Interview

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.

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.

Shimokawa:
In 1985, I went to the U.S. and studied at Mayo Clinic to conduct further research. That was when I started to study the vascular endothelium as my second research theme in addition to the first research theme of coronary spasm.

The vascular endothelium produces and releases relaxation factors collectively called the endothelium-derived relaxing factor (EDRF) in order to keep blood vessels relaxed. There are three types of EDRF. To mention them in the order of discovery, the first one is PGI2*9 identified in 1976. The second one is nitrogen monoxide (NO)*10, whose existence was reported by an American research group in 1980. This relaxing factor was identified in 1976, and the Nobel Prize was awarded to three researchers in 1998.

The third EDRF is the endothelium-derived hyperpolarizing factor (EDHF). It was discovered in 1988 independently by Dr. Vanhoutte, who supervised me while I was studying at Mayo Clinic, and a group led by Professor Hikaru Suzuki of the Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University. But its identity remained unknown for a long time. Is the EDHF less important than NO? That is not so. NO and EDHF play different roles, respectively, according to the vascular diameter. Regardless of animal species and blood vessel types, NO fulfills an important role where the vascular diameter is large and the EDHF becomes more important as the vascular diameter gets smaller. We showed this fact before the rest of the world. While researchers around the world were trying to identify the EDHF, we discovered ahead of them in 2000 that the identity of the EDHF is hydrogen peroxide (H2O2)*11 that the vascular endothelium produces and releases at the physiological concentration. Both NO and H2O2 are reactive oxygen species. And it became clear that not only NO but also H2O2 at the physiological concentration are good.

Many researchers in the world immediately argued against our H2O2/EDHF hypothesis because H2O2 had been used in experiments for many years as a major source of oxidant stress. This was the same kind of reaction seen when the identity of NO, considered as one of the nitrogen oxide pollutants, was determined. The NO hypothesis also met with a lot of objections at first, but the number of researchers who supported it gradually increased. As the hypothesis became more widely accepted over time, the Nobel Prize was finally awarded to the three researchers in 1998 who had led the NO research. What was interesting about the controversy over our H2O2/EDHF hypothesis was that, while researchers who supported the hypothesis used microvasculature, those who opposed it used relatively thick blood vessels. We also indicated that NO plays a big role in thick blood vessels, so the researchers of the latter case were right in a sense. The vascular diameter was not the decisive factor in everything. But, as the verification process progressed, the idea that H2O2 plays an extremely important role in microvasculature came to gain broad acceptance worldwide.

This is my second research theme, and I am still working on it. I have been involved in the research of coronary spasm, which I mentioned earlier, and the research of the EDHF. The William Harvey Lecture Award was given to me for my achievements in these two basic researches.

PR Office:
They gave enormous recognition for what you have done for years since you started your research life. Congratulations!

Notes:
*9: PGI2
A type of biologically active substance (substance in the body capable of physiological control) called prostaglandin deriving from fatty acid (arachidonic acid). It triggers a vasodilator action.
*10: Nitrogen monoxide
A chemical compound (NO) consisting of nitrogen and oxygen. It usually exists as a gas and triggers a vasodilator action.
*11: Hydrogen peroxide
A substance expressed by a chemical formula of H2O2. It is unstable and prone to release oxygen. The substance is a type of active oxygen that is highly reactive and has a strong oxidizing power.

PR Office:
What do you expect from the students and doctors of your lab?

Shimokawa:
First of all, I want them to have a strong sense of purpose. They should not be passive. I expect them to maintain a positive attitude, finding questions and trying to solve them on their own. When I give a lecture to soon-to-graduate students before graduation, I advise them that it be important to have their own dreams and stay committed to research whatever happens. It is also important to have a mentor whom you can think of as a goal. I have three mentors myself. One of them passed away, but I truly feel that the influence of education lasts even after the mentor is gone. I still communicate with this mentor in my heart almost every day.

As for clinical care, I always tell students, interns and teaching staff to think of patients as your parents. If they do, they do not give patients unnecessary diagnosis and treatment. I think it is essential for doctors to treat patients with this in mind.

Regarding research, I think it is important to do unique research. In research, being second after someone else is not good enough. The medical knowledge and techniques that we use daily without giving much thought are available because of the efforts that researchers of the past put in and the contributions of patients who cooperated. So, if our generation becomes complacent about the current situation and quits making research efforts, then the progress of medicine will stop, raising a question of responsibility for future generations.

Let me talk about my own researches now. The research of coronary spasm will open the door to a possibility of treatment as various Rho-kinase inhibitors are developed in the future. From the perspective of drug development, this research can be expected to be passed on to future generations. My second research of active oxygen and vascular function is difficult to apply for quick development of drugs, as the research of coronary spasm is. But I think that the results of this research have helped advance the research progress in this field as it revealed the mechanism of homeostasis of the vascular function, as well as the new roles played by NO and hydrogen peroxide.

Based on my own experience, I always tell younger people around me, “Don’t panic even when things get tough.” You should not try to solve problems hastily. It is true that, in some cases, the best way to solve a problem is to let time take care of it. If you keep working patiently and believing yourself, someone may lend you a helping hand and the situation may turn around in an unexpected way. From the perspective of becoming a good doctor, experiencing many setbacks is very important. Every patient is being faced with a setback in their life. It is impossible to become a good doctor without experiencing setbacks.

If you are in clinical training, I suppose you have two goals: to become a good clinician and to become a good researcher. In fact, you need to meet both of those goals. A job that joins these two things is that of a physician-scientist, and becoming a physician-scientist requires originality and vitality.

In my case, when I was assigned a research theme to create an animal model of coronary spasm, I began by carefully observing the clinical condition of humans and paying attention to the clinical fact that spasms occurred to atherosclerotic lesions. That was my original idea. I think that another big thing that helped me advance my future research smoothly was the bold change of the laboratory animal type that I decided to make while reading numerous papers of the past.

Backed by these experiences, I still stick to my research policy of observing clinical facts thoroughly, reading past research papers carefully, taking time to think well and, if necessary, making bold decisions based on the ideas acquired in the process. I couldn’t tell you about this today, but a good example is the research for developing noninvasive angiogenic therapy using sound waves (shock wave and ultrasound wave), on which I have been working for more than 15 years. I hope that undergraduate and graduate students who are starting their research life feel the same way about work as I do.

PR Office:
Thank you very much for sharing your useful experiences and perspectives with us.