March 2, 2000
Mutant gene may curb vascular disease
By A.J.S. Rayl
With medical adviser Stephen A. Shoop, M.D.
A Doctor In Your House.com
Giovanni Pomaroli could have had no idea he was destined for a place in medical science history. In fact, when he was born back in 1780 in Limone sul Garda, Italy, medical science was only just beginning.
But now - 220 years later - Pomaroli has turned out to be the catalyst of potentially revolutionary preventive advances which could save lives all over the world.
A mutation believed to have first occurred in one of Pomaroli's genes is the reason.
His descendants who have inherited the mutant gene are apparently protected from cholesterol-related heart disease and strokes, even though some of them have had less-than-healthy lifestyles.
Now, a team of physician-researchers headed up by Dr. P.K. Shah, director of cardiology at Cedars-Sinai Medical Center in Los Angeles, is working to transform that discovery into new treatments and strategies that will protect others from heart disease and strokes.
Surprising findings
The discovery process began in 1980 when one of Pomaroli's descendants, a male in his late 40s, went to the doctor because of irritable bowel syndrome. A routine blood test revealed an extremely high triglyceride level and a very low HDL (good cholesterol) level.
This type of lipid imbalance is ordinarily associated with premature coronary disease. Lipids are any of the free fatty acid fractions in the blood - including cholesterol, neutral fat, and triglycerides - which are elevated in various diseases such as atherosclerosis.
But Pomaroli's descendant showed absolutely no signs of vascular disease. It was such an uncanny profile that his physician referred him to Dr. Cesare Sirtori and Dr. Guido Franceschini, lipid researchers at the University of Milan. They confirmed the findings and agreed that the profile was quite remarkable.
At that point, they didn't know what they were dealing with. "But they suspected there might be a genetic defect," says Shah.
So Sirtori and Franceschini sent the samples for a more detailed analysis to Dr. Karl H. Weisgraber at the Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco (UCSF). It was there the genetic mutation was verified.
"They found the Apolipoprotein A-1 protein was different from the normal Apo A-1," says Shah. Apo A-1 is a major structural component of HDL cholesterol that serves to protect against vascular disease.
The mutated Apo A-1 protein differed from the normal Apo A-1 by a single amino acid at position 173. Every protein is comprised of amino acids that are numbered from one end to the other. In the Apo A-1 protein, there are 243 amino acids.
"Using standard techniques for mutation analysis, they discovered the normal amino acid, arginine, was replaced by a different amino acid called cysteine," Shah explains.
That information in hand, Sirtori and Franceschini researched church records, tracing the bloodline and the mutation back to Giovanni Pomaroli. Subsequently, they screened about 1,000 individuals and found that 35-40 relatives had the identical lipid profile - low HDL, high triglycerides, and no signs of vascular disease.
What they didn't know then was whether this mutation was actually conferring protective effects or if this was just an incidental finding.
Promising protein
Enter Shah, who has been named in various national peer surveys as one of the best cardiologists in the USA.
In 1992, Shah had become interested in HDL as a way of reversing heart disease. "Looking around the literature, I found out this mutant had been described, and there was no family history of coronary disease, and there was history of longevity," he recalls. Intrigued, he started making inquiries.
Shah found that Pharmacia-Upjohn had actually created an artificial copy of this mutant gene. Using genetic engineering techniques, they had begun to produce the AP-1 Milano protein in the laboratory.
"I got in touch with the company's scientific director," recalls Shah, "and asked if they would be interested in collaborating with my laboratory to determine if this protein was actually a more efficient form of HDL for vascular disease."
The company was very interested, and provided the protein linked to a lipid carrier, simulating an HDL-like particle. Before long, Shah was headlong into research.
He and Dr. Jan Nilsson set up the initial protocol to investigate the protein in rabbit models at Cedars-Sinai. They injected the protein intravenously into rabbits whose arteries had been injured to promote a rapid buildup of plaque via a high cholesterol diet.
"We were pleasantly surprised to find that these rabbits had about 70% less plaque buildup, even though their cholesterol levels remained in the 700-800 range," Shah reports. This was the first time anyone anywhere had described an intervention that would block the effects of cholesterol on arteries without changing cholesterol levels. They published their results in the journal Circulation in 1994.
