If a sharp pebble finds its way in between your shoe's inner sole and your sock, the ouch factor lets you know you'd better unlace, shake and relace that shoe. But large numbers of people throughout the world have advanced diabetes, and never feel that grating pebble. They suffer from polyneuropathy, a frequent side effect of diabetes mellitus - Types I and II alike - in which the lower extremities lose their sense of pain. Six percent of the U.S. population are diabetic, and 15 percent of them have foot ulcers.
More common than pebbles are ill-fitting shoes, which rub incessantly - but unfeelingly - against toes and heels, bruising, then breaking, the skin, which becomes infected and eventually ulcerated. These diabetic ulcers don't heal because the other protean metabolic disorders of the disease abrogate the healing process. In consequence, some 64,000 diabetics in the U.S. alone undergo lower-limb amputation annually. (See BioWorld Today, March 5, 1999, p.1.)
People with diabetes cannot produce insulin. Its lack causes blood vessels and tissue membranes to degenerate. This process interferes with normal healing by preventing the body's tissue-repair cells from getting into the injured area. Even in healthy individuals, wound healing is a complex, synchronized ballet of key proteins, led by fibronectin, integrin receptors and skin cells. "Plasma fibronectin," explained cell biologist Donna Livant, at the University of Michigan, Ann Arbor, "is a prevalent component of the blood, lymph and interstitial fluid between tissue cells, which circulates freely throughout the body.
"Right after a wound occurs," she continued, "enzymes cleave the fibronectin molecule into fragments, which diffuse from the wound. They function to summon adjacent skin cells to begin wound healing right away. These peptide fragments bind to integrin receptors on surrounding cells, stimulating them to invade the wound and repair the damage."
Early in this invasion process, enter the integrin receptors. "Cells in the body," Livant went on, "including the skin cells that form wound coverings - like epithelial cells, and the cells immediately underneath, like fibroblasts, as well as the endothelial cells that line the blood vessels - all express two integrin receptors that bind to plasma fibronectin. One is called alpha-5-beta-1, which binds to the PHSRN sequence of fibronectin. The other receptor, alpha-4-beta-1," she added, "binds a completely distinct sequence to repress the expression of the enzymes associated with invasion of the wound."
Sequence Hustles Repair Crew Into Wound
That PHSRN peptide - which Livant and her team isolated, then modified to augment its potency 100-fold - is a stretch of five amino acids, namely: proline (P), histidine (H), serine (S), arginine (R) and asparagine (N). Fibronbectin itself is a molecule of two identical chains, each numbering some 500 amino acids. "We believe," she observed, "that the sequence responsible for stimulating cell migration into wounds is PHSRN."
Livant is senior author of a research report in the June 2000 issue of The Journal of Clinical Investigation titled "The PHSRN sequence induces extracellular matrix invasion and accelerates would healing in obese diabetic mice."
"The db/db obese diabetic mice we work with," Livant told BioWorld Today, "are a good model of Type II diabetes because they aren't born with the late-onset phenotype that occurs when they're young adults. At three to seven months of age, these mice are young adults, and their delayed wound healing, obesity and late onset are all very characteristic of type II diabetes in humans. The Type I juvenile-onset nod - non-obese diabetic mouse strain - reportedly has delayed wound healing as well."
To simulate diabetic ulcers in their experimental mice, the co-authors made skin wounds on their backs with 4-millimeter biopsy punches to a depth averaging 1.7 millimeters. Into each of these lesions they pipetted a single drop of their enhanced PHSRN peptide solution.
"After eight days," Livant recounted, "skin wounds treated with the activated peptide in both diabetic and non-diabetic mice were completely closed and healed. Wounds in the untreated non-diabetic animals healed in nine days, but skin closure time in untreated diabetic mice ranged from 20 days to 42 days."
She went on: "When we examined sections of lesioned tissue under the microscope, we found that wound areas treated with PHSRN were invaded right away by cells involved in the healing process. This resulted in a rapid decrease in wound size, as well as rapid coverage by keratinocytes - skin cells. In consequence, this suggested that the peptide activates a pathway that functions in wound contraction."
She made the point that "in these diabetic mice, the untreated wounds heal very slowly, and they vary tremendously in the amount of time they take. But they all heal eventually. So this is not a model of chronic wounds.
"Since delayed cell migration is the reason wounds heal slowly in people with diabetes," Livant observed, "we believe our peptide could prove efficacious in preventing chronic wounds in diabetic patients. But in our work we have not yet studied chronic, non-healing wounds. We think our peptides might be very useful in these - or we hope they could be."
Non-Peptide Mimetic On The Way
At present, Livant and her co-authors "are very interested in how their PHSRN peptide could be used in conjunction with growth factors to further stimulate wound healing in diabetics. I think what people are doing now," she observed, "is mostly studying how to stimulate diabetic wound healing by applying various growth factors to their wounds."
She and her team are also engaged in "devising non-peptide mimetics of the PHSRN sequence, because it's so small - it's only five amino acids long, with a molecular weight of 600 or so. That's a pretty straightforward compound to model - to try to get a model that has a very similar structure, but is not an amino acid sequence.
"Such a compound," she explained, "would not be subject to degradation by a protease. And because chronic wounds have very high protease levels," Livant concluded, "a non-peptide mimetic of PHRSN could be very effective in stimulating those wounds to heal."
The university holds issued and pending patents covering use of the PHSRN peptide in wound healing, and Livant is in contact with potential biotech and pharmaceutical partners.