ACDI-D-12-00135-R2
EFFECT OF NORMALIZATION OF FASTING GLUCOSE BY INTENSIFIED INSULIN THERAPY AND INFLUENCE OF eNOS POLYMORPHISISMS ON THE INCIDENCE OF RESTENOSIS AFTER PERIPHERAL ANGIOPLASTY IN PATIENTS WITH TYPE 2 DIABETES: A RANDOMIZED, OPEN LABEL CLINICAL TRIAL.
PierMarco Piatti, Enrico Marone, Manuela Mantero, Emanuela Setola, Elena Galluccio, Pietro Lucotti, Ermal Shehaj, Valentina Villa, Francesca Perticone, Massimo Venturini, Alessio Palini, Flavio Airoldi, Ezio Faglia, Alessandro Del Maschio, Antonio Colombo, Roberto Chiesa, Emanuele Bosi, Lucilla D Monti.
SUPPLEMENTARY DATA
SUBJECTS AND METHODS
CLI diagnosis
CLI was defined as the presence of angiographic documentation of infrapopliteal arterial disease (stenosis >70% or occlusion) associated with persistent, recurring rest pain requiring analgesia and an ankle systolic pressure 50 mm Hg and/or toe systolic pressure 30 mm Hg or TcPO2 30 mm Hg and/orulceration, gangrene, or nonhealing wounds of the foot with ankle systolic pressure 50 mm Hg or toe systolic pressure 30 mm Hg or TcPO2 30 mm Hg and/orFontaine stages III-IV and Rutherford categories IV-Viand /or lifestyle-limiting claudication defined as Ruther-ford category II to III associated with jeopardized single vessel runoff or complete trifurcation vessel occlusion.
Glargine and Glulisine therapy algorithm
Glargine insulin was administered at bed time. The initial dose was determined as the total amount of iv insulin administered during the night (usually it is between 0.2 to 0.4 U/kg Body Weight) and changes weremade to keep fasting glucose levels between 80-100 mg/dl. The usual oral antidiabetic therapy was administered before meals. This therapy was maintained for one week before visit for randomisation.
The treatment goal for IIT was a fasting blood glucose level of 100 mg/dl and an HbA1c6.5% at the end of the follow-up. Patients maintained the same amount of long-acting insulin given at discharge while short acting insulin was the same total amount as insulin glargine but subdivided: 20% at breakfast, 40% at lunch and 40% at dinner since the total daily carbohydrate intake was divided into the same percentage. The treatment goal for SC was anHbA1c level of 7.0% at the end of the follow-up.Routine visits were scheduled after 1, 2, 4 weeks and then at 2, 4 and 6 months.
All patients were asked to monitor blood glucose levels each morning and document hypoglycemia and 1-day six point glucose profiles (before and 2 h after breakfast, lunch and dinner) before each clinic visit.
In the IIT arm, patients were contacted by telephone every week to target fasting glucose levels at 100 mg/dl and postprandial glucose levels to 110 and 140 mg/dlthrough titrated regimens.The titration of long-acting insulin and glulisine dosages was performed according with (1).
The protocol was written in accordance with the guidelines contained in the "Declaration of Helsinki 2" and with current national legislation.The protocol, the site's informed consent form and any other written information provided to the patients prior to any enrolmentwas approved by the local Ethical Committee.
Interventional technique
The controlateral or antegrade approach was used in all patients. Angioplasty was performed through an ipsilateral or controlateral femoral approach by using various sizes of introducer sheaths ranging from 5F to 8F. Before intervention, arteriography was performed to quantify the extent of the disease. After wire crossing balloon inflation was repeated routinely two to four times at the same segment. The anterior or posterior tibial arteries will be preferred targets if available. Interventions were performed with 135-cm low-profile coronary balloon catheters. Total occlusions were crossed with 0.014-inch hydrophilic wires. Lesions that need to be predilated were approached with a one-half millimetre balloon smaller than the reference vessel. The goal was to maximize runoff and establish continuous inline flow in at least one below-knee vessel to the ankle. If the primary angioplasty still resulted in residual stenosis, angioplasty was repeated with a balloon 1 mm larger than the previous one. A variety of balloon-expandable was used to obtain a 10% residual stenosis. Vascular closure devices were used when appropriate.
Antiplatelet therapy
Before angiography all patients started on acetyl salicylic acid 100 mg daily, ticlopidine 250 mg twice/daily or clopidogrel 75 mg daily. For patients not taking clopidogrel, a loading dose of 600 mg was given before the procedure. After arterial access, patients received unfractionated heparin at 70 UI/kg. Activated clotting times were not monitored. Patients were discharged on acetyl salicylic acid 100 mg daily and ticlopidine 250 mg twice daily or clopidogrel 75 mg/day for six months or longer if feasible.Patients who were intolerant to clopidogrel received ticlopidine 250 mg twice daily. GPIIB/IIIA utilization was considered in case of poor distal run-off, at operator’s discretion but not in case of complete tibial occlusion.
