Protease levels may be clue to slow healing

Proteins required for normal wound healing

Proteases are enzymes that cut proteins into fragments. Some researchers have hypothesized that these enzymes play a key role in wound healing — or, more accurately, in the inhibition of healing. That’s because the molecules that regulate wound healing are primarily proteins. If proteases do in fact destroy these crucial proteins, it follows that protease activity would impede the wound healing process.

"Proteases are important because the molecules that regulate wound healing and those that make up regenerative scar materials are proteins," says Greg Schultz, PhD, professor of OB/GYN at the University of Florida in Gainesville. "In fact, all things required for normal wound healing contain proteins."

Yet Schultz and other researchers have found that fluids produced by chronic wounds contain relatively high levels of matrix metalloproteinase (MMP), the class of protease molecules that are believed to attack proteins.

"We assumed early that chronic wounds do not produce enough growth factors. But we found that these wounds were actually producing lots of them," says Schultz.

He and his colleagues considered the possibility that endogenously produced growth factors were not working properly, because even though they found relatively large amounts of growth factor RNA contained in wound fluid, wounds still weren’t healing. Why?

"When we collected fluids from wounds, we noticed substantial amounts of degradation among proteins of higher molecular weight, and we noticed a loss of these proteins and a shifting from the standard profile associated with acute wounds," Schultz explains. This, Schultz and his colleagues hypothesized, resulted from the action of protein-destroying proteases.

An analysis of fluids collected from patients with acute healing wounds or chronic wounds showed that levels of pro-inflammatory cytokines (TNF-alpha and IL-1beta) were significantly elevated compared with the levels in fluids collected from acute wounds. In addition, protease levels were lower in healing wounds than in chronic wounds where fluid was collected prior to treatment — 50-fold lower, in fact.

When investigators added growth factors to fluids from healing wounds, the level of growth factors remained relatively stable. But when growth factors were added to fluids from nonhealing wounds, the growth factors rapidly degraded. This suggested that elevated levels of proteases in chronic wound fluids were contributing to the inability of the wound to heal because the proteases degraded essential growth factors and matrix proteins.

In one study, 15 patients with chronic venous/arterial leg ulcers that had failed outpatient treatment were admitted to the hospital. Samples of wound fluid were collected on admission and after two weeks of bed rest and standard therapy. All 15 patients showed clinical signs of granulation tissue formation and epithelialization, although two patients had no decrease in ulcer size. The average protease level in the 15 samples collected before therapy began was 38 +/- 10 micrograms of MMP eq/ml. After two weeks of treatment, the protease levels decreased in 12 of 15 patients to an average of 11 +/- 4 micrograms MMP eq/ml. Addition of an MMP inhibitor significantly decreased the average protease levels in samples taken before treatment. Levels of MMP and serine proteases both decreased significantly as the chronic wounds began to heal.

The investigators’ analysis revealed that the specific proteases that were found in elevated levels in chronic wound fluids have the ability to degrade matrix proteins present in the skin, and that their lack of specificity causes them to degrade other growth factors as well. This suggests that one reason the wounds failed to heal was that the proteases were destroying the growth factor receptors and extracellular matrix proteins to the point of keeping the wound from being repaired normally.

If this turns out to be true, it would offer one explanation for why externally applied growth factors work better in some wounds than in others.

Schultz noted that in various types of chronic wounds — pressure sores, venous stasis ulcers, diabetic ulcers — a similar rise in MMPs is noticed after the wound reaches a certain point. Other proteases also are elevated, but not as consistently as MMPs. He also says the observation of elevated protease activity in chronic wounds has been confirmed by other research groups in Germany and the United States, but adds that no one has conducted a study proving elevated protease levels cause wounds to stop healing.

"We only have correlation from the observations of other clinicians," he says. "The final proof that proteases cause wounds to remain chronic will require clinical trials showing that you can increase the rate of healing if you treat a wound with an MMP inhibitor combined with good clinical management."

Schultz hopes to establish clinical trials in which MMP inhibitors are applied to reduce the level of protease activity, and perhaps combine them with exogenous growth factors. He is currently looking for sponsorship from the pharmaceutical industry to explore these areas.