Impaired wound healing in mouse models of diabetes is mediated by TNF-α dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1)

Abstract

Aims/hypothesis The role of TNF-α in impaired wound healing in diabetes was examined by focusing on fibroblasts. Methods Small excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-α was specifically inhibited by pegsunercept. Results Diabetic wounds had increased TNF-α, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p<0.05). TNF-α inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-α was blocked (p <0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-α enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-α also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown. Conclusions/interpretation These studies indicate that TNF-α dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-α induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis.

Persistence of TNFα in diabetic wounds

“…In their study, anti-TNF treatment increased fibroblast density and improved wound healing. Of course, the usual caveat applies to such animal studies (the authors used streptozotocin-induced and db/db models of type 1 and type 2 diabetes, respectively) regarding whether they are relevant to human disease, but the results of Siqueira et al [1] are in close agreement with observations we have made in a human model of dermal wound healing employing cantharidin skin blisters.Cantharidin is a blistering agent used in the treatment of molluscum contagiosum and warts [2]. It is a protein phosphatase 1 and 2 alpha inhibitor that causes acantholysis [2].…”

Exercise, Obesity, and Cutaneous Wound Healing: Evidence from Rodent and Human Studies

“…Likewise, TNF-a impairs insulin signaling in wounds in ob/ob mice 37 and increases fibroblast apoptosis at the wound site in db/db mice. 38 Recent evidence has supported the hypothesis that leukocyte dysfunction mediates delayed healing in obesity and diabetes. Macrophages are responsible for clearing apoptotic PMNs from the wound site in order for healing to progress.…”

FOXO1 differentially regulates both normal and diabetic wound healing

“…Enhanced levels of advanced glycation end products (AGEs) and TNF are associated with altered diabetic wound healing. Impaired wound healing responses in diabetic animals are improved by blocking TNF, or AGEs, which reduces infiltration by proinflammatory macrophages, reduces fibroblast apoptosis, and improves collagen deposition (Goova et al, 2001;Goren et al, 2007;Siqueira et al, 2010;Ashcroft et al, 2012). Moreover, reducing inflammation by deletion of TLR4 improves the wound healing response (Dasu and Jialal, 2013).…”