Role of vitamin D in diabetic retinopathy: Pathophysiological and clinical aspects.

Epidemiological data predict a dramatic increase in the prevalence of diabetes and of diabetic retinopathy (DR) - the most common complication of diabetes-for which however we do not have so far effective tools for prevention and treatment. Since hypovitaminosis D is very frequent in patients with diabetes and vitamin D (VD) has vascular protective properties, several studies have addressed the association of VD deficiency with DR and its severity and progression, whereas the effects of VD supplementation on its natural history are largely unknown. Here we review the available evidence that supports the possible protective role of VD in DR and suggests to determine the VD levels in DR patients calling for a definitive randomized clinical trial to ascertain whether VD supplementation could protect against DR.

Risk of vertebral fractures in hypoparathyroidism.

Parathyroid hormone (PTH) exerts both anabolic and catabolic actions on bone,depending on the duration and periodicity of exposure. Hypoparathyroidism is defined by inadequate production of PTH in the presence oflow serum calcium. In hypoparathyroidism it has been reported an increase in corticaland trabecular bone mass, but it is still unknown if these quantitative variations maybe accompanied by qualitative ones and increased bone strength. Despite the extensive data available on the effects of hypoparathyroidism on bone, itseffect on the hard end point in this area which is the risk of fractures still remainsunresolved and highly debated. As a matter of fact no previous review has focused onthis relevant clinical topic. This review will deal with the various aspects of bone metabolism (turn-over,density, quality) in hypoparathyroidism, focusing on the few data available on therisk of fracture and in particular of morphometric vertebral fractures, the emerging way to assess actual skeletal fragility particularly in secondary forms of osteoporosis.

The Increased Transforming Growth Factor-ß Signaling Induced by Diabetes Protects Retinal Vessels.

The roles of transforming growth factor (TGF)-ß in extracellular matrix production and vascular remodeling, coupled with increased TGF-ß expression and signaling in diabetes, suggest TGF-ß as an important contributor to the microangiopathy of diabetic retinopathy and nephropathy. To investigate whether increased TGF-ß signaling could be a therapeutic target for preventing retinopathy, we used a pharmacologic approach (SM16, a selective inhibitor of the type 1 TGF-ß receptor activin receptor-like kinase 5, orally active) to inhibit the increased, but not the basal, Tgf-ß signaling in retinal vessels of diabetic rats. At the level of vascular gene expression, 3.5 months' diabetes induced minimal changes. Diabetes + SM16 for 3 weeks caused widespread changes in gene expression poised to enhance vascular inflammation, thrombosis, leakage, and wall instability; these changes were not observed in control rats given SM16. The synergy of diabetes and SM16 in altering gene expression was not observed in the lung. At the level of vascular network morphology, 7 months' diabetes induced no detectable changes. Diabetes + SM16 for 3 weeks caused instead distorted morphology and decreased density. Thus, in diabetes, retinal vessels become dependent on a small increase in TGF-ß signaling via activin receptor-like kinase 5 to maintain early integrity. The increased TGF-ß signaling may protect against rapid retinopathy progression and should not be a target of inhibitory interventions.

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.

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.

Emerging drugs for the treatment of diabetic ulcers.

INTRODUCTION: Diabetic ulcers are chronic nonhealing ulcerations that despite the available medical tools still result in high amputation rates. Growing evidence suggests that alteration of the biochemical milieu of the chronic wound plays a significant role in impaired diabetic wound healing. AREAS COVERED: The basic pathophysiology and the conventional treatment strategy of diabetic foot ulcers have been reviewed in the first section. In the second part, the most up-to-date bench and translational research in the field are described. The third section focuses on the drugs currently under development and the ongoing clinical trials evaluating their safety and efficacy. Finally, the major drug development issues and the possible scientific approaches to overcome them are analyzed. EXPERT OPINION: Significant strides in understanding the chronic wound development have led to the development of topical therapies to address aberrant expression of growth factors and overexpression of inflammatory cytokines. Current research in the laboratory suggests that while decreased growth factor expression occurs at the local wound level, increased systemic serum levels of growth factors suggest growth factor resistance.

