Study subject enrollment
Research was performed in accordance with study protocols approved by Maine Medical Center Institutional Review Board, which is accredited by the Association for the Accreditation of Human Research Protection Programs (AAHRPP). The study cohort consisted of 23 subjects recruited to undergo intra-operative myocardial biopsy at the time of scheduled coronary artery bypass grafting surgery at Maine Medical Center (MMC) in Portland, Maine. All subjects provided informed consent approved by the MMC Institutional Review Board. All subjects were over 18 years of age. Subjects with known active myocarditis, hypertrophic cardiomyopathy, constrictive pericarditis, significant valvular and/or pericardial disease, severe pulmonary hypertension, significant hepatic disease or renal impairment (creatinine > 2.5 mg/dL), severe ventricular arrhythmias, malignancy other than non-melanoma skin cancers, expected survival less than one year and inability to provide informed consent were excluded. Of the 25 patients that were enrolled in this study, two were excluded for missing LV epicardial biopsies, leaving 23 patients for the data analysis.
Reagents
EBM-2 Basal Medium and EGM-2 SingleQuot Kit supplement/growth factors were purchased from Lonza Walkersville, Inc. (Walkersville, MD), and EGM Cell Growth Medium-2 was prepared according to the manufacturer's instructions. NRG-1 (ECD, 377-HB) and EGF (236-EG) were purchased from Bio-Techne/R&D Systems. TAK-165 and GI 254023X were obtained from Tocris Bioscience (Bristol, UK) and diluted in dimethyl sulfoxide (Cell Culture grade, Sigma). Collagenase II (345 units per mg, CLS-2) was purchased from Worthington Biochemical Corporation (Lakewood, NJ), dispase II (04942078001) was from Roche Life Science (Indianapolis, IN). D-glucose and DNase I was from Sigma (St. Louis, MO). For experiments involving cell stimulation, final concentrations of dimethyl sulfoxide did not exceed 0.1%.
Human microvascular endothelial cells (HMEC-1)
HMEC-1 cells were cultured as previously described 19, 20. In brief, cells were maintained in M-199 medium supplemented with 15% (v/v) fetal bovine serum and 0.3 μg/ml endothelial cell growth supplement (ECGS, Sigma) under a humidified atmosphere of air-CO2 (19:1) at 37°C.
For high glucose experiments, HMEC-1 cells were transferred into a 15% FBS M-199 medium without ECGS containing either 5 mM or 25 mM D-glucose.
Blood sample collection
Blood samples from subjects were obtained immediately before surgery, post-anesthesia induction, but prior to skin incision. Venous blood (10 ml) was collected using BD Vacutainer ACD tubes. Small aliquots (50 µl) were used for flow cytometry analysis. Platelet-free blood plasma was prepared at room temperature using two-step centrifugation, each at 2,000 x g for 20 minutes.
LV epicardial biopsy
All procedures for tissue procurement were performed in compliance with institutional guidelines for human research and an institutional review board-approved protocol at MMC. Anterior LV free wall epicardial biopsies (average weight 26.4±1.8 mg) were obtained during planned coronary artery bypass surgery soon after the patient was placed on cardiopulmonary bypass, as described 21, 22. The epicardial biopsy was placed in a serum-free DMEM medium and kept on wet ice. All samples were processed within 3 hours of collection. All patients were followed postoperatively until discharge. No adverse effects or post-operative complications ascribable to the biopsy were detected, and all patients were discharged alive.
Preparation of cell suspension from LV epicardial biopsy
Preparation of cell suspension was performed according to a protocol published previously 23. In brief, minced tissue was incubated in digestion solution (10 mg/ml collagenase II, 2.5 U/ml dispase II, 1 μg/ml DNase I, and 2.5 mM CaCl2) for 45 minutes at 37°C. After passing through a 70-μm cell strainer, the resulting myocyte-free single-cell suspension was centrifuged at 500 x g, washed with Dulbecco's PBS, and resuspended in PBS/0.5%BSA/2mM EDTA. Cells were counted, and the cell suspension was divided into two parts. One part was immediately used for flow cytometric analysis, and the other part was used for cell isolation.
