Adeno-Associated Viral Vector Delivered Cardiac-Specific and Hypoxia-Inducible VEGF Expression in the Ischemic Mouse Hearts

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.

[1]  S. Solomon,et al.  Hypoxia-regulated therapeutic gene as a preemptive treatment strategy against ischemia/reperfusion tissue injury. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Loda,et al.  Potential for germ line transmission after intramyocardial gene delivery by adeno-associated virus. , 2004, Biochemical and biophysical research communications.

[3]  P. Krieg,et al.  Molecular regulation of cardiac chamber-specific gene expression. , 2004, Trends in cardiovascular medicine.

[4]  Y. Kan,et al.  Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Henry,et al.  Pharmacological Treatment of Coronary Artery Disease With Recombinant Fibroblast Growth Factor-2: Double-Blind, Randomized, Controlled Clinical Trial , 2002, Circulation.

[6]  K. Schmidt-Ott,et al.  Vigilant Vector: Heart-Specific Promoter in an Adeno-Associated Virus Vector for Cardioprotection , 2002, Hypertension.

[7]  E. Manseau,et al.  Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery. , 2001, The American journal of pathology.

[8]  Y. Kan,et al.  Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Pearlman,et al.  Intracoronary basic fibroblast growth factor (FGF-2) in patients with severe ischemic heart disease: results of a phase I open-label dose escalation study. , 2000, Journal of the American College of Cardiology.

[10]  F. Agani,et al.  Expression of hypoxia-inducible factor-1alpha in the brain of rats during chronic hypoxia. , 2000, Journal of applied physiology.

[11]  H. Blau,et al.  VEGF gene delivery to myocardium: deleterious effects of unregulated expression. , 2000, Circulation.

[12]  L. Kedes,et al.  Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat--angiogenesis and angioma formation. , 2000, Journal of the American College of Cardiology.

[13]  Dian Feng,et al.  Heterogeneity of the Angiogenic Response Induced in Different Normal Adult Tissues by Vascular Permeability Factor/Vascular Endothelial Growth Factor , 2000, Laboratory Investigation.

[14]  J. Isner,et al.  Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. , 1998, Circulation.

[15]  J. Isner,et al.  Direct intramuscular injection of plasmid DNA encoding angiopoietin-1 but not angiopoietin-2 augments revascularization in the rabbit ischemic hindlimb. , 1998, Circulation.

[16]  H. Blau,et al.  VEGF gene delivery to muscle: potential role for vasculogenesis in adults. , 1998, Molecular cell.

[17]  P. Opolon,et al.  Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors. , 1998, Human gene therapy.

[18]  L. Villarreal,et al.  Adeno-associated virus vectors can be efficiently produced without helper virus , 1998, Gene Therapy.

[19]  B. V. von Specht,et al.  Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease. , 1998, Circulation.

[20]  H. Katus,et al.  Analysis of tissue-specific gene delivery by recombinant adenoviruses containing cardiac-specific promoters , 1997 .

[21]  Takayuki Asahara,et al.  Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb , 1996, The Lancet.

[22]  R. Harvey,et al.  An HF-1a/HF-1b/MEF-2 combinatorial element confers cardiac ventricular specificity and established an anterior-posterior gradient of expression. , 1996, Development.

[23]  Peipei Ping,et al.  Intracoronary gene transfer of fibroblast growth factor–5 increases blood flow and contractile function in an ischemic region of the heart , 1996, Nature Medicine.

[24]  J. Pearlman,et al.  Magnetic resonance mapping demonstrates benefits of VEGF–induced myocardial angiogenesis , 1995, Nature Medicine.

[25]  J. Isner,et al.  Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. , 1994, Circulation.

[26]  W Grossman,et al.  Basic fibroblast growth factor improves myocardial function in chronically ischemic porcine hearts. , 1994, The Journal of clinical investigation.

[27]  K. Chien,et al.  Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice , 1994, Molecular and cellular biology.

[28]  K. Chien,et al.  Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. Brem,et al.  Myosin light chain-2 luciferase transgenic mice reveal distinct regulatory programs for cardiac and skeletal muscle-specific expression of a single contractile protein gene. , 1992, The Journal of biological chemistry.

[30]  K. Chien,et al.  A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and alpha-adrenergic-inducible expression in cultured neonatal rat myocardial cells , 1991, Molecular and cellular biology.

[31]  B W Kimes,et al.  Properties of a clonal muscle cell line from rat heart. , 1976, Experimental cell research.