Type 2 Diabetes Mellitus and Atherosclerosis
Type 2 Diabetes Mellitus and Atherosclerosis
In individuals with Type 2 Diabetes (T2D) it has been obtained an outstanding improvement in
 the management of hyperglycemia, but it has not been achieved a similar result in the
 reduction of the atherosclerotic syndrome. The comprehension of the mechanisms that link over
 nutrition to inflammation and innate immune response can be important to understand the
 relationship between insulin resistance, diabetes mellitus and endothelial dysfunction. It
 will be investigated: 1) the role of Toll Like Receptors (TLR)s in the pathophysiology of T2D
 and associated atherosclerosis; 2) the role of aspirin and nicotinamide adenine dinucleotide
 phosphate (NADPH) oxidase inhibitor/s in the production of TxA2 and F2-isoprostanes in T2D
 patients; 3)new biomarkers associated to Diabetes and atherosclerosis including markers of
 endothelial dysfunction and cytokines.

 It will be analyzed in isolated platelets from normal controls and/or diabetic patients the
 production of TxA2, isoprostanes and pro-inflammatory/thrombotic cytokines using aspirin and
 NADPHoxidase inhibitors.
Study design: prospective, controlled, randomized study. All eligible patients will be
 randomly assigned in a 1:1 manner to receive aspirin or placebo.

 In each recruited subjects we will do:

 1. Anamnestic clinical information and Anthropometric measurements

 2. Electrocardiogram, echocardiogram and ultrasound assessment of carotid intima-media
 thickness (IMT) and flow-mediated dilatation (FMD)

 3. Ankle-Brachial Index measurement (ABI)

 4. blood samples from an antecubital vein after an overnight fast and urine samples at
 baseline, after 3 days and after 30 days of aspirin (100 mg/day) administration.

 Laboratory Methods: Blood samples will be immediately centrifuged at 2,000 rpm for 20 min at
 4°C, and the supernatant was collected and stored at -80°C until measurement. All
 measurements will be done blinded. Samples will be tested in duplicate, and those showing
 values above the standard curve will be re-tested with appropriate dilutions.Analysis of
 urinary and platelet isoprostane:Urinary PGF2α-III was measured by a previously described and
 validated EIA assay method. Ten millilitre urine were extracted on a C-18 SPE column; the
 purification was tested for recovery by adding a radioactive tracer (tritiated PGF2α-III)
 (Cayman chemical). The eluates were dried under nitrogen, recovered with 1ml of buffer, and
 assayed in a PGF2α-III specific EIA kit (Cayman chemical). Urinary PGF2α-III concentration
 was corrected for recovery and creatinine excretion and expressed as pg/mg of creatinine. PRP
 was then centrifuged 20 min at 800 g to concentrate platelets and the pellet was suspended in
 Tyrode buffer to obtain a final platelet concentration of 5x108/mL. PGF2α-III content was
 measured by a validated EIA assay method as previously described and expressed as pg/mg
 platelet protein.
Inclusion Criteria:

 -  Type 2 Diabetes Mellitus (defined according to the criteria of the American Diabetes
 Association)

 -  Sign of a written informed consent to participate to the interventional study

 Exclusion Criteria:

 -  Liver disease

 -  Serious renal disorders (serum creatinine >2.5 mg/dL)

 -  History or evidence of previous major vascular events (myocardial infarction,
 transient ischemic attack, stroke)

 -  History of major bleeding

 -  Autoimmune diseases

 -  Cancer or present or recent infections

 -  Use of non-steroidal anti-inflammatory drugs, drugs interfering with cholesterol
 metabolism, or vitamin supplements or antiplatelet drugs in the previous 30 days