This is very similar to the findings in human at early stages of the cardiac disease

24 hours in 200 ml EGM-2MV medium or medium without serum and growth factors either supplemented with HDL or bovine serum albumin. Next, the medium was removed and cells were extensively washed. Adherent cells were fixed with a 4% paraformaldehyde solution and stained with FITC-labeled isolectin for 1 hour. The number of positive cells per microscopy field was quantified in a blinded fashion. relaxation were measured. The end-diastolic LV pressure was calculated manually from the pressure in function of time curves. The time constant of isovolumetric LV pressure fall was calculated using the method of Weiss et al.. Arterial blood pressure measurements were obtained after withdrawal of the catheter from the LV to the ascending aorta. Data were registered with a Powerlab Bridge Amplifier and Chart Software. Bone Marrow EPC Isolation and Quantification Bone marrow mononuclear cells were isolated by density gradient centrifugation using Histopaque-1077 as described. Immediately following isolation, cells were plated onto fibronectin-coated 24-well plates at a density of 46106 cells/well and cultured in EGM-2MV BulletKit medium. After 7 days of culture, the number of EPCs, identified as Dil-ac-LDL isolectin double positive cells, was quantified in randomly selected microscopy fields. Real-time Quantitative Reverse Transcriptase Polymerase Chain Reaction Analysis Six weeks after gene transfer or saline injection, hearts were dissected, briefly rinsed with saline buffer, snap-frozen, and stored at 280uC until use. RNA was extracted from the left ventricular myocardium using TRIzol reagent and the PurelinkTM RNA Mini Kit. An on-column DNase treatment was performed using PurelinkTM DNase according to the manufacturer’s protocol. Total RNA was reverse transcribed using the QuantiTect Reverse Transcription kit. Real-time quantitative reverse transcriptase-polymerase chain reaction was performed on a 7500 FAST real-time PCR system using the TaqMan Fast Universal PCR Master Mix and a premade mix containing primers and MGB probes to quantify Atp2a2 and Nos3 cDNA levels. The glyceraldehyde 3phosphate dehydrogenase housekeeping gene was used as endogenous control. Data analysis was performed using DDCtbased fold-change calculations. Tissue Preparation for Histological Analysis Hearts were harvested for histological analysis 6 weeks after gene transfer or saline injection. Mice were perfused via the abdominal aorta with phosphate-buffered saline and hearts were arrested in diastole by CdCl, followed by perfusion fixation with 1% paraformaldehyde in PBS. After dissection, hearts were post-fixated overnight in a 1% paraformaldehyde solution, embedded in paraffin, and 6 mm thick cross-GFT505 cost sections at 130 mm spaced intervals were made extending from the apex to the basal part of the left ventricle. Morphometric Analysis of the Myocardium Laminin staining was performed with rabbit anti-mouse laminin antibodies. Cardiomyocyte cross-sectional area was analyzed on laminin stained sections by measuring at least 200 randomly selected cardiomyocytes in the myocardium. Two mid-ventricular cross-sections were analyzed per mouse. Cardiomyocyte density was determined on the same laminin stained sections by counting the number of cross-sectioned round shaped cardiomyocytes per mm2 of LV myocardium. Relative vascularity of the myocardium was determined as and was assessed on sections double stained for rat anti-mouse CD31 and rabbit antimouse laminin. Computer-assisted imag