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Winner of the IUPAC Prize
for Young Chemists - 2002

 

Stefan Lorkowski wins one of the first 4 IUPAC Prize for Young Chemists, for his Ph.D. thesis work entitled "Differential Gene Expression in Human Macrophages During Foam Cell Formation."

Current address (at the time of application)

Augustastrasse 36, D-48153 Münster, Germany

E-mail: stefan.lorkowski@uni-muenster.de

Academic degrees

  • Ph.D. in Biochemistry, University of Münster, March 2001
  • M.S. in Chemistry and Biochemistry, University of Münster, Sep. 1997

  • Accreditation as a Member of the Institute of Biology and as a Chartered Biologist, Dec. 2001
  • Accreditation as a Associate Member of the Royal Society of Chemistry; Aug. 2001
  • Accreditation as a Clinical Biologist of the Society of Clinical Biology and Bioanalytics, Feb. 2001
Ph.D. Thesis

Title Differential Gene Expression in Human Macrophages During Foam Cell Formation
Adviser Prof. Dr. Erwin Arno Galinski, Institute of Biochemistry, University of Münster
Thesis Committee Priv.-Doz. Dr. Dr. Paul Cullen, Institute of Arteriosclerosis Research, Prof. Dr. Erwin Arno Galinski (adviser); Prof. Dr. Bernt Krebs, FRSC, Institute of Inorganic Chemistry, Prof. Dr. Ernst-Ulrich Würthwein, Institute of Organic Chemistry, University of Münster.

Essay

Introduction
Atherosclerosis is a very common chronic inflammatory disease of the subintimal space of the medium-sized and large arteries. Sites of predilection are the coronary arteries supplying the heart, the carotid vessels supplying the brain, and the arteries of the legs. The full-blown atherosclerotic plaque takes many years to develop. Characteristically, it takes the form of an eccentric lesion consisting of a lipid-rich core containing necrotic cellular debris that is surrounded by areas of smooth muscle cell proliferation and inflammatory cellular infiltrates. This lesion may partially obstruct the artery, causing a painful lack of oxygen in downstream tissue with the symptoms of angina pectoris (tightness and pain in the chest) or intermittent claudication (leg pain). The thin fibrous cap overlying the lesion may also rupture, triggering formation of a blood clot that totally obstructs the artery. If this occurs in a coronary artery, the result is infarction (death) of the cardiac muscle or myocardium, a condition that, despite advances in medical care, is still fatal in half of all cases. If the clot occurs in a carotid artery, the result is stroke that may be fatal or lead to a life of profound disability. Overall, atherosclerosis and its complications are responsible for about 40% of deaths in the developed world and for about 30% of fatalities in developing nations.

A main feature of the atherosclerotic plaque is the presence of large numbers of macrophages. These macrophages ingest in unregulated fashion chemically and/or enzymatically altered low-density lipoproteins (LDL) that have become trapped beneath the arterial intima. Storage of the cholesterol from this LDL as lipid droplets imparts a foamy appearance to the cytoplasm of the macrophages that are then termed foam cells. Much in vitro data and pathological findings suggest that the process of macrophage foam cell formation has an important influence on the genesis and progression of atherosclerosis.

Aim of thesis project
The aim of my research was to determine how the process of foam cell formation affects the pattern of gene expression within the macrophage. We hoped that this would enable us to understand more about how atherosclerosis develops and maybe even point the way towards the development of new anti-atherosclerotic therapies.

Methods and results
The first task was to secure a supply of good-quality human macrophages. To do this, monocytes, the precursors of macrophages, were isolated by a process called countercurrent cell elutriation from the blood of healthy volunteers, usually medical students. All donors had normal blood lipids and all were selected to be homozygous for so-called E3 variant of a macrophage protein called apolipoprotein E. This was because previous work by our group had shown that the way macrophages handle cholesterol is profoundly influenced by what variant of apolipoprotein E they produce.

The monocytes were allowed to differentiate into macrophages by growing them in culture medium supplement with 20% human serum for 14 days. To transform them into foam cells, we added chemically modified LDL to the culture medium for two days. This modified LDL is taken up by macrophages in large amounts. To monitor foam cell formation, we measured the accumulation of cholesterol within the cells using a high-performance liquid chromatography (HPLC) technique developed in our laboratory.

To analyse gene expression, we used the method of differential display reverse transcription polymerase chain reaction (DDRT-PCR). In this technique, very short stretches of DNA are used as so-called primers to amplify the messenger ribonucleic acid (mRNA) molecules within the cell. Because these stretches occur commonly throughout the genome, it is reckoned that DDRT-PCR amplifies over 90% of all the mRNAs in a cell. Unfortunately, DDRT-PCR is a difficult and unforgiving technique, and is beset by the problem of false-positive results, i.e. differences in gene expression are found where none exist. We therefore began by improving the technique. We developed novel DDRT-PCR protocols that increased signal intensity, reduced non-specific results, and isolated longer stretches of mRNAs that were easier to characterize.

Using DDRT-PCR, we identified 36 mRNAs that appeared to be differentially expressed during foam cell formation. In three cases, this regulation was confirmed by independent techniques. Further studies showed that these three mRNAs coded respectively for an acidic calcium-independent type A2 phospholipase, the adenosine triphosphate (ATP)-binding cassette transporter G1 (ABCG1), and a novel gene product that had not previously been identified.

ABC proteins form a huge family that occurs in all known organisms. Most are membrane-spanning transporters that actively move molecules between cell compartments or cells using energy derived from the breakdown of ATP.

