Interview

On the Winning of the William Harvey Lecture Award

Research life started with an effort to create an animal model of spasm

PR Office:
First of all, we would like to congratulate you on winning the William Harvey Lecture Award*2 of the European Society of Cardiology (ESC)*1. We are here today to hear from you about the research achievements that have earned you the award.

Shimokawa:
Let me begin by telling you about the research life that I have led over the past nearly three decades. It was in 1981 that I started my research career after two years of clinical training. The first research topic I worked on was spasm of the coronary arteries of the heart, which remains one of my research themes. The heart has three coronary arteries on its surface, and a coronary artery anomaly causes angina pectoris and acute myocardial infarction. One of the causes of these diseases is coronary artery spasm. The professor assigned me, a novice researcher who had just joined the lab, to create an animal model for reproducing coronary spasm.

To tell you the truth, the same assignment had been given to senior members of the lab, who had been experimenting with dogs with little success. When I took over the research, my boss said, "If you guys fail, we will stop the research." Knowing that I had been given the last chance, I started by studying related researches and past research papers very hard in order to figure out what was wrong.

Doing these researches, I came to realize that spasm is never experienced by normal blood vessels and occurs only to blood vessels hardened by one type of arteriosclerosis or another. Clinical findings also revealed that coronary spasm results from various degrees of coronary atherosclerotic lesion. This made me conclude that, in order to create an animal model of coronary spasm, it would be necessary to produce coronary atherosclerotic lesions in laboratory animals. But, as I read more papers, I found out that it had been reported that atherosclerotic lesions similar to those observed in humans were difficult to develop in dogs. On the other hand, I came across some papers stating that atherosclerotic lesions similar to those of humans could be produced in pigs. So I thought that the use of pigs might make it possible to create an animal model. Assuming a long-term experiment, I decided to use miniature pigs, which would not gain much in weight as they grew. I went to the professor and asked him to buy us miniature pigs. It was over 30 years ago, and doing long-term research of coronary arteriosclerosis using miniature pigs was rare at the time.

PR Office:
How did the professor react?

Shimokawa:
He was surprised at first at my decision to switch the subject to miniature pigs, which had never been used in the lab before. The bigger problem, though, was the price of the animal. It cost 130,000 yen per pig 30 years ago. When I told the price to the professor, he asked if I had mistakenly added two zeros. His reaction was understandable. We could get a mixed-breed dog for free or, at the highest, for less than 1,500 yen those days. So the professor turned down my request right away. But I kept asking, and eventually he permitted us to buy two miniature pigs. We had only two pigs, but I was told that the research would be stopped if we didn't deliver results.

We conducted the experiment like this: First, we produced injury to the intima of a coronary artery using a balloon and fed the pig with cholesterol-rich food for a long period of time (three to six months) in an effort to develop a coronary atherosclerotic lesion. Feeding cholesterol-rich food for a long period of time cost a lot of money, too. When we performed coronary arteriography, the image of the coronary artery was almost normal, which disappointed us all. But applying contractile stimulation using histamine and serotonin induced coronary spasm very similar to that seen in humans with high levels of reproducibility. I still vividly remember that, when we confirmed the reproducibility, I jumped in joy with my lab mates I was working with.

After succeeding in creating a coronary spasm model quite similar to that of humans, we cut the coronary artery into serial sections and examined them in detail histologically. We found that early coronary atherosclerotic lesions not visualized by coronary arteriography had formed in some parts and that coronary spasms had occurred in those parts with high levels of reproducibility. After that, the professor provided support for our research. This was more than 30 years ago, and the molecular mechanism of coronary spasm was not understood at all. Anyway, we succeeded in experimentally demonstrating using an animal model that coronary spasms occur in parts of a coronary artery with early coronary atherosclerotic lesions. Our paper was published in Science magazine in 1983 (without going through a peer review process). We received a letter directly from the editor in chief who praised us, saying "This is a rare thing that happens only two or three times a year." I remember that the letter flattered us a lot. The Internet did not exist back then. So people had to request copies of the paper by postcard. We received nearly 1,000 requests at a time, and we were surprised that half of them came from veterinarians who were troubled by the problem of sudden deaths of pigs.

