By David N. Leff
Human beings aren't the only mammals threatened with atherosclerosis.
Meet the Watanabe rabbit, which is born with familial hypercholesterolemia (FH). This means that the homozygotic animal has inherited from both parents a mutant gene that deprives it of the receptor for low-density lipoprotein (LDL) needed to expel surplus cholesterol from its blood.
Instead, that lipid piles up in their coronary arteries and aorta, until atherosclerotic plaques dam them up, and starve the beating heart of oxygen-laden blood. These rabbits have a deletion in their LDL receptor gene, which prevents it from binding to LDL in the liver.
This genetic fate makes Watanabe rabbits ideal models for studying FH, even though precious few humans come into the world with the homozygous (two-mutant) form of the disease. A scant 100 cases of homozygous FH exist in the U.S. Most of them are children, doomed to an early death from myocardial infarction. (See BioWorld Today, Nov. 20, 1995, p. 1.)
Heterozygous FH -- in which only one parent passes on the defect -- is another, less lethal, story. Lacking only one LDL receptor, it deposits less excess cholesterol, so it takes years to reach the climax of heart attack.
"About one American in 500 is heterozygous for the mutant LDL receptor gene," said cell biologist Albert Edge. Of all patients who sustain their first heart attack before age 60, he told BioWorld Today, 5 percent owe their cardiovascular event to heterozygous FH.
Lipid-lowering drugs mitigate their danger of atherosclerosis, he pointed out, citing a recent survey in which these compounds decreased total cholesterol build-up by 25 percent.
Edge is senior director of molecular and cellular biology at Diacrin Inc., in Charlestown, Mass., and senior author of an article in Nature Medicine for January 1997. Its title: "Reduction of serum cholesterol in Watanabe rabbits by xenogeneic hepatocellular transplantation."
Homozygous FH is all but untreatable; the only therapy is to replace the patient's liver, harboring the double-mutant LDL genes, with a healthy donor liver transplant.
At Diacrin, Edge and his co-authors are trying a less dangerous and onerous alternative: giving donor pig liver cells instead of the entire organ to a series of Watanabe rabbits.
"We transplanted the porcine cells, about 100 million per treatment," he said, "by infusing them slowly into the portal vein, which supplies blood to the liver. The trick was to get them to seed into the organ's hepatic parenchyma. These are the plates of liver cells where all the hepatocytes reside, and carry out their metabolic processes."
Within two days of the infusion, the team could detect the donor porcine cells integrating into their new host's organ, of which they represented as much as 2 percent of the native hepatocytes
But that small fraction of cells, bearing the LDL receptor gene, was sufficient to decrease the enormous cholesterol build-up in those homozygous FH animals by a good 40 percent -- on par with what drugs such as Lovastatin accomplish in heterozygous patients. And the rabbits maintained their functioning LDL receptor for a full 100 days, the duration of the trial.
"That result was very encouraging," Edge observed, "because it means that the pig cells can actually enter into metabolism alongside the rabbit hepatocytes. And that's what we attribute the cholesterol lowering to. The cells are able to actually bind LDL, pick it up, metabolize it and then -- although we don't have direct evidence for this -- it appears that they are also excreting the cholesterol steroid nucleus as bile."
This is the final step in scavenging cholesterol from the blood and removing it from the body.
Diacrin must still overcome one obstacle -- graft rejection -- before its porcine hepatocyte transplantation strategy is ready for human trials.
To manage donor graft rejection by the rabbit's immune system, it added cyclosporin.
Transplanting cells rather than whole organs, he added, does seem to "have something going for it. Hyperacute rejection [HAR] could potentially be a problem for vascularized organs rather than liver cells. It's cells in blood-vessel walls that pull the trigger on HAR. But the jury's not in on that yet."
Edge's group has already advanced from rabbits to monkeys in trying their porcine hepatocyte transplant protocol.
First Aid Against Liver Killers
"Our target for the first patients would be acute, fulminant liver failure. The reason is," he explained, "that these people are critically ill and in imminent danger of dying. They don't really have any good alternative, other than a whole-organ liver transplant, which is hard to come by."
Besides mushroom poisoning, which is a liver-killer, Edge cited such candidate diseases as acute hepatitis, alcoholic liver disease and Tylenol poisoning.
He expects to start his first clinical trials "within a year."
Porcine donor hepatocytes, he pointed out, would greatly relieve that shortage. It could treat people suffering from such metabolic liver deficiencies as PKU (phenylketonuria) and Crigler-Najjar disease.
But Edge and Diacrin are thinking farther into the future, applying their pig liver experience to such approaches as repairing damaged and dead heart muscle with porcine cardiomyocytes.
The company already has Phase I trials well advanced for treating Parkinson's and Huntington's diseases with pig-derived neurons. (See BioWorld Today, Jan. 2, 1996, p. 1.) *