PARACENTRAL ACUTE MIDDLE MACULOPATHY IN CENTRAL RETINAL VEIN OCCLUSION COMPLICATING AMYLOID LIGHT-CHAIN AMYLOIDOSIS.

PURPOSE: To describe a case of paracentral acute middle maculopathy associated with central retinal vein occlusion in a patient affected by amyloid light-chain amyloidosis. METHODS: One patient with confirmed diagnosis of amyloid light-chain amyloidosis, displaying paracentral acute middle maculopathy and central retinal vein occlusion, was recruited. The patient underwent complete ophthalmologic examination and multimodal imaging, including: fundus autofluorescence, fluorescein angiography, indocyanine green angiography, spectral-domain optical coherence tomography, and optical coherence tomography angiography. RESULTS: Fundus autofluorescence showed a ferning pattern, corresponding to linear hypofluorescence in late-phase indocyanine green angiography and delayed venous filling, detected by fluorescein angiography. Structural optical coherence tomography revealed a hyper-reflective line located in the outer plexiform layer, corresponding to the prominent middle limiting membrane, along with several placoid lesions. Optical coherence tomography angiography found that the superficial capillary plexus was preserved, whereas vessel density was reduced in both the deep capillary plexus and the choriocapillaris. After 1 year of follow-up, the patient achieved an almost complete morphological recovery. CONCLUSION: Multimodal imaging described in depth the morphological features of a case of combined paracentral acute middle maculopathy and central retinal vein occlusion in a patient affected by amyloid light-chain amyloidosis.

Cutaneous alterations in diabetes mellitus.

 Dermatological problems occur with increased frequency in individuals with diabetes mellitus (DM). Cutaneous manifestations may be the first presenting sign of DM or even precede the diagnosis by many years. The main changes in the skin are due to alterations of microcirculation, the nervous system, and collagen. The most common skin problems in DM are acanthosis nigricans, necrobiosis lipoidica, diabetic dermopathy, scleredema, and granuloma anulare. The purpose of this review is to describe the molecular and anatomopathological alterations occurring at the skin during DM, and to illustrate the most important and common clinical skin manifestations in patients with DM. .

Patrolling Monocytes Are Recruited and Activated by Diabetes to Protect Retinal Microvessels.

In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6Clow patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1-/- mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention.

The Effect of Continuous Positive Airway Pressure on Vascular Function and Cardiac Structure in Diabetes and Sleep Apnea. A Randomized Controlled Trial.

Rationale: Although both type 2 diabetes mellitus (T2DM) and obstructive sleep apnea (OSA) are independently recognized as risk factors for cardiovascular disease, little is known about their interaction.Objectives: We hypothesized that T2DM and OSA act synergistically to increase vascular risk, and that treatment of OSA would improve vascular reactivity in patients with T2DM plus OSA.Methods: Cross-sectional study of 141 adults with T2DM, OSA, T2DM plus OSA, and control subjects, followed by a 3-month, parallel-arm, randomized, placebo-controlled trial comparing active and sham continuous positive airway pressure (CPAP) in 53 adults with T2DM plus OSA. Endothelium-dependent macro- and microvascular reactivity (flow-mediated dilation [FMD] of the brachial artery and acetylcholine-induced dilation of forearm microvasculature, respectively) and cardiovascular magnetic resonance to assess left- and right-ventricular mass/volume.Results: Mean (±SD) FMD was 6.1 (±4.0)%, 7.3 (±3.6)%, 6.8 (±4.5)%, and 4.8 (±2.9)% in control subjects, T2DM only, OSA only, and T2DM plus OSA, respectively. We observed a significant T2DM × OSA interaction on FMD, such that the mean effect of OSA in those with T2DM was 3.1% (95% confidence interval [CI], 0.6 to 5.6) greater than the effect of OSA in those without T2DM. A total of 3 months of CPAP resulted in a mean absolute increase in FMD of 0.3% (95% CI, -1.9 to 2.5; primary endpoint), with a net improvement of 1.1% (95% CI, -1.4 to 3.6) among those with adherence of 4 h/night or greater. A significant T2DM × OSA interaction was found for both left ventricular (LV) and right ventricular end-diastolic volume, such that OSA was associated with a 22.4 ml (95% CI, 3.2 to 41.6) greater LV end-diastolic volume and 23.2 ml (95% CI, 2.6 to 43.8) greater right ventricular end-diastolic volume in those with T2DM compared with the impact of OSA in those without T2DM. We observed a net improvement in LV end-diastolic volume of 8.7 ml (95% CI, -7.0 to 24.4).Conclusions: The combination of T2DM plus OSA is associated with macrovascular endothelial dysfunction beyond that observed with either disease alone. CPAP for 3 months did not significantly improve macrovascular endothelial function in the intent-to-treat analysis; however, cardiovascular magnetic resonance results suggest that there may be a beneficial effect of CPAP on LV diastolic volume.Clinical trial registered with www.clinicaltrials.gov (NCT01629862).

