CARDIAC MYOCYTE p300 LEVELS REGULATE MYOCARDIAL ANGIOGENESIS Mônica Pessanha, Jian Qin Wei, Lina Shehadeh, Joanne Faysal, Keith A. Webster and Nanette H. Bishopric* Univ. of Miami School of Medicine, Dept. of Molecular and Cellular Pharmacology, RMSB 6026, PO Box 016189 (R-189), Miami, FL 33101. *
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INTRODUCTION The transcriptional coactivator p300 is a nuclear histone acetyltransferase (HAT) that modifies chromatin in a sequence-specific manner to permit transcriptional activation, and is required for tissue-specific transcription in many tissues, including the heart. We have previously shown that cardiac myocyte growth is exquisitely sensitive to p300 levels, that p300 is upregulated during hypertrophic growth of the heart, and that p300 overexpression is necessary and sufficient to induce cardiac myocyte hypertrophy. Here we demonstrate that targeted postnatal expression of p300 in cardiac myocytes results in marked increase in the density and size of cardiac blood vessels. METHODS A hemagglutinin-tagged full length p300 cDNA transgene driven by the murine α-myosin heavy chain promoter was used to generate transgenic mouse strains expressing p300 at 2.5-fold above wild type levels in a cardiac myocyte-restricted manner. Tissue levels of p300 were verified by Western blotting. Heart weight, body weight and tibia length were measured at 1, 2 and 3 months of age, and myocardial samples were harvested and processed for histology and quantitative morphometry. Figure 1. Enlarged coronary arteries induced by elevated myocyte p300 levels.TG=female transgenic mouse aged 6 mo. WT = wild type female littermate. Arrow indicates prominent epicardial coronary artery. (LA, RA = left and right atria. LV = left ventricle.
RESULTS Myocardial levels of p300 were increased by ~2.5-fold over non-transgenic littermates by 2 weeks of age. At sacrifice, gross cardiac hypertrophy and enlargement of the epicardial arteries were noted (Figure 1). Histology and morphometric analysis of hematoxylin and eosin-stained myocardial sections performed on 1,2 and 3 month old wt and p300 transgenic (p300Tg) littermates revealed marked cardiac myocyte hypertrophy as early 1 mo of age (Fig. 2A). Vessel density and length were each significantly increased at all time points(Figs 2B and 2C)). Overall collagen content was not increased, but perivascular elastin and collagen were prominent, suggesting growth of mature vessels (Fig D). Microarray analysis of myocardial tissue at 1 month indicated upregulation of several angiogenic regulatory factors, including a 1.4-fold increase in Angpt4 (angiopoietin-like protein 4). Western analysis showed a 1.7-fold increase in
myocardial levels of vascular endothelial growth factor (VEGF) and 2.0-fold increase in Jun (AP-1), a transcriptional regulator of VEGF.
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Figure 2. Enlargement of both myocytes and blood vessels in p300 transgenic mouse hearts. AC: Quantitative morphometry of 1,2 and 3 month old wt and p300 transgenic (p300Tg) myocardium. A. Myocyte cross-sectional area. B. Vessel density, calculated as percentage of total tissue volume. C. Vessel length in mm. D. Picrosirius Red staining of myocardial tissue from WT (upper ) and Tg (lower) animals at age 3 months, revealing increased perivascular collagens I and III. R = brightfield, L = fluorescence. For all groups, N= 5, p< 0.001 at 2 and3 mo for comparisons of WT and Tg mice.
DISCUSSION AP-1 and HIF-1 are both coactivated by p300 and are important transcriptional regulators of VEGF. Although p300 expression is restricted to myocytes in this model, increased p300 availability enhances myocyte production of VEGF, angiopoeitin-like-4 and possibly other factors under both aerobic and hypoxic conditions. Importantly, the signal gradient generated by p300-expressing myocytes promotes growth of large and medium-sized, mature blood vessels. The observation that intrinsic myocyte growth signals can drive compensatory paracrine growth of the blood vessel supply may shed light on angiogenic processes occurring in other tissues and during tumor growth. 1. Yao, T. P. et al. (1998) Cell 93, 361-372 2. Slepak, T. I. et al. (2001) J. Biol. Chem. 276, 7575-85 3. Wei JQ, et al. (2003) Nature Biotechnology Short Reports, 14:51-52
