Volume 16 Number 4, SUMMER 1999

branching out

by ruthann richter

More than a decade ago, when he was working in his lab at Harvard Medical School, Stanford cardiologist Tom Quertermous, MD, turned his interest to a seemingly obscure biological process that few scientists considered worthy of attention.

There were perhaps a dozen labs in the country at that time that paid any mind at all to this process, known as angiogenesis -- the body's natural way of growing blood vessels. When studying angiogenesis first struck his fancy, Quertermous could hardly know that the process would become one of medicine's hottest topics, the intense focus of researchers at hundreds of academic and commercial labs, all betting that it will open the way to new treatments for heart disease and cancer, among other ailments.

"At that time, there was no hint that angiogenesis might have any clinical application," says Quertermous, Stanford professor and chief of the Division of Cardiovascular Medicine. "There were maybe 10 or 15 labs that cared. Now, of course, everybody cares. Half the world wants to start angiogenesis, and half the world wants to stop it."

Angiogenesis (from the Greek word "angio" for vessel) is the process by which the body grows new blood vessels for various purposes, such as wound healing and menstruation. The developing embryo employs a similar process, known as vasculogenesis, to grow its entire circulatory system, a feat that mice embryos accomplish in just a few days. In theory, if doctors could find a way to imitate this process in a therapeutic setting, their patients could grow small networks of vessels to serve as miniature bypasses, carrying blood to oxygen-hungry hearts or legs. This would allow patients to avoid major procedures, such as coronary artery bypass surgery, and regain the use of damaged limbs.

"Therapeutic angiogenesis is going to change the face of cardiovascular medicine," predicts John Cooke, MD, PhD, Stanford associate professor of medicine. "There will be fewer angioplasties and fewer surgeries as biological bypasses replace them."

Some 30 percent of patients with coronary artery disease, or millions of Americans, ultimately could be treated with the angiogenic drugs, Quertermous says. But that prospect is still several years away.

"It's a brilliant idea, and we've seen enough to know that it will work," he says. "All we need is a good study to point us in the right direction."

The first physician to give any serious thought to angiogenesis was Judah Folkman, MD, a researcher at Boston Children's Hospital, who was subject to ridicule when he first began investigating the process some 25 years ago. Early on, Folkman observed that once a tumor grows beyond a few hundred thousand cells, it begins sending out chemical signals to help build a blood supply. These signals promote the growth of capillaries, small blood vessels that enable the tumor to thrive and spread. Folkman began searching for the chemical switch that set the process in motion and in 1983 his lab identified the first angiogenic growth factor. Folkman's goal was to find a way to turn off this molecule and others like it to halt the progression of cancer. His work ultimately led to the discovery of two factors, called angiostatin and endostatin, which he found to be powerful growth inhibitors, at least in laboratory mice. These widely publicized compounds, while they show promise in animals, have yet to be tested in human patients.

Folkman's early work opened the door to the discovery of dozens of other growth factors, which are structurally different but work in similar ways. Perhaps the most-studied of these factors is

fibroblast growth factor (FGF), a powerful little protein, cloned in 1986. In the early 1990s, scientists in both the United States and Japan began testing a bioengineered version of FGF in laboratory dogs and pigs with constricted blood flow to the heart. When researchers injected the drug into the coronary arteries of the animals, they found evidence of vessel growth and significantly improved blood flow.

With early animal results in hand, researchers in Germany at University Hospital Freiburg launched the first small-scale clinical trial of FGF in 20 patients who had blockages in at least three coronary arteries. The researchers injected FGF into the bottlenecked area and observed the growth of capillaries in and around the injection site within three months. Some of these capillaries functioned as bypasses, new channels for carrying blood to the heart, the researchers reported in February 1998 in the journal Circulation.

In November 1998, Chiron Corp. in Emeryville, Calif., launched the first large, multi-center trial of FGF in 300 heart patients, including 30 or more patients from Stanford and the University of California, San Francisco. Seventy-five percent of the patients in the trial will receive FGF, while the other 25 percent will receive a placebo. To qualify for the experiment, patients must suffer from severe angina, or chest pain caused by arterial blockages, and cannot be candidates for traditional therapy, says Stanley Rockson, MD, Stanford assistant professor of cardiovascular medicine and a principal investigator in the trial.

The treatment involves a relatively simple, one-hour procedure that is performed in the cardiac catheterization lab. Doctors guide a small catheter over a thin wire from the thigh into the chest and infuse the drug for 10 minutes into two separate arteries that feed the heart.

Rockson says the trial is based on the knowledge that growth factors such as FGF have an affinity for tissues that lack oxygen. These oxygen-deprived tissues, like those found in clogged arteries, put out a distress call through receptors that attract the growth factors.

"It's like a big sponge waiting for these factors to arrive," Rockson says. "When we infuse the drug, it theoretically should be completely absorbed in the abnormal area of the heart."

It's conceivable that the injected drug could travel to other parts of the body, he notes. For that reason, patients are carefully screened to be sure that they have no hidden cancer, which could be stimulated by the drug, or any retinal or other eye problems, which also could be aggravated by the treatment. But he believes the risk to patients is very small and notes that there have been no reports thus far of any malignancies related to growth factor treatment.

"I'm very optimistic, not so much on the basis of early results, but because I believe very much in the biology," Rockson says. In three to five years, he predicts, it's likely that some version of angiogenesis will be a routine form of therapy for many heart patients.

"I think angiogenesis is here to stay. It's a question of working out the details of how to utilize its power," he says.

