Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers.

BACKGROUND: To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing. METHODS: We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing. RESULTS: All EPC phenotypes except the kinase insert domain receptor (KDR)(+)CD133(+) were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34(+)KDR(+) count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding. CONCLUSIONS: Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34(+)KDR(+) reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.

The evolving natural history of neurophysiologic function in patients with well-controlled diabetes.

This study aimed to investigate prospective changes to neurophysiologic function over 3 years in patients with well-controlled diabetes. Sixty-two subjects had neurologic examinations, symptom scores, autonomic testing, nerve conduction studies, quantitative sensory testing, and laser-Doppler flowmetry at 18-month intervals for 3 years. During the study, there was a 1 µV decrease in sural amplitude (p < 0.05), an increase in monofilament detection threshold of 0.36 g (p < 0.001), and a decrease in the axon-reflex vasodilation in the foot (p < 0.005) and forearm (p < 0.05). There was an increase in symptoms of distal hypersensitivity (p < 0.005) but no change in neuropathy frequency or severity. Our findings suggest that laser-Doppler flowmetry, a test of small fiber function, can detect the largest neurophysiologic change over time in groups of patients with diabetes. Sural nerve amplitude and monofilament thresholds may be more effective at detecting change in individual patients. Other tests of neurophysiologic function may require longer periods of time and greater numbers of participants to detect a difference. We conclude that patients with well-controlled diabetes and optimal medical management of comorbid risk factors have low rates of neuropathy development and progression although the clinical relevance of this finding to the general population of individuals with diabetes is unknown.

Postexercise phosphocreatine recovery, an index of mitochondrial oxidative phosphorylation, is reduced in diabetic patients with lower extremity complications.

OBJECTIVE: To identify differences in postexercise phosphocreatine (PCr) recovery, an index of mitochondrial function, in diabetic patients with and without lower extremity complications. METHODS: We enrolled healthy control subjects and three groups of patients with type 2 diabetes mellitus: without complications, with peripheral neuropathy, and with both peripheral neuropathy and peripheral arterial disease. We used magnetic resonance spectroscopic measurements to perform continuous measurements of phosphorous metabolites (PCr and inorganic phosphate [Pi]) during a 3-minute graded exercise at the level of the posterior calf muscles (gastrocnemius and soleus muscles). Micro- and macrovascular reactivity measurements also were performed. RESULTS: The resting Pi/PCr ratio and PCr at baseline and the maximum reached during exercise were similar in all groups. The postexercise time required for recovery of Pi/PCr ratio and PCr levels to resting levels, an assessment of mitochondrial oxidative phosphorylation, was significantly higher in diabetic patients with neuropathy and those with both neuropathy and peripheral arterial disease (P < .01 for both measurements). These two groups also had higher levels of tumor necrosis factor-α (P < .01) and granulocyte colony-stimulating factor (P < .05). Multiple regression analysis showed that only granulocyte colony-stimulating factor, osteoprotegerin, and tumor necrosis factor-α were significant contributing factors in the variation of the Pi/PCr ratio recovery time. No associations were observed between micro- and macrovascular reactivity measurements and Pi/PCr ratio or PCr recovery time. CONCLUSIONS: Mitochondrial oxidative phosphorylation is impaired only in type 2 diabetes mellitus patients with neuropathy whether or not peripheral arterial disease is present and is associated with the increased proinflammatory state observed in these groups.

Mechanisms involved in the development and healing of diabetic foot ulceration.

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.

Foot muscle energy reserves in diabetic patients without and with clinical peripheral neuropathy.

OBJECTIVE To investigate changes in the foot muscle energy reserves in diabetic non-neuropathic and neuropathic patients. RESEARCH DESIGN AND METHODS We measured the phosphocreatinine (PCr)/inorganic phosphate (Pi) ratio, total (31)P concentration, and the lipid/water ratio in the muscles in the metatarsal head region using MRI spectroscopy in healthy control subjects and non-neuropathic and neuropathic diabetic patients. RESULTS The PCr/Pi ratio was higher in the control subjects (3.23 +/- 0.43) followed by the non-neuropathic group (2.61 +/- 0.36), whereas it was lowest in the neuropathic group (0.60 +/- 1.02) (P < 0.0001). There were no differences in total (31)P concentration and lipid/water ratio between the control and non-neuropathic groups, but both measurements were different in the neuropathic group (P < 0.0001). CONCLUSIONS Resting foot muscle energy reserves are affected before the development of peripheral diabetic neuropathy and are associated with the endothelial dysfunction and inflammation.