To return to what I was saying, angiospasm occurs when the vascular smooth muscle*3 contracts abnormally. And, a layer of squamous cells called the vascular endothelium that covers the inner surface of blood vessels produces and releases a relaxation factor. It had been considered that spasm is caused by the disruption of the balance between the relaxant response of the vascular endothelium and the contractile response of the vascular smooth muscle. As a result, the vascular endothelial dysfunction hypothesis and vascular smooth muscle hypercontraction hypothesis were proposed as the mechanism for coronary spasm, which eventually led to medical controversy.

PR Office:
Did the animal model that you created open the door to the possibility of settling the controversy?

Shimokawa:
In our first model, both an endothelial dysfunction resulting from the regeneration of balloon-injured endothelium and excessive contraction of the vascular smooth muscle caused by the balloon injury were observed. So the mechanism could not be explained fully with this model alone. In 1996, based on pathological findings for humans (findings regarding the inflammation of the coronary adventitia of regions of coronary spasm)*4, I proposed a second coronary spasm model demonstrating that coronary spasm would also be induced with high levels of reproducibility by applying an inflammatory stimulus chronically from the coronary adventitia of a pig. Since the endothelial function remained normal in this model, it became more probable that the excessive contraction of the vascular smooth muscle was the main cause of coronary spasm. As coronary spasm could be reproduced using an animal model, it became possible to examine changes in regions of coronary spasm on the molecular level. Through a series of basic and clinical researches, we discovered in 2000 for the first time in the world a molecular mechanism of coronary spasm in which the expression and activity of a protein kinase*6 called Rho-kinase*5, which served as the molecular switch for the vascular smooth muscle, were increased.

The contraction of the vascular smooth muscle is caused by the myosin light chain*7 receiving phosphorylation and interacting with actin*8. This is suppressed by myosin phosphatase and promoted by myosin phosphoenzyme. We found that, in regions of coronary spasm, Rho-kinase was active and myosin phosphatase was suppressed, resulting in significantly enhanced phosphorylation of the myosin light chain and thus high-level prolonged abnormal contraction (spasm) of the vascular smooth muscle. From this result, we concluded that it might be possible to prevent coronary spasm by using a drug capable of suppressing Rho-kinase. So, we checked the existing drugs to see if there was any having an inhibitor effect on Rho-kinase and came across a drug called fasudil. This drug was used only in Japan to treat cerebral vasospasm after subarachnoid hemorrhage although its mechanism remained unknown. We found that fasudil is metabolized to hydroxy fasudil in the human body and that it triggers selective Rho-kinase action. Also, through a series of researches, we revealed for the first time in the world that Rho-kinase is deeply related to the causes of a wide range of circulatory diseases. Today, a number of domestic and foreign pharmaceutical companies are in fierce competition to develop a selective Rho-kinase inhibitor.

Photo:Shimokawa

Notes:
*1: European Society of Cardiology
A society of cardiology founded in 1950 with a membership of over 80,000 (nonprofit organization)
*2: William Harvey Lecture Award
A prestigious award named after the English anatomist William Harvey famous for developing the theory of the circulation of blood
*3: Vascular smooth muscle
Muscle surrounding blood vessels. When the vascular smooth muscle contracts, the blood vessels become narrower in diameter, resulting in poor blood flow. The muscle cannot be moved consciously.
*4: Pathological findings
Description of results of observing samples of cells, tissues, or organs using a microscope or other tool and opinions about such results
*5: Rho-kinase
A type of enzyme that adds a phosphate group to protein. It controls the contraction of smooth muscles.
*6: Protein kinase
An enzyme that adds a phosphate group to protein. When a phosphate group is added to a protein, its nature and activity change.
*7: Myosin light chain
A type of protein that makes up a muscle. It functions in conjunction with actin.
*8: Actin
A type of protein that makes up a muscle. It functions in conjunction with myosin.

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