Roberta Assaloni

Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress.

Aims/hypothesis. Oxidative stress plays an important role in diabetic vascular complications. It has been shown that an imbalance in the ratio of nitric oxide: superoxide anion, because of a prevalence of superoxide anion, leads to an alteration in vascular reactivity. In this condition peroxynitrite production, resulting from the reaction between nitric oxide and superoxide, could increase. Peroxynitrite is responsible for nitration of tyrosine residues in proteins. Therefore, the presence of nitrotyrosine in plasma proteins is considered indirect evidence of peroxynitrite production. The aim of this study was to demonstrate the presence of nitrotyrosine in the plasma of patients with Type II (non-insulin-dependent) diabetes mellitus and to correlate its concentrations with the plasma concentrations of glucose and antioxidant defenses. Methods. A total of 40 Type II diabetic patients and 35 healthy subjects were enrolled, and glycaemia, plasma nitrotyrosine, total antioxidant parameter and glycated haemoglobin were measured. Nitrotyrosine was detected by ELISA with a detection limit of 10 nmol/l. Results. Nitrotyrosine was found in the plasma of all diabetic patients (means ± SD = 0.251 ± 0.141 μmol/l), whereas it was not detectable in the plasma of healthy control subjects. Nitrotyrosine plasma values were correlated with plasma glucose concentrations (r = 0.38, p < 0.02) but not with total antioxidant parameter or glycated haemoglobin. Total antioxidant parameter was reduced in diabetic patients (p < 0.01). Conclusions. The presence of nitrotyrosine in the plasma of diabetic patients indicates that peroxynitrite is generated in diabetes, suggesting a possible involvement of peroxynitrite in the development of diabetic complications. [Diabetologia (2001) 44: 834–838]

Effect of Atorvastatin and Irbesartan, Alone and in Combination, on Postprandial Endothelial Dysfunction, Oxidative Stress, and Inflammation in Type 2 Diabetic Patients

Background—Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction and inflammation through oxidative stress. Statins and angiotensin type 1 receptor blockers have been shown to reduce oxidative stress and inflammation, improving endothelial function. Methods and Results—Twenty type 2 diabetic patients ate 3 different test meals: a high-fat meal, 75 g glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, endothelial function, nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6 were assayed during the tests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan 300 mg/d, both, or placebo for 1 week. The 3 tests were performed again between 5 and 7 days after the start of each treatment. High-fat load and glucose alone produced a decrease in endothelial function and increases in nitrotyrosine, C-reactive protein, intercellular adhesion molecule-1, and interleukin-6. These effects were more pronounced when high-fat load and glucose were combined. Short-term atorvastatin and irbesartan treatments significantly counterbalanced these phenomena, and their combination was more effective than either therapy alone. Conclusions—This study confirms an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function and inflammation, suggesting oxidative stress as a common mediator of such an effect. Short-term treatment with atorvastatin and irbesartan may counterbalance this phenomenon; the combination of the 2 compounds is most effective.

Constant and intermittent high glucose enhances endothelial cell apoptosis through mitochondrial superoxide overproduction

It has been previously shown that hyperglycemia enhances free radical production, inducing oxidative damage, which in its turn activates the death pathways implicated in cell apoptosis and necrosis. But the possible involvement of this pathway in the hyperglycemia‐induced apoptosis of endothelial cells has not yet been reported.

Intermittent high glucose enhances ICAM‐1, VCAM‐1, E‐selectin and interleukin‐6 expression in human umbilical endothelial cells in culture: the role of poly(ADP‐ribose) polymerase