Several mutations in ABC proteins have been identified as the cause of such diverse diseases as adrenoleukodystrophy, a disorder of the nervous system, and cystic fibrosis, a disease that affects the lungs, the pancreas and the liver and that renders affected men infertile. In 1999, Rust et al. from our institute together with two other groups showed that mutations in the human ABCA1 gene cause the rare disorder of lipid disorder known as Tangier disease, which is characterized by the accumulation of cholesterol within macrophages. Based on this finding, and on the results of DDRT-PCR experiments, we concluded that ABC proteins may play a role in the lipid metabolism of cells and might also contribute to the development of atherosclerosis.

A second hallmark of Tangier disease is the lack of high-density lipoprotein (HDL) particles in the circulation. HDL is thought to be the major particle responsible for transport of excess cholesterol from peripheral tissues back to the liver where it can be excreted. This is important because mammals have no way of breaking down the cholesterol molecule once it has been formed. The only way it can be removed is to excrete it intact from the liver in the form of bile salts. In tissue culture, HDL removes cholesterol from cells. In population studies, high levels of HDL in the blood were associated with a decreased risk of atherosclerosis. Conversely, the risk of atherosclerosis is increased in persons with Tangier disease.

While we were performing our experiments, other groups showed foam cell formation in macrophages switches on the gene for ABCA1. What we found, however, was that increase in expression of the ABCG1 gene that occurs the foam cell formation is far greater than that of ABCA1. At the time, however, the function of ABCG1 was unknown, and even today no inherited disease caused by mutations in the ABCG1 gene has been identified.

We therefore decided to dig a little deeper. First, we investigated how the expression of ABCG1 mRNA was affected by different techniques of foam cell formation and found that it increased to about the same extent irrespective of whether foam cells were produced using oxidized or acetylated LDL or just so-called liposomes as vesicles containing cholesterol. Conversely, we found that expression of ABCG1 decreased when we removed cholesterol from the macrophages by adding HDL. In addition we found that the expression of ABCG1 differed depending on the variant of apolipoprotein E produced by the cells.

All these findings were clues that like ABCA1, AGCG1 was somehow involved in how the cell dealt with cholesterol. This suspicion increased when we found that the gene for ABCG1 was constitutively switched on in the macrophages of patients with Tangier disease, which contain more cholesterol than normal macrophages.

At this stage we formulated the hypothesis that like its cousin ABCA1, ABCG1 also plays a role in removing cholesterol from cells. To further investigate the function of ABCG1 we made antibodies directed towards a loop of the ABCG1 protein we knew from computer modelling was likely to project outside the cell. Using these antibodies, we found out a number of things. First, we showed that the changes in the mRNA for ABCG1 were mirrored by changes in the amount of ABCG1 protein. Second, we used the antibodies to track down ABCG1 within the cell and found that in macrophages it was concentrated mainly in the outer cell membrane and in membrane structures around the cell nucleus. In fibroblasts, the principal cells of connective tissue, by contrast, ABCG1 seemed to be diffusely scattered about within the cell. Third, we found that in atherosclerotic arteries, ABCG1 is present mainly in foamy macrophages and in bundles of nerves coursing in the outermost layer of the artery, the adventitia.

So what exactly was ABCG1 doing? We knew from studies in the fruit fly, Drosophila melanogaster, that a related protein called "white" was involved in the transport of the amino acid tryptophan. Tryptophan is the raw material for the pigment in the eye of the fly, and the gene derives its name from the fact that its deficiency leads to a lack of pigment and an eye that is white in color. We therefore measured the uptake of radioactive tryptophan in relation to ABCG1 expression in macrophages, but found no relationship, although foamy macrophages did accumulate 5-hydroxyindoleacetic acid (5-HIAA), a breakdown product of tryptophan. However the accumulation of 5-HIAA was not influenced by inhibition of ABCG1 protein formation using so-called antisense oligonucleotides that prevented the translation of ABCG1 mRNA.

A second avenue to investigating the role of ABCG1 was to determine the structure of the ABCG1 gene and in particular to characterize the promoter, the region that regulates gene activity. To do this, we used advanced PCR-based techniques to identify the stretches of DNA upstream (5') and downstream (3') of the mRNA. We were surprised to uncover an unusual degree of complexity. We found that the gene contains five coding regions or exons that had not previously been described, and that it codes for a very large number of so-called splice variants with different amino acid sequences. In addition, we found that these alternatively spliced ABCG1 transcripts are controlled by different promoters.

To investigate the function of these promoters, we exposed the cells to compounds which are known to bind to the liver X receptor (LXR) and the retinoid X receptor (RXR), proteins regulating gene expression that reside within the cell nucleus. Both types of compound increased the activity of the ABCG1 gene.

Conclusions
Taken together, my results indicate that ABCG1 is involved both in the transport of cholesterol within macrophages and in foam cell formation. The strong expression of ABCG1 in macrophages in the arterial wall indicates that it may also play a role in the development of atherosclerosis. Results have also been obtained by our collaborator, Prof. Dr. Arnold von Eckardstein, that lend further support to this hypothesis. Dr. von Eckardstein's group showed that reducing the production of ABCG1 protein by means of antisense oligonucleotides decreased the secretion of apolipoprotein E, which, as noted above, is a very important player in removing cholesterol from cells and may well protect against atherosclerosis. Therefore, ABCG1 might actually turn out to be an interesting target for the development of new anti-atherosclerotic therapies.

Epilogue
Since completion of my PhD thesis, further work by our group has led to the identification of a new ABC transporter, ABCG4, that is highly homologous to ABCG1 and that is also expressed in human monocyte-derived macrophages. ABCG4 gene expression seems to be regulated in the same fashion as ABCG1 gene expression.


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