Scientists Identify Scar-forming Mechanisms With Implications for Scleroderma

Scientists have identified a mechanism of scar formation in the skin, and demonstrated in mouse models a way to heal wounds to create normal skin instead of scar tissue,  a study revealed.

Their work could have implications for treating people with scleroderma.

Modulating — or modifying the activity — of a gene called engrailed-1 may prevent tissue scarring, according to the scientists, who said that successful preclinical work in other animals may lead to clinical trials.

The study, “Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring,” was published in the journal Science. 

In scleroderma, also known as systemic sclerosis, cells known as fibroblasts secrete excess amounts of the protein collagen, which leads to thick and hardened scar-like skin. Scarring, or fibrosis, can accumulate in the organs, including the heart, lungs, and kidneys.

Typically, after an injury to the skin from accidents or surgery, fibroblasts secrete collagen to heal the wound rapidly. But skin scar tissue is different from normal skin because it has no hair follicles, no sweat glands, and is inflexible and weaker. 

“If you heal slowly, you might get an infection or bleed to death,” Michael Longaker, MD, the study’s co-senior author from Stanford Medicine, said in a university press release.

“A scar is a spot weld — it covers the wound quickly, but it compromises form and function,” Longaker said.

As a surgeon, Longaker wondered why wounds on the skin of unborn babies (fetuses) healed without scarring, but wounds on the skin of adults and children left scars. 

“That question occupied me for a year, which became four years, which became decades,” he said. “Since then, my research has expanded to many other areas, but the attempt to understand scar formation has always been an active area of interest.” 

Longaker noticed that skin tension — how “tight” it is — following surgery made a difference in scar formation, and by lowering the forces that pulled the edges of a wound, scar formation was reduced.

“Early in fetal development, when skin injury doesn’t result in scarring, the skin of the fetus is gelatinous and doesn’t really have the ‘tightness’ we associate with skin,” Longaker said. “At the other end of our lives, if a 95-year-old has been exposed to a lot of sun and has loose skin, scar formation is minimal because he or she doesn’t have that tension in the skin.” 

Further understanding of how scar tissue forms — and how it can be stopped or prevented — may allow for the development of therapies to treat or reverse skin and tissue scarring in scleroderma and other conditions, according to Longaker.

However, “there is currently no drug or molecular strategy for preventing or reversing the fibrotic process of scar formation,” he said. Additionally, little is known about the underlying mechanisms that block a normal, regenerative healing response. 

To find out, Longaker and his team focused on a gene called engrailed-1 (En1), which encodes a protein found in fibroblasts.

First, his team transplanted fibroblasts into a mouse model and traced En1 expression (activity) over time. The results revealed that fibroblasts in the skin began to produce the engrailed protein in a wound environment in up to 50% of scar-forming fibroblasts. 

To investigate the impact of skin tension on En1 gene activity, mouse fibroblasts that did not express engrailed were grown in varying ways: inside a soft gel without tension, on a stiff plastic dish that produced tension, and on the same tension-inducing plastic but in the presence of a chemical that blocked YAP, a signaling protein generated with tension. 

The team found that fibroblasts grown on the soft gel did not express engrailed. In contrast, fibroblasts grown on the stress-inducing plastic did. Also, cells grown on plastic did not express engrailed when treated with YAP blockers. Further experiments showed that engrailed regulated a wide array of genes related to scarring. 

In addition, the team found an increase in the number of fibroblasts expressing engrailed and thicker scars when tension was applied to healing wounds in mice.

Next, verteporfin (brand name Visudyne, by Bausch & Lomb), an approved medicine used to treat eye disease by blocking tension-induced signaling, was applied to surgical wounds in mice under anesthesia, and tension was applied. Verteporfin blocked En1 activation and led to skin regeneration in 30 days with the recovery of functional hair follicles and sweat glands.

“There must be three things for wound healing to be true skin regeneration,” Longaker said. “The skin needs to have normal hair follicles and glands, it needs to have a normal appearance under a microscope, and it needs to be just as strong as normal skin.”

According to Stanford Medicine, the results were “astounding.”

“The first thing we were shocked by was the all the hair in the healed wound,” Longaker said. “We were also able to see normal glands and showed that the skin was just as strong as unwounded skin.”

To confirm these results, an artificial intelligence algorithm compared microscopic images of skin to look for subtle differences unseen by the human eye. The program was unable to identify differences between skin regenerated with verteporfin and normal skin. 

Likewise, a mouse specially bred without YAP promoted recovery of normal skin, which “suggests that modulation of En1 activation, whether direct or indirect, can yield wound regeneration,” the team wrote. 

“These results are exciting because we have shown that we are able to intervene and stop fibroblasts from sensing mechanical force when healing a skin wound,” said Geoffrey Gurtner, MD, a professor of surgery at Stanford Medicine and the study’s co-senior author.

“Now we need to see if the same approach will work in preventing other kinds of scarring,” Gurtner said.

According to the scientists, it is possible that this approach could work for burns, bands of scar-like tissue that form inside the abdomen, and liver scarring.

“We have demonstrated fully regenerative skin healing in a postnatal mammal that normally scars; this finding has translational implications for the tens of millions of patients each year who develop scars and other fibroses,” the investigators concluded.

The implications could be far-reaching, according to Longaker.

“It’s estimated that 45% of Americans die from a disease that involves scarring in some form. So there are potentially many more applications,” he said.