Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium A.J. Siddiqui, a, * P. Blomberg, b E. W€ ardell, a I. Hellgren, a M. Eskandarpour, b K.B. Islam, b and C. Sylv en a a Department of Cardiology, Karolinska Institute, Stockholm, Sweden b Gene Therapy Center, Clinical Research Center, Stockholm, Sweden Received 9 September 2003 Abstract We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A 165 and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A 165 alone increased the myocardial cap- illary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A 165 and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A 165 boosts endogenous arterio- genesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Gene therapy; Vascular endothelial growth factor; Angiopoietin-1; Myocardial ischemia; Arteriogenesis The development of blood vessels in adult tissues is a complex process in which different growth factors and cytokines act in concert. Angiogenic growth factors targeting endothelial cells are believed to be the initial players in this complex multistage process. Vascular endothelial growth factor (VEGF) has been identified as a key component in most of the above conditions [1,2]. VEGF-A is an important isoform of the VEGF family that acts via two receptors (VEGFR-1 and VEGFR-2) specifically located in the vascular endothelial cell but also in vascular smooth muscle. Angiopoietins are one another class of important co-players with VEGF [3]. Over-expression of angiopoietin-1 (Ang-1), a Tie-2 li- gand and also an endothelial cell-specific growth factor, has been shown to produce highly branched and nu- merous leakage resistant blood vessels in the skin of transgenic mice [4]. Ang-1 also stimulates the formation of pericytes and smooth muscle cells with endothelial cells and thus provides maturity and stability of newly formed blood vessels. On the other hand, chronic VEGF overexpression in transgenic mice has been reported to produce numerous small, winding vessels lacking func- tional layers [4]. Combination gene therapy with several growth factors is a rational approach to creating more stable vessels for functional improvement in ischemic tissue. Co-administration of VEGF and Ang-1 modified angi- ogenesis and reduced vascular permeability in the skin of transgenic mice models and ischemic hind limb models [5–9]. Combined VEGF/VEGFR-2 and Ang-1/ Tie-2 signalling pathways also play an important role in the mobilisation and recruitment of hematopoietic stem cells and circulating endothelial progenitor cells [10]. Since there has been no report on the therapeutic effects of a combination of these growth factors in the myo- cardium, the present study was designed to investigate whether transient cardiac over-expression induced by phVEGF-A 165 and/or phAng-1 gene therapy could en- hance myocardial angiogenesis and whether there are * Corresponding author. Fax: +46-8-585-867-10. E-mail address: Anwar.J.Siddiqui@medhs.ki.se (A.J. Siddiqui). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.111 Biochemical and Biophysical Research Communications 310 (2003) 1002–1009 BBRC www.elsevier.com/locate/ybbrc