Indices of vascular success
Angiographic success was defined as dilation of all critical inflow lesions with a resulting residual stenosis of 30% and the absence of flow-limiting dissections on the final angiogram. Technical success was defined as a residual stenosis of 30% after balloon dilatation; Thrombolysis In Myocardial Infarction (TIMI) III antegrade flow; straight inline flow to the ankle in at least one tibial vessel; and absence of distal embolization, or need for unplanned corrective surgical intervention.Primary hemodynamic success was defined as an increase in resting ABI 0.1. In patients with falsely elevated ankle pressures, an increase in the TcPO2>13 mmHg was used to evaluate hemodynamic success.
DNA extraction and genotyping
Genomic DNA was obtained from all subjects participating into the study by established methods (2). Allelic discrimination of the single nucleotide polymorphism (SNP) was performed using the TaqMan chemistry with the ABI Prism 7700 apparatus (Applied Biosystems, Foster City, CA). The SNPs was: rs753482 (intron 19 A/C), at position 150337316. Primer and probe sets were designed and manufactured by Applied Biosystems as Custom TaqMan SNP genotyping assays. The forward and reverse primers and probes employed for the rs753482 discrimination were 5'-TGAGGACGACGGCTTTACC-3', 5'-CCAGGGTCAGGGTGTTCAG-3', 5'-VIC-CCCCCAACCCCTG-3', and 5'-FAM-CCCCCCACCCCTG-3', respectively. A final volume of 12 µl of polymerase chain reaction (PCR) fluid contained 50 ng of DNA, 6 µl of Master Mix, and 4 µl of H2O. The amplification conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 92°C for 15 s and 60°C for 1 min. Controls for each SNP were included in the assay, ~50% of samples were replicated with a concordance of 95%, and laboratory staff were blinded to case-control status.
Methods for isolation and evaluation of endothelial progenitor cells
A sample for endothelial progenitor cells isolation was taken from an antecubital vein of the forearm on the day of the angioplastic procedure. At the end of isolation procedure, FACS analysis was carried out to evaluate the presence of cell-surface markers, CD34+ andCD34+KDR+. The number of circulating CD34+ andCD34+KDR+ cells were measured to evaluate the EPC profile of each study subject before and during the study period.
In brief, 100 L peripheral blood was incubated with the following monoclonal antibodies: PE-conjugated anti-human CD34 (BD, FranklinLakes, NJ), allophycocyanin-conjugated anti-human KDR (R&D Systems, Minneapolis, MN), and PE-Cy7–conjugated anti-human CD45 (Beckman Coulter, Brea, CA). After incubation, 100 L Flow-Count beads (Beckman Coulter) was added to the stained whole blood. To avoid the loss of cells and/or counting beads, a lyse-no-wash technique was used as follows: erythrocytes were lysed with ammonium chloride buffer (155 mmol/L ammonium chloride, 10 mmol/L potassium bicarbonate, and 0.1 mmol/L EDTA), and the sample was analyzed immediately on a FACSCanto II flow cytometer with FACSDiva software (BD Biosciences, San Jose, CA). Circulating CD34+KDR+ cells were enumerated using a hierarchical gating strategy to count cells negative for the pan-hematopoietic marker CD45 or expressing only very low levels (CD45dim). Then, we gated CD34+ cells and then examined the resultingpopulation for expression of KDR. The listmode files obtained from these analyses were subsequently processed with FCS Express (De Novo Software, Los Angeles, CA), and the number of CD45dimCD34+KDR+ was determined and expressed 1*106 events. All analyses were performed by trained operators blinded to the patients’ status.
References
1 Riddle MC, Rosenstock J, Gerich J, on behalf of the Insulin Glargine Study Investigators (2003) The treat-to-target trial: randomized addition of glargine or human NPH insulin to oraltherapy of type 2 diabetic patients. Diabetes Care26: 3080-3086.
2.Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. USA: ColdSpringHarbor Laboratory Press, Plainview; 1989
Table I
Baseline clinical characteristics of type 2 diabetic patients (DMT2) affected by critical limb ischemia (CLI) before peripheral arterial revascularization (meanSD)
IIT / SCn. patients (male/female) / 23 (16/7) / 23 (15/8)
Age (years) / 70.210.4 / 73.08.0
Weight (kg) / 83.812.7 / 77.013.3
BMI (kg/m2) / 29.43.8 / 28.04.6
Waist (cm) / 102.58.6 / 104.68.5
Systolic blood pressure (mmHg) / 13519 / 13619
Diastolic blood pressure (mmHg) / 789 / 8020
Plasma Creatinine (mg/dl) / 1.10.2 / 1.10.3
GFR (ml/min/1.73 m2) / 68.24.43 / 71.14.53
AER (mg/dl) / 48.393.7 / 83.9135.6
Duration of Diabetes (years) / 18.08.5 / 20.88.1
Previous Myocardial infarction / 16/23 / 13/23
Previous carotid TEA / 10/23 / 14/23
Proliferative retinopathy / 14/23 / 14/23
Neuropathy / 15/23 / 16/23
Smoke (yes/no/ex) / 2/7/14 / 2/6/15
GFR: Glomerular Filtration Rate; AER: Albumin Excretion Rate
1