In addition to FGF, another related protein that has been heavily studied is VEGF, or vascular endothelial growth factor. VEGF, first isolated in 1985, acts in a very specific manner, leading new capillaries to grow from existing capillaries. FGF, on the other hand, promotes growth of larger vessels and aids in the growth of other kinds of tissues as well, Rockson says.

In 1998, Genentech Inc. in South San Francisco, Calif., launched the first major VEGF trial involving 178 patients nationwide. All of the patients had severe coronary blockages and were limited in their ability to exercise. Patients received two infusions through a catheter, followed by three intravenous injections of the drug. The researchers tested patient performance on a treadmill before and after they received the drug and looked for changes in the blood supply to the heart muscle on a nuclear medicine scan.

This past February, Genentech announced that the trial results had been disappointing and that there appeared to be no clear benefit to the patients who received the drug, versus those who received a placebo.

"Some patients responded, and some didn't," says Cooke, who was a principal investigator in the trial. "It may be that there were modulating factors, like high cholesterol, that played a role."

Cooke's own research, reported in February at the American College of Cardiology, has shown that there is a specific circulating factor in the blood that can inhibit angiogenesis in patients with high cholesterol. So it could be that patients who have clogged arteries and high cholesterol, which often go hand in hand, may be resistant to angiogenesis, he says.

"You may not be able to make a bypass if you have high cholesterol," he says.

The disappointing trial results also may have been related to the way in which the drug was delivered, Quertermous says. By squirting VEGF down the coronary arteries, he says, much of the drug gets dissipated or hits a dead end in the blocked vessel, so the effects are limited and hard to control.

"It doesn't say angiogenesis won't work," he says. "It says you have to be clever about how you go about it."

He says he believes angiogenic drugs are likely to work best if they are delivered directly into the heart muscle. Simon Stertzer, MD, a Stanford professor of medicine who performed the first balloon angioplasty, agrees. He is working on a new delivery method, a catheter-based system that would guide the material directly into the heart from the inside. He is now testing the system in pigs, using FGF and other agents.

"It is not known yet what delivery system will prevail, but we feel this method makes the most sense," as it gets the drug directly into the area where it is most needed without spillover into the system, Stertzer says.

He says Stanford is one of the few sites in the country where researchers are working on the two critical pieces of angiogenic treatment -- i.e. which growth factor and which delivery mechanism will prove most effective.

"Some people are working on the gun, and some people are working on the bullet," Stertzer says. "At Stanford, we're working on both."

Researchers are also working on gene therapy approaches that could prove to be ideal for getting regulated levels of angiogenic factors into oxygen-starved tissues. Rather than in-

jecting the protein alone, doctors theoretically could insert the gene for the protein into muscle cells in the heart or leg. The muscle then would be primed to pump out the factor over a sustained period of time, and patients would not have to receive repeated infusions.

"Once these molecules are expressed, theoretically you don't need to stimulate growth once it has occurred," Rockson says.

This year, Rockson expects to participate in a VEGF gene therapy trial in patients who are in danger of losing a limb because of severe arterial blockages. These patients typically experience intractable pain and have a limited ability to walk or exercise. The trial will involve use of disabled adenovirus, a common cold virus, as a method to insert the gene into the muscle cells around the clogged vessel. The trial is sponsored by drug maker Parke-Davis.

Part of the difficulty in gene therapy, however, is in regulating the amount of drug that gets delivered. Helen Blau, PhD, and her colleagues in molecular pharmacology used retroviruses as a vector, or delivery vehicle, to inject the VEGF gene into the leg muscles of laboratory mice. Within six weeks after the injection, the researchers were stunned to find that the protein had induced the formation of massive blood vessel growths in the animals' limbs.

"It says clinically that using it blindly and not knowing what dosage you're delivering could be dangerous," says Blau, professor and chair of molecular pharmacology.

Blau has since refined her delivery system, using the drug tetracycline as a tool to regulate the amount of gene that is expressed, giving clinicians the ability to move dosage levels up or down.

"We can give it again, turn it on and off and vary it. So this is really a breakthrough for regulatable systems. It could be used for all kinds of gene therapy," says Blau, who reported the results in December 1998 in the journal Nature Genetics. She has since received hundreds of inquiries from scientists about the vector, she says. She says she hopes to begin clinical trials next year, in collaboration with Quertermous and Cooke, on a regulatable delivery system in patients with arterial blockages in the leg.

While FGF and VEGF remain the most intensely studied of the growth factors, there might be others yet undiscovered that could prove to be even better choices for use as treatments. Quertermous has already identified several new factors which work through different biological pathways than either FGF or VEGF. One of these, which he has dubbed DEL1, is found in the mouse embryo but disappears at birth. It is then reactivated in the adult mouse when a tumor is present, he says. Quertermous, who first identified the factor in 1993, has been studying DEL1 to see what turns it on and what other molecules might be involved in the process. He has both National Institutes of Health and California Cancer Program grants to look at whether DEL1 can new induce new blood vessel growth in mice and whether it can be inhibited to stop cancer progression. If those experiments bear fruit, he could begin trials with the new factor by the end of next year, he says.

"It's unclear which of the factors will have the best potency in a clinical situation," he says. "That's really hard to tell until you get in there and do it."

For now, researchers note, the technology remains very much in its infancy. Only with time and careful scientific inquiry will its promise be proven -- or broken.

"Three years from now," Stertzer says, "angiogenesis will either be very important, or it will be falling by the wayside." SM