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Updated: Aug 12, 2025
Protocol to Create Chronic Wounds in Diabetic Mice
Published on: September 25, 2019
Neutralization of excessive CCL28 improves wound healing in diabetic mice
Zhenlong Chen1, Jacob M Haus2, Luisa A DiPietro3,4
1Department of Anesthesiology, College of Medicine, University of Illinois at Chicago, Chicago, IL, United States.
Abstract:
Introduction: Chronic, non-healing skin wounds such as diabetic foot ulcers (DFUs) are common in patients with type 2 diabetes mellitus (T2DM) and often result in limb amputation and even death. However, mechanisms by which T2DM and inflammation negatively impact skin wound healing remains poorly understood. Here we investigate a mechanism by which an excessive level of chemokine CCL28, through its receptor CCR10, impairs wound healing in patients and mice with T2DM. Methods & Results: Firstly, a higher level of CCL28 was observed in skin and plasma in both patients with T2DM, and in obesity-induced type 2 diabetic db/db mice. Compared with WT mice, adipose tissue from db/db mice released 50% more CCL28, as well as 2- to 3-fold more IL-1β, IL-6, and TNF-α, and less VEGF, as determined by ELISA measurements. Secondly, overexpression of CCL28 with adenovirus (Adv-CCL28) caused elevation of proinflammatory cytokines as well as CCR10 expression and also reduced eNOS expression in the dorsal skin of WT mice as compared with control Adv. Thirdly, topical application of neutralizing anti-CCL28 Ab dose-dependently accelerated wound closure and eNOS expression, and decreased IL-6 level, with an optimal dose of 1 μg/wound. In addition, mRNA levels of eNOS and anti-inflammatory cytokine IL-4 were increased as shown by real-time RT-PCR. The interaction between eNOS and CCR10 was significantly reduced in diabetic mouse wounds following application of the optimal dose of anti-CCL28 Ab, and eNOS expression increased. Finally, enhanced VEGF production and increased subdermal vessel density as indicated by CD31 immunostaining were also observed with anti-CCL28 Ab. Discussion: Taken together, topical application of neutralizing anti-CCL28 Ab improved dorsal skin wound healing by reducing CCR10 activation and inflammation in part by preventing eNOS downregulation, increasing VEGF production, and restoring angiogenesis. These results indicate anti-CCL28 Ab has significant potential as a therapeutic strategy for treatment of chronic non-healing diabetic skin wounds such as DFUs.
Insights
Excessive chemokine CCL28 impairs wound healing in type 2 diabetes. Neutralizing anti-CCL28 antibody therapy improved healing by reducing inflammation and restoring blood vessel formation in diabetic wounds.
Area of Science:
- Biomedical Science
- Immunology
- Wound Healing Research
Background:
- Chronic non-healing skin wounds, like diabetic foot ulcers (DFUs), are a significant complication of type 2 diabetes mellitus (T2DM).
- The precise mechanisms by which T2DM and inflammation impede skin wound healing are not fully understood.
- Elevated levels of chemokine CCL28 and its receptor CCR10 are implicated in impaired wound healing in T2DM.
Purpose of the Study:
- To investigate the role of chemokine CCL28 and its receptor CCR10 in T2DM-associated impaired skin wound healing.
- To evaluate the therapeutic potential of neutralizing anti-CCL28 antibodies in accelerating wound closure and restoring healing mechanisms.
Main Methods:
- Quantified CCL28 levels in skin and plasma of T2DM patients and db/db mice using ELISA.
- Overexpressed CCL28 in wild-type (WT) mice to assess its impact on cytokine and eNOS expression.
- Administered neutralizing anti-CCL28 antibody topically to diabetic mouse wounds and evaluated wound closure, protein expression (eNOS, VEGF, IL-6), and angiogenesis (CD31 staining).
- Analyzed mRNA levels of eNOS and IL-4 using real-time RT-PCR.
Main Results:
- Higher CCL28 levels were found in T2DM patients and db/db mice compared to controls.
- CCL28 overexpression increased pro-inflammatory cytokines and CCR10, while decreasing eNOS expression.
- Topical anti-CCL28 antibody treatment dose-dependently accelerated wound closure, increased eNOS and VEGF expression, reduced IL-6, and promoted angiogenesis.
- The antibody treatment decreased the interaction between eNOS and CCR10 and restored eNOS expression.
Conclusions:
- Excessive CCL28, via CCR10, exacerbates inflammation and impairs wound healing in T2DM.
- Neutralizing anti-CCL28 antibody therapy effectively promotes wound healing by reducing CCR10 activation and inflammation.
- This antibody treatment restores angiogenesis and has potential therapeutic value for chronic diabetic wounds like DFUs.
