In many ways, the process to heal a wound parallels the steps involved in repairing, for example, a pothole. Both require specific supplies: cells and platelets, or asphalt and sand. And both processes necessitate a way transport those supplies, like a safe and accessible roadway. For the body, that means a network of blood vessels that extend to and eventually permeate the wound site.
This process of blood-vessel formation, known as angiogenesis, is a crucial for wound repair, yet the molecules responsible for controlling it have remained only partly understood.
A new study led by researchers from the University of Pennsylvania School of Dental Medicine has identified a key player in the process, the molecule Foxo1. Already known to be involved in repairing an exposed wound by stimulating the movement of keratinocytes to cover the wounded area, Foxo1 was shown to be required for normal angiogenesis during healing. FOXO1 does this by inducing the production of a key factor, VEGFA. (Foxo1 refers to the protein and FOXO1 refers to the gene.)
“This is the first study that shows that FOXO1 organizes keratinocyte activity to promote VEGFA expression and wound angiogenesis,” says Hyeran Helen Jeon, an instructor in Penn Dental Medicine’s Department of Orthodontics and the first author on the paper, published in the Journal of Pathology. “Stimulating FOXO1 could induce cells to produce more VEGFA. Blocking Foxo1 could work in cases where reduced angiogenesis would be helpful, like in patients with tumors or diabetic retinopathy.” The study was recently highlighted in a commentary article in the journal as an important finding.
Jeon conducted the work in collaboration with Dana Graves, interim dean of Penn Dental Medicine and a professor of periodontics. They began when Jeon was enrolled in Penn’s Doctor of Science in Dentistry (DScD) Program, which Graves directs.
Previous studies explored the role of Foxo1 in keratinocyte migration in wounds, but turned up a link to angiogenesis when Jeon took a closer look at an older set of experiments and found that genes regulated by FOXO1 were strongly related with angiogenesis. Thus the team began investigating the role of Foxo1 in keratinocytes in blood-vessel formation.
In mice lacking Foxo1 in keratinocytes, the researchers found that the density of blood vessels in healing wounds was decreased by about 50 percent, compared to their littermates with normal levels of the protein. Proliferating endothelial cells were decreased by 45 percent, and formation of new connective tissue on the surface of the wound was reduced by 50 percent. The wounds did not heal nearly as quickly.
While it is known that keratinocytes express VEGFA, a protein recognized as important for angiogenesis, the mechanism of its regulation during wound healing had not been fully clarified. Looking specifically at VEGFA, they found that its expression levels in epithelium—the outer layer of different tissues—was significantly reduced when the gene FOXO1 was removed in these cells. Further, it was found that Foxo1 binds to the VEGFA promoter and can directly regulate its production in keratinocytes.
To confirm that this effect held in a larger animal model, the team collaborated with Paulo Coelho of New York University. Using a porcine model, they found that using a compound that inhibits Foxo1 reduced blood vessel density in healing wounds by around 40 percent.
“After confirming this effect, we felt very confident,” Jeon says. “Hopefully in the future we’ll further explore a clinical application of Foxo1.”
In addition to exploring its application in wound healing, Jeon, Graves, and their colleagues plan to look in detail at how Foxo1 is functioning in angiogenesis in diabetic wounds. Earlier work led by Graves showed that Foxo1 behaved differently in mice with diabetes compared to mice with normal blood sugar, having the opposite effect of cell migration. The researchers would like to see whether blood-vessel formation shows a similar variation in diabetes.
Jeon says the promising findings of their study underscore the benefits of Penn’s DScD Program, of which she was the first graduate in the Department of Orthodontics.
“In the health fields, most students experience patient treatment, but I think the clinical and research experiences are complementary and enhance each other,” she says. “That’s the way we can make the biggest difference in health. I am a real beneficiary of this program and sincerely appreciate it.”
Jeon and Graves coauthored the study with Penn Dental Medicine’s Quan Yu, Yongjian Lu, Evelyn Spencer, Chanyi Lu, Tatyana Milovanova, Yang Yang, Chenying Zhang, Olga Stepanchenko, and Rameen Vafa; and with Coelho of New York University.
The work was supported by the National Institute of Dental and Craniofacial Research and an International Team for Implantology grant.