Microvascular reactivity and inflammatory cytokines in painful and painless peripheral diabetic neuropathy.

OBJECTIVE: We investigated the association between inflammation, microvascular reactivity, and the development of peripheral diabetic neuropathy. RESEARCH DESIGN AND METHODS: We studied three groups: 55 healthy control subjects, 80 nonneuropathic patients, and 77 neuropathic diabetic patients. We also subdivided the neuropathic patients into a subgroup of 31 subjects with painless neuropathy and 46 with painful neuropathy. We measured the foot skin endothelium-dependent and -independent vasodilation, the nerve axon reflex-related vasodilation (NARV), and inflammatory cytokines and biochemical markers of endothelial function. RESULTS: The endothelium-dependent and -independent vasodilation and NARV were lower in the neuropathic group (P < 0.05). NARV was further reduced in the subgroup of painless neuropathy when compared to painful neuropathy (P < 0.05). Compared to the other two groups, the neuropathic group also had higher serum levels of PDGF AA/BB (P < 0.05), RANTES (P < 0.01), leptin (P < 0.0001), osteoprotegerin (P < 0.01), G-CSF (P < 0.05), sE-Selectin (P < 0.01), sICAM (P < 0.0001), sVCAM (P < 0.001), CRP (P < 0.0001), TNFalpha (P < 0.05), and fibrinogen (P < 0.05). Patients with painful neuropathy had higher sICAM-1 (P < 0.05) and CRP levels (P < 0.01) when compared to painless neuropathy. No major changes in the above results were observed in 78 diabetic patients who were seen for a second visit 21 months after the first visit. CONCLUSIONS: Peripheral diabetic neuropathy is associated with increased biochemical markers of inflammation and endothelial dysfunction. Painful neuropathy is associated with further increase in inflammation and markers of endothelial dysfunction and preservation of the nerve axon reflex.

The use of medical hyperspectral technology to evaluate microcirculatory changes in diabetic foot ulcers and to predict clinical outcomes.

OBJECTIVE: Foot ulceration is a serious complication of diabetes, and new techniques that can predict wound healing may prove very helpful. We tested the ability of medical hyperspectral technology (HT), a novel diagnostic scanning technique that can quantify tissue oxy- and deoxyhemoglobin to predict diabetic foot ulcer healing. RESEARCH DESIGN AND METHODS: Ten type 1 diabetic patients with 21 foot ulcer sites, 13 type 1 diabetic patients without ulcers, and 14 nondiabetic control subjects were seen up to 4 times over a 6-month period. HT measurements of oxyhemoglobin (HT-oxy) and deoxyhemoglobin (HT-deoxy) were performed at or near the ulcer area and on the upper and lower extremity distant from the ulcer. An HT healing index for each site was calculated from the HT-oxy and -deoxy values. RESULTS: Hyperspectral tissue oxygenation measurements observed changes in tissue immediately surrounding the ulcer when comparing ulcers that heal and ulcers that do not heal (P < 0.001). The sensitivity, specificity, and positive and negative predictive values of the HT index for predicting healing were 93, 86, 93, and 86%, respectively, when evaluated on images taken at the first visit. Changes in HT-oxy among the three risk groups were noted for the metatarsal area of the foot (P < 0.05) and the palm (P < 0.01). Changes in HT-deoxy and the HT healing index were noted for the palm only (P < 0.05 and P < 0.01, respectively). CONCLUSIONS: HT has the capability to identify microvascular abnormalities and tissue oxygenation in the diabetic foot and predict ulcer healing. HT can assist in the management of foot ulceration.

Talactoferrin alfa, a recombinant human lactoferrin promotes healing of diabetic neuropathic ulcers: a phase 1/2 clinical study.