Critical appraisal of definitions and diagnostic criteria for sarcopenic obesity based on a systematic review.

BACKGROUND: Sarcopenic obesity is a clinical and functional condition characterized by the coexistence of excess fat mass and sarcopenia. Currently, different definitions of sarcopenic obesity exist and its diagnostic criteria and cut-offs are not universally established. Therefore, the prevalence and sensitivity of this condition for any disease risk prediction is affected significantly. AIM: This work was conducted under the auspices of the European Society for Clinical Nutrition and Metabolism (ESPEN) and the European Association for the Study of Obesity (EASO). An international expert panel performed a systematic review as an initial step to analyze and summarize the available scientific literature on the definitions and the diagnostic criteria for sarcopenic obesity proposed and/or applied in human studies to date. METHODS: The present systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The search was conducted in April 2018 in three databases (PubMed, Scopus, Web of Science). Human studies conducted in both sexes, irrespective of ethnicity, and published from 2007 to 2018 were included; cohorts of individuals with obesity and acute or chronic conditions and treatments reported to negatively influence skeletal muscle mass and function independently of obesity were excluded from final analyses. The quality of the studies was evaluated using the Newcastle-Ottawa Scale (NOS) adapted for cross sectional studies. RESULTS: The electronic search retrieved 2335 papers of which 75 met the eligibility criteria. A marked heterogeneity in definitions and approaches to diagnose sarcopenic obesity was observed. This was mainly due to differences in the definitions of obesity and sarcopenia, in the methodologies used to assess body composition and physical function, and in the reference values for the variables that have been used (different cut-offs, interquartile analysis, diverse statistical stratification methods). This variability may be attributable, at least in part, to the availability of the methodologies in the different settings, to the variability in specialties and backgrounds of the researcher, and to the different settings (general population, clinical settings, etc.) where studies were performed. CONCLUSION: The results of the current work support the need for consensus proposals on: 1) definition of sarcopenic obesity; 2) diagnostic criteria both at the level of potential gold-standards and acceptable surrogates with wide clinical applicability, and with related cut-off values; 3) methodologies to be used in actions 1 and 2. First steps should be aimed at reaching consensus on plausible proposals that would need subsequent validation based on homogeneous studies and databases, possibly based on analyses of existing cohorts, to help define the prevalence of the condition, its clinical and functional relevance as well as most effective prevention and treatment strategies.

Treatment of hypoparathyroidism.

The goal of the clinical management of hypoparathyroidism is to correct acute and chronic hypocalcemia. Treatment of acute hypoparathyroidism via intravenous infusion of Ca++ salts, is necessary only in symptomatic patients, or in asymptomatic patients in the setting of a rapid decrease in ionized Ca++ levels. The treatment cornerstones of chronic hypoparathyroidism are oral supplementation of calcium and/or active vitamin D, that can be associated with dietary restriction of sodium and phosphates, thiazide diuretics, and phosphate binders. Notably, PTH replacement is emerging as a innovative treatment of chronic hypoparathyroidism. rhPTH (1-84) has been shown to safely reduce calcium and vitamin D dosage, and increase serum calcium levels in hypoparathyroid patients. Therefore, rhPTH (1-84) appears to represent a new option in patients with chronic hypoparathyroidism "resistant" to conventional treatment.

