MANHATTAN -- Blocked or closed blood vessels mean a medical emergency: for instance, in heart attacks and strokes, time is of the essence.
Since the blood delivers the nutrients and removes the waste from all tissues, when a vessel is blocked, the tissues beyond the blockage are endangered. Doctors have to work to restore the blood flow quickly, usually within a few minutes or hours.
They can use several modern medical technologies: inflatable balloons within a vessel or "clot busting" drugs have made it a common occurrence to provide quick relief for blocked blood flow. But there is a downside to having these capabilities. In one of the ironies of medicine, tissue damage can also occur after blood flow is restored.
"Even with these modern technologies, a doctor is put between a rock and a hard place," says a Kansas State University scientist, a member of a research team whose investigations may one day help resolve the quandary.
"Eventually, a blockage will damage tissue, but using the new technologies to open the vessels and restore the blood flow to the tissues often places the doctor in the position of creating tissue damage, too," said Chris Ross. "That's because a very complex biochemical process that closely resembles inflammation happens after blood flow is restored."
In medical parlance, the blockage is ischemia; the process of blood flow restoration is reperfusion.
Ross and K-State physiologist Frank Blecha are studying this so-called reperfusion injury, a condition that's a major contributor to many important disease syndromes including coronary artery disease and stroke.
The Kansas State University researchers have discovered a substance, a very small protein called PR-39, that suppresses production of the toxic oxygen metabolites that contribute to the characteristic inflammation-like response of reperfusion injury.
Ross said the worrisome mechanisms of reperfusion injury could have evolved as part of the body's response to invading microorganisms. Even though there are no microbes to respond to in the case of a blocked blood vessel, the response mechanism becomes accidentally, and unfortunately, triggered into action. Thus, it's the body's own cellular defenses that account for reperfusion injury.
"It's really an unhappy accident of physiology," Ross added.
The most prominent cell that contributes to the tissue damage is called the neutrophil, and it produces the toxic oxygen products. They are ordinarily used to kill microorganisms, but can be very damaging to tissues in reperfusion injury. "It's sort of the price we pay for being able to make potent antimicrobials, such as free radical oxygen," Ross said.
One of the more toxic of these metabolites is hypochlorite -- "that's the active ingredient in household bleach," said Blecha, professor of anatomy and physiology.
Neutrophils of some animals produce a second substance that suppresses or down-regulates the production of the toxic oxygen metabolites. This complex regulatory system is somewhat like an on-off switch.
In 1995, a K-State graduate student isolated PR-39 from pig neutrophils, and his subsequent experiments demonstrated PR-39's ability to block neutrophil oxidative activity.
Jishu Shi worked in the labs of Blecha and Ross in K-State's College of Veterinary Medicine.
He found that when PR-39 is added to isolated neutrophils their ability to produce the free radical oxygen is blunted to 30 percent of normal level. Such 'down-regulation' was a critical finding, said Blecha.
"That's when we began to wonder: Could PR-39 be useful as an inflammation fighter?" he added.
Since 1995, the KSU team has acquired collaborators who are extending the research from cellular studies to using animal models. PR-39 continues to look very promising.
Dr. Tom Leto of the National Institutes of Health's Laboratory of Host Defenses has been instrumental in defining the molecular mechanisms of action of PR-39. "Dr. Leto is an acknowledged expert in the neutrophil enzyme system that generates the free radical oxygen," Ross said.
Said Blecha, "The fact that Dr. Leto and others have repeated our work and extended our results adds a great deal to the credibility of this project. Their efforts make it a collaborative study involving some very well-known scientists from across the country."
Experiments carried out by Dr. Ron Korthuis at the Louisiana State University School of Medicine, Shreveport, have demonstrated that PR-39 can completely prevent reperfusion injury in an animal model. He uses a real-time video technique to observe events when a tiny artery is first blocked, then opened. He examines the biochemical and cellular events that take place during reperfusion injury.
Kortuis has found that the presence of PR-39 does several important things: it prevents the generation of the toxic oxygen products and the tissue damage that usually occurs when they are present; and it also blocks the adherence of neutrophils themselves to the blood vessel walls and their subsequent migration into tissues, which would be the typical inflammatory sequence.
"This ability of PR-39 to block multiple sites in the biochemical pathway that's involved in reperfusion injury is a very exciting finding," say the K-State scientists. "It suggests that PR-39's overall effectiveness in an actual disease situation might be increased and, if it can be, then someday perhaps this substance can be enlisted for use in human medicine."
Working with the KSU Research Foundation, Blecha, Ross and Shi have applied for several patents involving PR-39 and its anti-inflammatory and anti-microbial activities. Shi is now a post-doctoral researcher at the UCLA Medical School. The KSU research has been supported through several grants from the Kansas Affiliate of the American Heart Association.
Blecha and Ross point out that it will be years before PR-39 could be developed into a drug treatment for human diseases.
"It is always a long time from drug discovery and the first understanding of a basic biochemical activity of a substance to the day when human patients benefit from such research," they say. That said, the K-State researchers acknowledge, "It is exciting to be involved with a compound like PR-39 that has the potential to make a real difference in treating some rather devastating illnesses."
Since the research findings were first reported, the KSU researchers and their colleagues have published five papers and 17 abstracts. A 1996 paper, "PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47phox," was published in the Proceedings of the National Academy of Science. Three papers are now in review. Ross and Blecha have been invited to talk about PR-39 at the National Institutes of Health, the Harvard Medical School and the Louisiana State University School of Medicine.
Prepared by Kay Garrett, office of research and sponsored programs
For information contact:
Frank Blecha, professor of anatomy and physiology, at
(785) 532-4537
Christopher Ross, associate professor of anatomy and physiology, at (785)
532-4507
R.W. Trewyn, president of the KSU Research Foundation, at (785)
776-2014.
Journal
Proceedings of the National Academy of Sciences