Most people associate ischemia, if they associate it with anything at all, with stroke.
But a stroke is not the only injury that leads to ischemia, which is caused by lack of blood flow to an organ. Nor is the brain the only body part that can become ischemic. Atherosclerosis can lead to intestinal ischemia, and ischemia also results as a side effect of medical procedures "when you clamp off blood supply, as required in major surgery," said Michael Carroll, professor at Harvard Medical School's CBR Institute for Biomedical Research.
And as if ischemia alone wasn't bad enough, re-establishing blood flow to oxygen-derived tissues leads to an inflammatory response that causes further tissue damage: reperfusion injury.
The severity of reperfusion injury varies, but especially in short ischemic events, reperfusion injury damage can be as bad as or worse than the ischemic damage itself.
In previous studies, Carroll and his colleagues identified a natural antibody that is responsible for reperfusion injury. "Natural" meaning, in this case, that it is an antibody of the innate immune system, and it circulates constantly, not just in response to an infection.
When those natural antibodies come upon a foreign invader, they activate the complement system - "a cascade of proteins that is in our blood, much like the clotting cascade," Carroll said. And when the complement system becomes activated on the surface of a cell, "the end result is damage to that cell."
In new research, published in the Jan. 3, 2006, online Journal of Experimental Medicine by Carroll and his colleagues from Brigham and Women's Hospital, Massachusetts General Hospital, and DecImmune Therapeutics, all of Boston, they identified the complement system's target in reperfusion injury: a specific region of non-muscle myosin.
Like its better-known muscular colleague, non-muscle myosin is a protein motor that binds to actin and is involved in movement - in this case, cell motility. It also is involved in aspects of cell division and perhaps, Carroll said, "it has a function in a cell that becomes ischemic. That's what we are testing now."
By making a library of peptides with random sequences and testing their ability to bind different natural antibodies, the scientists first identified an artificial peptide that bound to the reperfusion IgM, but did not bind to a control IgM. Injecting mice with that artificial peptide before inducing intestinal ischemia reduced reperfusion injury, suggesting that the peptide was binding IgM and preventing it from binding to cells.
Sequence analysis of the peptide showed homologies with a region of non-muscle myosin heavy chain; the scientists made a synthetic version of the non-muscle myosin region to test whether it, too, would be effective in ameliorating reperfusion injury.
The synthetic peptide bound to the natural antibody that Carroll and his colleagues previously had identified as the culprit in reperfusion injury. Injecting mice with the synthetic peptide before inducing ischemia again reduced reperfusion injury, though higher concentrations were necessary of the synthetic peptide than of its library-derived random-sequence counterpart.
To see whether the peptide was effective in ischemia generally, or only in intestinal ischemia, the researchers tested it in hind-limb ischemia, which models what might occur during surgery. The peptide prevented reperfusion injury there, as well; its effectiveness in both intestinal and hind-limb ischemia "supports the notion that this is a general mechanism of reperfusion injury," Carroll said.
The group also is testing a cardiovascular model, and results, while preliminary, support the idea that the same mechanism is active here. The researchers believe that cellular myosin moves to the cell surface after ischemia, where it is found and bound by IgM. If the myosin were circulating after apoptosis, there is no reason why IgM should bind only the myosin but not the dozens of other pieces of cellular debris that would be floating around.
Carroll is co-founder and chief scientific officer of DecImmune, which holds exclusive licenses to patents on both the IgM and the peptide. The patents themselves are held by the CBR Institute, Brigham & Women's hospital and Harvard Medical School.
Carroll and colleague Francis Moore founded the company "as an attempt to use some of our laboratory findings in a therapeutic way," Carroll said. He added that there was not only a lack of therapeutics for reperfusion injury, but also a "lack of enthusiasm" to develop them by industry, because the notion that innate immunity is responsible for reperfusion injury is still controversial. So "we approached a few companies, saw that they weren't interested, and decided to found a company ourselves."
Carroll said that DecImmune was originally supported by a Small Business Innovation Research grant and is "presently searching for VC funding." Formerly named Natural Antibodies Inc., the company was renamed DecImmune for the 10 binding sites of IgM.
"I liked Natural Antibodies better - it was a more accurate description," Carroll said. "But my colleagues in the business world told me that's not how you name a company."