Influence of insulin and glargine on outgrowth and number of circulating endothelial progenitor cells in type 2 diabetes patients: a partially double-blind, randomized, three-arm unicenter study

73 patients with type 2 diabetes mellitus according to the American Diabetes Association (ADA) criteria were enrolled in this prospective partially double-blind, randomized, three-arm mono-center trial (EudraCT-Nr: 2006-006573-24). The study took place at the University Hospital in Heidelberg, Germany. The main inclusion criteria were treatment with oral diabetes medication, HbA1c values < 6.5% and ≤ 9% and age between 35 and 70 years. The primary endpoint of the trial was the relative change of number of circulating EPC 4 weeks after start of therapy compared to baseline as detected by FACS analysis. Secondary endpoints included: i) change of number of circulating EPC 4 months after start of therapy compared to baseline as detected by FACS analysis ii) change in circulating EPC as detected by in vitro outgrowth iii) intima media thickness iv) skin microvascular function (as measured by laser Doppler perfusion upon heat stimulation) v) long-term Glucose control (HbA1c) vi) short-term Glucose control (fasting glucose). Glitazone and erythropoietin treatment was not permitted during the trial since they were previously shown to influence EPC [19],[20]. All other oral antidiabetics as well as lipid and blood pressure medications were allowed. Patients with statin treatment were allowed to enter the study, yet statins were not added during the 4 months of treatment due to known effect on EPC [21]. Patients were randomized in a 1:2:2 ratio in 3 groups. The first treatment arm, consisting of patients receiving oral medication only, was treated openly. The other two treatment arms, consisting of patients receiving bedtime Insulin once daily in addition to existing oral medication, were double blinded with glargine controlled by NPH insulin. Randomisation of patients and monitoring of the trial was performed according to the Standard Operating Procedures from the Coordination Centre for Clinical Trials (KKS) in Heidelberg. Ready to use NPH insulin or glargine pens were packed and blinded by the pharmacy of the University Hospital in Heidelberg according to the randomization list. Anamnestic data (Table 1) in addition to blood tests, and EPC quantification, were documented/performed at the entry visit (visit 1), after 4 weeks (visit 2) and after 4 months (visit 3). Whereas intima media thickness (IMT) and laser Doppler data were documented/performed at the entry visit (visit 1) and after 4 months (visit 3).

On the day of visit 1, all patients randomized into one of the groups receiving insulin were educated by a qualified diabetes nurse or a physician on the following topics: i) use of the insulin pen ii) injection techniques and dosage iii) hypoglycemia awareness and self treatment according to the guidelines of the German Diabetes Association [22]. Patients receiving oral medication were educated concerning hypoglycemia awareness and self treatment only. All patients entering this trial were supplied with a device for blood glucose self monitoring (Accu-Check Aviva ®,, Roche Diagnostics, Mannheim Germany). In patients randomized to receive either glargine or NPH insulin, therapy was initiated with subcutaneous injection of 4 international units at bedtime (usually around 10 p.m.). The dose was adjusted according to morning fasting glucose levels using a titration scheme that was handed out to each participant and aiming at fasting glucose levels < 100 mg/dl. Patients on oral medication were titrated to maximum dose using the following combinations: metformin alone, DPP4-inhibitors alone, sulfonylurea alone, metformin + DPP4-inhibitors, metformin + sulfonylurea, metformin + glinides, acarbose could be added to any of these combinations. All patients were provided with a diabetes diary at visit 1 in which daily insulin dose, daily morning fasting glucose self- measurements, one diurnal glucose profile weekly (4 pre-meal values) and possible hypoglycemic episodes were documented.

In order to discuss possible adverse events and to further adjust treatment telephone calls of the investigators with the participants took place 2 weeks and 2 months after the entry visit.

The study complied with the Declaration of Helsinki and was approved by the Ethics Committee of the University of Heidelberg (Ethic-Committee-No: AFm0-023/2007); all patients entered the study according to the guidelines after giving informed consent.

On the day of all three visit time-points, ~ 20 ml of venous whole blood was drawn from the participants for measurement of the following routine parameters: HbA1c, glucose, hemoglobin, leukocyte count, erythrocyte count, triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol, electrolytes (Sodium, Potassium), liver transaminases (GOT, GPT), creatinine and urea. The samples for routine parameters were sent to the department of clinical chemistry for analysis within 60 minutes after blood was drawn.

