Mast Cells Regulate Wound Healing in Diabetes.

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.

Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA-26a.

Wound healing is a physiological reparative response to injury and a well-orchestrated process that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix deposition, and wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. Herein, we investigate the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to high glucose in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~3.5-fold) four days post-wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to ~80%), increased granulation tissue thickness (by 2.5-fold) and accelerated wound closure (53% after nine days) compared to scrambled anti-miR controls in db/db mice. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene SMAD1 in ECs nine days post-wounding of diabetic mice. In addition, high glucose reduced activity of the SMAD1-3'-UTR. Diabetic dermal wounds treated with LNA-anti-miR-26a had increased expression of ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy.

Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies.

Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.

Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds. We also studied the impact of deficiency in SP or its receptor, neurokinin-1 receptor, on wound healing in mouse models. SP treatment improved wound healing in mice and rabbits, whereas the absence of SP or its receptor impaired wound progression in mice. Moreover, SP bioavailability in diabetic skin was reduced as SP gene expression was decreased, whereas the gene expression and protein levels of the enzyme that degrades SP, neutral endopeptidase, were increased. Diabetes and SP deficiency were associated with absence of an acute inflammatory response important for wound healing progression and instead revealed a persistent inflammation throughout the healing process. SP treatment induced an acute inflammatory response, which enabled the progression to the proliferative phase and modulated macrophage activation toward the M2 phenotype that promotes wound healing. In conclusion, SP treatment reverses the chronic proinflammatory state in diabetic skin and promotes healing of diabetic wounds.

Aliskiren improves vascular smooth muscle function in the skin microcirculation of type 2 diabetic patients with normal renal function.

OBJECTIVE: The objective of this paper is to study the effect of aliskiren on metabolic parameters and micro- and macrovascular reactivity in individuals diagnosed with or at high risk for developing type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: We studied 47 T2DM and 41 at-risk individuals in a randomized, double-blinded, placebo-controlled trial. All subjects were treated with 150 mg aliskiren or placebo daily for 12 weeks. Twenty-six (55%) of T2DM and four (8%) at-risk subjects were also treated with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers. RESULTS: Aliskiren treatment was associated with improvement in systolic and diastolic blood pressure and endothelium-independent vasodilation at the skin microcirculation in those with T2DM but not in those at risk. There were no incidences of hypotension and no significant changes in serum potassium or creatinine levels with aliskiren treatment in either study group. CONCLUSIONS: Aliskiren improves blood pressure and vascular smooth muscle function in the skin microcirculation of T2DM patients.

Expression of neuropeptides and cytokines in a rabbit model of diabetic neuroischemic wound healing.

OBJECTIVE: The present study is designed to understand the contribution of peripheral vascular disease and peripheral neuropathy to the wound-healing impairment associated with diabetes. Using a rabbit model of diabetic neuroischemic wound healing, we investigated rate of healing, leukocyte infiltration, and expression of cytokines, interleukin-8 and interleukin-6, and neuropeptides, substance P, and neuropeptide Y. METHODS: Diabetes was induced in New Zealand White rabbits by administering alloxan while control rabbits received saline. Ten days later, animals in both groups underwent surgery. One ear served as a sham, and the other was made ischemic (ligation of central+rostral arteries) or neuroischemic (ischemia+ resection of central+rostral nerves). Four 6-mm punch biopsy wounds were created in both ears and wound healing was followed for 10 days using computerized planimetry. RESULTS: Nondiabetic sham and ischemic wounds healed significantly more rapidly than diabetic sham and ischemic wounds. Healing was slowest in neuroischemic wounds, irrespective of diabetic status. A high M1/M2 macrophage ratio and a high proinflammatory cytokine expression, both indicators of chronic proinflammatory state, and low neuropeptide expression were seen in preinjury diabetic skin. Postinjury, in diabetic wounds, the M1/M2 ratio remained high, the reactive increase in cytokine expression was low, and neuropeptide expression was further decreased in neuroischemic wounds. CONCLUSIONS: This rabbit model illustrates how a combination of a high M1/M2 ratio, a failure to mount postinjury cytokine response as well as a diminished neuropeptide expression, contribute to wound-healing impairment in diabetes. The addition of neuropathy to ischemia leads to equivalently severe impaired wound-healing irrespective of diabetes status, suggesting that in the presence of ischemia, loss of neuropeptide function contributes to the impaired healing associated with diabetes.

Increased skin inflammation and blood vessel density in human and experimental diabetes.

Systemic inflammation is associated with impaired wound healing in diabetes mellitus (DM) patients. Using immunohistochemistry techniques, the authors investigated changes in skin inflammation and skin blood vessels in human and experimental diabetes. Comparing to the non-DM human subjects, the total number of inflammatory cells per biopsy and the number of inflammatory cells around blood vessels, a strong indication of inflammation, were higher in DM subjects irrespective of their risk for developing diabetic foot ulcer. Inflammatory cell infiltration was robustly increased in all DM animal models compared with their non-DM controls. The number and density of blood vessels and CD31 positive proliferating endothelial cells around preexisting skin vessels was also higher in the DM patients. However, there were no differences in the skin blood flow between the non-DM and DM subjects. The number of skin blood vessels was also increased in the DM animals; however, these differences were less obvious than the ones observed for inflammatory cells. We conclude that skin inflammation and skin blood vessel density is increased in diabetic human subjects and in rodent and rabbit models of diabetes.