DNA Microarray and Quantitative Analysis Reveal Enhanced Myocardial VEGF Expression with Stunted Angiogenesis in Human Tetralogy of Fallot

Tetralogy of Fallot (ToF) is a cyanotic congenital heart disease with prominent right ventricular hypertrophy (RVH) associated with impaired myocardial oxygen and nutrient supply. Consequently, the right ventricle may manifest in altered molecular phenotype with a number of adaptive and inherited gene profiles which are largely unknown. The aim of the present study was to investigate the myocardial differential gene expression profile and to assess myocardial vascularisation in patients with ToF. DNA microarray analysis on right ventricular biopsies from ToF-patients operated for primary corrective surgery (referred as ToF-1; n = 12, mean age 0.5 year) and age matched controls (n = 6) was validated by Northern hybridisation and RT-PCR. Employing immunohistochemistry and video image analysis expression of vascular endothelial growth factor (VEGF), vascular density (by α-SMA and CD31 staining) and myocyte cross sectional area (Gomori's reticuline staining) were assessed in ToF-1 and adult patients (referred as ToF-2, n = 12, mean age 30 years) who underwent surgery for pulmonary regurgitation and compared the data with respective age matched controls (n = 6/12). DNA microarray analysis revealed altered expression pattern for 236 genes including enhanced (1.5-2.2-fold) expression of angiogenic factors and their receptors including; VEGF, flt-1, flk-1 angiopoietin-2, FGF-2, FGF-R1, PDGF-A, whereas, flt-4, Tie, TGF-β, TGF-β3R showed decreased (1.6-3.4-fold) expression in ToF-patients. Northern blot analysis verified the expression patterns of VEGF and flk-1 in both ToF-1 and ToF-2 patients. VEGF staining in cardiomyocytes was increased in ToF-1 (1.5-fold, p < 0.05) as compared to ToF-2. Video image analysis revealed enhanced vascular density (p < 0.01) with enlarged myocyte cross sectional area (p < 0.01), but vascular wall thickness remained unchanged in ToF-1 patients as compared to age matched controls. Our data suggest that RVH is associated with profound changes in gene profile for a number of genes, where VEGF/VEGF-R system contributes to enhance, but stunted myocardial angiogenesis in patients with ToF.

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