Isolation of CD105+CD31-CD45- cells
Endothelial cells and immune cells were labeled with APC-conjugated CD31 (WM59) and CD45 (HI30) and removed from epicardial cell suspension using anti-APC MicroBeads and MS columns (Miltenyi Biotec, Inc.). The CD105+ cells were magnetically isolated from CD31, and CD45 depleted cell suspension using PE-conjugated CD105 (43A3) and anti-PE MicroBeads (Miltenyi Biotec, Inc.). Cells were grown in M199-EGM-2 (3:1, v/v) supplemented with SingleQuotsTM Supplements (CC-4176, Lonza), 10% FBS and 1× antibiotic-antimycotic solution. Cells between passages 2-5 were used for experiments.
For the experiments with high glucose, epicardial CD105+CD31-CD45- cells were transferred into a 10% FBS M199-EBM-2 medium without growth factor supplement, containing either 5 mM or 25 mM D-glucose.
Flow cytometric analysis
Cells (106 or less/ml) were treated with Human TruStain FcX™ (Biolegend, San Diego, CA) to prevent non-specific binding, followed by incubation with relevant antibodies for 25 minutes at 4˚C. Cell-surface antigen expression was examined using the following antibodies: CD31 (WM59), CD45 (HI30), and CD105 (43A3) (all from BioLegend, San Diego, CA).
ErbB receptors were assayed using PE-conjugated anti-human ErbB2 (Fab1129P) and IgG2b isotype-matched control (IC0041P), ErbB3 (Fab3481P) and IgG1 control (IC002P), ErbB4 (Fab11311P) and IgG2a (IC003P) isotype control. All anti-ErbB antibodies and controls were purchased from R&D Systems (Minneapolis, MN). All antibodies were titrated to establish high separation between positive and negative cell populations while maintaining a low level of background. Isotype-matched control antibodies were used to determine the level of undesired non-specific binding.
For intracellular staining, cells were fixed and permeabilized using DB Cytofix/Cytopermä (554714, BD Biosciences). The total level of ErbB2 protein was determined using mouse monoclonal Anti-Neu/ErbB2/HER2 Antibody (3B5) (sc-33684, Santa Cruz Biotechnology) in combination with anti-mouse PE-conjugated IgG (715-116-150, Jackson ImmunoResearch Labs). The level of phosphorylated ErbB2 was measured using rabbit anti-phospho-HER2/ErbB2 (clone: D66B7, Cell Signaling Technology) in combination with anti-rabbit APC-conjugated IgG (711-136-152, Jackson ImmunoResearch Labs).
Fluorescence spillover in multiparametric multicolor flow cytometric analysis was corrected after including all relevant antibodies in the other channels along with an isotype control antibody in a single channel of interest. The expression of cell markers and ErbB receptors was determined after subtraction of the mean fluorescence intensity (MFI) of isotype-matched controls from the MFI of specific antibodies and represented as a delta mean fluorescence intensity (DMFI) as described elsewhere.
Data acquisition was performed on a MacsQuant Analyzer 10 (Miltenyi Biotec., Inc.), and the data were analyzed using WinList 5.0 software. Erythrocytes were lysed with ammonium chloride. Viable and non-viable cells were distinguished using DAPI (to detect dead nucleated cells) and LIVE/DEAD® Fixable Violet Stain kit for detection of non-nucleated cell debris (Life Technologies, Carlsbad, CA).
Analysis of circulating IL-6, IL-8 and TNFa.
The levels of IL-6, IL-8 and TNFa in platelets free blood plasma were measured using Human DuoSet ELISA kits (Bio-techne/R&D Systems).
Statistical analysis
Shapiro-Wilk test was used to determine variable distribution. Comparisons between two groups were performed using two-tailed unpaired t-tests (normal distribution) or Mann-Whitney for skewed distribution. Comparisons between three or more groups were performed using one-way or two-way ANOVA with Tukey's multiple comparisons test. For continuous variables, correlation analysis was performed using either Pearson correlation (normal) or Spearman's rank test (skewed distribution correlation). A P-value < 0.05 was considered significant.