The Acute and Chronic Effects of Remote Ischemic Conditioning on Endothelial Glycocalyx Integrity, Cardiovascular Function and Biomarkers

The present study is a prospective, randomized trial conducted at the Second University Department of Cardiology in Attikon University Hospital. A two hundred seventy patients with STEMI , within 48 hours after primary PCI are randomized in two remote conditioning (RIC) protocols or no intervention other than standard treatment (control group). The first protocol utilizes two ischemic stimuli by brachial cuff inflation of both arms at 200 mmHg for 5 minutes, separated by 15 minutes, after a baseline vascular function assessment (T0). Each ischemic stimulus is followed by a vascular function assessment (T1, T2), with a final assessment 25 minutes after the second cuff deflation (T3). The second protocol is identical with the first, except for the omission of the second ischemic stimulus. Both protocols are preceded by a sham conditioning procedure, by way of cuff inflation omission after their placement around the ordinary brachial position. The RIC protocol will be also performed in 30 healthy volunteers. Blood samples are drawn at baseline (T0) and at the termination of each protocol (T3). All patients are in sinus rhythm, while exclusion criteria include Killip class>2 during the index event, administration of nitrates, history of previous known coronary artery or other cardiovascular disease, previous PCI or coronary artery bypass surgery (CABG), as well chronic inflammatory and systemic disease. Furthermore, The investigators are going to conduct a two - year follow up in order to asess a) changes in left ventricular contractility via estimation of Left Ventricular End Systolic Volume( LVESV) by echocardiography study b) changes in endothelial glycocalyx and arterial stiffness.

Arterial stiffness is assessed by carotid-femoral pulse wave velocity (PWV) using arterial tonometry (Complior, Alam Medical, Vincennes, France); normal values <10 m/s. PWV is calculated as the distance between the carotid and femoral arterial pulse palpation site, divided by the transit time between waves (m/s). All measurements are performed by the same examiner, who is blind to the ischemic protocol exploited (intra-observer variability=5%).

The perfusion boundary region (PBR) of sublingual arterial microvasculature (diameter span from 5 to 25 μm) is measured using Sidestream Darkfield imaging (Microscan, Glycocheck, Microvascular Health Solutions Inc., Salt Lake City, UT, USA). The PBR is the cell-poor layer, resulting from the phase separation between the flowing red blood cells (RBC) and plasma on the microvessel luminal surface. The PBR includes the component of glycocalyx that does allow cell penetration. Thus, an increased perfused boundary region (PBR) is consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties and is a marker of reduced glycocalyx thickness. This constitutes a standardized, reproducible, operator-independent method of assessing arterial glycocalyx, and is thus proposed as a means to endothelial integrity evaluation.

Malondialdehyde (MDA) is determined spectrophotometrically with a commercial kit (Oxford Biomedical Research, Rochester Hills, Mich, colorimetric assay for lipid peroxidation; measurement range 1-20 nmol/L; 3.39% and 4.75% intra-assay and inter-assay variability respectively). IL-6 is measured by a high-sensitivity immunoassay [human IL-6 Quantikinine (high sensitivity)], that detects values as low as 0.094 (intra-assay variability <5%).

MicroRNAs (Mirs) are small, single stranded, non-coding RNA molecules comprising 19-25 nucleotides that regulate post-transcriptional gene expression in response to cellular or environmental stimuli [38]. Their non-invasiveness and stability in serum allows prompt estimation of their expression using archived serum samples. Specific MiRs have been implicated in the pathogenesis of cardiovascular disease and IRI. MiR-144 serves as a pivotal RIC mediator, while miR-150 and miR-499 inhibit apoptosis and fibrosis in the setting of animal models of myocardial IRI [40, 41]. In addition, miR-21 has been demonstrated to reduce infarct size and early left ventricular (LV) remodelling after IRI in rats. On the contrary, miR-208 exerts deleterious effects by way of hypertrophy and adverse remodelling induction . Serum miRNA are obtained from samples using the NucleoSpin miRNA Plasma Kit (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) according to instructions of the manufacturer. The expression patterns of the miRNAs tested and a housekeeping gene, U6sn, were quantitatively assayed using reverse transcription and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Stem-loop complementary DNAs (cDNAs) are synthesized using looped reverse transcription primers specific for each miRNA. Reverse transcription and quantification is performed with the Mir-X™ MicroRNA Quantification Kit (Clontech Laboratories, USA) according to the instruction of the manufacturer using Roche Light Cycler Fluorescence Quantitative PCR System (ABI, USA). All the samples are amplified in triplicate and each experiment was repeated three times to confirm reproducibility. The fold change in expression level is calculated using the 2-ΔΔCt method. The primers for PCR are: