"It's very exciting to think that Pcsk9 might play a large role in the pathway to regulate the uptake of bad cholesterol from blood," said Kara Maxwell, the lead researcher of the study published this week in the journal Proceedings of the National Academy of Sciences. Maxwell is an M.D.-Ph.D. student in the Laboratory of Biochemical Genetics and Metabolism headed by Jan L. Breslow, M.D., the senior author of the study.
High levels of LDL cholesterol in the blood lead to heart attacks because the waxy LDL molecules build up inside the walls of arteries, causing damage to the blood vessels and leading to clots that block the follow of blood to the heart muscle.
Maxwell's cholesterol study in mice is highly relevant to humans because mutant forms of the Pcsk9 gene have been linked to one form of autosomal dominant hypercholesterolemia, a group of genetic disorders characterized by excessive levels of cholesterol in the bloodstream.
To identify genes that regulate cholesterol levels in the blood in response to high cholesterol diets, Maxwell examined gene activity in mice that were fed normal diets and mice that were fed high-cholesterol diets. In this experiment she found a previously unknown gene, now called Pcsk9, that was expressed at a much lower level in mice that were fed high-cholesterol diets compared to mice that were fed normal diets.
Last year, a research team led by Nabil Seidah and Catherine Boileau at the Hospital Necker-Enfants Malades in France independently found that mutations in the Pcsk9 gene were linked to autosomal dominant hypercholesterolemia. But the function of Pcsk9 was not determined until now.
To find out what Pcsk9 does, Maxwell inserted the Pcsk9 gene into a virus that targets the liver and injected the virus into normal mice, where it made Pcsk9 protein inside liver cells.
With abnormally high levels of Pcsk9 in their livers, the mice developed high cholesterol. Four days after injection, the animals' LDL levels were five times the normal level, and their levels of total cholesterol in the bloodstream had doubled. (Total cholesterol measures LDL plus HDL plus another type of cholesterol.)
Maxwell suspected that the Pcsk9 protein was raising LDL levels by acting on the liver cells' LDL receptors, which snatch up LDL molecules from the blood.
To test this hypothesis, Maxwell injected the Pcsk9-carrying virus into a strain of mice that do not have LDL receptors because of a genetic defect. When Maxwell examined the special high-cholesterol mice after they had been injected with Pcsk9, she found that their cholesterol levels had not changed -- LDL levels did not increase as they had in normal mice. This suggests that Pcsk9 has no effect on cholesterol levels unless LDL receptor molecules are present.
In addition, Maxwell found that normal mice injected with the Pcsk9 lost their liver LDL receptors. This indicates that Pcsk9 directly affects LDL receptors.
"LDL receptors are the main way of getting LDL out of the blood," said Maxwell. "I think knowing that Pcsk9 modulates LDL receptors is important for understanding how LDL levels are regulated."
Maxwell and Breslow said it is unclear why Pcsk9 gene mutations cause high cholesterol in humans but this work in mice should help to figure this out.
"If the same mechanism Maxwell found in mice pertains to humans and we could inhibit Pcsk9 with drugs, this should increase the number of LDL receptors on cells and in this way lower LDL levels in the blood. This might offer another therapeutic approach to the high blood cholesterol levels that are a major risk factor for heart disease," said Breslow, a former president of the American Heart Association.
Out of all diseases, heart disease is the number one killer of Americans, followed by cancer. People with high levels of LDL, the "bad" cholesterol, and low levels of HDL, the "good" cholesterol are at high risk for developing heart disease.
"The question is, is your cholesterol at the right level?" said Breslow. "There are a huge number of people that fall into the category in which their LDL should be lowered. A lot can be done with diet and exercise but many people also need drugs."
The American Heart Association recommends that people maintain their total cholesterol level below 200 mg/dL, their LDL cholesterol below 130 mg/dL and their HDL cholesterol above 40 mg/dL.
Currently, about 105 million American adults have total cholesterol levels above 200 mg/dL. The average LDL cholesterol level among Americans is 140 mg/dL, which is higher than the recommended level.
Breslow is a pioneer in the genetics of heart disease. In 1992, his lab produced the first mouse model of heart disease by knocking out, or deleting, the apo E gene. The mouse provided the scientific community with the first small animal model to study heart disease in a controlled fashion that can render discoveries relevant for the human condition.
This research was supported by the National Institutes of Health. Maxwell is supported by a grant from the NIH's Medical Scientist Training Program.