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dcyphr | Impaired Wound Repair in Adult Endoglin Heterozygous Mice Associated with Lower NO Bioavailability

Abstract

This study assesses Endoglin’s (Eng) role in wound healing by comparing heterozygous Eng+/- mice and normal Eng+/+ mice. The area of the wound was higher in Eng+/- mice and took longer to heal compared to the Eng+/+ mice. The Eng+/- mice had slower growing and elongated in shape keratin cells. Decreased nitric oxide (NO) slowed the healing of normal Eng+/+ mice, but had no effect on Eng+/- mice. Increased NO caused faster wound closure in Eng+/-, but had no effect on the Eng+/+ mice. Stimulation with 12-O-tetradecanoylphrobol-13-acetate (TPA) enhanced Eng expression in tissue culture and in live mice. These results show that Eng plays an important role in wound healing and the availability of NO.


Introduction

Wound healing is a very complex process that includes specific signaling pathways. Processes like reepithelialization, inflammation, connective tissue contraction, angiogenesis, and tissue remodeling are needed in wound healing. The delay of wound healing in diabetic patients is caused by poor blood flow, low oxygen levels, too much inflammation, edema, and endothelial cell dysfunction.

Transforming growth factor-beta (TGF-beta) is one important factor throughout wound healing. Endoglin (CD105 or Eng) works to regulate how the cells respond to the molecules that bind to the TGF-beta family. Eng is seen in high levels in tissues that are undergoing angiogenesis. Inhibiting Eng stops angiogenesis. Genetically modified mice without any Eng do not survive through pregnancy, but heterozygous mice (Eng+/-) live full lives. Eng regulates nitric oxide synthase (NOS), and Eng+/- mice produce less nitric oxide (NO). After a wound, Eng expression increases along with inflammation.


Materials and Methods

Both the NOS inhibitor and NO source were given by mouth to the mice. TPA was applied to the skin of the mice, and then a skin sample was prepared for analysis. Immunohistochemistry, western blot, and RT-PCR were used to analyze the wound tissue.


Results

Temporal and Spacial Expression of Eng During Repair

Eng expression in normal mice was low at first, but increased, peaked, and decreased within 8 days after injury. Eng expression was highest at the edges of the wound and blood vessels near the wound.


Delayed Wound Closure in Eng-deficient Mice

Wound healing and closure was slower in Eng+/- mice compared to normal mice by 1 or 2 days. NOS inhibition, which decreases NO levels, slowed wound healing in normal mice, but did not affect Eng+/- mice. Interestingly, increased NO levels increased wound healing in Eng+/- mice, but had no effect on normal mice. The skin near the wound is thinner and longer than normal in Eng+/- mice, even after the wound has closed.


Disturbed Epidermal Proliferation in Eng-deficient Mice

Researchers tested Ki67 to show rapidly growing keratin cells. In unwounded tissue, there was no difference in Ki67 for Eng+/- or normal mice. But in wounds, Ki67 was reduced in Eng+/- mice.

Researchers tested K17 to see how many migrating keratin cells were present. K17 was higher in Eng+/- than normal mice.

TPA is a chemical that promotes tumors. Researchers applied TPA to the skin of mice, and this area had increased Eng and Ki67. But tumor growth was lower in Eng+/- mice than in normal mice.


Discussion

This study provides support that Eng plays a huge role in wound healing. Decreased Eng can cause an increase in a molecule called transforming growth factor-beta (TGF-beta). High expression of TGF-beta will cause angiogenesis to stop, so Eng and TGF-beta work together to balance angiogenesis.

Nitric oxide (NO) has been shown to increase wound healing in previous studies, and this study supports those findings. This study related low NO levels with poor wound healing, and that this is the case in Eng+/- mice. Low NO levels caused by Eng+/- mice may cause low growth of keratin cells and low expression of vascular endothelial growth factor. Decreased NO can also decrease angiogenesis, which is closely regulated by Eng and TGF-beta. Even though Eng increases wound healing, high amounts are also seen in many skin diseases.