BACKGROUND: Talactoferrin alfa, a recombinant form of human lactoferrin, is a novel immunomodulatory protein with demonstrated ulcer healing properties in animal models. METHODS: A phase 1/2 clinical study was conducted at 7 clinical sites to determine if talactoferrin can improve wound healing in diabetic patients with foot ulceration. Fifty-five patients with diabetic neuropathic foot ulcers participated in this 2-phase study. In phase 1, groups of 3 patients each received open-label 1%, 2.5%, or 8.5% talactoferrin gel twice daily, in a sequential design, to their ulcer for 30 days. No drug-related adverse events were found at any dose level. Phase 2 was a randomized, placebo-controlled, single-blind study of 2.5% and 8.5% gels, with patients equally divided between the 3 groups. In combination with good wound care, treatment was administered topically twice daily to the ulcers for 12 weeks. The primary endpoint was the incidence of > or = 75% healing (relative to baseline size). RESULTS: The study, which in phase 2 was powered to detect a difference between the placebo and combined talactoferrin arms with P < .1, met the primary objective. The groups receiving the 2.5% (n = 15) and 8.5% (n = 15) gels had twice the incidence of > or = 75% reduction in ulcer size compared with the placebo group (n = 16): 47%, 53%, and 25%, respectively. On an intent-to-treat basis, the combination of the 2 active groups when compared with the placebo group showed a strong trend toward statistical significance (P = .09). There were no talactoferrin-related adverse events or laboratory abnormalities. CONCLUSIONS: Topical talactoferrin appears to be safe and well tolerated and improves healing of diabetic neuropathic ulcers.

Early changes in the skin microcirculation and muscle metabolism of the diabetic foot.

BACKGROUND: Changes in the large vessels and microcirculation of the diabetic foot are important in the development of foot ulceration and subsequent failure to heal existing ulcers. We investigated whether oxygen delivery and muscle metabolism of the lower extremity were factors in diabetic foot disease. METHODS: We studied 108 patients (21 control individuals who did not have diabetes, 36 patients with diabetes who did not have neuropathy, and 51 patients with both diabetes and neuropathy). We used medical hyperspectral imaging (MHSI) to investigate the haemoglobin saturation (S(HSI)O2; % of oxyhaemoglobin in total haemoglobin [the sum of oxyhaemoglobin and deoxyhaemoglobin]) in the forearm and foot; we also used 31P-MRI scans to study the cellular metabolism of the foot muscles by measuring the concentrations of inorganic phosphate and phosphocreatine and calculating the ratio of inorganic phosphate to phosphocreatine (Pi/PCr). FINDINGS: The forearm S(HSI)O2 during resting was different in all three groups, with the highest value in controls (mean 42 [SD 17]), followed by the non-neuropathic (32 [8]) and neuropathic (28 [8]) groups (p<0.0001). In the foot at resting, S(HSI)O2 was higher in the control (38 [22]) and non-neuropathic groups (37 [12]) than in the neuropathic group (30 [12]; p=0.027). The Pi/PCr ratio was higher in the non-neuropathic (0.41 [0.10]) and neuropathic groups (0.58 [0.26]) than in controls (0.20 [0.06]; p<0.0001). INTERPRETATION: Our results indicate that tissue S(HSI)O2 is reduced in the skin of patients with diabetes, and that this impairment is accentuated in the presence of neuropathy in the diabetic foot. Additionally, energy reserves of the foot muscles are reduced in the presence of diabetes, suggesting that microcirculation could be a major reason for this difference.

Diabetic foot complications: diagnosis and management.

Foot complications in patients with diabetes mellitus are a challenge to the health care industry. A great deal of expenditure is due to the management of diabetic foot complications. This places a great burden on the health care industry. It also places a great burden on those diabetic patients with foot complications and their families. Therefore, their effective management in an efficient manner is crucial to our patients. To deal with these problems, a dedicated, knowledgeable, and experienced multidisciplinary team is key. Intervention at the earliest possible time yields the best outcome. Prevention is the focus for those with no ulcerations. For those with ulcerations, prompt recognition and treatment is key. The importance of classifying ulcerations according to size, depth, presence or absence of infection, and vascular status can not be overstated. Proper offloading is vital for those with neuropathic lesions. Recognition of patients with a component of ischemia and vascular intervention to increase perfusion will aid in wound healing. Of course deep infection requires immediate drainage. All efforts of those in the multidisciplinary team are directed at the restoration and maintenance of an ulcer-free foot which is important in enabling our patients to maintain their ambulatory status.