We examined the role of vascular function and inflammation in the development and failure to heal diabetic foot ulcers (DFUs). We followed 104 diabetic patients for a period of 18.4 ± 10.8 months. At the beginning of the study, we evaluated vascular reactivity and serum inflammatory cytokines and growth factors. DFUs developed in 30 (29%) patients. DFU patients had more severe neuropathy, higher white blood cell count, and lower endothelium-dependent and -independent vasodilation in the macrocirculation. Complete ulcer healing was achieved in 16 (53%) patients, whereas 13 (47%) patients did not heal. There were no differences in the above parameters between the two groups, but patients whose ulcers failed to heal had higher tumor necrosis factor-α, monocyte chemoattractant protein-1, matrix metallopeptidase 9 (MMP-9), and fibroblast growth factor 2 serum levels when compared with those who healed. Skin biopsy analysis showed that compared with control subjects, diabetic patients had increased immune cell infiltration, expression of MMP-9, and protein tyrosine phosphatase-1B (PTP1B), which negatively regulates the signaling of insulin, leptin, and growth factors. We conclude that increased inflammation, expression of MMP-9, PTP1B, and aberrant growth factor levels are the main factors associated with failure to heal DFUs. Targeting these factors may prove helpful in the management of DFUs.

Purpose. Nitric oxide (NO) is involved in leukostasis and blood-retinal barrier (BRB) breakdown in the early stages of diabetic retinopathy (DR), but it is unclear which NO synthase (NOS) isoforms are primarily involved. In this study, the authors aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO-mediated leukostasis and BRB breakdown. methods. Diabetes was induced with streptozotocin for 2 weeks. Mice were treated with a NOS inhibitor, N ^G-nitro-L-arginine methyl ester (L-NAME), which shows a preference for constitutive isoforms over iNOS. Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat-mounted retinas with confocal microscopy, in vivo with a scanning laser ophthalmoscope, and in vitro in a retinal endothelial cell line. ICAM-1, occludin, and ZO-1 levels were assessed by Western blot, flow cytometry, or immunohistochemistry. Nitrotyrosine content was assessed by immunohistochemistry. results. Diabetes increased leukostasis within retinal vessels and BRB permeability, which were reduced by L-NAME. Similar effects were observed in diabetic iNOS knockout mice. In diabetic mouse retinas, ICAM-1 protein levels increased, whereas the immunoreactivity of tight junction proteins, occludin and ZO-1 decreased, in correlation with increased protein levels of all NOS isoforms. Those effects were prevented by L-NAME and also in diabetic iNOS knockout mice. High glucose and nitrosative/oxidative stress also increased leukostasis caused by ICAM-1 upregulation. conclusions. These results indicate that the iNOS isoform plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM-1 upregulation and tight junction protein downregulation.

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.