Blockade Of Ogfr Enhances Full-thickness Diabetic Wound Healing
Open Access
- Author:
- Immonen, Jessica Ann
- Graduate Program:
- Anatomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 07, 2014
- Committee Members:
- Dr Patricia Mc Laughlin, Dissertation Advisor/Co-Advisor
Dr Patricia Mc Laughlin, Committee Chair/Co-Chair
Samuel Shaomin Zhang, Committee Member
Alistair J Barber, Committee Member
Joseph W Sassani, Committee Member
Timothy Cooper, Special Member - Keywords:
- diabetes
wound healing
opioids
naltrexone - Abstract:
- Diabetes mellitus affects over 25 million individuals in the United States. Complications related to diabetes cause financial burden, as well as loss of quality of life. More than 25% of patients with either Type 1 or Type 2 diabetes will experience one or more episodes of delayed cutaneous wound closure, a serious complication that if untreated, can lead to ulceration, systemic infection, and possible amputation. Understanding the pathophysiology associated with diabetes and wound closure, as well as design of new, non-toxic therapies, is needed. The hypothesis of this thesis project is that naltrexone (NTX), an opioid receptor antagonist, blocks opioid receptor binding to accelerates DNA synthesis, cell proliferation and wound healing. The opioid growth factor (OGF)- OGF receptor (OGFr) axis is involved in cell replication. OGF, chemically termed [Met5]-enkephalin, interacts with the OGFr to depress DNA synthesis by upregulating p16 and p21 to halt the cell cycle at the G1/S interface. To address how the OGF-OGFr pathway modulates wound healing, the first aim investigated angiogenesis, an integral phase of wound repair. Using a rat model of Type 1 diabetes, along with age-matched normal rats as controls, full-thickness cutaneous wounds were created on the dorsum and treated with topical applications of 10-5 M NTX dissolved in moisturizing cream. Wound closure was monitored photographically and tissue samples were collected for histopathology. Blood vessel formation, measured by VEGF, FGF-2, and α-SMA staining, was enhanced in rats treated with topical NTX relative to those receiving only vehicle. Specific aim 2 evaluated the long-term effects of NTX on skin and the remodeling phase of wound healing. Full-thickness wounds generated in diabetic rats treated with NTX revealed normal collagen deposition and normal remodeling compared to controls. Tensile strength of the wounds was restored, and no adverse histopathology was noted following 3 weeks of NTX treatment. The mechanism of action associated with NTX treatment was investigated in Specific aim 3. The initial studies determined the specific and selective opioid receptor modulated by the nonspecific antagonist NTX. Using molecular technology and the NIH 3T3 fibroblast cell line, OGFr protein expression was depressed by siRNAs. Application of NTX to the fibroblast cultures revealed that suppression of OGFr with siRNA blocked the response to NTX. Studies with specific ligand agonists demonstrated that only OGF, and not DAMGO, DPDPE, or EKC, altered growth of cultured fibroblasts. Further studies with NIH 3T3 cells, and primary auricular fibroblasts, confirmed the presence of OGF and OGFr in fibroblasts. These data confirm that the OGF-OGFr axis mediates cell replication in fibroblasts, a primary cell involved in cutaneous wound healing, and that the OGF-OGFr axis is the specific biological pathway blocked by NTX. In Specific Aim 4, studies examined the efficacy of NTX in a mouse model of Type 2 diabetes. The genetic mouse model, using db/db mice, was established and full-thickness cutaneous wounds were generated. Wound closure in diabetic mice was significantly delayed relative to normal C57Bl/6 mice, and topical application of NTX significantly accelerated wound closure relative to db/db mice receiving only vehicle. In conclusion, delays in cutaneous wound closure are related to cellular proliferation and may be modulated by the OGF-OGFr pathway. Blockade of this regulatory pathway by the opioid antagonist NTX enhances all stages of wound repair, and may serve as a safe and efficacious treatment for wound closure complications found in individuals with Type 1 or Type 2 diabetes.