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Recruitment of leucocytes to atherosclerotic lesions in vivo

Institution: University of California, San Diego
Investigator(s): Daniel Steinberg, M.D., Ph.D.
Award Cycle: 2000 (Cycle 9) Grant #: 9RT-0050 Award: $175,102
Subject Area: Cardiovascular Disease
Award Type: Research Project Awards

Initial Award Abstract
More cigarette smokers die of a heart attack than die of lung cancer or any other single cause. There is very good evidence from studies in animals that oxidative damage to the large, cholesterol-carrying molecules in the blood stream, especially low density lipoproteins (LDL), plays an important part in atherosclerosis, the underlying disease of the arteries that ultimately leads to a heart attack. The oxidatively damaged LDL (OxLDL)is then taken up by cells in the wall of the artery, and that is how the cholesterol concentrations build up. Another effect of OxLDL is to attract circulating white blood cells (mainly monocytes) into the damaged artery wall, which further accelerates the disease process. While a good deal has been learned about the molecules to which monocytes stick and the molecules that draw them into the artery wall, the relative importance of these has yet to be properly assessed in the whole animal. Methods simply have not been available that are sensitive enough for this kind of study. We in this laboratory have now developed such a method, one that is sensitive enough to detect even one genetically marked cell in the presence of 1 million genetically unmarked cells. The method takes advantage of the remarkable sensitivity of the polymerase chain reaction (PCR) which uses an enzyme in such a way as to amplify a single copy of a gene into millions of copies in just a few hours. We take advantage of the fact that only males have a copy of the testis-determining gene (sry gene) which resides on the Y chromosome, the chromosome that is exclusively found in males. The "trick" is to transfuse monocytes taken from a male donor mouse into the blood stream of a female recipient mouse and determine how many male cells appear in the wall of the artery after a given time interval. Using this method we have shown for the first time in vivo that 2 of the compounds suspected on the basis of in vitro studies to be important in recruiting monocytes to lesions are in fact involved also under in vivo conditions. These are tumor necrosis factor-and interleukin-1. Our theory is that the very first thing that happens is a buildup of OxLDL in the artery wall, which in turn causes monocytes from the blood to enter the artery wall. Since these monocytes can then themselves contribute to further oxidation of LDL and, indirectly, speed up the recruitment of still more monocytes, the damage to the artery might well spiral out of control. We will test directly whether administration of compounds that block oxidation of LDL decreases the rate at which monocytes enter the artery wall. We will also explore the role of MCP-1 and the nature of the regulation of MCP-1. Specifically, we will test whether a new class of drugs, the thiazolidinones, inhibits monocyte recruitment and whether that leads to inhibition of the formation of new lesions in the artery wall. Finally, we will test the hypothesis that cigarette smoking increases the rate of monocyte entry into lesions of LDL receptor deficient mice and by so doing accelerates the progression of atherosclerotic lesions.

Final Report
The major cause of death in the United States, both among smokers and among non smokers; is disease of the coronary arteries leading to heart attacks. The blood vessel disease is called atherosclerosis and it is a progressive degeneration that begins with the entry of white blood cells (monocytes) into the artery wall. The present studies were designed to measure the rate at which monocytes enter the artery wall using a highly sensitive procedure developed in this laboratory over the last few years. The method capitalizes on the sensitivity with which genetic DNA can be amplified using the polymerase chain reaction (PCR). Originally we introduced monocytes from a male donor mouse into female recipients. Because male cells, and only male cells, contain the Y chromosome we could amplify a gene present on that chromosome and totally absent from the DNA in the female recipient. That method worked very nicely and the feasibility, sensitivity and reproducibility of the method were established using that method. It allowed us to measure for the first time the rates at which monocytes enter atherosclerotic lesions in the intact animal, without uncertainties about whether or not in vitro systems behave in a manner that mirrors the situation in the intact animal.

During these studies we showed that one of the drugs currently widely used in the treatment of diabetes (rosiglitazone) could inhibit the progression of atherosclerosis in male mice but we saw no effect in females. It became important to be able to measure the effects of the drug separately in male and female animals, which meant devising a new strategy. Over the past year we have done that, taking advantage of the availability of a strain of mice into which a new gene has been introduced - the gene for a bacterial enzyme, (3-galactosidase. All of the cells in the body of this animal, including of course their blood monocytes, contain about 20 copies of this gene. That makes it possible now to transfer monocytes from either males or females of this strain into normal recipients of either sex and trace the injected cells by using PCR that amplifies the P-galactosidase gene. The sensitivity of this method, because of the 20 copies/cell, is remarkable, allowing us to detect the recruitment of monocytes into the artery wall even before any lesions are visible. If we find that monocyte recruitment is inhibited by rosiglitazone in male mice as well as females, we will need to choose between two hypotheses: 1. That the drug down regulates the production of a chemoattractant (MCP-1), which has been demonstrated; 2. That it down regulates the expression of the receptor for MCP-1, something demonstrated by Dr. O. Quehenberger in our laboratory very recently.

The availability of this new, highly sensitive method should be a powerful general tool for investigators interested in atherosclerosis or in other inflammatory processes.