Defective Myogenic Response of Retinal Vessels Is Associated With Accelerated Onset of Retinopathy in Type 1 Diabetic Individuals.

PURPOSE: We seek to identify pathogenic mechanisms for diabetic retinopathy that can become therapeutic targets beyond hyperglycemia and hypertension. We investigated if a defective myogenic response of retinal arteries to increased perfusion pressure, which exposes capillaries to increased pressure and flow, is associated with the onset of clinical retinopathy. METHODS: We examined prospectively the incidence of retinopathy in type 1 diabetic individuals tested 4 years earlier for the retinal arterial myogenic response, and in a cross-sectional study the prevalence of defective myogenic response in type 1 patients who had diabetic retinopathy. Among these, we contrasted early-onset (after 15 ± 2 years of diabetes, E-DR; n = 5) to late-onset (after 26 ± 3 years of diabetes, L-DR; n = 7) retinopathy. We measured the myogenic response using a laser Doppler blood flowmeter after a change in posture from sitting to reclining, which increases retinal perfusion pressure. RESULTS: Five of seven participants who 4 years prior had a defective myogenic response had now developed clinical retinopathy; as compared with only one of six participants who 4 years prior had a normal response (P = 0.10). In the cross-sectional study, all participants had normal retinal hemodynamics at steady state. In response to the postural change, only the E-DR group showed defective myogenic response (P = 0.005 versus controls, P = 0.02 versus L-DR) and abnormally high retinal blood flow (P = 0.016 versus controls). CONCLUSIONS: In type 1 diabetic patients, a defective myogenic response of retinal arteries to pressure is not required for the development of clinical retinopathy, but is prominently associated with an accelerated onset of retinopathy.

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.

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.

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.

Molecular biomarkers of vascular dysfunction in obstructive sleep apnea.

Untreated and long-lasting obstructive sleep apnea (OSA) may lead to important vascular abnormalities, including endothelial cell (EC) dysfunction, hypertension, and atherosclerosis. We observed a correlation between microcirculatory reactivity and endothelium-dependent release of nitric oxide in OSA patients. Therefore, we hypothesized that OSA affects (micro)vasculature and we aimed to identify vascular gene targets of OSA that could possibly serve as reliable biomarkers of severity of the disease and possibly of vascular risk. Using quantitative RT-PCR, we evaluated gene expression in skin biopsies of OSA patients, mouse aortas from animals exposed to 4-week intermittent hypoxia (IH; rapid oscillations in oxygen desaturation and reoxygenation), and human dermal microvascular (HMVEC) and coronary artery endothelial cells (HCAEC) cultured under IH. We demonstrate a significant upregulation of endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha-induced protein 3 (TNFAIP3; A20), hypoxia-inducible factor 1 alpha (HIF-1α?? and vascular endothelial growth factor (VEGF) expression in skin biopsies obtained from OSA patients with severe nocturnal hypoxemia (nadir saturated oxygen levels [SaO2]<75%) compared to mildly hypoxemic OSA patients (SaO2 75%-90%) and a significant upregulation of vascular cell adhesion molecule 1 (VCAM-1) expression compared to control subjects. Gene expression profile in aortas of mice exposed to IH demonstrated a significant upregulation of eNOS and VEGF. In an in vitro model of OSA, IH increased expression of A20 and decreased eNOS and HIF-1α expression in HMVEC, while increased A20, VCAM-1 and HIF-1αexpression in HCAEC, indicating that EC in culture originating from distinct vascular beds respond differently to IH stress. We conclude that gene expression profiles in skin of OSA patients may correlate with disease severity and, if validated by further studies, could possibly predict vascular risk in OSA patients.

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.

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.