FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 71Specific Aim 3: Identify differences in microRNAs in EF and SFobtained from WV patients with CAD and non-CAD: Microarray for microRNAs (small noncodingRNAs that regulate gene expression pathways by degrading mRNA) will be performed on EF andSF obtained from patients with CAD and non-CAD to identify unique microRNAs as potentialbiomarkers or targets for therapy.Methods/ Study Design: With help of Dr. Christopher Adams, Dr. Paulette Wehner and Dr. ToddGress (Dept. Cardiovascular Services) and Dr. Chowdhury (Dept. Thoracic Surgery) we havesuccessfully recruited 60 subjects (30 men and 30 women) undergoing coronary artery bypassgraft surgery (CABG) at the St. Mary’s Heart Center, Huntington, WV (IRB approved study). We arein the process of recruiting control patients (no coronary artery disease) undergoing valve typesurgery. We collected blood, epicardial/perivascular fat and subcutaneous fat from all subjects atthe time of the surgery.Blood: Blood was centrifuged and plasma was separated immediately and frozen at -800C for furtheranalysis. Circulating levels of adipokines, [adiponectin (high molecular weight, low molecular weightand total adiponectin), human TNF alpha, IL-6 and insulin] using ELISA assay was performed. Wealso performed a cytokine-chemokine array using Millipore Multiplexing assay that measures 39cytokines simultaneously in a Luminex 100 system.Epicardial and Subcutaneous fat: Total mRNA and miRNA were isolated from epicardial andsubcutaneous fat using Tri reagent. Gene expression levels of pro-inflammatory markers(fractalkine, MDC and TARC, IL-6) and anti-inflammatory markers (adiponectin and PPAR gamma)were determined in the isolated mRNA from both the fat tissues using Bio-Rad MyiQ real time PCRsystem.Superarray RT2 miRNA array (SA Biosciences) consisting of whole genome human miRNAs (800miRNAs) are performed to assess miRNA changes between the Epicardial and subcutaneous fat.In addition, circulating miRNA isolated from the blood of a sub-set of patients will be performed inthe future. The correlation between the circulating miRNA and tissue miRNA will be performed.Target mRNA for the significantly altered miRNAs will also be determined in the tissues.Statistics and Bio-informatics: Dr. Todd Gress (Department of Medicine, MUSOM) helped withstatistical analysis of the data and helped perform correlation studies between laboratorybiochemical data to clinical parameters (data obtained from the Society of Thoracic Surgerydatabase) of each patient.RESULTS:Aims 1 and 2: Our preliminary results indicated sex differences in both circulating protein levels ofadiponectin (total and high molecular weight-HMW) and tumor necrosis factor-α and tissue mRNAlevels of adiponectin, peroxisome-proliferator activated receptor γ and interleukin-6 in epicardial fatcompared to subcutaneous fat. Correlation analysis of biochemical findings to clinical outcomes(using Society of Thoracic Surgeon’s database) showed an inverse correlation between circulatingadiponectin levels (both total and HMW) to previous myocardial infarction or congestive heart failureand serum creatinine and a positive correlation with high density lipoprotein. The patients whounderwent urgent CABG procedure had lower adiponectin levels compared to an electiveprocedure.The multiplex analysis (39 chemokines/cytokine array) of the plasma samples obtained from CADpatients revealed that several cytokines/chemokines were altered in a sex specific manner. Inparticular, we observed differences in the circulating protein levels of the linked chromosome16q13chemokines (fractalkine, macrophage derived chemokine (MDC)) in patients with CAD. Similarchanges in the tissue mRNA levels of 16q13 chemokines in the epicardial fat compared tosubcutaneous fat using real time PCR were obtained which correlated with the circulating levels ofPHS 2590 (Rev. 06/09)Continuation Format Page