Arterial tissues were obtained from explanted hearts from nine patients recruited as subjects for heart transplantation at the University of Colorado Anschutz Medical Campus. Tissues were collected under the Human Heart Tissue Bank protocol (COMIRB 01-568 PI P. Buttrick, Chief Cardiology Division; co-I for vascular studies K. Moulton). All appropriate informed consent was received from subjects and confirmation was obtained that they were aware that their samples would be used in research. For artery harvest, immediately after explant, left and right, left anterior descending, left marginal, and left circumflex coronary arteries as well as aortic tissues were dissected from diseased hearts, cleaned of fatty and cardiac muscle tissues, and processed for histology; a total of 24 individual vessels were analysed. After histological processing and haematoxylin and eosin staining, vascular sections were reviewed by Dr Moulton and characterized based on relative size of atheroma, presence of plaque neovascularization, composition of cells, lipid and matrix and media attenuation. De-coded and de-identified arterial tissues sections were obtained from Dr Moulton and were immunohistochemically or immunofluorescently stained for PTEN; patient clinical data were not available for these tissues. For immunohistochemistry, formalin-fixed, paraffin-embedded tissues were deparaffinized, rehydrated and underwent antigen retrieval by heating for 20 min at 100 °C in a decloaking chamber (Biocare). Antigen/antibody complexes were visualized using kits from Vector Laboratories and sections lightly counterstained with haematoxylin. Sections were imaged using an Olympus BX41 microscope equipped with SPOT software. Antibody used was monoclonal anti-PTEN (1:100; Cascade Bioscience, Winchester, MA). For immunofluorescent double staining, tissues were pre-treated as described above and sequentially incubated with a polyclonal anti-PTEN antibody (1:50; Millipore) followed by incubation with a cy3-conjugated monoclonal anti-αSMA antibody (Sigma). To detect PTEN expression, a TSA Biotin amplification system was used (Perkin Elmer). Briefly, following incubation with anti-PTEN, sections were sequentially incubated with secondary biotinylated anti-rabbit IgG (1:400), HRP-conjugated Streptavidin (1:100), BiotinylTyramide (1:50) and Alexa-488-conjugated Streptavidin. Sections were imaged using a laser-scanning confocal microscope (510 META NLO) and analysed using LSM 510 software.
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