2000 IRISH SCIENTIST YEAR BOOK

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Vascular homeostasis - a cholesterol balancing act?

University College Dublin
Orlaith Lawler* & Therese Kinsella

Figure 1.

Cardiovascular disease is responsible for over 42% of premature deaths in Ireland annually. Worldwide, it accounts for 34% and, hence, Ireland has one of the highest known fatality rates due to this disease.

A major cause of cardiovascular disease is thrombosis, or unwanted clotting, which reduces the flow of oxygenated blood through blood vessels. Blood clotting is, however, necessary for blood vessel repair in response to injury, but unwanted blood clotting must be avoided to prevent thrombosis. This balance between blood clot formation and prevention is referred to as vascular homeostasis.



In our laboratory, we examine two protein receptors that are involved in controlling the balance of this essential clotting process. These receptors, which are acted upon by specific hormone like compounds referred to as thromboxane A 2 and prostacyclin, send signals to platelets, the blood cells most important in this clotting process, and thereby control their participation in the blood clotting process. Thromboxane A 2 , an activator, induces platelet aggregation or clotting and contraction of muscle cells within the walls of the blood vessels. Prostacyclin, an inhibitor, prevents platelet aggregation and induces vasodilation or relaxation of muscle cells in the walls of the blood vessels. In general, the actions of prostacyclin counteract those of thromboxane A 2 and, therefore, the relative levels of these two compounds are essential in controlling platelet participation in this clotting process. Imbalances of these compounds may contribute to a number of cardiovascular disorders such as thrombosis and artheriosclerosis.

Artheriosclerosis is caused by a build up of lipids, such as cholesterol, that can lead to the formation of plaque. Excess cholesterol may be either derived through the diet or synthesised by the body's own mechanisms. In artheriosclerosis, cholesterol deposits on the walls of arteries causing plaque to develop and, thus, causes a physical obstruction to blood flow, resulting in a narrowing of the vessels. An embolus, or a free flowing clot, is thus more likely to become trapped in such a vessel than in a vessel that has no plaque. In severe cases, reductions in blood flow of 90% have been recorded, and it is in this situation that a major coronary event occurs, such as a heart attack. The Statins are a family of cholesterol lowering drugs which are prescribed clinically to counteract this plaque build up in the arteries. The Statins act by inhibiting cholesterol synthesis, thus reducing the amount of free cholesterol in the body available to deposit on the artery walls (Figure 1) .

Prostacyclin, as an inhibitor of thrombosis, acts by interacting through a specific cell surface receptor - termed the prostacyclin receptor - and it is this receptor that I focus much of my work on. Prostacyclin, by interacting with its prostacyclin receptor on the surface of the platelet, transmits signals to within the platelet and, in turn, leads to an increase in second messengers within the platelet itself. This pathway is involved in the inhibition of platelet aggregation and vascular smooth muscle relaxation.


Figure 2.The effect of Statins, the cholesterol lowering drugs, on prostacyclin receptor signalling.

The prostacyclin receptor itself is made up of approximately 400 amino acids or building blocks. Analysis of the prostacyclin receptors from a number of species showed that they each contain a conserved end region. This end region consists of four identical amino acids, the order and location of which predicted a site where a cholesterol subunit may be attached. To investigate whether this cholesterol attachment is necessary for the prostacylin receptor to signal, we mutated the first of these four amino acids where it was thought the cholesterol subunit may be attached. This produced a mutated prostacyclin receptor that was found not to signal efficiently: upon measuring the mutated receptor's signalling, we found that it was reduced by 82% compared to that of the normal or wild type prostacyclin receptor. This provided evidence that the amino acid we mutated is essential for efficient prostacyclin receptor signalling. To establish whether the prostacylin receptor's inability to signal was due to the lack of the cholesterol subunit, we examined the receptor's ability to signal in the presence or absence of the Statin cholesterol lowering drugs. Our experimental data demonstrated that prostacyclin receptor signalling was reduced in the presence of the Statins in a dose dependent manner, with up to 80% inhibition of receptor signalling recorded, depending on the particular Statin under study (Figure 2) . This data confirmed that the prostacyclin receptor requires cholesterol to signal efficiently and that the amino acid we mutated is necessary for this cholesterol attachment to occur.

In summary, our studies show that cholesterol is necessary for prostacyclin receptor signalling: this signalling causes a decrease in platelet aggregation and muscle contraction and, hence, plays a central role in preventing unwanted blood clotting or thrombosis. However, too much cholesterol can lead to an increase in the build up of plaque in the blood vessels, leading to atheriosclerosis. The scientific question that remains to be fully answered is whether or not too little free cholesterol in the body interrupts our body's natural ability to maintain vascular homeostasis.

This work was funded by the Irish Heart Foundation and Enterprise Ireland.

Contact:
Orlaith Lawler,
Department of Biochemistry,
UCD, Dublin 4;
E-mail: Orlaith.Lawler@ucd.ie


* Orlaith Lawler was one of the winners in the Merville Lay Seminars held in UCD in February 2000. This is a summary of her presentation.