On the day of all three visits, ~ 10 ml of venous whole blood was drawn from the participants and peripheral blood mononuclear cells (pBMCs) were isolated by density gradient centrifugation (lymphocyte separation; PAA Laboratories, Pasching, Austria). EPCs were characterized by flow cytometric analysis as previously described in detail [17] using anti-CD34-FITC (Miltenyi Biotec GmbH, Germany) and VEGFR-2-PE (R & D Systems, Minneapolis, MN, USA). All measurements were normalized for auto fluorescence and unspecific receptor binding using FITC- (Pharmingen BD Biosciences) and PE-labeled isotype controls. EPC were counted and analyzed using FACS Calibur cell sorter (Becton Dickinson, Heidelberg Germany) and Cell Quest Pro Software (Becton Dickinson, Heidelberg, Germany) and expressed as a percentage of gated mononuclear cells.

The outgrowth assay of EPCs in vitro was performed by a standard method indicating clonogenic potential of circulating EPCs as described in detail [3],[23],[24]. In brief, mononuclear cells were isolated from 30-40 mL whole blood by density gradient centrifugation (lymphocyte separation; PAA Laboratories, Pasching, Austria). PBMCs were preplated in tissue culture flasks (Sarstedt, Nuembrecht, Germany) in Medium199 (Invitrogen, Karlsruhe, Germany) supplemented with 20% fetal calf serum (PAA Laboratories) and penicillin/streptomycin/glutamine (Bio Whittacker, Walkersville, MD, USA) to allow adhesion of fast-adherent, differentiated cells. After 48 h, nonadherent cells were counted, and 10⁶ cells were plated on 24-cell plates coated with 25  μg/mL fibronectin (Sigma, St. Louis, MO, USA) and cultured in the same growth medium (see above) without any additional growth factors. Medium was changed after 3 days. Outgrowth of EPCs was quantified by counting of colony-forming units (CFUs).

Intima media thickness was detected as previously described [25],[26] at visits 1 and 3 by ultrasound in the right and left common carotid artery approximately 1-2 cm distal of the carotid bulb. Measurements were taken at the far wall of the artery at 4 different loci and in the end-diastolic phase of the heart cycle as documented by real time electrocardiogram.

Microvascular responsiveness was studied at visit 1 and 3 using laser Doppler perfusion measurement with PeriFlux System 5000 (Perimed, Järfälla, Sweden) upon heat provocation as described in detail [27]. For this, 2 probes were placed on the dorsum of the right foot. Basic capillary perfusion was documented at 37°C skin temperature for 10 minutes and at 44°C for additional 10 minutes to study heat-provoked increase in perfusion as a marker of microvascular function. The data were stored on a computer and analyzed offline with signal processing software (PeriSoft 2.50, Perimed). No current laser Doppler instrument can provide absolute perfusion values. Measurements are expressed as Perfusion Units (PU), which are arbitrary.

According to [17] and assuming a 0.01, 0.05, and 0.04 per cent of peripheral mononuclear cells change in number of circulating EPC after 4 weeks in the oral treatment group, glargine, and NPH-insulin group, respectively, with a common standard deviation of 0.04 randomisation of 75 patients was initially planned to achieve a power of 80% with a two-sided global F test with alpha of 5%. The trial was stopped after 73 patients, erroneously assuming that the full sample size of 75 patients had been obtained. With the patient number in the intention to treat (ITT) population (n = 55) a power of 69.9% is reached under the initial assumptions. Pair-wise group comparisons were planned to be carried out according to the closed-testing principle only after showing significance in the global test. The confirmatory analysis was primarily based on the ITT population. If a patient discontinued the trial prematurely, missing data were not replaced. In addition to the evaluation of the ITT population, a per-protocol (PP) analysis was performed including all randomized patients without major protocol violations as sensitivity analysis. Secondary variables were tabulated using appropriate descriptive measures of the empirical distributions and descriptive P values for treatment group comparisons.

For evaluation of the primary endpoint, a global F-test was performed to test for differences between the three groups. No pair-wise group comparisons could be carried out. Secondary endpoints were analysed using descriptive measures and graphical approaches. Homogeneity of treatment groups was described by comparison of demographic data and baseline values. Comparisons of binary data were carried out using Pearson ’ s chi square test. In case of three groups a global F-test was performed, two groups were compared using the t-test. All additional evaluations were described and fixed in the statistical analysis plan before database closure. All analyses were performed using SAS ® 9.2 (SAS Inc., Cary/NC, USA).