Shah and colleagues Dr. Bojan Cercek and Dr. Sanjay Kaul then conducted additional studies in genetically altered mice that had very high cholesterol levels ranging from 1,000-2,000. "We gave them 18 injections of this same preparation intravenously, and were either able to halt the progression of atherosclerosis or in very high doses able to reverse it," Shah says. That study was published two years ago, also in Circulation.
At that point, Shah says, "We felt that we had something that could have a significant impact . . . a very exciting new way to look at atherosclerosis and its reversal or stabilization. We were thrilled."
Protein power
They discovered that the mutation causes both a structural and a functional change in HDL. "In fact, two molecules link together and create what we call dimers," explains Shah. "We think the dimerization may play a role in changing the function of the Apo A-1 Milano protein."
The altered protein shares three functional effects of HDL. Shah explains:
"It promotes removal of cholesterol from the arteries. Called 'reverse cholesterol transport,' it extracts the cholesterol out of the arteries and takes it to the liver where it is disposed."
"It has anti-inflammatory effects. We know that inflammation plays a critical role in the initiation and progression of atherosclerosis, so this effect may be even more important than its cholesterol-depleting effect."
"It has potent antioxidant effects."
"We think that maybe the AP-1 Milano protein has multiple potential mechanisms of actions that could all produce a favorable affect on the blood vessels," Shah says.
For example, under Shah's direction, Kaul is currently conducting further studies in pigs to see if restenosis (reblocking) after angioplasty and stenting can be prevented. "Whenever we do coronary angioplasty or stenting, about 20%-30% of the time the arteries reblock within 6-9 months," Shah explains. Preliminary results in the pilot studies are promising.
Trials ahead
"The ultimate goal is to bring this intervention into clinical testing using the recombinant protein linked to a lipid carrier in humans," says Shah. He hopes to begin testing within the next year or so.
A key goal of the trials will be to determine how often the protein must be administered to achieve therapeutic effect, depending upon which condition is being treated.
Ideally, a one-time treatment during angioplasty would do the trick. More likely, patients whose heart disease resists today's most effective therapies would probably receive a treatment once a week - maybe once every two weeks - to prevent progression or maybe even reverse it.
The therapy might also help certain patients, such as those with familial hypercholesterolemia, who find the current therapies are problematic. "Even using plasma apheresis to remove their LDL cholesterol is still a pain in the neck. Every two to three weeks they have to go on a dialysis type machine for 3-4 hours, which is expensive," says Shah. "If we could achieve the same thing with an intravenous injection every three weeks, then that could be potentially very exciting for people like this who have no other way to go."
Gene therapy
The long-range goal is gene therapy - inserting the gene itself into humans so they can produce their own A-1 Milano.
In recent weeks, the death of a human patient in a gene therapy trial has turned the still-experimental procedure into a hotly debated and controversial issue. "Gene therapy has not yet fulfilled its promise, partly because we have not really figured out an effective and safe way of delivering the genes," offers Shah. But virtually no one in science and medicine views that as an insurmountable problem.
Shah is proceeding on a five-year gene therapy protocol with funding from the National Heart, Lung, and Blood Institute (NHLBI). "We're taking a unique type of virus - the adeno-associated virus - and using it as a vector to deliver the gene," he says.
Shah, Kaul and Dr. Tripathi Rajavashisdh have established a collaboration with Dr. K.K. Wong and Dr. Saswati Chatterjee from the City of Hope, who have been successfully testing this approach to deliver treatments for other diseases, including cancer and AIDS.
So far, the gene therapy work has primarily been carried out in tissue cultures, but, says Shah, "we're moving rapidly to mice and pigs." Once they're able to establish its efficacy and safety, they'll move to human trials.
"The potential is very exciting," says Shah. "There are many things we must do before we can say this is really ready for human testing." He projects such testing is "realistically about five years away."
"We are cautiously optimistic," says Shah. "The proof, as always, remains in the pudding."
If the pudding does provide the proof, what a dessert this mutant gene will be.
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