Summary.  It has been previously reported that endothelial cells exposed to constant high concentrations of glucose upregulate the expression of adhesion molecules. Moreover, it has been suggested that this phenomenon is related to generation of oxidative stress. It has also been suggested that oxidative injuries, related to high glucose, induce the activation of the enzyme poly ADP ribose polymerase (PARP), which can promote the expression of adhesion molecules and the generation of inflammation. Recent in‐vivo and in‐vitro evidence suggests that oscillation of glucose may play an autonomous and direct role in favoring the development of cardiovascular complications in diabetes. In this study we have investigated the effects of constantly high and intermittently high glucose on nitrotyrosine formation (a marker of nitrosative stress) and adhesion molecule (ICAM‐1, VCAM‐1 and E‐selectin), as well as on interleukin (IL)‐6 expression in human umbilical vein endothelial cells, either in the presence or in the absence of PJ34, a potent inhibitor of PARP. We found that oscillating glucose was more effective in triggering the generation of nitrotyrosine and inducing the expression of adhesion molecules and IL‐6 than stable high glucose. Pharmacological inhibition of PARP suppressed both nitrotyrosine formation, adhesion molecule expression and IL‐6 to the levels seen in the normal glucose conditions. Thus, PARP activation appears to be involved in both promoting nitrosative stress and upregulating adhesion molecules and inflammation in endothelial cells exposed to oscillating high glucose conditions.

Long-term Outcomes after Angioplasty of Isolated, Below-the-knee Arteries in Diabetic Patients with Critical Limb Ischaemia

BACKGROUND It has been shown that concomitant percutaneous transluminal angioplasty (PTA) of above-the-knee (ATK) and below-the-knee (BTK) arteries is highly beneficial for limb salvage in patients with critical limb ischaemia (CLI), but few published studies have specifically investigated outcomes in diabetic patients with CLI associated with isolated small BTK-vessel disease. This study aimed to evaluate the long-term results of successful PTA for limb salvage in such patients. MATERIALS AND METHODS From among the 634 patients with CLI in our database, we retrospectively selected a consecutive series of 101 diabetics (16%) with 107 critically ischaemic limbs (33 Rutherford 5 and 74 Rutherford 6) and no critical ATK lesion, who underwent PTA on isolated BTK lesions. RESULTS The limb salvage rate was 93% after a mean follow-up of 1048+/-525 days (2.9+/-1.4 years). Transcutaneous oxygen tension significantly increased after 1 month (18.1+/-11.2 vs. 39.6+/-15.1; p<0.05). After 1 year, target-vessel re-stenosis had occurred in 42% of the non-amputated limbs, nine patients (9%) had died because of medical conditions unrelated to PTA and three patients had undergone repeat PTA for recurrent CLI. CONCLUSIONS In our selected patient population with ischaemic diabetic foot and isolated BTK lesions, a successful endovascular procedure led to a high percentage of limb salvage at long-term follow-up.

The post‐prandial state in Type 2 diabetes and endothelial dysfunction: effects of insulin aspart

Objective  Recently, much attention has been focused on the possibility that the post‐prandial state may be a cardiovascular risk factor in diabetes. The aim of the present study was to evaluate whether the post‐prandial state is associated with endothelial dysfunction in patients with diabetes and to explore the effect on this aspect of managing post‐prandial hyperglycaemia by insulin aspart.

Use of a retrograde nail for ankle arthrodesis in Charcot neuroarthropathy: a limb salvage procedure.

Background: Charcot neuroarthropathy is a serious complication associated with diabetic neuropathy. This complication probably is most serious when the ankle is involved because of the instability and progressive deformity, which often leads to ulceration, osteomyelitis, and amputation. Arthrodesis before the ulcerated lesion appears is considered a limb salvage treatment. One of the most effective techniques for an unstable ankle in Charcot neuroarthropathy is retrograde transcalcaneal nailing. Methods: Eighteen diabetic patients, without a history of ulceration, were treated from July, 2003, to November, 2005, with panarthrodesis of the ankle using intramedullary retrograde transcalcaneal nailing. The average follow up was 14 ± 10.1 months. All patients completed the unloaded postoperative period with a fiberglass cast (3 months nonweightbearing and 3 months partial weightbearing) and commenced walking in shoes with a stiff rocker sole and a molded insole. Results: During the followup period there were no major complications. In three patients, removal of one of the proximal screws used for anchoring the nail to the tibia was done because of protrusion causing skin breakdown. Fourteen patients had a stable fusion and four patients had fibrous union. The percentage of limb salvage was 100% in the followup period. Conclusions: Our study confirms that this operative technique is effective and safe.

The preventive anti-oxidant action of thiazolidinediones: a new therapeutic prospect in diabetes and insulin resistance.

Background  Hyperglycaemia‐derived oxygen free radicals may be mediators of diabetic complications.