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Researchers in San Diego and the United Kingdom have identified a protein segment and the mechanism that underlies excess fibrous tissue growth leading to conditions such as liver fibrosis and cirrhosis. In addition, they’ve developed a mutated version of the protein that blocks this excess scar tissue in mice.
The study is published in the Oct. 26, 2001 edition of the journal Molecular Cell by researchers at the University of California, San Diego (UCSD) School of Medicine, the VA San Diego Healthcare System, the Salk Institute for Biological Studies, and the University of Dundee, United Kingdom.
Lead author Martina Buck, Ph.D., a UCSD and VA research scientist, found that a small piece of an important regulatory protein called C/EBP beta was responsible for fibrous tissue growth – which is excessive scar tissue – following injury or chronic illness. An appropriate amount of fibrous tissue growth is valuable, such as the formation of a scar over a skin wound. When normal healing goes awry, however, excessive build up of fibrous tissue can produce disfiguring scars externally or clog vital internal organ functions and lead to serious complications. By isolating a small section of C/EBP beta and changing one of its molecular components – amino acids, the building blocks of proteins – Buck and her team developed a mutated protein that stopped excessive fibrous tissue growth.
Although the study focused on liver injury in mice, the findings may also apply to fibrous tissue growth in other organs, such as the kidneys, lungs and skin, said the study’s senior author Mario Chojkier, M.D., UCSD professor of medicine and a liver specialist at the VA Medical Center.
"In some individuals," he added, "injury or a chronic illness causes fibrous tissue to accumulate. In severely burned patients, there could be terrible skin scarring. In the liver, this can lead to cirrhosis and serious, life threatening medical complications such as internal bleeding, fluid accumulation, and an inability to handle medications or environmental toxins. However, until now we haven’t known how this process occurred at the molecular level and how we might prevent it."
The researchers’ discoveries began with an investigation of the known events leading to excess tissue growth. With liver injury or a chronic illness such as viral hepatitis, a molecular chain of events leads to stimulation of specific liver cells called stellate cells, causing them to grow in number and size, producing the excessive amounts of fibrous tissue that interferes with normal function.
The team determined that injury or chronic illness activates a phosphorus molecule which attaches to an amino acid sequence within the C/EBP beta protein. The centerpoint for the team’s study became that amino acid sequence, KTVD, which consists of lysine (K), threonine (T), valine (V) and aspartic acid (D). With phosphorylation of KTVD – the addition of a phosphorus molecule – the amino acid sequence blocked the normal activity of another group of enzymes called caspases, which ordinarily would prevent the overproduction of fibrous tissue, resulting in excessive scar tissue growth.
To block the development of excess fibrous tissue, the researchers bred mice with KAVD, a modified version of the amino acid sequence that substituted an amino acid called alanine (A) for threonine (T). With this mutated amino acid sequence, the mice responded normally to liver injury and damage, without excessive scar tissue build up. Specifically, KAVD blocked the phosphorylation that had occurred with the KTVD amino acid sequence and allowed normal activation of caspases.
Noting that the KAVD peptide was shown to be safe in mice, Chojkier said that further refinements might allow for the development of a peptide that could be administered orally or inhaled by people who are susceptible to cirrhosis and other diseases where excessive scarring is a problem. He added that the research team hopes to study the peptide in human clinical trials in the next year or two.
In their study, the investigators also reported that mice bred without the C/EBP beta protein and mice with the KAVD version could be given another mutated protein, one with the phosphorylated properties of KTVD, to re-stimulate tissue growth. Although further studies are needed, Chojkier noted that this approach may hold promise for conditions requiring tissue or organ regeneration, such as in liver failure.
In addition to Buck and Chojkier, additional authors of the Molecular Cell paper were Valeria Poli, Ph.D., University of Dundee, United Kingdom; and Tony Hunter, Ph.D., the Salk Institute for Biological Studies. Funding came from the National Institutes of Health, the Department of Veterans Affairs, and the American Liver Foundation.
Journal
Molecular Cell