By David N. Leff
Science Editor
Being a couch potato and smoking cigarettes, observed vascular biologist Una Ryan, is hard to put into an animal model of atherosclerosis. Hypertension, she added, is known to be an additional risk factor.
Ryan is president and CEO of Avant Immunotherapeutics Inc. in Needham, Mass., and a research professor at the Whitaker Cardiovascular Institute of Boston University (BU).
Three years ago, she recalled, I was talking with Vicky Herrera [also of Whitaker] about how interesting it would be to look at two risk factors simultaneously, to develop an animal model that was larger than a transgenic mouse.
Herrera picks up the story. Avant wanted to develop a vaccine against CETP [a pro-atherogenic enzyme] and had been working with rabbits, she told BioWorld Today. I said to Dr. Ryan, `I know a way to give you a better animal model than rabbits, to facilitate your atherosclerosis studies. I proposed to her that we would make a transgenic model in the rat. And she said, `Well put our human CETP gene into your rat, so that when Avant develops the vaccine, we can test it in that animal model.
Pediatric cardiologist Victoria Herrera is lead author of a research paper in the December 1999 issue of Nature Medicine. Its title: Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival in Dahl salt-sensitive hypertensive rats transgenic for human cholesteryl ester transfer protein [CETP]. Its senior author is molecular geneticist Nelson Ruiz-Opazo, associate professor of medicine at BU; Ryan is a co-author.
This animal model, Ruiz-Opazo pointed out, combines for the first time hypertension and a pro-atherogenic component the CETP enzyme. The human CETP gene, he went on, resides on the long arm of chromosome 16, and is expressed mainly in the liver and intestine. The normal physiological function of the enzyme is still not well characterized. It promotes the interchange of cholesterol between different lipoprotein particles.
In fact, controversy continues in the cardiovascular research community as to whether CETP promotes atherosclerosis or protects against it. The co-authors of the Nature Medicine paper hope their rat model will lay this debate to rest.
High-Pressure Rats Risk Heart Disease
Herrera recounted how she and her team constructed their transgenic rat model: We took the human CETP cDNA, linked it up to an apolipoprotein promoter gene, which would express it in the rats liver, and copy what it does in humans. In in vitro tissue cell culture, it showed it could produce protein. So we injected the construct into one-cell rat embryos, as is usual in transgenesis. In the rat, she pointed out, its a little bit trickier than in the mouse; its more like witchcraft to do transgenesis in rats.
The reason we chose the Dahl salt-sensitive hypertensive rat, Herrera continued, is because we wanted a really robust atherosclerosis coronary heart disease model, and we knew that in humans hypertension worsens coronary heart disease, including myocardial infarctions and other hallmarks.
The co-authors developed four lines of their transgenic rats two low-expressers, two high-expressers. The difference was in the gene copy number. Each rat line retained a certain number of the genes the team introduced.
At six months of age, the rat models displayed the human features of fulminating atherosclerosis: Their cholesterol was high, Herrera recounted, the triglycerides were high, and HDL was low. Then we looked in their hearts for coronary artery atherosclerotic lesions. And we found lesions that were advanced in the sense they would stick out into the lumen of the blood vessel and partially occlude it. It was simulating the descriptions of human lesions, and thats what got us really excited.
Beyond what we have just published, Herrera said, they looked as if they were following what the human lesions are doing, including thrombosis. This distinguished the rats from current mouse atherosclerosis models, in the sense that they developed myocardial infarctions, and decreased survival.
So we predicted that these rats were likely to die earlier than their hypertensive, but non-transgenic control, littermates. And that is exactly what happened decreased survival. A lot of them died suddenly, but we were able to monitor and catch them when they started getting too distressed. Then we documented that we actually did see large occluded arteries with thrombosed atherosclerotic lesions. So we know from pathological evidence that they died of coronary artery disease rather than terminal hypertension
Team Welcomes Allies In Industry, Academia
The groups growing colony of human atherosclerotic rat models, Herrera said, will be made generally available to all academic investigators. Were open to collaborations, Herrera observed. In fact I sought collaborators while we were still characterizing the model, because were new in the atherosclerosis field; were hypertension people. If its going to take collaborations with both academia and industry, why not?
Commercially, she disclosed, BU has made an agreement with BioModels Inc. of Wellesley, Mass., with which we are forming a separate little entity called NoMI LLC. We signed the agreement late last week. NoMI stands for `No myocardial infarctions!
There are a lot of things to be done with this model, Herrera continued. Combining as it does hypertension and coronary heart disease, I see it mainly for its impact on human atherosclerosis. I think this model will really facilitate the gene target identification, which will then enable drug discovery.
We developed the animal model with Avant for their vaccine program, Herrera recalled. Our rats provided them with proof of concept that CETP is a good target. Now Avant has released us, and given BU the freedom to do whatever we went to do to commercialize this animal model.
Meanwhile, Avant recently began a single-dose Phase I safety trial of its vaccine. There are 48 volunteers in this whole study, Ryan told BioWorld Today. We have four dosage groups, and are now into the third one. (See BioWorld Today, April 20, 1999, p. 1.)