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Recruitment of leukocytes to atherosclerotic lesions in vivo

Institution: University of California, San Diego
Investigator(s): Daniel Steinberg, M.D., Ph.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6RT-0136 Award: $352,405
Subject Area: Cardiovascular Disease
Award Type: Research Project Awards

Initial Award Abstract
More cigarette smokers die of a heart attack than die of cancer or any other single cause. The exact sequence of events that link cigarette smoking to an increased susceptibility to athero-sclerosis, the blood vessel disease that triggers the heart attack, remains uncertain but there is some evidence that smoking cigarettes may increase oxidative damage to the cholesterol-carrying molecules, the lipoproteins (more specifically, the low density lipoproteins--LDL). Uptake of oxidized LDL by cells in the artery wall may be the main way in which cholesterol builds up. Another effect of oxidized LDL is to play a part in the recruitment of circulating white blood cells into the damaged artery. While a good deal has been learned about the several molecules to which white blood cells stick and the molecules that can attract them into the artery wall, the relative importance of these has yet to be properly tested in the whole animal. This is because methods have not been available that are sensitive enough and specific enough.

Molecular biologists have developed a remarkable method that makes it possible to make literally millions of exact copies of a gene (DNA) in just a few hours. Although currently available methods for measuring DNA are not nearly sensitive enough to measure the small quantities of DNA present in individual cells, we can easily measure the quantity present in three or four million copies of DNA molecules. This method, known as the polymerase chain reaction (PCR), can be carried out in such a way that only one specific gene in a large mixture of genes will be copied. For example, if two cells have differently constructed forms of a particular gene, we can set things up so that only one form of the gene is copied. The basic trick then is to introduce a known number of white blood cells from a donor animal into the blood of another animal that totally lacks one of the genes present in the donor’s white blood cells. The genes are otherwise as nearly as possible identical. The PCR test is set up so that it will detect only the donor white blood cells. Using this method we have shown that we can detect even one donor white blood cell in the presence of a million other cells and can in fact measure the very small number of white blood cells that enter a particular damaged area of a blood vessel in the course of a day.

Using this novel approach, we believe we will be able to demonstrate for the first time the natural history of the entry of white blood cells into atherosclerotic lesions, determine how long they persist in a lesion, and identify the key molecules that control this entry of cells and their survival. If we could identify the most important molecules involved, there would be the hope of developing drugs that target those molecules and thus slow the process of atherosclerosis. Such drugs could be used in conjunction with cholesterol-lowering regimens and should give additive protection. Finally, we will test the hypothesis that smoking increases the heart attack rate by increasing the oxidation of lipoproteins, as discussed above. We will test this possibility by exposing mice to cigarette smoke just before injecting white cells from a donor and see if the rate of entry of white cells into the lesions is increased. If so, we will then determine whether or not pre-treatment with antioxidants prevents this increase in white blood cell entry into the damaged artery.

Final Report
More cigarette smokers die of a heart attack than die of cancer or any other single cause. Atherosclerosis is the disease of the blood vessels that triggers heart attacks. One of the earliest events in causing damage is the entry of circulating white blood cells (monocytes) into the artery. If we better understood the mechanisms causing monocyte buildup we might be able to block that and slow the progression of the disease. We undertook to study this process in mice using an exquisitely sensitive method (PCR) that can detect just a handful of cells by hugely amplifying their genetic DNA. Our "trick" is to capitalize on the fact that males have some genes that are totally absent from females. We introduce monocytes from a male into a female and then measure with PCR the number of monocytes that have entered the blood vessel wall.

We have demonstrated the feasibility, sensitivity and reproducibility of the method. We developed a new method for purifying monocytes from mouse blood and developed methods for quantifying DNA extracted from atherosclerotic lesions.

Cell surface molecules that may cause monocytes to adhere to the vessel lining have been identified but only under static conditions. One of our first studies was to test whether agents that increase the-number of these cell surface molecules in vitro would increase monocyte accumulation in vivo. We showed that injection of TNF-a and IL=1(3 markedly stimulated monocytes recruitment measured by our new method. Recruitment was more than doubled. These studies also showed, unexpectedly, that the rate of monocyte recruitment decreased as the extent of atherosclerosis increased.

A new class of drugs used to treat diabetes, the thiazolidinediones, have multiple effects on monocyte/macrophages. Dr. Glass recently showed that one of these drugs, rosiglitazone, ameliorates atherosclerosis in mice. The mechanism of the protection is not known. We showed that rosiglitazone inhibits monocyte recruitment. Whether or not this is the major mechanism for its inhibition atherosclerosis will require further studies.

Polymerase chain reaction-based method for quantifying recruitment of monocytes to mouse atherosclerotic lesions in vivo
Periodical: Arteriosclerosis, Thrombosis, and Vascular Biology Index Medicus:
Authors: Kim C-J, Khoo JC, Gillotte-Taylor K, et al ART
Yr: 2000 Vol: 20 Nbr: Abs: Pg: 1976-1982

Is there a potential therapeutic role for vitamin E or other antioxidants in atherosclerosis?
Periodical: Current Opinion in Lipidology Index Medicus:
Authors: Steinberg D ART
Yr: 2000 Vol: 11 Nbr: Abs: Pg: 603-607

The oxidative modification hypothesis of atherogenesis: an overview
Periodical: Free Radical Biology and Medicine Index Medicus:
Authors: Chisolm GM, Steinberg D ABS
Yr: 2000 Vol: 28 Nbr: 12 Abs: Pg: 1815-1826