan Dietz, Ph.D., and hematologist, Dennis Gastineau, M.D., who are experts in isolating blood cells and modifying them in culture. The team supplies technical support to facilitate the translational component-solving the practical problems required to manufacture cells for use in the treatment of humans.

"Reagents that are used to prepare cellular products for laboratory research are usually unsuitable for use in human treatments," says Dr. Vuk-Pavlovic. "Our role, which will increase once Dr. Simari's project goes to clinical trial, is to produce FDA-certifiable cells. Such cells must be engineered in the absence of animal products and other reagents that do not meet clinical and industry standards."

Biologic Devices: Coating Stents

One risk of metallic devices such as stents, mechanical valves and heart assist devices is that blood clots form on them. When a metal stent is placed in a diseased vessel to keep it open, it is followed by a high risk period for blood clot formation of six weeks-the time it takes for cells to grow over it. If this time period could be shortened, it would make the stent less susceptible to clotting and perhaps lessen the need for anticoagulant therapy.

Gupreet Sandhu, M.D., Ph.D., is pioneering the development of stents with magnetic properties that promote healing without forming blood clots. In collaboration with Mayo Medical Ventures, Dr. Sandhu has developed stents that contain metal, a carrier and a magnetic material.

"Our intent is to magnetize the stent and place a magnetic particle in the cell so that it will be attracted to the surface of the stent," says Dr. Sandhu. "Metallic particles are commonly used for identifying tissues during magnetic resonance scans to track tissue cells. It's exciting to anticipate being able to induce coating of the stent within six minutes instead of six weeks."

The group thinks of this as a cellular bandage because, like a bandage, the cells are temporary-they help the wound heal without becoming part of it. Cells make factors that cause normal cells to grow and replace them.

If all goes well, the technique could also be used on synthetic grafts and lead to a new generation of cardiac surgical devices.

"The idea would be to weave in a treated fiber that would encourage the growth of cells to coat the graft," says Dr. Simari. "It would revolutionize cardiac surgery if we could develop a biologic therapy for a 4 mm synthetic graft for a bypass that would resist clotting."

How the Endothelium Regulates Thrombosis

The Simari lab's interest in how vessels heal, led the group to try to understand the role of blood clotting in vessel healing. They theorize that an imbalance within diseased vessels predisposes the formation of thrombosis. They have identified a key player, tissue factor pathway inhibitor (TFPI)-a protein produced in vascular cells that inhibits tissue factor, which is the primary initiator of thrombosis in the vessel wall. The endothelium is thought to be the site of TFPI regulation, which is expressed in relatively minor amounts in diseased vessels when compared to its protagonist tissue factor.

Through a series of studies designed to further the understanding of how the endothelium regulates blood clotting, the group has shown that they can increase or decrease TFPI expression and regulate important processes in the vasculature, including thrombosis, re-stenosis and artherosclerosis. To study these diseased states, they collaborated with molecular biologist, Jan van Deursen, Ph.D., to develop genetically modified mice and methods of genetic overexpression.

"We floxed the TFPI locus, which means we put in genetic elements on either side of the mouse gene so that TFPI expression in tissues could be specifically deleted," explains Dr. Simari. "For the first time, we have a mouse that has no endothelial TFPI that we can use to understand the biology. It's been really exciting."

The group studies several models of artherosclerosis and thrombosis to see if lower levels of endothelial TFPI affect the development of the two conditions.

"We have a broad range of projects in our lab and are getting some very exciting results," says Dr. Simari. "There has never been a better time to be involved in biomedical research."